Deconstructed plant virus components for tissue culture free plant transformation

Abstract

Provided herein are nucleic acids and vectors including geminivirus-based replicons and nanovirus-based replicons. Also disclosed are plant cells, whole plants, and plant parts including the nucleic acids or vectors. Further provided are methods of producing or expressing a nucleic acid or modifying the genetic material of a plant cell.

Description

Docket No. P15048WO00DECONSTRUCTED PLANT VIRUS COMPONENTS FOR TISSUE CULTURE FREE PLANT TRANSFORMATIONRELATED APPLICATIONS

[0001] This international patent application claims benefit of U.S. Provisional Patent Application Serial Nos.: 63 / 843,789, filed July 14, 2025; filed; 63 / 789,862, filed April 16, 2025; and 63 / 745,193, filed January 14, 2025; which are each incorporated herein by reference in its entireties.SEQUENCE LISTING XML

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on January 13, 2026, is named P15048WO00.xml and is 407,200 bytes in size. Also incorporated herein by reference in its entirety is the sequence listing named P15048US02.xml which was created on June 10, 2025, which was filed in U.S. Provisional Patent Application Serial No. 63 / 843,789 on July 14, 2025, and which is 407,116 bytes in size.TECHNICAL FIELD

[0003] This relates to agricultural biotechnology, more specifically compositions and methods for modifying plants.BACKGROUND

[0004] Plant genetic transformation strategies are essential tools for genetic engineering to produce transgenic and gene edited plants. Most plant transformation methods using bacteria such as Agrobacterium involve the conversion of Agrobacterium-infected dedifferentiated calli into regenerated plantlets through tissue culture. These methods are limited by numerous factors, including plant species, culture conditions, genetic and technical instabilities, expensive and complicated processes for culture of transformed cells, long duration, and low transformation efficiency.

[0005] Transient expression of a nucleic acid to transform or obtain gene edits in whole plants is a convenient tool for plant molecular biology, as it is a simplified process that avoids tissue cultureDocket No. P15048WO00steps and is less labor intensive than generating stable transgenic lines. However, delivery to preexisting meristems is a challenge and achieving a sufficient amount of exogenous nucleic acids (e.g., sufficient copy number) to effect transformation or gene edits in whole plants can be difficult in transient systems. Thus, improved compositions and methods for transforming or obtaining gene edits which avoid tissue culture steps are needed.SUMMARY

[0006] Disclosed herein are Gemini virus-based (Gemini) replicons and Nanovirus-based (Nano) replicons that have superior production of nucleic acids of interest in plant host cells without forming an active virus. The replicons are useful, for example, for transforming cells in the subepidermal mesophyll (also called L2) cell layer of plant shoot apical meristems which gives rise to the immediate underlying subepidermal plant tissue.

[0007] The Gemini replicons include a transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to DNA encoding a plant geminivirus coat protein (CP), a replication initiator protein (Rep), a first geminivirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents which can modify genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS.

[0008] The Nanovirus replicons include a transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to DNA encoding a plant nanovirus coat protein (CP), a replication initiator protein (Rep), a first nanovirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents which can modify genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS.

[0009] In various embodiments, the replicons comprise gene editing reagent(s) comprising: (i) an RNA directed DNA endonuclease and a guide RNA; (ii) a zinc finger nuclease; (ii) a transcription activator-like effector nuclease (TALEN); or (iv) a meganuclease. The geminivirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein to form a gemini-like viral particle (GLVP). Also provided are Gemini replicons further comprising nucleic acids encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS. In some embodiments, the gene editing reagents comprise a CRISPR-Cas effector protein m associationDocket No. P15048WO00with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.

