Methods of transformation and genome editing of plants

Abstract

Disclosed herein are methods for rapid transformation, genome editing, and regeneration of dicot plants. The methods disclosed herein involve transforming plant cells and explants with recombinant DNA to achieve genome edits. The methods enhance transformation and regeneration efficiency and reduce the time to produce transgenic plants.

Description

METHODS OF TRANSFORMATION AND GENOME EDITING OF PLANTS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 642,444, filed May 3, 2024, which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to methods for rapid transformation and genome editing of diverse plant dicot speciesSEQUENCE LISTING STATEMENT

[0003] This application contains a computer readable Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file was created on May 2, 2025, is named 266655000101 .xml, and is 70,745 bytes in size.BACKGROUND

[0004] The ability to generate CRISPR-edited plants through targeted genome editing is a crucial factor in the applied and basic gene function studies and genetic improvement of specific plants. The utilization of available protocols to transform and regenerate transgenic plants by Agrobactermm-mediated transformation and applying this system for targeted gene-editing are limited by the challenges of inefficient tissue culture systems in terms of rapid regeneration of plant tissues, and long timelines of experiments that may cause unwanted variationsin regenerated plants. This may negatively impact novel transgenic plant development and targeted decisions to obtain such plants in a desired, specific timescale.

[0005] The available standard transformation and regeneration methods have multiple drawbacks that limit the speed and efficiency with which transgenic plants can be produced and screened. For example, many standard methods of transformation and regeneration require the use of high auxin or cytokinin levels and require steps involving either embryogenic callus formation or organogenesis, leading to procedures that take many weeks before producing plants for growth in a greenhouse setting following transformation. These methods can take 12-23 weeks to produce plants, which include the steps of supplying 2, 4-D (auxin) to stimulate somatic embryo formation in (taking up to 8 weeks), production of embryogenic callus from the primary somatic embryos (taking up to an additional 8 weeks), forming shoots (taking up to an additional 3 weeks), and finally rooting (taking up to an additional 1 to 3 weeks). Other methods immediately supply a cytokinin along withthe auxin to stimulate direct morphogenesis to produce shoots and direct plant formation from 8 to 28 weeks after transformation. There remains a need for transformation methods that deliver significantly higher transformation frequencies and significantly more quality events (events containing one copy of a trait gene cassette with no vector (plasmid) backbone) in multiple inbred lines using a variety of starting tissue types (e.g. seeds, shoots, leaves, etc), landraces, inbreds representing a range of genetic diversity and having significant commercial utility.

[0006] This disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY

[0007] One aspect of the present disclosure is directed to a method of genome editing and regenerating a dicot plant. This method involves providing an explant from a dicot plant and transforming the explant with a microbe comprising recombinant DNA, where the recombinant DNA comprises a CRISPR-based genome editing system, and where said transforming is carried out under conditions effective for the CRISPR-based genome editing system to target and edit a gene of interest in the explant. The method also involves incubating the transformed explant and selecting a regenerated plant from the transformed explant, where the regenerated plant comprises the edit.

[0008] Another aspect of the present disclosure is directed to a method of genome editing and regenerating a plant of the family Cannabaceae. This method involves providing a hypocotyl and / or a cotyledon from a seed of a plant of the family Cannabaceae; culturing the hypocotyl and / or the cotyledon in a callus induction medium; dissecting out callus from the cultured hypocotyl and / or the cultured cotyledon; re-culturing green calli from the dissected out callus; and transforming the re-cultured calli with a microbe comprising recombinant DNA, where the recombinant DNA comprises a CRISPR-based genome editing system capable of targeting and modifying a gene of interest in the re-cultured calli to produce a transformed calli. The method also involves incubating the transformed calli in darkness and then light and selecting a regenerated plant from the transformed calli, where the regenerated plant comprises the edit.

[0009] A further aspect of the present disclosure relates to genome edited plants or plant parts produced by the methods disclosed herein.

[0010] Another aspect of the present disclosure relates to plants produced from the genome edited plants described herein.

[0011] In some embodiments, the present disclosure describes a rapid and efficient transformation and regeneration protocol using mature seeds as explants in multiple plant varieties that can be easily adapted for other diverse plants. For example, improvements described hereinclude 1) explant selection, 2) a rapid process for transformation and direct organogenesis resulting in the recovery of transgenic and edited plants in 8-9 weeks, 3) a new selectable marker, spectinomycin, for transgenic plant selection, and 4) CRISPR-based genome editing systems using the combination of all the above.

[0012] Transformation steps and standardization of different tissue culture and regeneration media, including identification of the best explant, growth hormones, selection, and gelling agents are described herein. In some embodiments, the inventive regeneration protocols described herein following direct organogenesis from mature seeds results in the production of transgenic shoots from the cotyledon and hypocotyl explants. Methods described herein take significantly shorter times (averaging approximately 55-60 days, compared to approximately 100 days reported previously by Zhang et al., “Establishment of an Agrobacterium-mediated Genetic Transformation and CRISPR / Cas9-mediated Targeted Mutagenesis in Hemp (Cannabis sativaL.) ” Plant Biotechnology Journal 19:1979-1987 (2021), which is hereby incorporated by reference in its entirety, to produce transgenic and gene-edited plants in Cannabis and other plants. Examples described herein followed a similar timeline that is much shorter than published protocols (Sharma and Anjaiah, “An Efficient Method for the Production of Transgenic Plants of Peanut (Arachis hypogaea L.) Through Agrobacterium tumefaciens-Mediated Genetic Transformation,” Plant Science , 159(1), 7-19 (2000); Tohidfar et al., “Agrobacterium-Meddated Transformation of Alfalfa (Medicago saliva) Using A Synthetic Cry3a Gene to Enhance Resistance Against Alfalfa Weevil” PlantCell, Tissue and Organ Culture (PCTOC), 773:227-235 (2013), each of which is hereby incorporated by reference in its entirety).

[0013] Methods described herein further pertain to direct organogenesis in, which is a more straightforward method for plant regeneration than the protocols previously reported in the literature that generally used callus-based somatic embryogenesis (Zhang et al., “Establishment of an Agrobacterium-mediated Genetic Transformation and CRISPR / Cas9-mediated Targeted Mutagenesis in Hemp (Cannabis sativa L.),” Plant Biotechnology Journal 19:1979-1987 (2021), which is hereby incorporated by reference in its entirety). Furthermore, methods described herein have been shown to result in optimal transformation efficiencies (e.g., ranging from 3% -23% for direct organogenesis) for the multiple plant varieties tested. Genome editing has been successfully demonstrated in multiple plant types demonstrating broad applicability to different plant taxa.

[0014] The rapid, robust, and repeatable protocols developed and disclosed herein overcome major limitations in genetic transformation and genome editing of diverse plant varieties. In someembodiments, utilizing mature seeds as explants ensures the continuous availability of plant material. In addition, the direct organogenesis protocols for transformation combined with effective transgenic plant selection results in fewer steps in the culture medium. These advances result in a rapid, efficient, and reproducible transformation and genome editing protocols in multiple plant varieties, offering the potential for novel trait development and crop enhancement.

[0015] Establishing efficient Agrobacterium-medda ed transformation protocols, in combination with optimized regeneration systems, is critical to advancing CRISPR-based functional genomics and unlocking transformative applications across Cannabis, hops, peanut, alfalfa and other plant types. As members of the Rosids, these particular species share evolutionary traits that underpin their resilience, metabolic diversity, and potential for genetic improvement. The urgent need to harness this potential through next-generation breeding underscores the importance of developing robust tissue culture systems and precise genome-editing platforms.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1. is a schematic illustration of a plasmid map showing the pCAMBIA2300 Cas9-gRNA dsRED vector.

[0017] FIG. 2 is a schematic illustration of a plasmid map showing the pPZP2000 Cas9- gRNA mGFP5 vector.

[0018] FIG. 3 is a schematic illustration of a plasmid map showing the pPZP200-dsRED- spcN vector.

[0019] FIG. 4 is a schematic illustration of a plasmid map showing the pCAMBIA2300 OPEN CRISPR-1 vector.

[0020] FIG. 5 is a schematic representation of the timeline of transformation and regeneration of the various steps of one embodiment of a direct organogenesis transformation procedure described herein.

[0021] FIGs. 6A-I are a series of photographs showing different steps in a transformation method via direct organogenesis from hypocotyl and cotyledons. Cannabis and hemp transformation is shown as an example. The steps include explant preparation, infection, selection of putative transgenic events, and subsequent transfer to tissue culture tubs. FIG. 6A shows germinating sterile seedlings dissected out of their seed coats. FIG. 6B shows seedlings co-cultivated with Agrobacterium. FIG. 6C shows germinating seedlings on co-cultivation medium. FIG. 6D shows cotyledons on regeneration selection medium. FIG. 6E shows hypocotyls on regeneration selection medium. FIG. 6F shows putative transgenic shoots regenerating from hypocotyls. FIG. 6G showsputative transgenic shoots regenerating from cotyledons. FIGs. 6H and 61 show putative transgenics in culture tubs.

[0022] FIGs. 7A-L are a series of photographs showing different steps in a transformation method via somatic embryogenesis from hypocotyl and cotyledon explants. Hemp transformation is shown as an example. FIG. 7A shows callus induction from hypocotyl explants. FIG. 7B shows callus induction from cotyledon explants. FIG. 7C shows calli in suspension culture. FIG. 7D shows growing calli in suspension culture. FIG. 7E shows greening calli in suspension culture. FIG. 7F shows calli in co-cultivation with Agrobacteria. FIG. 7G shows calli in selection medium. FIG. 7H shows dsRED reporter gene expression in calli indicating successful transformation. FIG. 71 shows somatic embryos on calli. FIG. 7J shows dsRED expression in somatic embryos. FIG. 7K shows growing somatic embryos. And FIG. 7L shows rooting of somatic embryos.

[0023] FIG. 8 is a photograph of a PCR-based detection of putative hemp transgenic plants transformed with a gene-editing construct. Top row: Putative transgenics were screened by PCR using Cas 9 primers (SEQ ID NOs:5-6). Bottom row: Putative transgenics were screened by PCR with VirD2 primers (SEQ ID NOs:3-4).

[0024] FIG. 9 is a sequence alignment showing genome edits in the Stomagen gene of Cannabis sativa. The sequence alignment shows the allele frequency table of amplicon sequencing of the Stomagen gene targeted by Cas9-sgRNA in Cannabis sativa. The top row shows the wild-type reference sequence (SEQ ID NO:24; a partial sequence of the Stomagen gene target with the position of the guide RNA (SEQ ID NO:42) shown underneath). The bottom rows show two separate genome edited alleles of Stomagen (SEQ ID NOs:25-26) and their respective abundance of reads.

[0025] FIGs. 10A-E are a series of photographs showing the different steps in a transformation method via direct organogenesis from cotyledon explants of peanut. FIG. 10A is a photograph showing peanut cotyledons immersed in Agrobacterium suspension. FIGs. 10B-D are photographs showing multiple putative transgenic shoots regenerating in selection medium. FIG. 10E is a photograph showing regenerated putative transgenic peanut plants.

[0026] FIGs. 11 A-B are photographs of regenerated hemp plants. FIG. 11 A is a photograph of a control plant. FIG. 1 IB is a photograph of a plant edited for target gene Phytoene Desaturase (PDS) showing albino leaves.

[0027] FIGs. 12 A-B are photographs of regenerated Cannabis plants. FIG. 12A is a photograph of a control plant. FIG. 12B is a photograph of a plant edited for target gene Phytoene Desaturase (PDS) showing albino leaves.

[0028] FIGs. 13A-B are photographs of regenerated peanut plants. FIG. 13 A is a photograph of a control plant. FIG. 13B is a photograph of a plant edited for target gene Phytoene Desaturase (PDS) showing albino leaves.

[0029] FIGs. 14A-B are photographs of regenerated Cannabis plants. FIG. 14A is a photograph of a control plant. FIG. 14B is a photograph of a plant edited for target gene, Stomagen, showing shriveled leaf phenotypes.

[0030] FIG. 15 is a sequence alignment showing genome edits in the Phytoene Desaturase gene of Cannabis sativa. The sequence alignment shows the allele frequency table of amplicon sequencing of the Phytoene Desaturase gene targeted by Cas9-sgRNA in Cannabis sativa. The top row shows the reference sequence (SEQ ID NO:36; a partial sequence of the Phytoene Desaturase gene target with the position of the guide RNA (SEQ ID NO:43) shown underneath). The bottom rows show five separate genome edited alleles of Phytoene Desaturase (SEQ ID NOs:37-41) and their respective abundance of reads.DETAILED DESCRIPTION

[0031] This disclosure is directed to methods for a rapid, efficient, and reproducible transformation and genome editing protocols in plants for the development of novel traits and crop improvement.

[0032] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure described for which they are suitable as would be understood by a person skilled in the art.

[0033] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0034] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0035] The term “about” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value or range.

[0036] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of thestated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” The methods and / or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0037] Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0038] A “reference sequence” means a nucleic acid or amino acid used as a comparator for another nucleic acid or amino acid, respectively, when determining sequence identity. A reference sequence can be a wildtype sequence.

[0039] “Sequence identity”, “percent identity” or “% identical” refers to the exactness of a match between a reference sequence and a sequence being compared to it when optimally aligned. For example, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Multalin program (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucleic Acids Res. 16:10881-90 (1988), whichis hereby incorporated by reference in its entirety) or the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Sequences may also be aligned using algorithms known in the art including, but not limited to, CLUSTAL V algorithm or the BLASTN or BLAST 2 sequence programs.Preferences and options for a given aspect, feature, embodiment, or parameter of the disclosure should, unless the context indicates otherwise, be regarded as having been disclosed in combinationwith any and all preferences and options for all other aspects, features, embodiments, and parameters of the disclosure.Recombinant DNA, Vectors, & Genome Editing

[0040] The methods disclosed herein comprise the use of recombinant DNA to introduce heterologous DNA (e g., a CRISPR -based genome editing system) into plants to perform genome editing. Recombinant DNA comprises non-naturally occurring polynucleotides produced by combining DNA segments from different sources into a contiguous stretch of DNA. In some embodiments, polynucleotides are present in a recombinant DNA construct comprise one or more expression cassettes that provide for transcription of a DNA sequence of interest. In some embodiments, recombinant DNA constructs incorporated into a plant genome express components required for genome editing.

