WO2022081864A1 - Methods and compositions for increasing genetic diversity in cannabis plants - Google Patents

Abstract

Disclosed herein are methods for inducing mutations of interest in Cannabis plants, seeds, and other plant tissue or cells, and populations of mutated Cannabis seeds, plants, and other plant tissue or cells prepared by the methods disclosed herein.

Description

METHODS AND COMPOSITIONS FOR INCREASING GENETIC DIVERSITY IN CANNABIS PLANTS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/091,754, filed October 14, 2020, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present application relates to methods and compositions for increasing genetic diversity in Cannabis by introducing human-induced mutations into Cannabis plants, seeds, and other plant tissue or cells.

BACKGROUND

[0003] Cannabis refers to a genus of flowering plants in the family Cannabaceae. and includes at least three recognized species: Cannabis saliva. Cannabis indica. and Cannabis ruderalis. Various types of Cannabis plants can exist within the same species, including narrow leaf and broad leaf types, as well as medicinal and non-medicinal types. Cannabis is a versatile plant producing products ranging from fiber extracted from stems for paper and textiles, seeds used for food and oil, and flowers producing secondary metabolites called cannabinoids. Cannabinoids include a range of compounds, including delta-9 tetrahydrocannabinol (“THC”) used for its psychoactive properties and cannabidiol (“CBD”), which is used for therapeutic purposes such as the treatment of certain types of epilepsy, nausea, and for pain and inflammation.

[0004] Cannabis varieties can be classified into five classes of cannabinoid content referred to as chemotypes or chemovars. Chemotype 1 (marijuana) has high THC and low CBD content. Chemotype 2 has approximately equal amounts of THC and CBD. Chemotype 3 (hemp) has high CBD and low THC. Chemotype 4 has high cannabigerol (“CBG”), a precursor of THC and CBD. Chemotype 5 does not produce cannabinoids. Hemp is defined by the Agricultural Improvement Act of 2018 as any Cannabis plant, or derivative thereof, that contains not more than 0.3% THC on a dry-weight basis.

[0005] Cannabis is typically dioecious, having male (XY, staminate) and female (XX, pistillate) plants, which develop male or female flowers on separate plants. Some Cannabis plants are monoecious, producing both male and female flowers. Female Cannabis flowers are characterized by pistils protruding from a calyx. The resinous glandular trichomes of the calyx are the primary site of cannabinoid synthesis. The ovaries are contained within the female calyx and, therefore, the calyx is site of seed development after fertilization by pollen. [0006] Unlike many agricultural crops that have been bred and selected for traits of interest over many decades, research and breeding of Cannabis have lagged in comparison due to limited access to plants for breeding. There is a need to accelerate Cannabis breeding by producing large amounts of genetic diversity to allow the selection and development of new and improved traits.

[0007] The present application is directed to overcoming these and other deficiencies in the art.

SUMMARY

[0008] A first aspect of the present application is directed to a method for introducing mutations of interest in a Cannabis plant. This method involves mutating a Cannabis plant by providing a plurality of Cannabis seeds with cracked seed coats and contacting the Cannabis seeds with a mutagenic agent to mutate the Cannabis seeds to produce a first population of mutated (Ml) seeds. The method further involves planting and growing the mutagenized Ml seeds into Ml plants, fertilizing the Ml plants with pollen under conditions effective for the Ml plants to produce a second population of mutated (M2) seeds. The method further involves planting and growing the M2 seeds into M2 plants, collecting DNA from the M2 plants, and harvesting progeny of the M2 plants, where the progeny comprise mutations.

[0009] Another aspect of the present application is directed to a method for introducing mutations of interest in a Cannabis plant. This method involves mutating a Cannabis plant by providing a plurality of Cannabis seeds with cracked seed coats; contacting the Cannabis seeds with a mutagenic agent to mutate the Cannabis seeds to produce a first population of mutated (Ml) seeds; planting and growing the mutagenized Ml seeds into Ml plants; collecting mutated (M2) pollen from the Ml plants; fertilizing Cannabis plants with M2 pollen under conditions effective for the Cannabis plants to produce mutated (M2) seeds; planting and growing the M2 seeds into M2 plants; collecting DNA from the M2 plants; and harvesting progeny of the M2 plants, where the progeny comprise mutations.

[0010] Another aspect of the present application relates to a population of mutated Cannabis seeds prepared by the methods described herein.

[0011] A further aspect of the present application relates to a method for introducing mutations of interest in a Cannabis plant. This method involves mutating a Cannabis plant, where said mutating comprises: providing a cell from a Cannabis plant; and contacting the cell with a mutagenic agent to mutate the cell to produce a first mutated (Ml) cell; culturing the Ml cell to produce an Ml plant; fertilizing the Ml plant with pollen under conditions effective for the Ml plant to produce a mutated (M2) cell; culturing the M2 cell to produce an M2 plant; collecting DNA from the M2 plant; and harvesting progeny of the M2 plant, where the progeny comprise mutations.

[0012] Another aspect of the present application relates to a method for introducing mutations of interest in a Cannabis plant. This method involves providing a cell from a Cannabis plant; contacting the cell with a mutagenic agent to mutate the cell to produce a mutated (Ml) cell; culturing the Ml cell to produce an Ml plant; collecting mutated (M2) pollen from the Ml plant; fertilizing a Cannabis plant with M2 pollen under conditions effective for the fertilized Cannabis plant to produce a mutated (M2) cell; culturing the M2 cell to produce an M2 plant; collecting DNA from the M2 plant; and harvesting progeny of the M2 plant, where the progeny comprise mutations.

[0013] Described herein are methods effective to increase genetic diversity in Cannabis plants, seeds, pollen, and other plant tissues or cells. Novel conditions, including seed pretreatment, and mutagen types and duration of treatment have been identified, which effectively induce genetic diversity while producing a viable population upon which to discover novel alleles in genes of interest. Various schemes to generate a population of mutagenized plants of defined gender depending on the source material used for mutagenesis and crosses are described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGs. 1 A-F are summaries of schemes representing different embodiments of the methods of introducing mutations of interest in a Cannabis plant of the present application. The abbreviations used include: pollen donor “PD”, pollen receiver, “PR”, mutation of interest, “MOI”, wild type, non-mutagenized, “WT”, first mutagenized generation, “Ml”, second mutagenized generation, “M2”, third mutagenized generation, “M3”. FIG. 1 A lists the starting material, Ml plants selected, the origin of pollen used for crossing, the origin of females used in crossing, and any discarded plants in the Ml generation for Schemes 1-13. FIG. IB lists the gender of M2 plants selected, the origin of the pollen used for crossing, the origin of females used for crossing, and any discarded plants in the M2 generation for Schemes 1-13. FIG. 1C indicates if there is an M3 pollen library created, and the gender of the M3 plants for Schemes 1- 13. FIG. ID lists the starting material, Ml plants selected (if any), origin of pollen used for crossing, origin of females used for crossing, and any discarded plants in the Ml generation for Schemes 14-25. FIG. IE lists the gender of Ml plants selected, the origin of the pollen used for crossing, the origin of females used for crossing, and any discarded plants in the Ml generation for Schemes 14-25. FIG. IF indicates if there is an M2 pollen library created and the gender of the M2 plants for Schemes 14-25. [0015] FIG. 2 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 1.