[0010] Also provided are methods for modifying genomic DNA of a plant comprising: (a) contacting a whole plant or part thereof with a bacterial plant transformation culture comprising a plant transforming bacterium containing a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to: (i) DNA encoding a plant geminivirus or nanovirus coat protein (CP) and a plant geminivirus or nanovirus replication initiator protein (Rep); and (ii) a first geminivirus or nanovirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents which can modify genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS; and (b) selecting a propagule of the plant comprising the modification of the genomic DNA. In various embodiments, the plant transforming bacterium is (i) an Agrobacterium sp. and the transfer DNA is an Agrobacterium T-DNA; or (ii) a Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., or Phyllobacterium sp. In some embodiments, the bacterial plant transformation culture contacts the plant part comprising a meristem of the plant, such as a shoot apical meristem, intercalary meristem, a vegetative meristem, a vegetative apical meristem, and / or a floral meristem. In some embodiments, the LI layer of the meristem is contacted by the bacterial transformation culture. In some embodiments, the L2 layer of the meristem is not directly contacted by the bacterial transformation culture,

[0011] In some embodiments, the plant is a dicot plant and the geminivirus is: (i) a Begomovirus and the RRS is a Begomovirus common region; (ii) a Curtovirus or a Topocuvirus and the RRS is a Curtovirus or a Topocuvirus intergenic region; or (iii) a Mastrevirus and the RRS is a Mastrevirus Large Intergenic region (L1R). In some embodiments, the plant is a monocot plant and the geminivirus is a Mastrevirus.

[0012] In some embodiments, the plant is a dicot plant and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roll virus (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV). subterranean clover stunt vims (SCSV), or cow vetch latent virus (CVLV). In some embodiments, the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV), cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV).

[0013] Also provided are non-human cells comprising a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to: (i) DNA encoding a plantDocket No. P15048WO00geminivirus coat protein (CP) and a plant geminivirus Rep protein; and (ii) a first geminivirus large intergenic region (RRS). DNA encoding one or more gene editing reagents, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS. In some embodiments, the first transfer DNA may comprise DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.

[0014] Also provided are plant gene editing systems comprising a whole plant wherein a part of the plant comprising a meristem is contacted with a bacterial transformation culture comprising a plant transforming bacterium containing a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to: (i) DNA encoding a plant geminivirus coat protein (CP) and a plant geminivirus replication initiator protein (Rep); and (ii) a first geminivirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS. In some embodiments, the first transfer DNA further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.

[0015] The foregoing and other features of this disclosure w ill become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE FIGURES

[0016] Fig. 1 is an illustration of plant Shoot Apical Meristem (SAM) layers reached by Agrobacterium-based delivery of gene editing components and a combination of Agrobacterium and geminivirus gene editing components.

[0017] Fig. 2 is a schematic map of a transfer DNA (T-DNA) encoded in a Gemini or nanovirus replicon. The T-DNA comprises a plant geminivirus or nanovirus coat protein (CP) and a plant geminivirus or nanovirus replication initiator protein (Rep). The T-DNA comprises gene editing reagents (Cas + guide) located between two geminivirus or nanovirus Rep recognition sites (RRS). The transfer of the T-DNA into plant genome is initiated from the right border (RB) and terminated at the left border (LB).

[0018] Fig. 3 is an illustration of a T-DNA comprising nucleic acid sequences encoding gene editing components delivered into the LI SAM layer by Agrobacterium. Gene expression of theDocket No. P15048WO00T-DNA occurs in the LI cell layer resulting in production of gemini virus or nanovirus derived coat protein (CP), replication initiator protein (Rep), and the replicon. A Gemini like particle or nanovirus like particle forms which comprises CP and the replicon with editing reagents and moves from the L1 cell layer into the L2 cell layer where gene editing occurs.

[0019] Fig, 4 is a schematic map of a transfer DNA (T-DNA) encoded in a Gemini replicon. The T-DNA comprises a plant gemini virus coat protein (CP) and a plant gemini virus replication initiator protein (Rep). The T-DNA comprises gene editing reagents (Cas + guide) located between two geminivirus Rep recognition sites (RRS) shown as long intergenic regions (LIR) The T-DNA also comprises Rep / C3 between two LIR sequences. The transfer of the T-DNA into plant genome is initiated from the right border (RB) and terminated at the left border (LB).