[0041] Recombinant DNA may be comprised in an expression cassette or a vector system (e.g., a linear or a closed circular plasmid). A “vector” is a nucleic acid, plasmid or virus used to transfer coding information to a host cell. A “cloning vector” is a small piece of DNA into which a foreign DNA fragment is inserted. Typically, the vector contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. The vector can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Useful selectable markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. The vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun, Ti-mediated gene transfer, calcium phosphate transfection, DEAE-Dextran-mediated transfection, lipofection, or electroporation. Examples of vectors include, but are not limited to, viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA, such as vaccinia and adenovirus, Pl -based artificial chromosomes, yeast plasmids, Agrobacterhim vectors Bacillus vectors, and Aspergillus vectors. Examples of bacterial vectors include, but are not limited to, T-DNA vectors, pQE vectors, pBluescript plasmids, pNH vectors, and lambda-ZAP vectors. Examples of eukaryotic vectors include pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 vectors.

[0042] A “gene” refers to an assembly of nucleotides that encode a polypeptide and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expressesa specific functional RNA, protein, or polypeptide, optionally including regulatory sequences preceding (5' noncoding sequences) and following (3' non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “heterologous” or “exogenous” gene refers to DNA not naturally located in the cell, or if naturally from the cell, the gene is heterologous if it is introduced into a different location than the original location in the cell. Heterologous DNA can be introduced into the host organism by gene transfer. Heterologous genes can comprise native genes inserted into a non-native organism, or chimeric genes, such as a native gene under control of a different promoter than its endogenous promoter. Heterologous DNA can include a gene or other polynucleotides such as promoters and / or terminators foreign to the cell.

[0043] “Operably linked” means an association between nucleic acid sequences on a single nucleic acid molecule such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0044] In some embodiments, a vector comprises a recombinant DNA comprising at least one regulatory sequence operably linked to one or more polynucleotides. The one or more polynucleotides can be expressed under the control of the regulatory sequence(s) in a plant cell or plant part, such as a promoter.

[0045] A “promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment. A promoter is a non-coding genomic DNA sequence, usually upstream (5') to and operably linked to the relevant coding sequence, and its primary function is to act as a binding site for RNA polymerase to initiate transcription by the RNA polymerase. A promoter may also include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The terms “capable of controlling expression” or “initiating transcription”, refer to the primary function of a promoter. Additionally, there is “expression” of RNA, including functional RNA, or the expression of a polypeptide for operably linked encoding nucleotide sequences, as the transcribed RNA ultimately may be translated into the corresponding polypeptide. Promoters vary in their “strength” (i.e., their ability to promote transcription). The nucleotide sequence of the promoter determines the nature of the RNA polymerase binding and other related protein factors that attach to the RNA polymerase and / or promoter, and the rate of RNA synthesis.

[0046] A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. A “tissue-specific” promoter is a promoter that drives expression primarily in certain tissue types of a plantbut may also be active to a lower level in other tissue cell types. For example, a “root-specific” promoter is a promoter that drives expression primarily in root tissues of a plant but may also be active to a lower level in other tissue cell types.

[0047] Nonlimiting examples of promoters that may be useful in embodiments disclosed herein, include the parsley ubiquitin promoter (Kawalleck et al., “Polyubiquitin Gene Expression and Structural Properties of the ubi4-2 Gene in Petroselinwn crispum," Plant Mol. Biol. 21 :673-684 (1993), which is hereby incorporated by reference in its entirety); Arabidopsis HSP70 promoter (Sung et al., “Comprehensive Expression Profile Analysis of the Arabidopsis HSP70 Gene Family,” Plant Physiol. 126:789-800 (2001), which is hereby incorporated by reference in its entirety); nopaline synthase (NOS) promoter (Ebert and An, “Identification of an Essential Upstream Element in the Nopaline Synthase Promoter by Stable and Transient Assays,” Proc. Natl. Acad. Sci. USA 84:5745-5749 (1987), which is hereby incorporated by reference in its entirety); the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens , the caulimovirus promoters, such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., “Expression of a Soybean P-conclycinin Gene Under the Control of the Cauliflower Mosaic Virus 35S and 19S Promoters in Transformed Petunia Tissues,” Plant Mol. Biol. 9:315-324 (1987), which is hereby incorporated by reference in its entirety); the CaMV 35S promoter (Odell et al., “Identification of DNA Sequences Required for Activity of the Cauliflower Mosaic Virus 35S Promoter,” nn? 313:810-812 (1985); U.S. Patent Nos. 5,322,938; 5,352,605; 5,359,142; and 5,530,196, which are hereby incorporated by reference in their entirety); the figwort mosaic virus 34S (FMV34S) promoter (Walker et al., “DNA Sequences Required for Anaerobic Expression of the Maize Alcohol Dehydrogenase 1 Gene,” Proc. Natl. Acad. Sci. USA 84:6624-6628 (1987); U.S. PatentNos. 6,051,753 and 5,378,619, which are hereby incorporated by reference in their entirety); the sucrose synthase promoter (Yang and Russell, “Maize Sucrose Synthase-1 Promoter Directs Phloem Cell-Specific Expression of GUS Gene in Transgenic Tobacco Plants,” Proc. Natl. Acad. Sci. USA 87:4144-4148 (1990), which is hereby incorporated by reference in its entirety); the R gene complex promoter (Chandler et al., Two Regulatory Genes of the Maize Anthocyanin Pathway are Homologous: Isolation ofB Utilizing R Genomic Sequences,” Plant Cell 1 :1175-1183 (1989), which is hereby incorporated by reference in its entirety); the chlorophyll a / bbinding protein gene promoter; a PCI SV promoter (U.S. Patent No. 5,850,019, which is hereby incorporated by reference in its entirety); the SCP1 promoter (U.S. Patent No. 6,677,503, which is hereby incorporated by reference in its entirety); and AGRtu.nos promoters (GenBank Accession No. V00087; Depicker et al., “Transgenic Carnations Obtained by Agrobacterium tumefaciens- Mediated Transformation by Leaf Explants,” J. Mol. Appl. Genet. 1 :561-573 (1982); and Bevan et al., “A Chimaeric Antibiotic Resistance Gene as a Selectable Marker for Plant Cell Transformation,” Nature 304:184-187 (1983), which are hereby incorporated by reference in their entirety).

[0048] Additional regulatory sequences that may be present in a recombinant DNA include, without limitation, 5' UTRs located between a promoter sequence and a coding sequence that function as a translation leader sequence. The translation leader sequence may be present in the fully-processed mRNA, and it may affect processing of the primary transcript, and / or RNA stability. Examples of translation leader sequences include maize and petunia heat shock protein leaders (U. S. Patent No. 5,362,865, which is hereby incorporated by reference in its entirety), plant virus coat protein leaders, plant rubisco leaders, and others. See, e.g., Turner and Foster, “The Potential Exploitation of Plant Viral Translational Enhancers in Biotechnology for Increased Gene Expression,” Molecular Biotech. 3(3):225-236 (1995), which is hereby incorporated by reference in its entirety. Nonlimiting examples of 5' UTRs include GmHsp (U.S. Patent No. 5,659, 122, which is hereby incorporated by reference in its entirety); PhDnaK (U.S. Patent No. 5,362,865, which is hereby incorporated by reference in its entirety); AtAntl; 7EF (Carrington and Freed, “Cap- Independent Enhancement of Translation by a Plant Poty virus 5’ Nontranslated Region,” J. Virol. 64:1590-1597 (1990), which is hereby incorporated by reference in its entirety); and AGRtunos (GenBank AccessionNo. V00087 and Bevan et al., “A Chimaeric Antibiotic Resistance Gene as a Selectable Marker for Plant Cell Transformation,” Nature 304:184-187 (1983), which are hereby incorporated by reference in their entirety).

[0049] In some embodiments, the recombinant DNA comprises a “gene cassette”. A “gene cassette” or an “expression cassette” is DNA construct that may be within a vector that contains all the necessary regulatory elements for transcription and translation of a cloned gene. The gene cassette typically includes a promoter, which initiates transcription; operably linked to a coding sequence, which is the actual gene or genes to be expressed; and a terminator sequence, which signals the end of transcription. The gene cassette is designed to be inserted into a host organism, where it can be transcribed and translated to produce the desired protein or other product. Thespecific elements and their arrangement can be varied to control the level, timing, and location of gene expression in the host organism.

[0050] A “terminator sequence” refers to a 3' non-translated sequence which may include 3' transcription termination regions and polyadenylation regions. These are genetic elements located downstream and operably linked to a polynucleotide sequence of interest and include polynucleotides that provide polyadenylation signal and / or other regulatory signals capable of affecting transcription or mRNA processing. The polyadenylation sequence can be derived from a variety of plant genes or from T-DNA genes. A nonlimiting example of a 3 ' transcription termination region is the nopaline synthase 3' region (nos 3'; Fraley et al., “Expression of Bacterial Genes in Plant Cells,” Proc. Natl. Acad. Sci. USA 80:4803-4307 (1983), which is hereby incorporated by reference in its entirety). An example of the use of different 3' non-translated regions is provided in Ingelbrecht et al., “Different 3 ’ End Regions Strongly Influence the Level of Gene Expression in Plant Cells,” Plant Cell 1 :671-680 (1989), which is hereby incorporated by reference in its entirety. Nonlimiting examples of polyadenylation signals include one from Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., “Tissue-Specific and Light-Regulated Expression of a Pea Nuclear Gene Encoding the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase,” EMBO J. 3 :1671-1679 (1984), which is hereby incorporated by reference in its entirety) and AGRtu.nos (GenBank Accession No. E01312, which is hereby incorporated by reference in its entirety).

[0051] The recombinant DNA and / or vector may comprise a selectable marker that confers a selectable phenotype on a transformed cell, such as a plant cell. Selectable markers may also be used to select for plants or plant cells that comprise a recombinant nucleic acid molecule. The marker may encode biocide resistance, antibiotic resistance (e.g., kanamycin, Geneticin (G418), bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate, glufosinate, etc.). Examples of selectable markers include, but are not limited to, a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate resistance; a nitrilase gene which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS) which confers imidazolinone or sulfonylurea resistance; and a methotrexate resistant DHFR gene. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin, tetracycline, and others. Examples of selectable markers are illustrated in, e.g., U.S.Patent Nos. 5,550,318; 5,633,435; 5,780,708; and 6, 118,047, which are hereby incorporated by reference in their entirety.

[0052] A recombinant DNA and / or vector may additionally or alternatively include a screenable marker. Screenable markers may be used to monitor expression. Exemplary screenable markers include, without limitation, a P-glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known (Jefferson et al., “Assaying Chimeric Genes in Plants: The GUS Gene Fusion Sy stem,” Plant Mol. Biol. Rep. 5:387-405 (1987), which is hereby incorporated by reference in its entirety); a gfp gene from A. victoria which encodes for green fluorescent light emission under UV light (Chalfie et al., “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263 :802-805 (1994), which is hereby incorporated by reference in its entirety); an Rlocus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaportaet al., “Molecular Cloning of the Maize R-Nj Allele by Transposon Tagging With Ac,” In 18 Stadler Genetics Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp. 263-282 (1988), which is hereby incorp orated by reference in its entirety); a P-lactamase gene (Sutcliffe, “Nucleotide Sequence of the Ampicillin Resistance Gene of Escherichia coli Plasmid pBR322,” Proc. Natl. Acad. Sci. USA 75 :3737-3741 (1978), which is hereby incorporated by reference in its entirety); a gene which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); a luciferase gene (Ow et al., “Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants,” Science 234:856-859 (1986), which is hereby incorporated by reference in its entirety); ax IE gene that encodes a catechol dioxygenase that can convert chromogenic catechols (Zukowski and Miller, “Hyperproduction of an Intracellular Heterologous Protein in a sacUh mutant of Bacillus subtilis ” Gene 46(2-3):247-255 (1983), which is hereby incorporated by reference in its entirety); an amylase gene (Ikuta et al., “The a Amylase Gene as a Marker for Gene Cloning: Direct Screening of Recombinant Clones,” Bio / TechnoL 8:241-242 (1990), which is hereby incorporated by reference in its entirety); a tyrosinase gene which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to melanin (Katz et al., “Cloning and Expression of the Tyrosinase Gene from Streptomyces antibioticus m' Streptomyces lividans,” J. Gen. Microbiol. 129:2703-2714 (1983), which is hereby incorporated by reference in its entirety); and an a-galactosidase.

[0053] A recombinant DNA may include or be present in a vector for use in Agrobacterium- mediated transformation, such as a Ti or Ri plasmid. Such vectors are known in the art and includesuch elements as T-DNA, vir regions, TDNA border regions, and may also contain selectable or screenable markers providing for efficient recovery of transformed plant cells, plant parts, or whole transformed plants. In some embodiments, an Agrobacterium binary vector is used. In some embodiments a recombinant DNA may include or be present in a vector for use in Rhizobia- mediated transformation.

[0054] A “transgene” or “chimeric gene” refers to a genetic locus comprising a DNA sequence, such as a recombinant DNA, which has been introduced into the genome of a plant by transformation, such as by Agrobacterium-m^ia eA transformation. A plant cell or plant comprising a transgene stably integrated into its genome is referred to as “transgenic plant cell” or a “transgenic plant”.

[0055] Genome editing” or “gene editing” is a type of genetic engineering in which DNA is inserted, replaced, or deleted, or any combination thereof, from a genome using artificially engineered nucleases, or “molecular scissors”. The nucleases typically create double-stranded breaks (“DSBs”) at desired locations in the genome and harness the cell’s endogenous mechanisms to repair the induced break by processes of homology dependent repair (“HDR”) or nonhomologous endjoining (“NHEJ”). Any method of genome editing may be used in the embodiments of the present disclosure. For example, genome editing nucleases may be artificially engineered, for example in methods where zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), or meganucleases are employed, or may be non-altered endonucleases where an RNA- guided endonuclease, such as from a CRISPR-based genome editing system, is used. The use of genome editing nucleases can be used, without limitation, for generating gene knockouts or knockdowns.

[0056] ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences, and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. ZFNs are made of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the I 'ok\ restriction endonuclease. ZFNs can be used to induce double-stranded breaks (DSBs) in specific DNA sequences and thereby promote site-specific homologous recombination with an exogenous template. The exogenous template contains the sequence that is to be introduced into the genome.