[0016] FIG. 3 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 2.

[0017] FIG. 4 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 3.

[0018] FIG. 5 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 4.

[0019] FIG. 6 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 5.

[0020] FIG. 7 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 6.

[0021] FIG. 8 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 7.

[0022] FIG. 9 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 8.

[0023] FIG. 10 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 9.

[0024] FIG. 11 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 10.

[0025] FIG. 12 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 11. [0026] FIG. 13 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 12.

[0027] FIG. 14 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 13.

[0028] FIG. 15 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 14.

[0029] FIG. 16 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 15.

[0030] FIG. 17 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 16.

[0031] FIG. 18 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 17.

[0032] FIG. 19 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 18.

[0033] FIG. 20 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 19.

[0034] FIG. 21 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 20.

[0035] FIG. 22 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 21.

[0036] FIG. 23 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 22. [0037] FIG. 24 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 23.

[0038] FIG. 25 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 24.

[0039] FIG. 26 is a schematic illustration of one embodiment of a method of introducing mutations of interest in a Cannabis plant according to the present application, which is referred to as Scheme 25.

[0040] FIG. 27 is a graphical representation of mutations discovered by evaluating 31,172 gene sequences in Ml generation EMS and ENU mutagenized Cannabis plants compared to controls.

[0041] FIG. 28 is a graphical representation of mutations discovered by evaluating 31,172 gene sequences in M2 generation EMS and ENU mutagenized Cannabis plants compared to controls.

[0042] FIG. 29 is a photograph of Ml Cannabis plants from mutagenized seed at approximately 1 week old.

[0043] FIG. 30 is a photograph of Ml Cannabis plants from mutagenized seed at approximately 3 weeks old.

[0044] FIG. 31 is a photograph of an Ml Cannabis plant at approximately 6 weeks old showing chlorotic leaves due to the mutagenic treatment. Ml plants are chimeric for mutations.

[0045] FIG. 32 is a photograph of another example of an Ml Cannabis plant at approximately 4 weeks old showing chimerism for chlorotic leaves.

[0046] FIG. 33 is a photograph of a chimeric leaf from an Ml Cannabis plant at approximately 3 weeks old.

[0047] FIG. 34 is a photograph of an example of an Ml Cannabis plant at approximately 10 weeks old showing an altered inflorescence phenotype with long internodes and long pistils.

[0048] FIG. 35 is an alignment of a partial protein sequence showing the position of a leucine to isoleucine mutation at amino acid 664 (L664I) identified in an ENU treated Ml plant (Q27) and in the M2 progeny of that plant (M217) compared to a reference wild type sequence of LOCI 15697098 of the NCBI Cannabis sativa Annotation Release 100.

[0049] FIG. 36 is an alignment of a partial protein sequence showing the position of a serine to glycine mutation at amino acid 333 (S333G) identified in an Ml plant (Q27) and in the M2 progeny of that plant (M217) compared to a reference wild” type sequence of LOCI 15724176.

[0050] FIG. 37 is an alignment of a partial protein sequence showing the position of a glutamate to lysine mutation at amino acid 407 (E407K) identified in an M2 plant (M218) and a different mutation of a tryptophan to a stop codon at amino acid 483 in an unrelated Ml plant (QI 1) compared to a reference wild type sequence of LOCI 15701262. A stop codon can be indicated with a dash

for example, in the alignment of FIG. 37. Alternatively a stop codon can be indicated with an asterisk (“*”) when referring to the stop codon in the text. For example, the tryptophan to stop codon at amino acid 483, can be written as W483*.

[0051] FIG. 38 is an alignment of a partial protein sequence showing the position of a tryptophan to a stop codon at amino acid 257 (the stop codon is indicated with a dash in the figure, W257*) in an Ml plant (Q20), a glycine to serine mutation at amino acid 311 (G311 S) identified in a different Ml plant (QI 8), and an alanine to a glycine mutation at amino acid 323 (A323G) in an unrelated M2 plant (M220) as compared to a reference wild type sequence of LOCI 15705217.

DETAILED DESCRIPTION

[0052] A first aspect of the present application is directed to a method for introducing mutations of interest in a Cannabis plant. This method involves mutating a Cannabis plant by providing a plurality of Cannabis seeds with cracked seed coats and contacting the Cannabis seeds with a mutagenic agent to mutate the Cannabis seeds to produce a first population of mutated (Ml) seeds. The method further involves planting and growing the mutagenized Ml seeds into Ml plants, fertilizing the Ml plants with pollen under conditions effective for the Ml plants to produce a second population of mutated (M2) seeds. The method further involves planting and growing the M2 seeds into M2 plants, collecting DNA from the M2 plants, and harvesting progeny of the M2 plants, where the progeny comprise mutations.

[0053] Another aspect of the present application is directed to a method for introducing mutations of interest in a Cannabis plant. This method involves mutating a Cannabis plant by providing a plurality of Cannabis seeds with cracked seed coats; contacting the Cannabis seeds with a mutagenic agent to mutate the Cannabis seeds to produce a first population of mutated (Ml) seeds; planting and growing the mutagenized Ml seeds into Ml plants; collecting mutated (M2) pollen from the Ml plants; fertilizing Cannabis plants with M2 pollen under conditions effective for the Cannabis plants to produce mutated (M2) seeds; planting and growing the M2 seeds into M2 plants; collecting DNA from the M2 plants; and harvesting progeny of the M2 plants, where the progeny comprise mutations.

[0054] Most Cannabis plants are photoperiod sensitive. Photoperiod refers to a plant's response to the amount of light and darkness to which it is exposed. Depending on the genetics, light exposure events will trigger transcription factors, which activate flowering genes within plants. For example, ‘short-day’ plants, will grow vegetatively during long days (typically more than 12-14 hours of light), and only begin flowering once the light hours are reduced to a certain number. Typically, short-day plants will flower when the day is less than 12 hours (z.e., the night is longer than 12 hours) regardless of plant age or size. Most Cannabis sativa plants flower when the length of continuous darkness exceeds about 10-12 hours per 24-hour period or when daylight lengths only last about 12-14 hours. Under greenhouse or indoor growing conditions, Cannabis plants can be kept in vegetative growth by providing long day light conditions through natural and/or supplemental lighting, which is typically provided for 3-6 weeks. When a switch to flowering is desired, the lighting can be reduced to short day conditions (i.e., 12 hours of light).