[0020] Fig, 5 is an illustration of a T-DNA comprising nucleic acid sequences encoding gene editing components delivered into the LI SAM layer by Agrobacterium. Gene expression of the T-DNA occurs in the LI cell layer resulting in production of geminivirus derived coat protein (CP), replication initiator protein (Rep), Rep / C3 protein, and the replicon. Rep / C3 protein performs rolling circle replication to the replicon to enhance replicon accumulation. Gemini like particles which comprises CP and the replicons encoding the editing reagents or Rep / C3 protein form and moves from the LI cell layer into the L2 cell layer to provide gene edits in the L2 cell layers.DETAILED DESCRIPTION

[0021] Described herein are compositions and methods for Geminiviral and Nanoviral vectors used for direct editing of plant meristem cells (e.g., Shoot Apical Meristem (SAM) cells). In certain embodiments, Geminivirus or Nanovirus components and gene editing nucleic acids encoded on T-DNA and delivered through Agrobacterium or other gene delivery methods effectively deliver and generate high copies of a nucleic acid encoding gene editing components to the L2 cell layer of the SAM. In certain embodiments, edited lines can be obtained by harvesting seed obtained from the meristematic cells of the plant which were edited rather than through tissue culture processes.

[0022] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B. and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).Docket No. P15048WO00

[0023] The term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or non-traditional pairing types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively).

[0024] The term “contacting” refers to placing an agent in direct physical association; includes both in solid and liquid form, and can take place either in vivo or in vitro. Contacting includes contacting a cell (e.g., a soybean cell) by placing an agent (e.g., a nucleic acid or vector) in direct physical association with the cell.

[0025] The term “control” refers to a reference standard. A control can be a positive or negative control. In some examples, the control is a measurement (e.g., expression of a target) obtained prior to modifying a host cell e.g., introducing an exogenous nucleic acid). In some examples, the control is a historical control or a standard reference or range (a typical measurement or range observed for a particular population, such as a typical measurement (e.g., gene expression) or range for an unmodified plant (eg., soybean)).

[0026] The term “expression” refers to expression of a nucleic acid including transcription and / or translation of the nucleic acid.

[0027] The phrase “expression cassette: refers to a nucleic acid fragment designed for expression of a particular gene (or genes) in a host cell. Expression cassettes can be included in a vector. An expression cassette can include regulatory elements, such as promoters and / or terminators.

[0028] The term “exogenous” refers to originating from a different source. For example, a nucleic acid molecule that is exogenous to a cell is a nucleic acid that originated from a source other than the cell itself (e.g.. a synthetic nucleic acid that is introduced into a cell). In another example, a nucleic acid molecule that is exogenous to a replicon is a nucleic acid that originated from a source other than the replicon itself.

[0029] The term “Geminiviruses” refers to a large family of plant viruses that contain circular, single-stranded DNA genomes. Examples of geminiviruses include the cabbage leaf curl vims, tomato golden mosaic virus, bean yellow dwarf virus, African cassava mosaic virus, wheat dwarf vims, miscanthus streak mastrevirus, tobacco yellow dwarf virus, tomato yellow leaf curl virus, bean golden mosaic virus, beet curly top virus, maize streak virus, and tomato pseudo-curly top vims. Replication of the geminivims genome occurs in a host nucleus using a rolling circle mechanism. Replication initiator protein (Rep) and RepA are encoded on the complementary-sense transcript (C transcript). Rep mRNA is generated when a short intron is spliced in the CDocket No. P15048WO00transcript, while unspliced mRNA allows translation of RepA. Rep mediates nicking and ligating functions during rolling circle replication. A Rep recognition site (RRS) contains a bi-directional promoter and a stem-loop structure that initiates rolling-circle replication. Rep recognition sites can be called a long intergenic region (LIR) in Mastreviruses, an intergenic region (IR) in Curtoviruses and Topocuviruses, or a common region (CR) in Begomoviruses with bipartite genomes (Baltes et al. Plant Cell. 2014 Jan 17; 26(1): 151 - 163. doi: 10.1105 / tpc.l 13.119792). The RRS includes a bi-directional promoter and a stem-loop structure that initiates rolling-circle replication Examples of Gemini viruses include, but are not limited to: Soybean Mild Mottle Virus (SbMMV; see, e.g., NCBI Reference Sequence NC 014140.1), Soybean Blistering Mosaic Virus (SbBMV; see, e.g., NCBI Reference Sequence NC 038463), Bean Yellow Dwarf Vims (BeYDV; see. e.g.. GenBank Reference Sequence Y11023), Chickpea chlorotic dwarf virus (CpCDV; originally submitted as Bean yellow dwarf virus, GenBank Reference Sequence DQ458791.1). Tomato yellow leaf curl virus (TYLCV; see, e.g.. GenBank Reference Sequence X15656.1), Maize Streak Virus (MSV; see, e.g., NCBI Reference Sequence: NC_075135.1), Wheat dwarf virus (WDV; see, e.g.. GenBank Reference Sequence: X02869.1), Tomato golden mosaic virus (TGMV: see. e.g., GenBank Reference Sequence: K02029.1), Cabbage leaf curl virus (CaLCuV; see, e.g., GenBank Reference Sequence: U65529.2). Sri Lankan cassava mosaic virus (SLCMV; see, e.g., GenBank Reference Sequence: AJ314737.1), Tomato leaf curl virus (ToLCV; see, e.g., NCBI Reference Sequence NC_003896.1),