[0057] TALEN is a sequence-specific endonuclease that is made of a transcription activatorlike effector (TALE) and a / < A I endonuclease. The transcription activator-like effector is a DNA binding protein that has a highly conserved central region with tandem repeat units of 34 amino acids. The base preference for each repeat unit is determined by two amino acid residues called the repeat-variable di-residue, which recognizes one specific nucleotide in the target DNA. Arrays of DNA-binding repeat units can be customized for targeting specific DNA sequences. As with ZFNs, dimerization of two TALENs on targeted specific sequences in a genome, results in FoH-dependent introduction of double stranded breaks, stimulating homology directed repair (HDR) and Nonhomologous end joining (NHEJ) repair mechanisms.

[0058] Meganucleases with re-engineered homing nucleases can also be used to effect genome modification in Cannabis in the methods described herein. Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). This site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-Scel meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance. Meganucleases are considered to be the most specific naturally occurring restriction enzymes. Among meganucleases, the LAGLID ADG family of homing endonucleases has become a valuable tool for the study of genomes and genome engineering. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed.

[0059] CRISPR / Cas type RNA-guided endonucleases provide an efficient system for inducing genetic modifications in genomes of many organisms. In some embodiments, a CRISPR- based genome editing system comprises a polynucleotide encoding a CRISPR-based genome editing nuclease and a polynucleotide encoding a guide RNA targeting a gene of interest. Non-limiting examples of genome editing nucleases include Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a (Cpfl), Csyl , Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, Mad7, OpenCRISPR-1, or homologs, modified versions, and endonuclease inactive versions thereof. In some embodiments, the genome editing nuclease is Cas9. In some embodiments, the genome editing nuclease is OpenCRISPR-1. An example of a fusion protein to Cas9 is a cytidine deaminase-Cas9 fusion protein used in cytidine base editing to mutate nucleotides in target genes without generating double-strand breaks as described in Komor et al., “Programmable Editing of aTarget Base in Genomic DNA without Double-Stranded DNA Cleavage,” Nature 533 :420-424 (2016), which is hereby incorporated by reference in its entirety. The use of CRISPR guide RNA in conjunction with CRISPR / Cas technology to target RNA is also described in Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR / Cas Systems,” Science 339:819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR / Cas-based Methods for Genome Engineering,” Cell 31 :397-405 (2013), each of which is hereby incorporated by reference in its entirety. OpenCRISPR-1 is described in Ruffolo et al., “Design of Highly Functional Genome Editors by Modeling the Universe of CRISPR-Cas Sequences,” BioRxiv 2024-04 (2024), which is hereby incorporated by reference in its entirety.

[0060] There are typically two distinct components to a CRISPR-based genome editing system, a guide RNA (“gRNA”) and a genome editing endonuclease. The gRNA uses a CRISPR RNA (“crRNA”) comprising a DNA targeting segment that can be engineered to contain a complementary stretch of nucleotide sequence (e.g., at least 10 nucleotides) to target a DNA site (e.g., a gene target) for binding and subsequent modification by CRISPR genome editing nuclease. The length of a crRNA may range from about 15 nucleotides to about 60 nucleotides. The crRNA can be chemically synthesized and can also be engineered to include a ribonucleotide analog or a modified form thereof, or an analog of a modified form, or non-natural nucleosides.

[0061] A guide RNA targeting a gene of interest may be developed using the sequence of the gene target according to any method known in the art. A software tool can be used to optimize the choice of gRNA within a target sequence, and to minimize total off-target activity across the rest of the genome. A non-limiting example of software to select a gRNA targeting a gene of interest is Geneious software (Geneious, San Diego, CA).

[0062] Although insertion of recombinant DNA into the plant genome may be used to generate genome modifications using a CRISPR-based genome editing system, such genome modificationscan be achieved without inserting recombinant DNA into the plant genome. In some embodiments, the CRISPR-based genome editing system that induces a modification is an RNP that is preassembled and delivered to a target explant. Furthermore, where recombinant DNA is inserted into the plant genome to effect genome editing, plants having edited genomes, but lacking recombinant DNA, may be obtained by segregation using standard breeding techniques such as crossing and backcrossing. Alternatively, transient expression of CRISPR-based genome editing system components, including guide RNA and a native or modified endonuclease throughrecombinant DNA in cells can also result in genome editing, since gene editing components are no longer required once the desired edit is generated. In such embodiments, genome edits are effected in the plant cell genome as a result of transient expression of CRISPR-based genome editing system constructs, and such modifications are inherited in future generations without the need to segregate away transgenes used to express the genome editing components required for genome edits.

[0063] In some embodiments, the genome edit is a mutation. A “mutation” is a human- induced change in the genetic sequence compared to a wild type sequence ( / .< ., a sequence that exists in nature). The mutation may be, without limitation, one or more nucleotide insertions, one or more nucleotide substitutions, one or more nucleotide deletions, or any combination thereof. In some embodiments, mutations are those that cause the gene to not be expressed or not be properly expressed, or those that inactivate the protein, such as from an amino acid substitution or deletion, a splice junction mutation, or the introduction of a frameshift to the coding region that may lead to a premature stop codon. A mutation that leads to a change in the expression of the gene such that the function of the encoded protein is eliminated or substantially decreased, such as a premature stop codon, is referred to herein as a “knockout” mutation. In some embodiments, CRISPR-based genome editing is used to generate a plant gene mutation by causing one or more point mutations, insertions, deletions, or any combination thereof.Delivering Recombinant DNA & CRISPR-Based Genome Editing Systems

[0064] One aspect of the present disclosure is directed to a method of genome editing and regenerating a dicot plant. This method involves providing an explant from a dicot plant, transforming the explant with a microbe comprising recombinant DNA, where the recombinant DNA comprises a CRISPR-based genome editing system, and where said transforming is carried out under conditions effective for the CRISPR-based genome editing system to target and edit a gene of interest in the explant. The method also involves incubating the transformed explant and selecting a regenerated plant from the transformed explant, where the regenerated plant comprises the edit.

[0065] Another aspect of the present disclosure is directed to a method of editing and regenerating a plant of the family Cannabaceae. This method involves providing a hypocotyl and / or a cotyledon from a plant of the family Cannabaceae, culturing the hypocotyl and / or the cotyledon in a callus induction medium, dissecting out callus from the cultured hypocotyl and / or the cultured cotyledon, re-culturing green calli from the dissected out callus, transforming the re-cultured calli with a microbe comprising recombinant DNA, where the recombinant DNA comprises a CRISPR-based genome editing system capable of targeting and modifying a gene of interest in the re-cultured calli to produce a transformed cell. The method also involves incubating the transformed calli in darkness and then light and selecting a regenerated plant from the transformed calli, where the regenerated plant comprises a modification in the gene of interest.

[0066] These aspects of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0067] In the methods described herein, plant cells or plant parts are used for transformation, genome editing, and regeneration. In some embodiments, recombinant DNA is introduced into the calli or explant using transformation. In some embodiments, a calli or explant is transformed.“Transformation” refers to the introduction of a nucleic acid, such as a recombinant DNA, into a host organism. Host organisms containing a transformed recombinant DNA construct or DNA fragment are referred to as “transgenic” or “recombinant” organisms. In some embodiments, the methods described herein comprise transforming the calli or explant with a microbe comprising recombinant DNA. In some embodiments, the recombinant DNA comprises a CRISPR-based genome editing system, as discussed supra. In some embodiments, said transforming is carried out under conditions effective for the CRISPR-based genome editing system to target and edit a gene of interest in the explant. The phrase “under conditions sufficient to” is used to describe any environment that permits the desired reaction to take place.

[0068] As used herein, the term “plant cell” includes cells, protoplasts, cell tissue cultures from which plants can be regenerated, callus, clumps, and cells that are intact in plants or parts of plants including, but not limited to seeds, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, hypocotyls, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, nu cellar tissue, ovaries, explants, and other plant tissues or cells.

[0069] Plant parts for use in the methods disclosed herein comprise cells and tissue that are capable of regeneration (“regenerable”) into a plant. In some embodiments, the plant part comprising regenerable cells is present on a growing plant or explant. In some embodiments, the plant part is an isolated, e.g., excised cluster of regenerable cells. In certain embodiments, the isolated regenerable cell cluster is an explant. In some embodiments, the plant part is an isolated regenerable cell cluster, e.g., an explant that has been dissected from a plant or a plant part, such as a seed. In some embodiments, the isolated regenerable cell cluster is a hypocotyl meristem or axillary bud. In some embodiments, the isolated regenerable cell cluster is an apical bud.

[0070] In some embodiments, the method disclosed herein comprises providing an explant from a dicot plant. In some embodiments, the explant from the dicot plant is suitable for transformation, genome editing and regeneration. An “explant” is a tissue or a piece of tissue taken from a plant to be used in a culture medium for transformation, growth, development, genome editing, and regeneration. An explant can be any part of the plant, e.g., without limitation, a cotyledon, a hypocotyl, a leaf, a stem, a root, or a seed, that may be excised and placed in a controlled, sterile environment to initiate the growth of new plants or tissues. Explants can be used to propagate plants, study plant development, or produce genetically modified organisms through techniques such as direct organogenesis and / or somatic embryogenesis.

[0071] In some embodiments, an explant is a mature seed. In some embodiments, an explant is derived from a mature seed (e.g., a tissue excised from a mature seed). A mature seed is a plant seed that has completed its development and is capable of germination. In some embodiments, an explant is an immature seed or a tissue excised from an immature seed. In some embodiments, a hypocotyl and / or a cotyledon is provided from a seed of a dicot plant. In some embodiments, the mature seed is germinated. In some embodiments, the mature seed comprises emerging cotyledons and hypocotyl tissues.

[0072] In some embodiments, the methods of the present disclosure comprise sterilizing a surface of the seed. For example, the surface of the mature seed may be sterilized, as a non-limiting example, by an initial wash in water followed by incubation in a hydrogen peroxide solution. In some embodiments, the initial wash in water is for 0.1, 0.5, 1, 2, 3, 4, or 10 hours, or any number or range therein, or more than 10 hours. In some embodiments, the hydrogen peroxide solution is 0.1%, 0.2%, 0.5%, 1%, 2%, 5% hydrogen peroxide or any number or range therein, or more than 5% hydrogen peroxide. In some embodiments the incubation in hydrogen peroxide is for 1 hour, 8 hours, 16 hours, 24 hours, 48 hours, 72 hours, or any number or range therein, or more than 72 hours. In some embodiments, the incubation in hydrogen peroxide is in the dark. In some embodiments, the methods of the present disclosure comprise incubating the seed in darkness before dissecting the hypocotyls and / or the cotyledons. In some embodiments, the methods of the present disclosure comprise incubating the seed in darkness before transforming. In some embodiments, the incubation in the dark before transforming is for 1 hour, 8 hours, 16 hours, 24 hours, 48 hours, 72 hours, or any number or range therein, or more than 72 hours. In some embodiments, incubating in the dark before transforming is carried out for 24-48 hours. In some embodiments, incubating in the dark before transforming is carried out for about 1-2 weeks, 2-3 weeks, 3-4 week, or any number or rangetherein. In some embodiments, incubating in the dark before transforming is carried out for about 2- 3 weeks. In some embodiments, the seeds are sterilized, e.g., by dry sterilization, such as by exposure to chlorogenic gas. In some embodiments, seeds are selected for cleanliness prior to sterilization.

[0073] In some embodiments, the mature seed is germinated until hypocotyls emerge. A seed can be incubated for germination for any suitable length of time. In some embodiments, a seed is germinated for about 12-24 hours, 24-48 hours, 48-72 hours, 72-96 or any number or range therein, or more than 96 hours. In some embodiments, a seed is germinated for about 48-72 hours. In some embodiments, hydrogen peroxide treatment sterilizes the seed and also enhances germination. In some embodiments, the methods of the present disclosure include removing a seed coat from the mature seed. In some embodiments methods of the present disclosure include splitting the cotyledons into separate parts prior to transforming (e.g., cutting the cotyledons into slices and separating the hypocotyl, which can be cultured separately). In some embodiments, the root is dissected out prior to transforming.

[0074] In some embodiments, the methods described herein include inducing callus tissue for use in transformation and regeneration. In some embodiments, the methods disclosed herein comprise culturing the hypocotyl and / or cotyledon in a callus induction medium to form callus. In some embodiments, callus induction medium and other media (e.g., without limitation, Agrobacterium induction medium (also known as induction medium), liquid resuspension medium, co-cultivation medium, resting medium, regeneration selection medium, and rooting medium) used in the methods described herein comprise Murashige-Skoog (“MS”) salts and vitamins or Driver and Kuniyuki Walnut medium (“DKW”) salts and vitamins. In some embodiments, the MS or DKW salts and vitamins are used at 0.5x or lx strength in the media. In some embodiments, the callus induction medium or other media described herein uses full strength (e.g., lx) MS salts and vitamins. In some embodiments, the callus induction medium or other media described herein comprise 2-(N-morpholino) ethanesulfonic acid (“MES”), a buffering agent used to maintain a stable pH in the culture medium. In some embodiment, the medium includes 0.01 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.5 g / L, 1 g / L, 2 g / L MES, or any number or range therein, or more than 2 g / L MES. In some embodiments, the callus induction medium or other media described herein include MES at 0.1 g / L. In some embodiments, the callus induction medium or other media described herein include sucrose. In some embodiments, the medium includes sucrose at 10 g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60 g / L, 68 g / L, 70 g / L, 80g / L, lOOg / L, or any number or range therein, or more than 100 g / L. In someembodiments, the callus induction medium or other media described herein include sucrose at 60 g / L or 68 g / L. In some embodiments, the media described herein include maltose at 10 g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60 / L, 68 g / L, 70 g / L, 80g / L, lOOg / L, or any number or range therein, or more than 100 g / L. In some embodiments, the callus induction medium or other media described herein includes coconut water. Coconut water is rich in vitamins, minerals, and growth-promoting substances such as cytokinins, which can enhance cell division and growth. In some embodiments, the callus induction medium or other media described herein include coconut water at 1 ml / L, 2 ml / L, 3 ml / L, 4 ml / L, 5 ml / L, 6 ml / L, 7 ml / L, 8 ml / L, 9 ml / L, 10 ml / L, 15 ml / L, 20 ml / L, or any number or range therein, or more than 20 ml / L. In some embodiments, the callus induction medium or other media described herein include 10 ml / L coconut water. In some embodiments, the callus induction medium or other media described herein include 2,4-dichlorophenoxyacetic acid (“2,4- D”). 2,4-D is a synthetic auxin, a class of plant hormones that promotes cell division and callus formation. In some embodiments, the callus induction medium or other media described herein include 0.01 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 5 mg / L, 10 mg / L 2,4-D, or any number or range therein, or more than 10 mg / L 2,4-D. In some embodiments, the callus induction medium or other media described herein include 2,4-D at 0. 1 mg / L. In some embodiments, the callus induction medium or other media described herein include 2,4-D at 3 mg / L In some embodiments, the callus induction medium or other media described herein include zeatin. Zeatin is a naturally occurring cytokinin, a class of plant hormones that promotes cell division and shoot formation. In some embodiments, the callus induction medium or other media described herein include includes 0.01 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.5 mg / L, 1 mg / L zeatin, or any number or range therein, or more than 1 mg / L zeatin. In some embodiments, the callus induction medium or other media described herein include includes zeatin at 0.5 mg / L. In some embodiments, the callus induction medium or other media described herein include naphthaleneacetic acid (“NAA”). NAA is a synthetic auxin, a class of plant hormones that primarily influences cell elongation, root formation, and overall plant growth. In some embodiments the callus induction medium or other media described herein include 0.01 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.5 mg / L, 1 mg / L NAA, or any number or range therein, or more than 1 mg / L NAA. In some embodiments, the callus induction medium or other media described herein include includes NAA at 0.5 mg / L. In some embodiments, the callus induction medium or other media described herein include indole-3 -acetic acid (“IAA”). IAA is a naturally occurring auxin. In some embodiments the callus induction medium or other medium includes 0.01 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.5 mg / L, 1 mg / L IAA, or any numberor range therein, or more than 1 mg / L IAA. In some embodiments, the callus induction medium or other media described herein include IAA at 0.5 mg / L.