[0055] Cannabis plants are typically dioecious meaning that plants are either male (XY, staminate) or female (XX, pistillate) plants, which develop male or female flowers, respectively. However, pollen can be induced from female (XX) plants by treatment of plant axillary meristems with silver compounds or by subjecting the plant to stress conditions (sub-optimal growth conditions). For example, a female plant can be treated with a silver containing solution. Such a treatment may be carried out through hand held spray bottles, standard agricultural or greenhouse spray or fog systems or devices. In one embodiment, a female plant can be sprayed with a silver containing solution one time or over a period of time. For example, a female plant can be sprayed with a silver containing solution every day, every other day, every 3 days, every 4 days, every 5 days, every 6 days, or more, over a period of 1, 2, 3, 4, or more applications. In one specific example, a female plant is sprayed or treated with a colloidal silver solution at a concentration amount of >30 ppm. In another embodiment, a female plant may be treated with a concentration of silver thiosulfate one or more times. In some embodiments, a female plant can be sprayed with 2 mM silver thiosulfate every 5 days starting 10 days before the start of short days for 3-4 applications. After several sprays or treatments, the female plants will start forming XX male flowers. Other forms of silver, including silver nitrate and silver thiosulfate can also be used. In some embodiments, the silver solution to trigger pollen formation is 2 mM silver thiosulfate, although other silver containing solutions and concentrations may also be used. In some embodiments, the silver solution to trigger pollen formation is about 0.1, 0.2, 0.3., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mM silver thiosulfate. Hormones such as gibberellins can also be used to induce male flowers on XX female Cannabis plants. Methods of inducing male flowers can be found in Ram and Sett, “Induction of Fertile Male Flowers in Genetically Female Cannabis sativa Plants by Silver Nitrate and Silver Thiosulfate Anionic Complex,” Theor. Applied Genetics, 62:369-375 (1982) and Ram and Jaiswal, “Induction of Male Flowers on Female Plants of Cannabis sativa by Gibberellins and its Inhibition by Abscisic Acid,” Planta 105:263-266 (1972), each of which is hereby incorporated by reference in its entirety.

[0056] By inducing female plants to develop male flowers, pollen that is genetically female (XX) can be produced and used to fertilize female flowers. In this way “feminized” (XX) seeds can be produced. As used herein, feminized seeds are seeds that are genetically female having been produced by fertilization of female flowers with female pollen.

[0057] In some embodiments (discussed infra), a plurality of feminized Cannabis seeds are mutagenized. In other embodiments, a plurality of mixed male and female seeds are mutagenized. In yet other embodiments, other plant tissue is mutagenized, such as tissue or cells from leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries, explants, axillary meristems, or protoplasts. In some embodiments, pollen is mutagenized. In some embodiments, feminized XX pollen is mutagenized. In some embodiments, XY pollen is mutagenized.

[0058] In some embodiments, a plurality of feminized or mixed gender seeds are germinated until their seed coats are cracked. In some embodiments, seeds are germinated by soaking in water for 24 hours +/- 2 hours at room temperature (RT) until germination of the seedling started to split the seed coat. In some embodiments, seeds are germinated for a time sufficient to split the seed coats of the majority of seeds. In some embodiments greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, greater than 70%, greater than 65%, greater than 60%, or greater than 50% of seeds have cracked seed coats. In some embodiments, greater than 90% of seeds have cracked seed coats. In some embodiments, greater than 95% of seeds have cracked seed coats.

[0059] After seed coats are cracked, seeds are contacted with a mutagenic agent. Any suitable mutagenic agent can be used for embodiments of the present application. Mutagens creating point mutations, deletions, insertions, rearrangements, transversions, transitions, or any combination thereof may be used. Suitable radiation mutagens include, without limitation, ultraviolet light, x-rays, gamma rays, and fast neutrons. Suitable chemical mutagens include, but are not limited to, ethyl methanesulfonate (“EMS”), methylmethane sulfonate (“MMS”), N- ethyl-N-nitrosourea (“ENU”), triethylmelamine (“TEM”), N-methyl-N-nitrosourea (“MNU”), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N’-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12 dimethyl-benz(a)anthracene (“DMBA”), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (“DEO”), diepoxybutane (“DEB”), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro- ethyl) aminopropylamino] acridine dihydrochloride (“ICR- 170”), sodium azide, formaldehyde, or combinations thereof.

[0060] In some embodiments, the mutagenic agent is EMS. In some embodiments, the mutagenic agent is EMS at a concentration less than 3.5%. In some embodiments, the mutagenic agent is EMS at a concentration less than 0.7%. In some embodiments, the mutagenic agent is EMS at concentrations between 0.1% and 0.7%. In some embodiments, the mutagenic agent is EMS at a concentration less than 0.1%. In some embodiments, the mutagenic agent is ENU. In some embodiments, the mutagenic agent is ENU at a concentration less than 3.5%. In some embodiments, the mutagenic agent is ENU at a concentration less than 0.7%. In some embodiments, the mutagenic agent is ENU at concentrations between 0.1% and 0.7%. In some embodiments, the mutagenic agent is ENU at a concentration less than 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01%. In some embodiments, the mutagenic agent is ENU at concentrations between 0.025% and 0.07%. The concentration of mutagenic agent may depend on the specific mutagenic agent being used and the type of plant tissue or cell being mutagenized.

[0061] In some embodiments, the contacting with the mutagenic agent occurs for 15 hours. In some embodiments the contacting with the mutagenic agent occurs for 10 hours. In some embodiments, the contacting with the mutagenic agents occurs for 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. In some embodiments the contacting with the mutagenic agent occurs for more than 20 hours.

[0062] In some embodiments, Cannabis seeds that are mutagenized are called “Ml seeds”. In these embodiments, Ml generation seeds are the first generation with human-induced mutations. Ml seeds are planted and grown into “Ml plants”. Ml plants produced from mutagenized Ml seeds are chimeric for mutations since different cells of the embryo including somatic and meristematic cells were subjected to mutagenesis in the seeds. Mutations in cells that contribute to the reproductive organs pass on mutations to the next generation. However, clonal propagation of Ml mutations in somatic tissue is possible in Cannabis. The Ml generation can be assessed for induced mutations such as chlorotic leaves to determine efficacy of mutagenic treatment.

[0063] Mutation frequency may be estimated by sequencing of mutagenized plant tissue, or by any other means suitable for detecting novel changes in DNA sequence. Suitable methods include, without limitation, Sanger sequencing, next generation sequencing approaches, mismatch enzyme cleavage detection using CEL I endonuclease (Colbert et al, “High- Throughput Screening for Induced Point Mutations,” Plant Physiology 126:480-484, (2001), which is hereby incorporated by reference in its entirety), denaturing high pressure liquid chromatography (“dHPLC”), constant denaturant capillary electrophoresis (“CDCE”), temperature gradient capillary electrophoresis (“TGCE”), among other methods. In another embodiment, human-induced mutations may be identified by next generation sequencing such as described in Krasileva et al., “Uncovering Hidden Variation in Polyploid Wheat,” Proc. Nat. Acad. Sci. 114-E913-E921 (2017), which is hereby incorporated by reference in its entirety.

[0064] The term “progeny” refers to any plant, seed, or gamete resulting from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance a progeny plant may be obtained by cloning or selfing of a parent plant or by crossing two parent plants. Progeny gametes such as pollen can also be produced.

[0065] The present application provides methods for crossing a first plant with a second plant. As used herein, the term “cross”, “crossing”, or “cross pollination” refers 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 back to one of the parents of the hybrid progeny. Crossing and backcrossing can be used to introduce one or more single mutations from one genetic background into another.

[0066] Pollen, ovules, or seeds produced from Ml plants derived from mutagenized seeds are considered the “M2” generation. Mutations in the M2 generation are not chimeric, and can be propagated in further generations. Fertilization of plants to produce the next M2 generation of seeds, can occur by fertilization of female flowers on the Ml plant or a wild-type plant, or by using Ml or wild-type pollen (male XY pollen or feminized XX pollen) on Ml or wild-type plants. After M2 generation seeds are collected, they can be planted. M2 plants can be individually barcoded to track both the DNA sampled from plant tissue of the M2 generation and progeny from each M2 plant. The resulting M2 DNA can be sequenced or used for any type of mutation detection methods to identify mutations in a gene of interest. Once a mutation of interest (“MOI”) is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the gene of interest.

[0067] In another embodiment, DNA or RNA from plants with induced or naturally occurring mutations can be screened with or without PCR by next generation sequencing methods such as exome capture or TILLING by sequencing (King et al., “Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing,” PloS One 10:e0137549 (2015); Tsai, et al., “Discovery of Rare Mutations in Populations: TILLING by Sequencing,” Plant Physiology 156: 1257-1268 (2011); Liu et al., “Gene Mapping via Bulked Segregant RNA-Seq (BSR-Seq),” PLoS One 7:e36406 (2012), each of which is hereby incorporated by reference it their entirety). [0068] As shown in Scheme 1 (FIG. 2), according to one embodiment seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, Ml males are selected and M2 pollen is collected from individual Ml males. M2 pollen is used to pollenate wild-type (unmutagenized) females. M2 seeds, which are heterozygous for human-induced mutations, are planted. Again M2 males are selected, individually tracked and used to collect M3 pollen, which is stored for an M3 pollen library. DNA from the individually tracked M2 males is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0069] In another embodiment, as shown in Scheme 2 (FIG. 3), seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, both Ml male and female plants are grown. Pollen is collected from individual Ml males and crossed to individual Ml females. M2 seeds are collected. Pollen from individually tracked M2 males is collected, individually barcoded and stored for an M3 pollen library. DNA from the individually tracked M2 males and females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0070] In yet other embodiments, as shown in Scheme 3 (FIG. 4), seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, only Ml female plants are grown. Pollen is collected from wild-type males and crossed to individual Ml females. M2 seeds are collected and planted. Individually tracked M2 females are pollinated with pollen from wild-type males. M3 seed is collected, individually barcoded and stored for an M3 library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0071] In a further embodiment, as shown in Scheme 4 (FIG. 5), seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, only Ml female plants are grown. Pollen is induced and collected from wild-type females and crossed to individual Ml females. M2 seeds are collected and planted. Individually tracked M2 females are pollinated with pollen from wild-type females. M3 seed is collected, individually barcoded and stored for an M3 library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0072] In another embodiment, as shown in Scheme 5 (FIG. 6), seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, only Ml female plants are selected. Pollen is induced and collected from Ml females and used to fertilize the same Ml female plants to self-pollinate them. Feminized M2 seeds are collected and planted, and female M2 plants are selected. Pollen from each M2 female is induced, collected and individually barcoded to form the M3 pollen library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0073] In another embodiment, as shown in Scheme 6 (FIG. 7), seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, only Ml female plants are selected. Pollen is induced, collected from Ml females and used to fertilize wild-type female plants. Feminized M2 seeds are collected and planted, and female M2 plants are selected. Pollen from each M2 female is induced and collected to form the M3 pollen library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0074] In some embodiments, as shown in Scheme 7 (FIG. 8), seeds of mixed gender are mutagenized and grown into Ml plants. In this embodiment, only Ml female plants are selected. Pollen is induced and collected from half the Ml female plants and used to fertilize the noninduced Ml female plants. Feminized M2 seeds are collected and planted, and female M2 plants are selected. Pollen from each M2 female is induced, collected, and individually barcoded to form the M3 pollen library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0075] In one embodiment, as shown in Scheme 8 (FIG. 9), feminized (XX) seeds are mutagenized and grown into Ml plants. Pollen from wild-type males is used to fertilize the Ml female plants. Mixed gender M2 seeds are collected and planted, and male M2 plants are selected. Pollen from each M2 male collected to form the M3 pollen library. DNA from the individually tracked M2 males is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0076] In a further embodiment, as shown in Scheme 9 (FIG. 10), feminized (XX) seeds are mutagenized and grown into Ml plants. Pollen from wild-type males is used to fertilize the Ml female plants. Mixed gender M2 seeds are collected and planted, and female M2 plants are selected and individually barcoded. Pollen from wild-type male plants is used to fertilize the M2 female plants. M3 seed is collected to form the M3 seed library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 seed library can be accessed to allow breeding of the mutation of interest.

[0077] In another embodiment, as shown in Scheme 10 (FIG. 11), feminized (XX) seeds are mutagenized and grown into Ml plants. Pollen is induced from wild-type females and used to fertilize the Ml female plants. Feminized M2 seeds are collected and planted, and female M2 plants are selected and individually barcoded. Pollen from wild-type female plants is again used to fertilize the M2 female plants. M3 seed is collected to form the M3 seed library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 seed library can be accessed to allow breeding of the mutation of interest.

[0078] In another embodiment, as shown in Scheme 11 (FIG. 12), feminized (XX) seeds are mutagenized and grown into Ml plants. Pollen is induced from Ml females and used to selfpollinate the Ml female plants. Feminized M2 seeds are collected and planted, and female M2 plants are selected and individually barcoded. Pollen is induced from M2 female plants and used to form the M3 pollen library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0079] In yet another embodiment, as shown in Scheme 12 (FIG. 13), feminized (XX) seeds are mutagenized and grown into Ml plants. Pollen is induced from Ml females and used to cross to wild-type female plants. Feminized M2 seeds are collected and planted, and female M2 plants are selected and individually barcoded. Pollen is induced from M2 female plants and used to form the M3 pollen library. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0080] In a further embodiment, as shown in Scheme 13 (FIG. 14), feminized (XX) seeds are mutagenized and grown into Ml plants. Pollen is induced from half the Ml females and used to cross-pollinate the other Ml female plants that were not induced to make pollen.

Feminized M2 seeds are collected and planted, and female M2 plants are selected and individually barcoded. Pollen is induced from half the M2 female plants and used to form an M3 pollen library and also to fertilize the other half of the M2 female plants. DNA from the individually tracked M2 females is collected for an M2 DNA library. In some embodiments, the M2 DNA library is screened for mutations in specific genes of interest. In other embodiments, the M2 DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M3 pollen library can be accessed to cross to females to allow breeding of the mutation of interest and the M3 seed library can be used to produce plants with mutations of interest.