[0075] In some embodiments, the callus induction medium or other media described herein include a gelling agent. In some embodiments, the gelling agent is agar. In some embodiments, the gelling agent is phytogel. In some embodiments, the callus induction medium or other media described herein are an agar-based medium. In some embodiments, the callus induction medium or other media described herein include 0.1 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, lOg / L, 15g / L agar, or any number or range therein, or more than 15 g / L agar. In some embodiments, the callus induction medium or other media described herein include includes 8 g / L agar. In some embodiments, the callus induction medium or other media described herein are a phytogel based medium. In some embodiments, the callus induction medium or other medium includes 0.1 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, lOg / L, 15g / L phytogel, or any number or range therein, or more than 15 g / L phytogel. In some embodiments, the callus induction medium or other media described herein include 10 g / L phytogel.

[0076] In this and any of the media described herein, the pH of the media described herein may be 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 7.0 7.5, or 8.0, or any number or range therein. In some embodiments, the pH of the media is 5.2, 5.7 or 5.8.

[0077] In some embodiments, the methods disclosed herein include culturing the hypocotyl and / or the cotyledon in a callus induction medium. In some embodiments, the dissected hypocotyls and cotyledons from germinated seeds are cultured on callus induction medium in the dark. In some embodiments, the culturing on callus induction medium in the dark is performed for 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, or any number or range therein, or more than 6 weeks. In some embodiments, the culturing on callus induction medium in the dark is performed for 2-3 weeks. In some embodiments, the culturing on callus induction medium is performed at room temperature.

[0078] In some embodiments, the methods include dissecting out callus from the cultured hypocotyl and / or the cultured cotyledon. In some embodiments, the methods include resuspending the dissected out callus in liquid suspension medium. In some embodiments, the liquid suspension medium is callus induction medium without any agar. In some embodiments, the callus is resuspended in liquid suspension medium in the dark. In some embodiments, the incubation of callus resuspended in liquid suspension medium in the dark is performed for 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, or any number or range therein, or more than 6 weeks. In someembodiments, the incubation of callus resuspended in liquid suspension medium in the dark is performed for 2-3 weeks. In some embodiments, after incubation in the dark, the callus resuspended in liquid suspension medium is incubated in the light. In some embodiments, the incubation of callus resuspended in liquid suspension medium in the light is performed for 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, or any number or range therein, or more than 6 weeks. In some embodiments, the incubation of callus resuspended in liquid suspension medium in the light is performed for 2-3 weeks. In some embodiments, the methods include resuspending the dissected out callus in darkness for about 2-3 weeks followed by 2-3 weeks in light. In some embodiments, the incubation of callus resuspended in liquid suspension medium is performed on a shaker. In some embodiments the shaker is used at 20-50 rpm, 50-75 rpm, 75-100 rpm, or 100-175 rpm or any number or range therein. In some embodiments, the shaker is used at 70 rpm.

[0079] In some embodiments, after the callus is incubated in the light in liquid suspension culture, the methods include re-culturing green calli from the dissected out callus. In some embodiments, re-culturing green calli from the dissected out callus is performed on solid callus induction medium. In some embodiments, reculturing green calli from the dissected out callus is performed for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or any number or range therein, or more than 6 weeks. I some embodiments, reculturing green calli from the dissected out callus is performed for about 3 weeks. In some embodiments, reculturing green calli from the dissected out callus occurs with transfer to fresh liquid suspension medium periodically, if needed. In some embodiments, reculturing the green calli from the dissected out callus occurs twice. In some embodiments, the re-cultured green calli from the dissected out callus are used for transformation.

[0080] In some embodiments, methods described herein may include infecting and / or incubating the calli or explant with a microbe, such as a Rhizobia microbe, e.g. , Ensifer adhaerens or an Agrobacterium species. In some embodiments, the microbe is a species of Rhizobia or Agrobacterium. As used herein, “infection” by the microbe may include an interaction between the microbe and a plant cell that is beneficial, or not pathogenic, to the plant cell. A suitable Rhizobia microbe may include, without limitation, proteobacteria that belong to the Rhizobiaceae or Phyllobacteriaceae family. In some embodiments suitable Rhizobia microbes include, without limitation, those of the genus Ensifer, Sinorhizobium, and Mesohrizobium. In some embodiments, the Rhizobia microbe is an / ■ / adhaerens, S. meliloti, xM. loti microbe. Suitable Rhizobia microbes are described in, e.g., Zuniga-Soto et al., “Ensifer-Mediated Transformation: An Efficient Non- Agrobacterium Protocol for the Genetic Modification of Rice,” SpringerPlus 4.600 (2015), which ishereby incorporated by reference in its entirety. A suitable Agrobacterium microbe includes, without limitation, Agrobacterium tumefaciens an Agrobacterium rhizogenes microbe. In some embodiment, the Agrobacterium species is Agrobacterium tumefaciens. In some embodiments, the Agrobacterium microbe includes strain EHA105, AGL1 or LBA4404. The microbe, such as Agrobacterium can be modified to carry a recombinant DNA configured to modify a genome, as described herein. The microbe can be any suitable microbe for delivering a recombinant DNA (e.g., delivering a T-DNA, a plasmid, a CRISPR-based genome editing system, a RNP structure, etc.) to the calli or explant.

[0081] Other methods to deliver a recombinant DNA for modification of a plant genome can be accomplished, without limitation, using electroporation, PEG-mediated DNA transfer, microinjection, particle bombardment, or vacuum infiltration to provide for stable or transient expression of the recombinant DNA. In some embodiments, wounding is employed in the plant transformation methods, including for example, microprojectile bombardment; treatment with glass beads; cutting, scratching or slicing; sonication; or silicon carbide fibers or whiskers.

[0082] In some embodiments, the calli or explant is pre-treated before infecting and / or incubating with a microbe. In some embodiments, the pre-treatment includes callus induction, as described supra. In some embodiments, pre-treatment includes explant preparation, e.g., sterilization and / or tissue excision.

[0083] In some embodiments, prior to infecting the calli or explant, the microbe, e.g., the Agrobacterium species, is streaked from a glycerol stock onto a Petri plate. In this and other embodiments, the microbe, e.g., the Agrobacterium species, is incubated for 1, 2, 3, 4, 5, 6, 7, 8 days or any number or range therein, or more than 8 days. In some embodiments, the microbe, e.g., the Agrobacterium species, is incubated for 3-4 days. In some embodiments, the microbe, e.g., the Agrobacterium species, is incubated at 18°C, 21°C, 24°C, 28°C, 30°C, 32°C, or any number or range therein. In some embodiments, the microbe, e.g., the Agrobacterium species, is incubated at 28°C. In some embodiments, the microbe, e.g., the Agrobacterium species, is incubated on YEP medium with appropriate antibiotic selection for 2-3 days. In some embodiments, the microbe, e.g., the Agrobacterium species, is then resuspended in Agrobacterium induction medium to form an induced Agrobacterium culture. In some embodiments, the microbe, e.g., the Agrobacterium species, is then resuspended in Agrobacterium induction medium at an OD of 0.6-0.7. In some embodiments, the microbe, e.g., the Agrobacterium species, is induced in induction medium for 1-2 hours prior toinfecting the plant cell or the explant. In some embodiments, the microbe, e.g., the Agrobacterium species, is incubated in induction medium on a shaking platform.

[0084] In some embodiments, the Agrobacterium induction medium (also called induction medium herein) includes 0.5x strength MS medium, (e.g., for use with certain plants such as Fabaceae). In some embodiments, the induction medium includes 0.5x strength DKW medium, (e.g., for use with certain plants such as Cannabaceae). In some embodiments, the induction medium includes glucose. In some embodiments, the induction medium includes glucose and / or sucrose at Ig / L, 5 g / L, 10 g / L, 20g / L, 50g / L, lOOg / L, or any number or range therein, or more than 100 g / L sucrose. In some embodiments, the induction medium includes glucose at 5 g / L. In some embodiments, the induction medium includes sucrose at 10 g / L.

[0085] In some embodiments, the induced microbe culture is resuspended in callus inoculation medium. In some embodiments, callus induction medium includes lx MS salts and vitamins. In some embodiments, callus induction medium includes sucrose and / or glucose. In some embodiments, callus induction medium or other medium includes KC1. In some embodiments, callus induction medium or other medium described herein includes 0.5 g / L, 1 g / L, 2 g / L 3 g / L, 4g / L, 5 g / L, or 10 g / L KC1 or any number or range therein, or more than 10 g / L KCl. In some embodiments, callus induction medium includes KC1 at 3 g / L. In some embodiments, callus induction medium or other medium described herein includes MgC . In some embodiments, callus induction medium or other medium described herein includes 0.5 g / L, 1 g / L, 2 g / L 3 g / L, 4g / L, 5 g / L, or 10 g / L MgCI2or any number or range therein, or more than 10 g / L MgCh. In some embodiments, callus induction medium includes MgCI2at 4 g / L.

[0086] In some embodiments, the methods disclosed herein involve transforming the plant cell or the explant with a microbe comprises infecting the calli or explant. In some embodiments, the methods involve transforming the explant with a microbe comprising recombinant DNA. In some embodiments, the methods involve transforming the re-cultured calli with a microbe comprising recombinant DNA. In some embodiments, transforming the calli or explant comprises immersing the calli or explant directly in the induced Agrobacterium culture. In some embodiments the re-cultured green calli are immersed in callus inoculation medium. In some embodiments, the calli or explant is immersed with the induced Agrobacterium culture in induction medium or callus inoculation medium for 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 16 hours or any number or range therein, or more than 16 hours. In some embodiments, the calli or explant is immersed with the induced Agrobacterium culture in induction medium or callus inoculation medium for 1-2 hours. Insome embodiments, immersing is performed in the dark. In some embodiments, immersing is performed at room temperature.

[0087] In some embodiments, transforming the calli or explant comprises infecting the calli or explant using an Agrobacterium species. Accordingly, a plant cell (e.g., callus) or explant is immersed in a culture comprising an Agrobacterium species. In some embodiments, the calli or explant is incubated with the Agrobacterium species in the dark. In some embodiments, the calli or explant is incubated with the Agrobacterium species for 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, or any number orrange therein, or more than 12 hours. In some embodiments, the calli or explant is incubated with the Agrobacterium species for 1-2 hours. In some embodiments, the calli or explant is incubated with the Agrobacterium species on a shaking platform (e.g., at 70 rpm).

[0088] In some embodiments, the methods comprise incubating the transformed explant. In some embodiments, the methods comprise incubating the transformed calli in darkness and then light. In some embodiments, transforming the plant cell or the explant comprises incubating or cocultivating the calli or explant with the microbe, e.g., the induced Agrobacterium culture. During cocultivation, also known as incubation, plant tissues or explants are exposed to the microbe under controlled conditions, allowing the microbe to interact closely with the plant cells to promote infection. As discussed supra , Agrobacterium is known for its ability to transfer DNA to plant cells, utilizing its Ti plasmid to introduce new genetic material, facilitating the integration of transfer of recombinant DNA to the plant genome.

[0089] In some embodiments, incubating the transformed explant is carried out in a cocultivation medium. In some embodiments, incubating the transformed calli is carried out in a cocultivation medium. In some embodiments, the co-cultivation medium includes an agar-based medium. In some embodiments, the plant part is incubated with the microbe on a first substrate, e.g., an agar gel. In some embodiments, the plant part is incubated with the microbe on a second permeable substrate, e.g., a filter paper, disposed on the first substrate. In some embodiments, the calli or explant is blotted on sterile filter paper and transferred to co-cultivation medium. In some embodiments, the calli or explant is incubated with the microbe in the dark. In some embodiments, the calli or explant is incubated with the microbefor 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, or any number or range therein, or more than 2 weeks. In some embodiments, the calli or explant is co-cultivated with the microbe for 2 days. In some embodiments, the calli or explantis co-cultivated with the microbefor 5-7 days. In some embodiments, the calli or explant isincubated with the microbe at a temperature of about 21 °C. In some embodiments, the calli or explant is co-cultivated with the microbe in the dark. In some embodiments, incubating is carried out in darkness for about 2 days. In some embodiments, incubating is carried out in darkness for about 5-7 days at a temperature of about 21°C.

[0090] In some embodiments, the co-cultivation medium or other medium described herein include benzylaminopurine (“BAP”). BAP is a synthetic cytokinin, a class of plant hormones that promotes cell division and shoot formation. In some embodiments the co-cultivation medium or other media described herein include 0.01 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.5 mg / L, 1 mg / L BAP, or any number or range therein, or more than 1 mg / L BAP. In some embodiments, the cocultivation medium or other media described herein include BAP at 0.2 mg / L.

[0091] In some embodiments, the co-cultivation medium or other media described herein include NAA at 0.5 mg / L. In some embodiments, the co-cultivation medium or other media described herein include NAA at 0.2 mg / L. In some embodiments, the co-cultivation medium or other media described herein include both BAP and NAA at any of the above-mentioned concentrations, respectively. In some embodiments, the co-cultivation medium or other media described herein include both BAP and NAA at 0.2 mg / L.

[0092] In some embodiments, the co-cultivation medium or other media described herein include lx strength MS or DKW salts and vitamins. In some embodiments, the co-cultivation medium or other media described herein include glucose at 10 g / L. In some embodiments, the cocultivation medium or other media described herein include sucrose at 20 g / L. In some embodiments, the co-cultivation medium or other media described herein include maltose at 30 g / L. In some embodiments, the co-cultivation medium or other media described herein include zeatin at 0.5 mg / L. In some embodiments, the co-cultivation medium or other media described herein include includes MES at 0.05 g / L. In some embodiments, the co-cultivation medium or other media described herein include 10 ml / L coconut water. In some embodiments, the co-cultivation medium or other media described herein include 2,4-D at 3 mg / L. In some embodiments, the co-cultivation medium or other media described herein include MES at 0. 1 g / L.

[0093] In some embodiments, the co-cultivation medium or other media described herein include acetosyringone. Acetosyringone is a phenolic compound that acts as a signaling molecule that enhances the virulence of Agrobacterium. In some embodiments the co-cultivation medium or other media described herein include 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM acetosyringone, or any number or range therein, or more than 200 mM acetosyringone. In someembodiments the co-cultivation medium or other media described herein include 50 mM acetosyringone. In some embodiments the co-cultivation medium or other media described herein include 150 mM acetosyringone.

[0094] In some embodiments, the induction or co-cultivation medium or other media described herein include a nonionic surfactant. In some embodiments, the nonionic surfactant is an organosilicone surfactant. Suitable nonionic surfactant include, without limitation, trisiloxane (e.g., heptamethyltrisiloxane), polysorbate (Tween®) (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80), Triton™ X-100 and polyethylene glycol (PEG). In some embodiments, the nonionic surfactant includes a modified heptamethyltrisiloxane, e.g., a polyalkyleneoxide-modified heptamethyltrisiloxane. In some embodiments, the nonionic surfactant is Silwet® L77. In some embodiments, the induction, inoculation, co-cultivation medium or other media described herein include 0.01% to 5%, e.g., 0.05% to 3%, 0.1% to 2%, 0.1% to 1%, 0.2% to 0.8% of the nonionic surfactant. In some embodiments, the inoculation medium co-cultivation medium or other media described herein include about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1 .4%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% or about 5%, or any number or range therein, or more than 5% of the nonionic surfactant.Regeneration via Direct Organogenesis & Somatic Embryogenesis

[0095] In some embodiments, a transformed plant is regenerated using direct organogenesis. “Direct organogenesis” is a process in plant tissue culture where new organs, such as shoots or roots, are formed directly from explant tissues without an intermediate callus formation stage. This method involves the regeneration of plant organs from specific tissues or cells, such as cotyledon, hypocotyl, leaves, stem, root, or seeds, under controlled conditions in a sterile culture medium. The process may be induced by the application of specific growth hormones and nutrients that promote the differentiation and development of new organs directly from the explant. Direct organogenesis is advantageous because it can reduce the risk of genetic variability and somaclonal variation, leading to more uniform and stable regenerated plants.

[0096] In some embodiments, a transformed plant is regenerated using somatic embryogenesis. “Somatic embryogenesis” is a process in plant tissue culture where explant tissues are cultured to induce callus formation, a mass of undifferentiated cells. Under controlled conditions and with the application of specific growth hormones, these callus cells can be stimulated to develop into somatic embryos.

[0097] In some embodiments, the methods of the present disclosure include selecting a regenerated plant from the transformed calli. In some embodiments, the methods of the present disclosure include selecting a regenerated plant from the transformed explant. In some embodiments, the regenerated plant comprises the genome edit in the gene of interest. In some embodiments, selecting a regenerated plant includes culturing the transformed calli or explant to induce shoot formation after co-cultivation with the microbe. In some embodiments, the method includes resting the transformed re-cultured calli or the transformed explant before culturing for shoot formation e.g., culturing with selection). Accordingly, the transformed re-cultured calli or the transformed explant are transferred to resting media. Resting media provides a nurturing environment that aids in the recovery and initial growth of plant tissues after transformation. In some embodiments, the method comprises transferring the incubated, transformed explant to a liquid resting medium. In some embodiments, the method comprises transferring the dissected out calli to a liquid resting medium. In some embodiments, the method comprises transferring the incubated, transformed calli to a liquid resting medium.

[0098] In some embodiments, the resting medium or other media described herein include one or more antimicrobial agents, e.g., an antibiotic. The medium may include any suitable antimicrobial agent to reduce, slow, and / or prevent growth of microbes (e.g., Agrobacterium'). Suitable antimicrobial agents include, without limitation, Timentin and / or Cefataxime. In some embodiments, the resting medium or other media described herein include an effective amount of one or more of the antimicrobial agent(s). In some embodiments the medium contains 1 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L or any number or range therein, or more than 500 mg / L Timentin and / or Cefataxime. Resting media or other media described herein include may also include any of the components discussed supra. In some embodiments, the transformed explant or the transformed re-cultured calli is incubated in resting medium for about 1 -3 days, 4-7 days, 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, or any number or range therein, or more than 6 weeks. In some embodiments, the transformed explant is incubated in resting medium for about 4-7 days. In some embodiments, the transformed calli is incubated in resting medium for about 2-3 weeks. In some embodiments, the incubation in resting media may be performed in the light or in the dark. In some embodiments, the methods of the present disclosure comprise incubating the explant in light for 4-7 days. In some embodiments, the transformed recultured calli is incubated in resting media for about 2 weeks in the dark. In some embodiments, themethods further comprise exposing the calli to light. In some embodiments, the incubation in resting media is performed at room temperature.

[0099] In some embodiments, selecting a regenerated plant from the transformed re-cultured calli or the transformed explant is performed using regeneration medium with selection (regeneration selection medium). In certain embodiments, selecting a regenerated plant involves culturing the transformed re-cultured calli or the transformed explant to induce shoot formation. In some embodiments, shoot formation includes one or more selection cycles for shoots that are genetically modified. In some embodiments, a selection cycle includes incubating the transformed re-cultured calli or the transformed explant on a medium that includes a selective agent that is specific for a selective marker included in the recombinant DNA with which the transformed re-cultured calli or the transformed explant was transformed. The regeneration selection medium or other media described herein may include any suitable selective agent. In some embodiments, the regeneration selection medium or other media described herein include spectinomycin or geneticin. In some embodiments, the regeneration selection medium includes 1 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, or any number or range therein, or more than 500 mg / L of spectinomycin or geneticin. In some embodiments, selecting comprises selecting the regenerated plant using spectinomycin.

[0100] Regeneration selection medium or other media described herein may also include any of the components discussed supra. In some embodiments the regeneration selection medium or other media described herein include 0.5x MS or 0.5x DKW salts and vitamins. In some embodiments, the regeneration selection medium or other media described herein include sucrose at 15 g / L. In some embodiments, the regeneration selection medium or other media described herein include maltose at 30 g / L. In some embodiments, the regeneration selection medium includes BAP at 0.3 mg / L. In some embodiments, the regeneration selection medium or other media described herein include NAA at 0.2 mg / L. In some embodiments, the regeneration selection medium or other media described herein include IAA at 0.2 mg / L. In some embodiments, the regeneration selection medium comprises a gelling agent.

[0101] In some embodiments of the methods of the disclosure, the method comprises transferring the explant to a regeneration selection medium and culturing the explant in light until multiple shoots form from the explant. In some embodiments, selecting a regenerated plant includes incubatingunder light with transfer to fresh regeneration selection medium periodically, if needed. In some embodiments, transfer to fresh regeneration selection medium occurs every 2 weeks. Insome embodiments, incubating is performed for 1-5 weeks, e.g., 1-2 weeks, 2-3 weeks, or 2-4 weeks, per selection cycle or any number or range therein, or more than 5 weeks per selection cycle. In some embodiments, those plant parts that grow under selective conditions are included in the next selection cycle. In some embodiments, the plant parts are subjected to 1, 2, 3, 4, 5, or 6 or more selection cycles. In some embodiments, the same amount of selection agents are used per selection cycle. In some embodiments, the amount of selection agents are used in one or more selection cycle is different from the amount used in one or more other selection cycles. In some embodiments, the amount of the selection agent is increased from the first to the second selection cycle.

[0102] In some embodiments, selecting a regenerated plant includes incubating under dark for 2-3 weeks, as a non-limiting example. In some embodiments, after incubation in the dark, selecting a regenerated plant includes incubating under light with transfer to fresh regeneration selection medium every 2 weeks, if needed, as a non-limiting example. In some embodiments, incubating is performed for 1-5 weeks, e.g., 1-2 weeks, 2-3 weeks, or 2-4 weeks, per selection cycle or any number or range therein, or more than 5 weeks per selection cycle.

[0103] In some embodiments, transformed shoots formed on the regeneration selection medium are transferred to germination cups or other suitable container.

[0104] In some embodiments, the methods of the present disclosure include elongating the shoot after selection.

[0105] In some embodiments, the regenerated plant is rooted. Accordingly, in some embodiments, the methods of the present disclosure include incubating a shoot on a suitable rooting medium. In some embodiments, the regenerated plants surviving selection are incubated on rooting medium to form roots. In some embodiments, the rooting medium or other medium includes indole- 3 -butyric acid (“IB A”). IBA is a naturally occurring auxin that promotes root formation. In some embodiments the rooting medium or other medium includes 0.01 mgL, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.5 mg / L, 1 mg / L IBA, or any number or range therein, or more than 1 mg / L IBA. In some embodiments, the rooting medium or other medium includes IBA at 0.1 mg / L. In some embodiments, the rooting medium includes a suitable selective agent. In some embodiments, the rooting medium includes spectinomycin or geneticin. In some embodiments, the rooting medium includes Timentin and / or Cefataxime.

[0106] The methods of the present disclosure are effective in producing regenerated plants by direct organogenesis in a shortened period of time (see, e.g., FIG. 5). In some embodiments, the methods of the present disclosure are effective to produce a regenerated plant in about 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days or any number or range therein. In some embodiments, the regenerated plant is produced in about 35-50 days. In some embodiments, the regenerated plant is produced in about 45 days. In some embodiments, the regenerated plant is incubated to produce roots. In some embodiments, the regenerated plant is rooted in soil. In some embodiments, the regenerated plant is a rooted regenerated plant. In some embodiments, the methods of the present disclosure are effective to produce a rooted regenerated plant in about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 95 days, or any number or range therein. In some embodiments the rooted regenerated plant is produced in about 40-60 days. In some embodiments, the method is effective to produce a rooted regenerated plant in about 45-60 days. In some embodiments, the method is effective to produce a rooted regenerated plant in about 55 days. In some embodiments, the methodis effective to produce a rooted regenerated plant in less than 70 days. In some embodiments, the regenerated plant comprises the edit in the gene of interest.

[0107] In some embodiments, the methods are effective to produce a transformed plant. Transformed plants are capable of growth on selective media (e.g., media with a selection agent). In some embodiments, regenerated plants that survive two rounds of selection on selective media are considered to be transformed. In some embodiments, a transformed plant is healthy and green on selective media in comparison to non-transformed plants that may be sick, necrotic, dead, or bleached under selection. In some embodiments, the transformation efficiency is about l%-80%, 3%-23%, 5-20%, 10%-50%, or any number or range therein. In some embodiments, transforming is carried out at an efficiency of at least 3-10%. In some embodiments, the transformation efficiency is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 23%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any number or range therein.

[0108] In some embodiments, the methods of the present disclosure are directed to regenerating a dicot plan. The method involves providing an explant from a dicot plant, transforming the explant with a microbe comprising recombinant DNA, where the recombinant DN A comprises a selectable marker, and where said transforming is carried out under conditions effective for expression of the selectable marker. The method also involves incubating the transformed explant and selecting a regenerated plant from the transformed explant, where the regenerated plant comprises the selectable marker.

[0109] In some embodiments, the methods of the present disclosure are directed to a method of regenerating a plant of the family Cannabaceae. This method involves providing a hypocotyl and / or a cotyledon from a seed of a plant of the family Cannabaceae, culturing the hypocotyl and / or the cotyledon in a callus induction medium, dissecting out callus from the cultured hypocotyl and / or the cultured cotyledon, re-culturing green calli from the dissected out callus, transforming the recultured calli with a microbe comprising recombinant DNA, where the recombinant DNA comprises a selectable marker, and where said transforming is carried out under conditions effective for expression of the selectable marker in the re-cultured calli. The method also involves incubating the transformed calli in darkness and then light, and selecting a regenerated plant from the transformed calli, where the regenerated plant comprises the selectable marker.Genome Edited Plants