[0081] Other embodiments start with mutagenesis of pollen. The mutagenized pollen is considered part of the Ml generation. The mutagenized Ml pollen is used to fertilize a female plant to produce seeds that are still considered the Ml generation. In this case, mutations in the Ml generation are not chimeric (having been directly induced in the gamete and not somatic tissue), and can be propagated in further generations. Ml plants derived from mutagenized pollen can be individually barcoded to track both the DNA sampled from plant tissue of the Ml generation and progeny from each Ml plant. The resulting Ml DNA can be sequenced or used for any type of mutation detection methods to identify mutations in a gene of interest. Fertilization to produce the next M2 generation of seeds can occur by fertilization of female flowers on the Ml plant or a wild-type plant, or by using Ml or wild-type pollen (male XY pollen or feminized XX pollen) on Ml or wild-type plants. Once a mutation of interest is identified in a gene of interest, the seeds of the Ml plant carrying that mutation are grown into adult M2 plants and screened for the phenotypic characteristics associated with the gene of interest.

[0082] As shown in Scheme 14 (FIG. 15), according to one embodiment, male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted, and Ml male plants are selected. M2 pollen is collected from individual Ml males and stored for an M2 pollen library. DNA from the individually tracked Ml males is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0083] In another embodiment, as shown in Scheme 15 (FIG. 16) male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Ml female plants are induced to form (XX) M2 pollen, the M2 pollen is collected from individual Ml females and stored for an M2 pollen library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 pollen library can be accessed to cross to females to allow breeding of the mutation of interest.

[0084] In a further embodiment, as shown in Scheme 16 (FIG. 17) male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Half the Ml female plants are induced to form (XX) M2 pollen and M2 pollen is collected from individual Ml females. The M2 pollen is stored for an M2 pollen library and also used for crosses to Ml female plants. M2 seeds from the crosses are stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed and pollen libraries can be accessed to allow breeding of the mutation of interest.

[0085] In another embodiment, as shown in Scheme 17 (FIG. 18) male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Wild-type female plants are induced to form (XX) pollen and used to fertilize the Ml female plants. M2 seeds from the crosses are stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed library can be accessed to allow breeding of the mutation of interest.

[0086] In some embodiments, as shown in Scheme 18 (FIG. 19) male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Wild-type male (XY) pollen is used to fertilize the Ml female plants. M2 seeds from the crosses are stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed library can be accessed to allow breeding of the mutation of interest.

[0087] In another embodiment, as shown in Scheme 19 (FIG. 20) male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. The Ml female plants are induced to form (XX) M2 pollen and M2 pollen is collected from individual Ml females. The M2 pollen is stored for an M2 pollen library and also used for self-pollination of the Ml female plants. M2 seeds from the crosses can also be collected and stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed and pollen libraries can be accessed to allow breeding of the mutation of interest.

[0088] In one embodiment, as shown in Scheme 20 (FIG. 21) male (XY) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml male and female plants are selected. The M2 pollen is collected from individual Ml males and used to fertilize the Ml females. The M2 pollen is stored for an M2 pollen library. M2 seeds from the crosses can also be collected and stored in an M2 seed library. DNA from the individually tracked Ml males and females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed and pollen libraries can be accessed to allow breeding of the mutation of interest.

[0089] In another embodiment, as shown in Scheme 21 (FIG. 22) female (XX) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. The Ml female plants are induced to form (XX) M2 pollen and M2 pollen is collected from individual Ml females. The M2 pollen is stored for an M2 pollen library and also used for fertilizing wild-type female plants. M2 seeds from the crosses can also be collected and stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed and pollen libraries can be accessed to allow breeding of the mutation of interest.

[0090] In a further embodiment, as shown in Scheme 22 (FIG. 23) female (XX) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Half of the Ml female plants are induced to form (XX) M2 pollen and M2 pollen is collected from these individual Ml females. The M2 pollen is stored for an M2 pollen library and also used for pollination of the Ml female plants. M2 seeds from the crosses can also be collected and stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed and pollen libraries can be accessed to allow breeding of the mutation of interest.

[0091] In a further embodiment, as shown in Scheme 23 (FIG. 24) female (XX) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. The Ml female plants are induced to form (XX) M2 pollen and M2 pollen is collected from individual Ml females. The M2 pollen is stored for an M2 pollen library and also used for self-pollination of the Ml female plants. M2 seeds from the crosses can also be collected and stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed and pollen libraries can be accessed to allow breeding of the mutation of interest.

[0092] In a further embodiment, as shown in Scheme 24 (FIG. 25) female (XX) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Wild-type male (XY) pollen is used to fertilize the Ml female plants. M2 seeds from the crosses are stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed library can be accessed to allow breeding of the mutation of interest.

[0093] In another embodiment, as shown in Scheme 25 (FIG. 26) female (XX) pollen is mutagenized and used to fertilize wild-type female plants to produce Ml seeds. In this embodiment, Ml seeds are planted and Ml female plants are selected. Wild-type female plants are induced to form (XX) pollen and used to fertilize the Ml female plants. M2 seeds from the crosses are stored in an M2 seed library. DNA from the individually tracked Ml females is collected for an Ml DNA library. In some embodiments, the Ml DNA library is screened for mutations in specific genes of interest. In other embodiments, the Ml DNA library is sequenced for mutations in many genes. As mutations of interest are identified, the M2 seed library can be accessed to allow breeding of the mutation of interest.

[0094] Mutations of interest can be brought to homozygosity by selfing a plant (i.e., to induce pollen on a female plant that can be used to self-pollinate that female plant or a sibling female plant with the same mutation, or by crossing a male and a female plant with the same mutation). In some embodiments, plants with different mutations in the same gene can be bred together. Plants with mutations of interest can also be bred to other varieties of Cannabis to develop new, unique Cannabis varieties and hybrids.

[0095] Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line which is the recurrent parent. The source of the trait to be transferred is called the donor parent, such as a Cannabis plant with a mutation of interest. After the initial cross of a plant with a mutation of interest to another plant, individuals possessing the mutations of interest from the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plants can be selfed to produce plants with homozygous mutations of interest. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait, such as the mutations of interest, transferred from the donor parent. Backcrossing methods can also be used with the Cannabis plants of the present application to improve or introduce one or more characteristics. [0096] A further aspect of the present application relates to a population of mutated Cannabis seeds prepared by any of the methods described herein.

[0097] A further aspect of the present application relates to a method for introducing mutations of interest in a Cannabis plant. This method involves mutating a Cannabis plant, where said mutating comprises: providing a cell from a Cannabis plant; and contacting the cell with a mutagenic agent to mutate the cell to produce a first mutated (Ml) cell; culturing the Ml cell to produce an Ml plant; fertilizing the Ml plant with pollen under conditions effective for the Ml plant to produce a mutated (M2) cell; culturing the M2 cell to produce an M2 plant; collecting DNA from the M2 plant; and harvesting progeny of the M2 plant, where the progeny comprise mutations.

[0098] Another aspect of the present application relates to a method for introducing mutations of interest in a Cannabis plant. This method involves providing a cell from a Cannabis plant; contacting the cell with a mutagenic agent to mutate the cell to produce a mutated (Ml) cell; culturing the Ml cell to produce an Ml plant; collecting mutated (M2) pollen from the Ml plant; fertilizing a Cannabis plant with M2 pollen under conditions effective for the fertilized Cannabis plant to produce a mutated (M2) cell; culturing the M2 cell to produce an M2 plant; collecting DNA from the M2 plant; and harvesting progeny of the M2 plant, where the progeny comprise mutations.