[0110] In some embodiments, the methods of the present disclosure are suitable for transformation, genome editing, and regeneration of a plant, including dicot plants. Exemplary dicots include, but are not limited to, plants from the Fabaceae family, including legumes such as alfalfa (Medicago sativa), chickpeas (Cicer arietinurri), peanuts (Arachis hypogaea), soybeans (Glycine max)' , and various beans including black beans, kidney beans, lima beans, navy beans, and pinto beans; plants from the Ro saceae family encompassing fruit-bearing plants such as almonds (Prunus dulcis), apples (Main s dome stica), apricots (Prunus armeniaca), cherries (Prunus avium), peaches (Primus persica), pears (Pyrus), plums (Prunus domestica), raspberries (Rubus idaeus), and strawberries (Fragaria x ananassa), plants from the Solanaceae family, including vegetables and fruits such as bell peppers (Capsicum annuum), eggplants (Solanum melongend), potatoes (Solanum tuberosum), and tomatoes (Solanum lycopersicum),' plants from the Brassicaceae family including broccoli (Brassica oleracea), cabbage (Brassica oleracea), cauliflower (Brassica oleracea), and canola (Brassica napus),' plants from the Cucurbitaceae family, including cucumbers (Cucumis sativus), melons (Cucumis melo), pumpkins (Cucurbita pepo , squash (Cucurbita), and watermelons (Citrullus lanatus),’ plants from the Asteraceae family, including artichokes (Cynara scolymus), chamomile (Matricaria chamomilla), chicory (Cichorium intybus), dandelion (Taraxacum , echinacea (Echinacea , endive (Cichorium endivia), and lettuce (Lactuca sativa),' plants from the Rutaceae family, including citrus fruits such as lemons (Citrus limori), limes (Citrus aurantiifolia), and oranges (Citrus sinensis), plants from the Lamiaceae family, including herbs such as lavender (Lavandula), maijoram (Origanum majorana), mint (Mentha), oregano (Origanum vulgar e),rosemary (Salvia rosmarinus), and sage (Salvia officinalis),' plants from theVitaceae family, including grapes (Vitis vinifera),' plants from the Moraceae family, including figs (Ficus carica), mulberries (Morns), and jackfruits (Artocarpus heterophyllus),' plants from the Anacardiaceae family, including cashews (Anacardium occidentale), mangoes (Mangifera indica), and pistachios (Pistacia vera). The Apiaceae family includes celery (Apium graveolens), cilantro (Coriandrum sativum), and parsley (Petroselinum crispuni),' plants from the Malvaceae family including cotton (Gossypium),' plants from the Cannabaceae family including Cannabis and hemp (Cannabis saliva) and hops (Humulus hipulus),' plants from the Ericaceae family, including blueberries (Vaccinium corymbosum) and cranberries (Vaccinium macrocarpon) plants from the Myrtaceae family including guavas (Psidium guajava) ; plants from the Lauraceae family including avocados (Persea americana),' plants from the Passifloraceae family, including passion fruits (Passiflora edulis),' plants from the Sapindaceae family includes lychees (Litchi chinensis),' plants from the Euphorbiaceae family, including cassava (Manihot esculenta), and plants from the Zingiberaceae family including turmeric (Curcuma longa).[0U1] In some embodiments, the dicot is a plant selected from alfalfa, almonds, apples, apricots, artichokes, asparagus, avocados, bell peppers, beets, blackbeans, blackberries, blueberries, breadfruits, broccoli, cabbage, canola, Cannabis, carrots, cashews, cauliflower, celery, chamomile, cherries, chicory, chickpeas, cilantro, cocoa, coffee, collard greens, cotton, cowpea, cranberries, cucumbers, dandelion, dragon fruits, durians, echinacea, eggplants, endive, escarole, figs, grapes, guavas, hazelnuts, hemp, hops, jackfruits, kale, kidney beans, kiwis, lavender, lemons, lettuce, lima beans, limes, lychees, mangoes, marjoram, melons, mint, mulberries, mustard greens, navy beans, okra, olives, oranges, oregano, papayas, parsley, passion fruits, peaches, peanuts, pears, peas, peppers, pinto beans, pistachios, plums, pomegranates, potatoes, pumpkins, quinces, radishes, rapeseed, raspberries, red clover, rhubarb, rosemary, sage, sesame, soybeans, spinach, squash, strawberries, sunflowers, sweet potatoes, Swiss chard, tobacco, tomatoes, turnips, vetch, walnuts, watermelons, white clover, and zucchini. In some embodiments, the dicot is of the species Cannabis saliva L. ox Humulus lupulusV. In some embodiments, the dicot is a plant selected from Cannabis, hemp, hop, peanut, or alfalfa.

[0112] In some embodiments, the dicot is a member of the Cannabaceae or Fabaceae family. The Cannabaceae or Fabaceae families are two distinct but economically important families of flowering plants within the Rosids clade. The Rosid clade is a major lineage of dicots that encompasses a broad diversity of agriculturally and industrially significant crops. Cannabaceaeincludes high-value multipurpose crops such as hemp, Cannabis (Cannabis sativa L.), and hop (Humulus lupulus L. known for their medicinal, fiber, nutritional, and biocomposite applications. As used herein, “Cannabis” refers to a genus of flowering plants in the family Cannabaceae, and includes at least three recognized species: Cannabis sativa, 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 nonmedicinal types. Cannabis is also classified based on cannabinoid content into 5 classes referred to as chemotypes or chemovars. Chemotype 1 (marijuana) has high amounts of tetrahydrocannabinolic acid (THC) and low amounts of cannabidiol (CBD), with amounts of THC as high as 30%. Chemotype 2 has approximately equal amounts of THC and CBD. Chemotype 3 (hemp) has high amounts of CBD, low amounts of THC, with amounts of CBD as high as 20%. Chemotype 4 has high amounts of cannabigerol (CBG), a precursor of THC and CBD. Chemotype 5 does not produce cannabinoids. The term “hemp” refers to a nonmedicinal strain of Cannabis sativa grown for industrial uses and is considered herein to be a Cannabis plant defined as hemp by the U.S. Government as Cannabis plants containing no more than 0.3% THC by dry weight. The Fabaceae family is one of the largest and most important legume families, comprising key crops like peanut (Arae his hypogaea Z.) and alfalfa (Medicago sativa L Members of the Fabaceae family are known for their roles in global food systems and regenerative agriculture. Peanut serves as a protein- and oil-rich food staple, whereas alfalfa excels as a high- yielding forage crop with strong nitrogen-fixation capabilities that enhance soil health and reduce fertilizer dependency.

[0113] In some embodiments, the methods described herein involve analyzing the regenerated plant to detect the genome edit. Detecting the genome edit can be performed using any method known in the art. For example, the region of a gene targeted by the CRISPR-based genome editing system can be PCR amplified and sequenced (see, e.g., FIGs. 9 & 15). Other forms of nucleic acid detection can include next generation sequencing methods such as DNA SEQ or RNA SEQ using any known sequencing platform including, but not limited to: Roche 454, Solexa Genome Analyzer, AB SOLiD, Illumina GA / HiSeq, Ion PGM, MiSeq, among others.

[0114] In some embodiments, molecular markers can be used to detect and track the genome edited mutations for the breeding or further propagation of plants comprising the genome edited mutations.

[0115] In non-limiting examples, a gene of interest targeted for genome editing, may include genes that affect plant architecture, disease resistance, flowering time, photoperiod response,nitrogen use efficiency, abiotic stress tolerance, drought tolerance, biotic stress tolerance, herbicide resistance, shelf life, flavor, aroma, color, texture, oxidation, yield, nutritional content, starch content and / or composition, oil content and / or composition, protein content and / or composition, amino acid content and / or composition, allergenicity, pest tolerance, and agronomic performance. In some embodiments, the gRNA sequence targets a gene selected from the group consisting of genes altering plant architecture, disease resistance, flowering time, photoperiod response, nitrogen use efficiency, abiotic stress tolerance, biotic stress tolerance, herbicide resistance, shelf life, flavor, aroma, color, texture, oxidation, yield, nutritional content, starch content and / or composition, oil content and / or composition, protein content and / or composition, amino acid content and / or composition, allergenicity, pest tolerance, and agronomic performance.

[0116] Plant cells, plant tissue, and plants comprising a genome edit according to the present disclosure may display improved traits in comparison to corresponding wild-type plants. In some embodiments, the genome edit may confer a visual trait in comparison to corresponding wild-type plant to facilitate transformation and selection.

[0117] In some embodiments, the gene of interest is a Stomagen gene or a Phytoene Desaturase gene. In some embodiments, the gene of interest is a Cannabis sativa Tetrahydrocannabinolic Acid THCA) Synthase (CsTHCA Synthase) gene. In some embodiments, the gene of interest is a Fatty Acid Desaturase gene (e.g., FAD-2 or FADS)' . In some embodiments, the gene of interest is a Phosphatase 2C gene. In some embodiments, the gene of interest is a Stomagen gene, a Phytoene Desaturase gene, a THCA Synthase gene, a Fatty Acid Desaturase-2 gene, a Fatty Acid Desaturase-3 gene, or a Phosphatase 2C gene.

[0118] A gene of interest in one dicot may be used to identity a gene of interest in another dicot. For example, a gene or interest can be identified using bioinformatic software (e.g., by performing sequence comparisons to identify sequence identity and motifs or domains in the nucleotide or amino acid sequences) and / or phylogenetic analysis.

[0119] The Stomagen gene encodes a peptide hormone known as stomagen, which plays a role in the regulation of stomatai development in plants. Stomata are small openings on the surface of leaves that facilitate gas exchange, allowing carbon dioxide to enter for photosynthesis and oxygen and water vapor to exit. Stomagen is produced in the mesophyll cells of the leaf and acts as a positive regulator of stomatai development. It promotes the formation of stomata by interacting with specific receptors on the surface of epidermal cells, leading to the activation of signaling pathways that result in the differentiation and development of stomatai cells. Alteration of the Stomagen genetarget by, e.g., genome editing, can lead to changes in stomatai development, potentially resulting in altered plant growth, making it useful as a visual indicator of a successful genome edit in the methods disclosed herein. In some embodiments, plants comprising edits in the Stomagen gene have improved abiotic stress resistance and / or biotic (pest) stress resistance.

[0120] In some embodiments, the Stomagen gene is from Cannabis sativa. In some embodiments, the Stomagen gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO: 15. In some embodiments the Stomagen gene comprises the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:15. In some embodiments, the Stomagen gene comprises a coding sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the Stomagen vae comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0121] In some embodiments, the plant has a genome edit in the Stomagen gene. In some embodiments the genome edit in the Stomagen gene is selected from any one of more of SEQ ID NOs: 25-26. In some embodiments, the plant with the genome edit in the Stomagen gene has an altered leaf phenotype compared to a plant without a genome edit in the Stomagen gene.

[0122] In some embodiments, the Stomagen gene and / or the Phytoene Desaturase gene is from Cannabis sativa Humuhis hipulus, Medicago sativa, or Arachis hypogaea.

[0123] Phytoene desaturase is an enzyme involved in the biosynthesis of carotenoids, which are essential pigments in plants. Carotenoidsplay a role in photosynthesis by protecting chlorophyll from photooxidative damage and by contributing to the light-harvesting process. Phytoene desaturase specifically catalyzes the conversion of phytoene to lycopene, a key step in the carotenoid biosynthetic pathway.

[0124] In some embodiments, alteration of the Phytoene Desaturase gene target by, e.g., genome editing, can lead to the disruption of carotenoid biosynthesis. This can lead to a deficiency in carotenoids, which in turn affects the stability and function of chlorophyll. As a result, the leaves of the plant may exhibit chlorosis, which is characterized by a yellowing of the leaves due to the breakdown of chlorophyll and the lack of green pigmentation making edits of Phytoene Desaturase useful as a visual indicator of a successful genome edit in the methods disclosed herein.

[0125] In some embodiments, the Phytoene Desaturase gene is from Cannabis sativa. In some embodiments, the Phytoene Desaturase gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO:21 . In some embodiments the Phytoene Desaturase gene comprises the nucleotide sequence of SEQ ID NO:21, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:21 . In some embodiments, the Phytoene Desaturase gene comprises a coding sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:22. In some embodiments, the Phytoene Desaturase gene comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of SEQ ID NO:23.

[0126] In some embodiments, the plant has a genome edit in the Phytoene Desaturase gene. In some embodiments the genome edit in the Phytoene Desaturase gene is selected from any one of more of SEQ ID NOs: 36-41. In some embodiments, the plant with the genome edit in the Phytoene Desaturase gene has a chlorotic tissue phenotype compared to a plant without a genome edit in the Phytoene Desaturase gene.

[0127] In some embodiments, the genome edit is in a Cannabis THCA Synthase (CsTHCA) gene. CsTHCA is an enzyme that catalyzes the conversion of cannabigerolic acid (CBGA) into tetrahydrocannabinolic acid (THCA), which is the precursor to tetrahydrocannabinol (THC), the primary psychoactive compound in Cannabis. This enzymatic reaction is a crucial step in the biosynthetic pathway of cannabinoids in the Cannabis plant.

[0128] In some embodiments, the THCA Synthase gene is from Cannabis sativa. In some embodiments, the THCA Synthase gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO: 18. In some embodiments the THCA Synthase gene comprises the nucleotide sequence of SEQ ID NO: 18, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the THCA Synthase gene comprises a coding sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the THCA Synthase gene comprises an amino acid sequence that has atleast 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or100% (or any number or range therein) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0129] In some embodiments, the plant has a genome edit in the THCA Synthase gene. In some embodiments, the plant with the genome edit in the THCA Synthase gene has an altered cannabinoid phenotype compared to a plant without a genome edit in the THCA Synthase gene.

[0130] In some embodiments, the genome edit is in a Fatty Acid Desaturase gene, such as FAD-2 and / or FAD-3 of Arachis hypogaea. FAD-2 and FAD-3 are enzymes involved in the biosynthesis of polyunsaturated fatty acids in peanuts, playing a crucial role in determining the oil composition of the seeds. FAD-2 is responsible for the desaturation of oleic acid to linoleic acid, while FAD-3 further desaturates linoleic acid to produce alpha-linolenic acid. Modifying the activity of these enzymes through genetic engineering can enhance the nutritional profile of peanut oil by increasing its oleic acid content.

[0131] In some embodiments, the FAD-2 gene is from Arachis hypogaea. In some embodiments, the FAD-2 gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO:30. In some embodiments the FAD-2 gene comprises the nucleotide sequence of SEQ ID NO:30, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:30. In some embodiments, the FAD-2 gene comprises a coding sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:31 . In some embodiments, the FAD-2 gene comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of SEQ ID NO:32.

[0132] In some embodiments, the FADS gene is from Arachis hypogaea. In some embodiments, the FAD-3 gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO:33. In some embodiments the FADS gene comprises the nucleotide sequence of SEQ ID NO:33, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:33. In some embodiments, the FAD-3 gene comprises a coding sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:34. In some embodiments, the FAD-3 gene comprises an amino acid sequence that has atleast 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of SEQ ID NO:35.

[0133] In some embodiments, the plant has a genome edit in the FAD-2 and / or FAD-3 gene. In some embodiments, the plant with the genome edit in the FAD-2 and / or FAD-3 gene has an altered fatty acid content in oil compared to a plant without a genome edit in the FAD-2 an / or FAD- 3 gene.

[0134] In some embodiments, the genome edit is in a Protein Phosphatase 2C (PP2C) gene. PP2C genes encode a group of enzymes that play a role in cellular signaling by dephosphorylating serine / threonine residues on target proteins. PP2C genes are involved in the abscisic acid (ABA) signaling pathway, helping plants respond to environmental stresses such as drought and salinity.

[0135] In some embodiments, the Protein Phosphatase 2C gene is from Medicago sativa. In some embodiments, the PP2C gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO:27. In some embodiments the PP2C gene comprises the nucleotide sequence of SEQ ID NO:27, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:27. In some embodiments, the PP2C gene comprises a coding sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:28. In some embodiments, the PP2C gene comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of SEQ ID NO:29.

[0136] In some embodiments, the plant has a genome edit in the PP2C gene. In some embodiments, the plant with the genome edit in the PP2C gene has improved drought tolerance compared to a plant without a genome edit in the PP2C gene.