[0099] Culturing the Ml cell to produce an Ml plant can include tissue culture and regeneration of tissue. Means for regeneration vary from species to species of plant, but generally a petri plate containing explants or a suspension of transformed protoplasts is first provided. Callus tissue is formed and transformation of callus tissue can be performed. Shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, genotype, and history of the culture. If the Ml cell is a pollen cell, culturing the Ml cell can include fertilization of a female gamete to produce a seed.

[0100] Specific embodiments disclosed can be used or combined with other embodiments, without limitation.

EXAMPLES

Example 1 - Mutagenesis of Cannabis Seeds

[0101] In accordance with one exemplary embodiment of the application, Cannabis seeds of variety, BaOx, were pre-germinated by soaking in water for 24 hours +/- 2 hours at room temperature (RT) until germination of the seedling started to split the seed coat. After soaking, most seeds (95%) had uniformly split their seed coats. Unexpectedly, BaOx seeds were found to be very sensitive to the ENU mutagen compared to EMS (Table 1). Batches of approximately 275, 1,100, or 2,200 seeds with split seed coats were treated with ethylmethane sulfonate (EMS, Sigma Aldrich, St. Louis, MO) at concentrations of 0.35%, 0.5% or 0.6% (v/v) (~ 28 mM to 48.3 mM) by gently agitating at 45 rpm on a shaker in a fume hood. Similar numbers of seeds with split seed coats were treated with ethyl nitrosourea (ENU, Sigma Aldrich, St. Louis, MO) at 0.02%, 0.025%, 0.04%, 0.05% or 0.06% (v/v) by gently agitating at 45 rpm on a shaker in a fume hood. Following a 10-15 hour incubation, the mutagenized seeds were rinsed under running water for about 4-8 hours. Finally, the mutagenized seeds were planted in a 1 : 1 mixture of Sunshine soil mixes 4 and 5 (Sungro, Agawam, MA) and allowed to germinate in a greenhouse under day/night temperatures of 29°C/20°C and 18 hours light/6 hours dark. A total of approximately 13,860 mutagenized seeds were planted. Cannabis seeds were much more sensitive to ENU treatment than EMS treatment, such that ENU was used at 10-fold lower concentrations for mutagenesis. The lower ENU survival ratios were attributed to cytotoxicity, not to genotoxicity. Mutagen cytotoxicity affects survival and depends on the species. Survival of Cannabis Ml seeds after mutagenic treatment is shown in Table 1.

Table 1. Survival of Cannabis Seeds after Mutagenic Treatment

Example 2 - Ml Mutation Frequency Evaluation

[0102] DNA was extracted from Cannabis leaves from a sample of 10 Ml EMS mutagenized plants (called Q11-Q20), 10 Ml ENU mutagenized plants (called Q21-Q30), and 10 control unmutagenized plants, called (Q01-Q10).

[0103] The Ml plant DNA was prepared using methods and reagents based on the Qiagen® (Valencia, CA) DNeasy® 96 Plant Kit. Approximately 50 mg of frozen leaf tissue was placed in a sample tube with a zinc-plated steel bead, frozen in liquid nitrogen and ground 2 times for 45 seconds each at 21.5 Hz using the Retsch® Mixer Mill MM 300. Next, 300 pl of lysis buffer [Buffer API, solution DX and RNAse (100 mg/ml)] at 65° C was added to the sample. The tube was sealed and shaken for 15 seconds then briefly centrifuged at 5,200 x g. Following the addition of 100 pl precipitation (P3-like) buffer, the tube was shaken for 15 seconds. The samples were placed in a freezer at -20° C for 5 min then centrifuged for 20 minutes at 5,200 x g. A 300 pl aliquot of supernatant was transferred to a Whatman hydophilic GF/C Filter plate which was placed on another sample plate and centrifuged for 10 min at 5,200 x g. A new filter plate was placed on a sample plate and 400 pl of binding (AW1- like) buffer was added. The flow through from the previous step was then transferred to the new filter plate with buffer and centrifuged for 10 min at 5,200 x. Next, 650 pl of wash (AW2-like) buffer was added to the filter plate with bound DNA, and centrifuged for 10 min at 5,200 x g. The filter plate was then placed on a new set of sample tubes and 90 pl of elution (AE-like) buffer was applied to the filter. It was incubated at room temperature for 1 minute and then spun for 3 minutes at 5,200 x g. The filter plate was removed and the tubes containing the pooled filtrates were capped.

[0104] Purified DNA samples were prepared into whole genome sequencing libraries by Novogene (8801 Folsom Blvd, Suite 290, Sacramento, CA 95826), and sequenced on Illumina NovoSeq S4 lanes. A 1 pg aliquot of genomic DNA was randomly fragmented by sonication, then DNA fragments were end polished, A-tailed, and ligated with the full-length adapters for Illumina sequencing, and followed by further PCR amplification with P5 and indexed P7 oligos. The PCR products as the final construction of the libraries were purified with AMPure XP system. Then libraries were checked for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and quantified by real-time PCR (to meet the criteria of 3 nM). All libraries passed quality control checks.

[0105] Each sample’s sequencing reads were mapped to an annotated reference genome (CS10; NCBI Refseq: GCF_900626175.2, GenBank Accession No. NC_044371, which is hereby incorporated by reference in its entirety) with bwa-mem2 (Vasimuddin et al., “Efficient Architecture- Aware Acceleration of BWA-MEM for Multicore Systems,” IEEE Parallel and Distributed Processing Symposium (IPDPS), (2019), which is hereby incorporated by reference in its entirety), and duplicate reads were marked with samtools markduplicates (Li et al., “The Sequence alignment/map (SAM) format and SAMtools,” Bioinformatics 25(16):2078-9 (2009), which is hereby incorporated by reference in its entirety). Haplotypes were identified per sample with GATK4 Haplotype Caller, and then nucleotide variants were jointly called for all samples with GATK4 Genotype GVCFs (McKenna et al., “The Genome Analysis Toolkit: A Map Reduce Framework for Analyzing Next-Generation DNA Sequencing Data,” Genome Research 20: 1297-303 (2010), DePristo et al., “A Framework for Variation Discovery and Genotyping Using Next-Generation DNA Sequencing Data,” Nature Genetics 43:491-498 (2011), and Van der Auwera et al., “From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline,” Current Protocols In Bioinformatics 43 : 11.10.1-11.10.33 (2013), each of which is hereby incorporated by reference in their entirety). All loci, with or without detected polymorphisms, were output as a variant call format file (“VCF”).

[0106] Steps 1-6 below were performed with BCFTOOLS (Li., “A Statistical Framework for SNP Calling, Mutation Discovery, Association Mapping and Population Genetical Parameter Estimation from Sequencing Data,” Bioinformatics 27(21):2987-2993 (2011), which is hereby incorporated by reference in its entirety), and step 8 with R statistical language (version 4.0.2; R Core Team R: “A language and environment for statistical computing,” R Foundation for Statistical Computing, Vienna, Austria (2020), which is hereby incorporated in its entirety).

1) The resulting VCF was filtered for genic regions (n = 31, 172) according to the NCBI Cannabis sativa Annotation Release 100.

2) Insertion and deletion (“Indel”) records were excluded from all analyses.

3) The VCF was then filtered for loci that had a minimum of 20 aligned reads for each sample. This assured that there was enough power to detect variants at each sample’s locus.

4) After filtering the VCF file, keeping only loci that had the minimum threshold of read coverage, and removing all indels, the total number of remaining rows (all loci, with or without single nucleotide polymorphism (“SNP”) variants) served as the base denominator when calculating the mutation rate in subsequent steps (see step 8).

5) Loci that were not polymorphic were removed from the VCF.

6) Multiallelic loci were expanded into multiple rows, one row for each allele found for every locus.

7) The VCF was then converted to a tabular text format with bcftools’ query command.

8) Variants were removed if more than one minor allele existed across all samples, except when the allele may have been inherited from Ml to M2 generations. This step removed alleles that were likely inherited from ancestral genetic variation. De-novo mutations shared among individuals were expected to be extremely rare. The count of removed variants was subtracted from the base denominator for variant rate calculations.

9) The resulting unique alleles were counted for each sample to serve as the estimated number of novel mutations.

[0107] Two samples in the Ml sample set were found to have high mutation rates, even in mutation categories that were not expected for that mutagen (QI 7 EMS (0.377 /10,000 bp AT:CG transversions) and Q26 ENU (0.277 /10,000 bp AT:CG transversions)). These excess variants were likely due to outcrossing from pollen contamination and were removed from further analysis.

[0108] Chemically induced mutation rates were estimated by subtracting the mean number of found mutations in the control samples from each mutagenized sample (Table 2). Mutation rates (mutations / 10,000 bp) for each treatment and generation calculated from whole genome sequences for each sample. EMS and ENU treated samples had elevated mutation rates in the Ml generation. Table 2. Summary of Mutations Per 10,000 Base Pairs For Each Ml Treatment

[0109] For EMS mutagenesis, GC to AT transitions were the most common type of mutation. For ENU mutagenesis, GC to AT transitions, AT to GC transitions and AT to TA transversions were all identified.

Example 3 - M2 and M3 Library Production from Mutagenized Seeds

[0110] As described in Scheme 10 (FIG. 11), feminized seed of the Cannabis variety BaOX was mutagenized with EMS or ENU at the concentrations shown in Table 1 to create an Ml mutagenized hemp seed population. Ml plants in this population were crossed with wild type, feminized pollen from Cannabis variety Berry Blossom to create M2 seed. M2 seed was harvested from Ml plants and planted to generate M2 plants. DNA was extracted from individual M2 plants to form a DNA library as described in Example 2. Mutations were detected in the M2 DNA library by sequencing as described in Example 4. M2 plants in this population were crossed with wild type, feminized pollen of variety BaOX to create M3 seed. M3 seed was harvested from M2 plants and were planted to generate M3 plants. DNA was extracted from individual M3 plants for sequencing for conformation of inheritance of induced mutations from the M2 to the M3 generation.

[OHl] For some plants of the Ml library, axillary meristems of Ml plants were sprayed with a solution of 2 mM silver thiosulfate every 5 days, starting 10 days before the switch to short days. The solution was applied 3-4 times to induce male flowers. The M2 pollen from these plants was then allowed to self-fertilize the Ml plant, to produce M2 seeds. This corresponds to Scheme 11 as shown in FIG. 12. M2 seeds were not planted or analyzed for mutation frequency for these individuals.

Example 4 - M2 Mutation Frequency Evaluation

[0112] DNA was extracted from Cannabis leaves from a sample of 10 M2 EMS mutagenized plants (one of which (M210) was a direct progeny of a sequenced Ml plant from Example 2 (QI 9)), 10 M2 ENU mutagenized plants (7 of which were direct progeny of sequenced Ml plants from Example 2 (Q24-Q30)) and 10 control non- mutagenized plants using the method described in Example 2. The relationship of these individuals is shown in Table 3. The Treatment Group column indicated if the sample was a control, or the type of mutagen used. The concentration indicates the concentration of mutagen and the duration of the mutagenic treatment in hours (“hrs”).

Table 3. Samples Evaluated

[0113] Purified DNA samples were normalized to 12.5 ng/pl then prepared into whole genome sequencing libraries using NEBNext® Ultra™ II FS DNA Library Prep Kit and indexed using NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) for unique barcoding on P5 and P7 according to manufacturer’s instructions. Prepared libraries were then normalized to 23 ng/pl and sent in to be sequenced at Novogene (8801 Folsom Blvd, Suite 290, Sacramento, CA 95826) on Illumina NovoSeq S4 lanes. Libraries were checked for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and quantified by real-time PCR (to meet the criteria of 3 nM). All libraries passed quality control checks.

[0114] Mutation frequency sequence analysis on the M2s was performed as described in Example 2. A high number of unique alleles were identified in samples from all treatment groups (FIG. 28 and Table 4). This is likely due to the experimental design that crossed two varietals (the mutagenized BaOx samples, and untreated Berry Blossom), to produce the M2 population. Offspring of two populations that have many unique alleles are expected to result in hybrid offspring heterozygous at many loci. These ancestrally inherited mutations were unable to be distinguished from those generated by mutagenesis in the Ml treatments.

Table 4. Summary of Mutations Per 10,000 Base Pairs For Each M2 Treatment

Example 5 - Inheritance of Ml mutations in M2 progeny

[0115] Variants identified in Example 4 for 7 of 10 ENU M2 plants were used to evaluate inheritance of Ml mutations in the M2 plants (Table 5). The proportion of inherited variants ranged from 0.01 to 0.23. The maximum inheritance possible would be 0.5 because the induced mutations in the Ml samples were heterozygous. As shown in Table 5, each row represents a parent-offspring pair. Ml variants indicate the count of unique heterozygous loci present in the 31,172 genes (chemically induced mutations). M2 variants indicate the count of the Ml variants that were also present in the M2 offspring.