[0137] In some embodiments of the present disclosure, the gRNA targets a gene involved in plant architecture. In some embodiments, the gRNA targets a gene involved in disease resistance. In some embodiments, the gRNA targets a gene involved in flowering time. In some embodiments, the gRNA targets a gene involved in abiotic stress resistance. In some embodiments, the gRNA targets a gene involved in oil composition. In some embodiments, the gRNA targets a gene involved in disease resistance. In some embodiments, the gRNA targets a gene involved in drought tolerance.

[0138] Once the genome modification is stably incorporated into the genome of a plant, the plant can be propagated in any number of ways, including for example by sexual reproductionthrough seed by selfing, and by clonal propagation using cuttings. The genome modification can also be introduced into other plants by breeding. Thus, such genome edited plants and the resultant modified traits can be used in a conventional breeding scheme or in in vitro plant propagation to produce more modified plants with the same characteristic(s), and / or can be used to introduce the same characteristic(s) in other varieties of the same or related species. . In some embodiments, the recombinant DNA comprises a CRISPR-based genome editing system. In some embodiments, the recombinant DNA comprises a CRISPR-based endonuclease. Optionally, the recombinant DNA comprises a second expression cassette encoding a guide RNA capable of directing the endonuclease to a target a gene of interest. In some embodiments, the recombinant DNA further comprises a third expression cassette encoding a selectable, wherein the third expression cassette is configured to express the marker in the plant cell. In some embodiments, the method includes identifying a plant having the genome edit and lacking the recombinant DNA by segregation, backcrossing or genotyping.

[0139] Another aspect of the present disclosure relates to a genome-edited plant, or a plant part thereof, produced by the methods described herein.

[0140] A further aspect of the present disclosure relates to a plant product derived from a genome-edited plant, or plant part thereof, described herein.

[0141] Yet another aspect is a plant comprising a genome edit in a gene of interest. In some embodiments, the gene of interest is any one or more of a Stomagen gene, a Phytoene Desaturase gene, a THCA Synthase gene, a Fatty Acid Desaturase-2 gene, & Fatty Acid Desaturase-3 gene, or a Phosphatase 2C gene.

[0142] Also provided herein are products derived from a genome-edited plant obtained using methods described herein. In some embodiments, the product is an extract derived from the genome- edited plant, where the extract exhibit properties distinct from a conventional extract derived from a non-edited plant. In some embodiments, the extract is an oil extract. In some embodiments, the oil extract is high oleic acid oil. In some embodiments, the extract includes one or more cannabinoids, such as THC and / or CBD. In some embodiments, the product is a plant fiber derived from the genome-edited plant.

[0143] Various types of genome edits are encompassed by the present disclosure, and include modifications resulting from insertion of recombinant DNA, into the plant genome to generate genome modified transgenic plants. Recombinant DNA incorporated into a plant genome can provide for expression of foreign proteins in plants, overexpression of proteins native to theplant, or for reduced expression of native plant genes. In other embodiments, recombinant DNA constructs incorporated into the plant genome express components required for genome editing such as a CRISPR-based genome nuclease and a guide RNA.

[0144] In some embodiments, recombinant DNA inserted into the plant genome provides for expression of foreign genes or overexpression of native genes. Such embodiments utilize recombinant DNA constructs, cassettes, and vectors such as described above and provide plants having an improved trait or traits as the result of expression of a nucleic acid sequence encoding a polypeptide or polypeptides of interest. In some embodiments, genome edits provide for modification of cannabinoid and / or terpene metabolic pathways to provide plants having increased or decreased levels of specific compounds. In some embodiments, genome edits provide for improved fiber and / or seed properties to enhance industrial uses associated with hemp varieties. In some embodiments, recombinant DNA inserted into a plant genome imparts traits that are beneficial to agronomic production of a crop plant, including, for example increased yield, enhanced nitrogen use efficiency, abiotic stress tolerance (such as tolerance to drought, cold, or osmotic conditions), herbicide tolerance, and reduction in damage caused plant pests or pathogens, such as insects, fungi, bacteria, and viruses.

[0145] The following examples are intended to exemplify the embodiments of the disclosure but are by no means intended to limit the scope thereof.EXAMPLESExample 1 - Agrobacterium Strains and Vectors

[0146] Two Agrobacterium strains, LBA4404 and AGL1, were used in the binary vector transformation system. The binary vector system contains the T-DNA binary vector (pPZP200 or pCAMBIA2300) and a helper Ti plasmid (for T-DNA transfer). The T-DNA binary vectors contain the ColEl and pVSl plasmid origins of replication for replication in E. coli and in Agrobacterium. The LBA4404 strain contains the helper Ti plasmid, pAL4404, while the AGL1 strain contains the helper Ti plasmid, pTiBO542. The Ti plasmids initiate the expression of genes that facilitate T-DNA transfer into the plant genome. All constructs were then subjected to nanopore sequencing for sequence confirmation before conducting transformation experiments. Exemplary vectors are shown in FIGs. 1-4.

[0147] A. tumefaciens strain LBA4404 vector system contained the T-DNA binary vector with either pZP200 or pCAMBIA2300 backbone. Strain AGL1 contained only the T-DNA binary vector backbone, pCAMBIA2300. Binary vectors contained either the neomycin selectable marker,NPTII, or the spectinomycin selectable marker, spcN. Two visual reporter cassettes were used: dsRED and GFP.

[0148] The pPZP200 binary vector T-DNA contains the NPTII plant selectable marker and the GFP reporter cassette. The pCAMBIA2300 binary vector T-DNA contains the NPTII plant selectable marker and the dsRED reporter cassette. An iteration of our pPZP200 binary vector also contains the spectinomycin plant selectable marker, spcN.

[0149] Binary vectors contained Cas9-NLS driven by double-enhanced CaMV 35S promoter and NOS terminator sequences. GuideRNAs (gRNAs) were cloned into T-DNA binary vectors for each target gene. For the set of vectors that are based on the pPZP2000 backbone, each guide was independently cloned into a pENTR / D-TOPO-based entry plasmid containing the Arabidopsis U6- 26 promoter to drive gRNA expression and a double enhanced 35 S promoter driving Cas9 expression. A Gateway™ LR reaction (Thermo Fisher Scientific, Waltham, MA) was used to move the gRNA and Cas9 cassette into a pPZP200-based binary vector. For the other set of vectors, single gRNAs were independently cloned into the pCAMBIA2300 binary vector containing the Arabidopsis U6-26 promoter and 35S promoter driving Cas9 expression.

[0150] Also developed was a vector design to evaluate spectinomycin as a plant-selectable marker, spcN in Cannabis and other crops. This marker was cloned into a binary vector with a desired visual reporter cassette. The spcN cassette was cloned into a cassette containing an eFl A promoter from Glycine max and a ubiquitin 10 terminator from Arabidopsis thaliana.Example 2 - Transformation Procedures

[0151] Agrobacterium preparation. 1 ) Master plate preparation : Agrobacterium was streaked from glycerol stocks on ABA medium with appropriate antibiotics and incubated at 28°C for 3-4 days. The master plates thus prepared were kept in the refrigerator, and working plates were prepared by re-streaking for up to a month. 2) Working plate preparation: Agrobacterium from master plates was re-streaked on YEP medium with appropriate antibiotics and incubated at 28°C for 2-3 days. 3) Inoculum preparation: Bacteria were scraped from the working plate using a sterile loop and resuspended in Agrobacterium induction medium (for Cannabaceae, half-strength Driver and Kuniyuki Walnut medium (DKW) salts and vitamins + lOg / L sucrose + 5g / L glucose + 0.05mM 2- (N-morpholino)ethanesulfonic acid (MES) + 50mM acetosyringone + 0.02% silwet, pH 5.8 was used, and for Fabaeceae, half-strength Murashige-Skoog (MS) salts and vitamins + lOg / L sucrose +5g / L glucose + 0.05mMMES + 50mM acetosyringone + 0.02% silwet, pH 5.8 was used) at an OD of 0.6-0.7 and induced for 1-2 hours on a slow shaker at 70 rpm at room temperature.Organogenesis-Based Protocol

[0152] Mature seed explant preparation. 1) Seed sterilization and germination: The whole seeds were surface sterilized by an initial wash for 1-2 hours in running tap water and then incubated in the dark (Petri dishes were sealed and wrapped in aluminum foil) in 2% hydrogen peroxide on a solution for 24-48 hours. 2) Explant preparation: Mature seeds with emerging hypocotyls were used as such for transformation. In an alternate protocol, mature seeds with emerging hypocotyls and the seed coat removed before transformation were used. And in another alternate protocol, the cotyledons from mature seeds with emerging hypocotyls and the seed coat removed were split into separate parts (e.g., the cotyledons and hypocotyls were separated / sliced and cultured separately, and the root was dissected out before transformation).

[0153] Agrobacterium inoculation. 1) Infection: The explants (whole seeds or dissected cotyledons and hypocotyls were immersed directly into the induced Agrobacterium culture in tall Petri dishes and incubated in dark (covered with aluminum foil) for 1-2 hours on a 70-rpm shaker. 2) Co-cultivation: The infected explants were blotted on sterile (autoclaved) filter paper and transferred to solid Co-cultivation medium (Full strength Murashige-Skoog / DKW salts and vitamins + 20g / L sucrose + lOg / L glucose + 0.2mg / L Benzyl Amino Purine (BAP) + 0.2 mg / L NAA + 0.05 mM MES + 50mM acetosyringone + 8g / L agar, pH 5.8) and incubated in dark at 21°C for 5-7 days. This cocultivation medium formulation (with 0.2mg / L Benzyl Amino Purine (BAP) + 0.2 mg / L NAA instead of 0.5mg / L Thiadiazuron + 0.01 mg / L meta-topolin, + 0.2mg / L IAA) increased efficiency of regenerated plant formation in terms of numbers and rigorous morphology.

[0154] Transgenic Plant regeneration . 1) Resting: The co-cultivated explants were transferred to liquid Resting Medium (Full strength Murashige-Skoog / DKW salts and vitamins + 20g / L sucrose + + 0.2mg / L BAP + 0.2mg / L NAA + 0.05 mM MES + 8g / L agar, pH 5.8 with1 OOmg / L cefatoxime + 1 OOmg / L Timentin), and incubated in light at room temperature for 4-7 days.2) Regeneration with selection: The explants from resting medium were transferred to solid Regeneration Selection Medium (Half strength Murashige-Skoog / DKW salts and vitamins + 15g / L sucrose + 0.3 mg / L Benzyl Aminopurine + 0.2mg / L NAA + 0.2mg / L IAA + 8g / L agar, pH 5.8 with 1 OOmg / L cefatoxime + 1 OOmg / L Timentin and appropriate selection marker antibiotics, namely, 30 mg / L geneticin or 25 mg / L spectinomycin). The cultures were incubated in light at room temperature and the formation of multiple shoots was monitored with transfers to fresh selectionmedia every two weeks. Multiple shoots surviving selection were individually transferred from Petri dishes to germination cups with Regeneration selection media and monitored for selection and survival. Leaf sampleswere taken at this stage for PCR testing for transgene integration. 3) Rooting: The explants surviving selection were transferred to solid Rooting Medium (half strength Murashige-Skoog / DKW salts and vitamins + 15g / L sucrose + 0.1 mg / L IBA + lOg / L phytagel, pH 5.7 with 100mg / L cefatoxime+ l OOmg / L Timentin and appropriate selection marker antibiotics). 4) Plantlet multiplication: Putative transgenic plants that were PCR positive were transferred to Hemp multiplication medium (Phytotech Labs, Lenexa, KS) for generating multiple clones of the transgenic plantlets.Somatic Embryogenesis-Based Protocol for Cannabaceae

[0155] Hemp mature seed explant preparation, callus induction, and suspension cultures. 1) Seeds were sterilized and germinated as described above, and dissected hypocotyls and cotyledons were cultured on callus induction medium (full strength Murashige-Skoog / DKW salts and vitamins + 60g / L sucrose + 0. Ig / L MES + lOml / L Coconut water + Img / L 2,4-D + 0.5mg / L zeatin + 0.5mg / L NAA + 8g / L agar, pH 5.8) and incubated in dark at room temperature for 2-3 weeks. 2) Callus was dissected out from explants and resuspended in liquid suspension medium (callus induction medium without agar) for 2-3 weeks in dark followed by 2-3 weeks in light on a shaker at 70 rpm. 3) Green calli were re-cultured on the solid callus induction medium for 3 weeks, and in some cases for 6 weeks, and used for transformation.

[0156] Agrobacterium inoculation. 1) Inoculum preparation: The Agrobacterium culture (prepared on plates as described above) was resuspended in callus inoculation medium (Murashige- Skoog / DKW salts and vitamins + 68g / L sucrose + 36g / L glucose + 3g / L KC1 + 4g / L MgCl2+15 OmM acetosy ringone + 0.02% silwet, pH 5.2) at an OD of 0.6-0.7 and induced for 1-2 hours on a slow shaker at 70 rpm at room temperature. 2.) Infection: The calli were immersed directly into the induced Agrobacterium culture in tall Petri dishes and incubated in dark (covered with aluminum foil) for 1-2 hours on a 70 rpm shaker.3) Co-cultivation: The infected calli were transferred onto sterile (autoclaved) filter paper overlaid on solid Co-cultivation medium (Full strength Murashige- Skoog / DKW salts and vitamins + 3 Og / L maltose + lOg / L glucose + 0. Ig / L MES + lOml / L Coconut water + 3mg / L 2,4-D + 0.5mg / L zeatin + 0.5mg / L NAA + 150mM acetosyringone + 8g / L agar, pH 5.8) and incubated in the dark at 21°C for 2 days.

[0157] Transgenic Hemp regeneration. 1) Resting: The co-cultivated explants were transferred to Resting Medium ( Murashige-Skoog / DKW salts and vitamins + 3 Og / L maltose +O. lg / L MES + lOml / L Coconut water + 3mg / L 2,4-D + 0.5mg / L zeatin + 0.5mg / L NAA + 8g / L agar, pH 5.8 with lOOmg / L cefatoxime + lOOmg / L Timentin), and incubated in dark at room temperature for 2 weeks. 2) Regeneration with selection: The calli from resting medium were transferred to solid Regeneration Selection Medium (Murashige-Skoog / DKW salts and vitamins + 30g / L maltose + O. lg / L MES + 1 Oml / L Coconut water + 3mg / L 2,4-D + 0.5mg / L zeatin + 0.5mg / L NAA + 8g / L agar, pH 5.8 with 1 OOmg / L cefatoxime+ 1 OOmg / L Timentin) and appropriate selection marker antibiotics, namely, 30 mg / L Geneticin or 25 mg / L spectinomycin). The cultures were incubated in dark for 2-3 weeks and then transferred to light at room temperature. The formation of multiple shoots was monitored with transfers to fresh selection media every two weeks.Example 3 - Direct Organogenesis is an Efficient Method for Dicot Transformation

[0158] Transformation was performed on multiple hemp varieties, including Futura 83, Yuma, and Puma, originally obtained from IHC Production LLC, Colorado. Peanut, hops and alfalfa experiments were conducted with commercially available seeds. The seeds were germinated in the laboratory and used directly as explants for all experimental procedures. Regenerated plants of Cannabis, hemp, hops, peanut and alfalfa were all successfully obtained on regeneration mediaunder selection.

[0159] The overall process of direct organogenesis using two types of explants, hypocotyl, and cotyledon, for Cannabis, peanut, and alfalfa transformation is described in FIG. 5. The time needed from the selection of an explant to the regeneration of a rooted plant took approximately 55- 60 days. The rooted plants were moved to the hardening step under a controlled environment.

[0160] FIGs. 6A-I illustrate the different steps in the transformation method via direct organogenesis from hypocotyl and cotyledon in Cannabis and hemp. The steps include explant preparation, infection, selection of putative transgenic events, and subsequent transfer to tissue culture tubs.

[0161] FIGs. 10A-E illustrate the different steps in the transformation method via direct organogenesis from cotyledon explants of peanut to regenerated putative transgenic peanut plants.Example 4 - Somatic Embryogenesis is an Efficient Method for Cannabis Transformation

[0162] The somatic embryogenesis pathway for plant transformation and regeneration is an effective way to overcome the dependency on seed material while also improvingthe transformation efficiency and gene-editing frequency in cannabis cultivars. A protocol was successfully developedfor establishing callus cultures, inducing somatic embryos, transgene expression in the somatic embryos, and producing shoot-like structures from hypocotyl and cotyledon explants (FIGs. 7A-L). The various steps involved in the somatic embryogenesis process are provided in FIGs. 7A-L.Example 5 - Direct Organogenesis Transformation Efficiencies

[0163] The plants transformed using direct organogenesis that survived two rounds of selection on appropriate media supplemented with geneticin or spectinomycin were used to determine the transformation frequency. Most of these plants were healthy and green, while the nontransformed plants were either sick, necrotic, dead, or bleached under selection, depending on the type of selection applied. The overall transformation efficiency varied by cultivar and ranged between 3% and 23%, as shown in Table 1, using Cannabis transformation as an example. The transformation efficiency for peanut was 8-10%.Table 1. Transformation Efficiencies Detected in Different Cannabis Cultivars.Example 6 - Genome Editing & Molecular Analysis of Transformed Plants Harboring the Gene Edit

[0164] After transformation, tissue samples of transformants were taken and genotyped for the presence of T-DNA using primers to amplify fragments of the coding regions of Cas9, NPTII, and gRNA (Table 2). To rule out the presence of Agrobacterium in tissue samples, samples weretested for presence of Agrobacterium using universal virD2 primers, a chromosomal gene present in the bacteria (Table 2). FIG. 8 represents a PCR gel with putative transgenic events that are free of Agrobacterium.

[0165] Genome editing was performed on gene targets including Stomagen in Cannabis and hemp, Phytoene Desaturase (PDS) in Cannabis, hemp, and peanut, and Fatty Acid Desaturase genes, such as FAD-2 and / or FAD-S in peanut. Genome edits have been confirmed in PDS in Cannabis, hemp, and peanut, and for Stomagen in Cannabis.Table 2. Genotyping Primers used for Detecting the Presence of Cas9 / Vird2 / NPTII Genes in the Plant Tissue SamplesExample 7 - Next-Generation Sequencing Identifies Stable Gene-Edited Events

[0166] For the tissues transformed with the constructs containing Cas9, genotyping for gene edits was performed by employing next-generation sequencing. A region spanning 300-500 bp around the predicted Cas9-induced cut site was amplified with primers (Table 3) adapted for Illumina sequencing.Table 3. Primers for Amplicon Sequencing to Detect Genome Editing

[0167] FIG. 9 represents the amplicon sequencing of a target gene, CsStomagen, which was successfully edited to create indels and / or deletions. Different gene targets were selected for genome editing; the sequence details of these gene targets are provided in Table 4.Table 4. Candidate Genes Targeted for Genome editing Along with the Target GenomicSequencesExample 8 - Phenotypic Evaluation of Stable Gene-Edited Events

[0168] Distinct phenotypic changes were observed in the gene-edited hemp, Cannabis and peanut plants that were edited to have mutations in either the PDS or Stomagen genes. Genome- edited PDS plants having inactivating mutations in the PDS gene showed albino leaves at varying degrees in hemp (FIGs. 11A-B), Cannabis (FIGs. 12A-B) and peanut (FIGs. 13A-B). In case of Stomagen genome edited hemp plants having inactivating mutations, a distinct change was observed in the leaf morphology (FIGs. 14A-B). A sequence alignment showing genome edited mutations in in the Phytoene Desaturase gene of Cannabis sativa are shown in FIG. 15.Prophetic Example 9 - Genome Editing of Additional Genes of Interest

[0169] Recombinant DNA constructs including guide RNAs and a CRISPR-based genome editing nuclease are made. In one recombinant DNA vector, a guide RNA is designed to target the alfalfa gene (Medicago sativa) Protein Phosphatase 2C (MsPP2C) for editing. Alfalfa plants are transformed with the recombinant DNA construct having the guide RNA targeting MsPP2C and alfalfa plants comprising genome edits in MsPP2C are regenerated using direct organogenesis with the methods disclosed herein. In another recombinant DNA vector, a guide RNA is designed to target the Cannabis gene (Cannabis sativa) Tetrahydrocannabinolic acid (THCA) synthase (CsTHCA synthase) for editing. Cannabis plants are transformed with the recombinant DNA construct having the guide RNA targeting CsTHCA synthase and Cannabis plants comprisinggenome edits CsTHCA synthase are regenerated using either direct organogenesis or somatic embryogenesis with the methods disclosed herein.

[0170] 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

WHAT IS CLAIMED IS:1 . A method of genome editing and regenerating a dicot plant, said method comprising: providing an explant from a dicot plant; transforming the explant with a microbe comprising recombinant DNA, wherein the recombinant DNA comprises a CRISPR-based genome editing system, and wherein said transforming is carried out under conditions effective for the CRISPR-based genome editing system to target and edit a gene of interest in the explant; incubating the transformed explant; and selecting a regenerated plant from the transformed explant, wherein the regenerated plant comprises the edit.

2. The method of claim 1, wherein the regenerated plant is rooted, and wherein the method is effective to produce a rooted regenerated plant in about 45-60 days.

3. The method of claim 1, wherein the regenerated plant is rooted, and wherein the method is effective to produce a rooted regenerated plant in less than 70 days.

4. The method of claim 1, wherein said transforming is carried out at an efficiency of at least 3-10%.

5. The method of any one of the preceding claims, wherein the explant is derived from a mature seed.

6. The method of claim 5, wherein the mature seed comprises emerging cotyledons and hypocotyl tissues.

7. The method of claim 5 or claim 6, further comprising: removing a seed coat from the mature seed prior to said transforming.

8. The method of claim 6 or claim 7, further comprising: splitting the cotyledons into separate parts prior to said transforming.

9. The method of any one of claims 5-8 further comprising: dissecting out a root prior to said transforming.

10. The method of any one of claims 5-9 further comprising: sterilizing a surface of the seed.

11. The method of any one of claims 5-10 further comprising: incubating the mature seed in darkness before said transforming.

12. The method of claim 11, wherein said incubating in darkness is carried out for 24-48 hours.

13. The method of any one of the preceding claims, wherein the microbe is a species of Agrobacterhim .

14. The method of any one of the preceding claims, wherein said incubating the transformed explant is carried out in a co-cultivation medium.

15. The method of any one of the preceding claims, wherein said incubating the transformed explant is carried out in darkness for about 5-7 days at a temperature of about 21°C.

16. The method of claim 15 further comprising: transferring the incubated, transformed explant to a liquid resting medium prior to said selecting.

17. The method of claim 16, wherein said incubating the transformed explant is carried out in light for about 4-7 days.

18. The method of claim 17 further comprising: transferring the explant to a regeneration selection medium and culturing the explant in light until multiple shoots form from the explant.

19. The method of any one of the preceding claims, wherein said selecting comprises selecting the regenerated plant using spectinomycin.

20. The method of any one of the preceding claims, wherein said CRISPR-based genome editing system comprises: a polynucleotide encoding a CRISPR-based genome editing nuclease and a polynucleotide encoding a guide RNA targeting the gene of interest.

21. The method of any one of the preceding claims, wherein said dicot plant is a plant selected from alfalfa, almond, apple, apricot, artichoke, asparagus, avocado, bell pepper, beet, black bean, blackberry, blueberry, breadfruit, broccoli, cabbage, canola, Cannabis, carrot, cashew, cauliflower, celery, chamomile, cherry, chicory, chickpea, cilantro, cocoa, coffee, collard greens, cotton, cowpea, cranberry, cucumber, dandelion, dragon fruit, durian, echinacea, eggplant, endive, escarole, fig, grape, guava, hazelnut, hemp, hops, jackfruit, kale, kidney bean, kiwi, lavender, lemon, lettuce, lima bean, lime, lychee, mango, marjoram, melon, mint, mulberry, mustard, navy bean, okra, olive, orange, oregano, papaya, parsley, passion fruit, peach, peanut, pear, pea, pepper, pinto bean, pistachio, plum, pomegranate, potato, pumpkin, quince, radish, rapeseed, raspberry, red clover, rhubarb, rosemary, sage, sesame, soybean, spinach, squash, strawberry, sunflower, sweet potato, Swiss chard, tobacco, tomato, turnip, vetch, walnut, watermelon, white clover, or zucchini.

22. The method of claim 21, wherein the dicot plant is a plant selected from Cannabis, hemp, hop, peanut, or alfalfa.

23. The method of any one of the preceding claims, wherein the gene of interest is any one or more of a Stomagen gene, a Phytoene Desaturase gene, a THCA Synthase gene, a Fatty Acid Desaturase -2 gene, a Fatty Acid Desaturase-3 gene, and a Phosphatase 2C gene.

24. The method of claim 23, wherein the gene of interest is the Stomagen gene comprising a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 15.

25. The method of claim 24, wherein the edit in the Stomagen gene is selected from any one of more of SEQ ID NOs:25-26.

26. The method of any one of claims 23-25, wherein the gene of interest is the Stomagen gene, and wherein the regenerated plant comprises an altered leaf phenotype compared to a plant without a genome edit in the Stomagen gene.

27. The method of claim 23, wherein the gene of interest is the Phytoene Desaturase gene comprising a nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO 21 .

28. The method of claim 27, wherein the edit in the Phytoene Desaturase gene is selected from any one of more of SEQ ID NOs:37-41.

29. The method of any one of claims 23 and 27-28, wherein the gene of interest is the Phytoene Desaturase gene, and wherein the regenerated plant comprises a chlorotic tissue phenotype compared to a plant without a genome edit in the Phytoene Desaturase gene.

30. The method of claim 23, wherein the gene of interest comprises the THCA Synthase gene comprising a nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:18; the Fatty Acid Desaturase-2 gene comprising a nucleotide sequence of SEQ ID NO:30 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:30; the Fatty Acid Desaturase- gene comprising a nucleotide sequence of SEQ ID NO:33 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:33; and the, Phosphatase 2C gene comprising a nucleotide sequence of SEQ ID NO:27 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:27.

31. A genome edited plant or a plant part thereof produced by the method of any one of claims 1-30.

32. A plant product from the genome edited plant of claim 31 .

33. A method of genome editing and regenerating a plant of the familyCannabaceae, said method comprising: providing a hypocotyl and / or a cotyledon from a seed of a plant of the family Cannabaceae; culturing the hypocotyl and / or the cotyledon in a callus induction medium; dissecting out callus from the cultured hypocotyl and / or the cultured cotyledon; re-culturing green calli from the dissected out callus; transforming the re-cultured calli with a microbe comprising recombinant DNA, wherein the recombinant DNA comprises a CRISPR-based genome editing system capable of targeting and modifying a gene of interest in the re-cultured calli to produce a transformed calli; incubating the transformed calli in darkness and then light; and selecting a regenerated plant from the transformed calli, wherein the regenerated plant comprises the edit.

34. The method of claim 33, wherein the plant is of the species Cannabis sativa L. or Humulus lupulus L.

35. The method of claim 33 or claim 34, wherein the seed is a mature seed.

36. The method of claim 35, wherein the mature seed comprises emerging hypocotyls.

37. The method of any one of claims 33 to 36 further comprising: sterilizing a surface of the seed prior to said dissecting.

38. The method of any one of claims 33 to 37 further comprising: incubating the seed in darkness before said dissecting.

39. The method of claim 38, wherein said incubating in darkness is carried out for about 2-3 weeks.

40. The method of any one of claims 33-39 further comprising:resuspending the dissected out callus in liquid suspension medium.

41. The method of claim 40, wherein said resuspending is carried out in darkness for about 2-3 weeks followed by 2-3 weeks in light.

42. The method of any one of claims 33 to 41 , wherein the microbe is a species of Agrobacterium.

43. The method of any one of claims 33 to 42, wherein said incubating the transformed calli is carried out in a co-cultivation medium.

44. The method of claim 43, wherein said incubating is carried out in darkness for about 2 days.

45. The method of any one of claims 33-44 further comprising: transferring the incubated, transformed calli to a liquid resting medium.

46. The method of claim 45 further comprising: incubating the transformed calli in darkness for about 2-3 weeks.

47. The method of claim 46 further comprising: exposing the transformed calli to light.

48. The method of any one of claims 33-47, wherein said selecting is carried out with spectinomycin.

49. The method of any one of claims 33-47, wherein said CRISPR-based genome editing system comprises: a polynucleotide encoding a CRISPR-based genome editing nuclease and a polynucleotide encoding a guide RNA targeting the gene of interest.

50. The method of any one of claims 33-49, wherein the gene of interest is any one or more of a Stomagen gene, a Phytoene Desaturase gene, a THCA Synthase gene, a Fatty Acid Desaturase-2 gene, a Fatty Acid Desaturase-3 gene, and a Phosphatase 2 C gene.

51. A genome edited plant or a plant part thereof produced by the method of any one of claims 33-50.

52. A plant product from the plant of claim 51 .

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