Table 5. Inheritance of Chemically Induced Mutations

[0116] Exemplary mutations identified in the TILLING library are listed in Table 6. DNA changes are given as the type of nucleotide in the reference sequence followed by the position of the nucleotide in the genetic LOCUS identified and then the type of nucleotide changed due to mutagenesis. For example, G1219A indicates a G at position 1219 in the reference sequence (LOCI 15701262, GenBank Accession No. XM_030629012, which is hereby incorporated by reference in its entirety) that was mutated to an A. Similarly, the amino acid mutations are given as the amino acid residue in the reference sequence followed by the position of the amino acid in the LOCUS identified and then the changed amino acid residue. For example, L664I of LOCI 15697098 (GenBank Accession No. XM_030624004, which is herein incorporated by reference) indicates a Leucine at amino acid position 664 of LOCI 15697098 was mutated to an Isoleucine. An asterisk (“*”) indicates a mutation to a stop codon. The nucleotide sequence and amino acid sequence of additional LOCI identified in Tables 6 and 7, including LOCI 15724176 (GenBank Accession No. XM_030653648) and LOCI 15705217 (GenBank Accession Nos. XM_030632485 and XM_030632484) are hereby incorporated by reference in their entirety. Table 6. Exemplary Mutations

[0117] Alignments of amino acid sequences of exemplary mutations of interest are shown in FIGs. 35-38. Individual amino acid sequences are shown in Table 7 (Reference Sequence (“Ref’) is considered wild type). Mutations that change the amino acid sequence are indicated with bold font. The LOCUS designators are from the annotated Cannabis reference genome (CS10; NCBI Refseq: GCF_900626175.2, GenBank Accession No. NC_044371, which is hereby incorporated by reference in its entirety) also called Cannabis sativa Annotation Release 100. LOCI 15697098 is an L-type lectin-domain containing receptor kinase IV.2, LOCI 15724176 is a zeatin O-xylosyltransferase, LOCI 15701262 is an RNA-dependent RNA polymerase 1, and LOCI 15705217 is a GATA transcription factor 8 gene.

Table 7. Exemplary Mutant Amino Acid Sequences

[0118] Mutations of interest can be identified in additional genes by extracting DNA from a larger number of mutagenized individuals and sequencing. [0119] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

- 33 -WHAT IS CLAIMED IS:

1. A method for introducing mutations of interest in a Cannabis plant, said method comprising: mutating a Cannabis plant, wherein said mutating comprises: providing a plurality of Cannabis seeds with cracked seed coats; and contacting the Cannabis seeds with a mutagenic agent to mutate the Cannabis seeds to produce a first population of mutated (Ml) seeds; planting and growing the Ml seeds into Ml plants; fertilizing the Ml plants with pollen under conditions effective for the Ml plants to produce a second population of mutated (M2) seeds; planting and growing the M2 seeds into M2 plants; collecting DNA from the M2 plants; and harvesting progeny of the M2 plants, wherein the progeny comprise mutations.

2. The method of claim 1, wherein said contacting the Cannabis seeds with a mutagenic agent is carried out for 10-15 hours.

3. The method of claim 1, wherein said mutagenic agent is EMS at a concentration of less than 3.5%.

4. The method of claim 1, wherein said mutagenic agent is ENU at a concentration less than 0.07%.

5. The method of claim 1, wherein the Ml plants have a gender selected from the group consisting of male plants, male and female plants, female plants, and female plants treated to produce pollen.

6. The method of any one of claims 1-5 further comprising: selecting female Ml plants for said fertilizing.

7. The method of claim 6, wherein said fertilizing is carried out with pollen selected from the group consisting of pollen from the Ml plant itself, pollen from a wild-type female plant, pollen from a wild-type male plant, pollen from a different Ml mutant plant, and combinations thereof. - 34 -

8. The method of any one of claims 1-5 further comprising: selecting male Ml plants or female Ml plants treated to produce pollen.

9. The method of claim 8 further comprising: fertilizing a Cannabis plant selected from the group consisting of a wild-type female plant, a different Ml mutant plant, and combinations thereof with pollen produced from the male Ml plants or female Ml plants treated to produce pollen.

10. The method of claim 8, wherein female M2 plants or wild-type Cannabis plants are fertilized using pollen selected from the group consisting of pollen from the M2 plant itself, pollen from a wild-type female plant, pollen from a wild-type male plant, pollen from a different M2 plant, and combinations thereof.

11. The method of any one of claims 1-10, wherein said harvesting comprises collecting a plant part selected from the group consisting of pollen, seeds, feminized pollen, and combinations thereof.

12. The method of any one of claims 1-11, wherein the plurality of Cannabis seeds are feminized.

13. The method of claim 12, wherein the Ml plants are fertilized using pollen selected from the group consisting of pollen from the Ml plant itself, pollen from a wild-type female plant, pollen from a wild-type male plant, pollen from a different Ml mutant plant, and combinations thereof.

14. The method of claim 13, wherein female M2 plants are fertilized using pollen selected from the group consisting of pollen from the M2 plant itself, pollen from a wildtype female plant, pollen from a wild-type male plant, pollen from a different M2 plant, and combinations thereof.

15. The method of claim 13, wherein male M2 plants are selected and said method further comprises: fertilizing unmutagenized female Cannabis plants with pollen from the male M2 plants.

16. A population of mutated Cannabis seeds prepared by the method according to any one of claims 1-15.

17. A method for introducing mutations of interest in a Cannabis plant, said method comprising: providing a plurality of Cannabis seeds with cracked seed coats; contacting the Cannabis seeds with a mutagenic agent to mutate the Cannabis seeds to produce a first population of mutated (Ml) seeds; planting and growing the Ml seeds into Ml plants; collecting mutated (M2) pollen from the Ml plants; fertilizing Cannabis plants with M2 pollen under conditions effective for the Cannabis plants to produce mutated (M2) seeds; planting and growing the M2 seeds into M2 plants; collecting DNA from the M2 plants; and harvesting progeny of the M2 plants, wherein the progeny comprise mutations.

18. The method of claim 17, wherein said contacting the Cannabis seeds with a mutagenic agent is carried out for 10-15 hours.

19. The method of claim 17, wherein said mutagenic agent is EMS at a concentration of less than 3.5%.

20. The method of claim 17, wherein said mutagenic agent is ENU at a concentration less than 0.07%.

21. The method of any one of claims 17-20 further comprising: collecting M2 pollen from male Ml plants or female Ml plants treated to produce pollen, and combinations thereof.

22. The method of claim 21 further comprising: fertilizing a Cannabis plant selected from the group consisting of a wild-type female plant, a different Ml female mutant plant, the same Ml female plant producing the pollen, and combinations thereof.

23. The method of any one of claims 17-22, wherein the plurality of Cannabis seeds are feminized.

24. A population of mutated Cannabis seeds prepared by the method according to any one of claims 17-23.

25. A method for introducing mutations of interest in a Cannabis plant, said method comprising: mutating a Cannabis plant, wherein said mutating comprises: providing a cell from a Cannabis plant; and contacting the cell with a mutagenic agent to mutate the cell to produce a first mutated (Ml) cell; culturing the Ml cell to produce an Ml plant; fertilizing the Ml plant with pollen under conditions effective for the Ml plant to produce a mutated (M2) cell; culturing the M2 cell to produce an M2 plant; collecting DNA from the M2 plant; and harvesting progeny of the M2 plant, wherein the progeny comprise mutations.

26. A method for introducing mutations of interest in a Cannabis plant, said method comprising: providing a cell from a Cannabis plant; contacting the cell with a mutagenic agent to mutate the cell to produce a mutated (Ml) cell; culturing the Ml cell to produce an Ml plant; collecting mutated (M2) pollen from the Ml plant; fertilizing a Cannabis plant with M2 pollen under conditions effective for the fertilized Cannabis plant to produce a mutated (M2) cell; culturing the M2 cell to produce an M2 plant; collecting DNA from the M2 plant; and harvesting progeny of the M2 plant, wherein the progeny comprise mutations.