[0030] The term ’‘Nanoviridae” or ‘'nanovirus'’ refers to a family of plant viruses with 6-8 multipartite, circular, single-strand DNAs. Examples of nanoviruses include cardamom bushy dwarf virus (CdBDV), abaca bunchy top virus (ABTV), banana bunchy top virus (BBTV) (genus babuvims), milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow leaf virus (FBYLV), faba bean necrotic yellow leaf virus (FBNYV), black medic leaf roll virus (BMLRV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV), subterranean clover stunt vims (SCSV), and cow vetch latent virus (CVLV). Replication of the nanovirus genome occurs in a host nucleus using a rolling circle mechanism. Replication initiator protein (Rep) is encoded by DNA component DNA-R. Rep is believed to have DNA cleavage and nucleotidyl transfer activity and initiate replication of all viral genomic DNA. The non-coding region contains a promoter and a stem-loop structure that initiates rolling-circle replication. Examples of Nanoviruses include, but are not limited to: cardamom bushy dwarf virus (CdBDV; see e.g, GenBank Reference Sequences: EF546812. EF546811, EF546808, EF546813, EF546810, EF546809), abaca bunchy top virus (ABTV; see e.g, GenBankDocket No. P15048WO00Reference Sequences: EF546812, EF546811, EF546808, EF546813, EF546810, EF546809, banana bunchy top virus (BBTV; see e.g.. GenBank Reference Sequences: L41578, L41575, L41577, S56276, L41574, L41576, milk vetch dwarf virus (MDV; see e.g., GenBank Reference Sequences: AB000923, AB000927, AB000925, AB027511, AB009046, AB000924, AB000926, AB255373), milk vetch chlorotic dwarf virus (MVCDV; see e.g., GenBank Reference Sequences: MN273332, MN273334, MN273335. MN273336, MN273333, MN273337, MN273338, MN273339). pea yellow stunt virus (PYSV; see e.g. GenBank Reference Sequences: KC979056, KC979057, KC979058, KC979054, KC979055, KC979062, KC979059, KC979061), faba bean yellow leaf virus (FBYLV; see e.g., GenBank Reference Sequences: HE654126, HE654125, HE654127. HE654123, HE654124, HE654128, HE654129, HE654130), black medic leaf roll virus (BMLRV; see e.g., GenBank Reference Sequences: KC978960, KC978961, KC978962, KC978958. KC978959, KC978963, KC978964, KC978965), faba bean necrotic yellows virus (FBNYV; see e.g, GenBank Reference Sequences: AJ132179, AJ132182, AJ132186. AJ132180, AJ132183, AJ132181, AJ132184, AJ749902), pea necrotic yellow dwarf virus (PNYDV; see e.g., GenBank Reference Sequences: JN133280. JN133281, JN133282, GU553134, JN133279. JN133283. JN133284, JN133285), faba bean necrotic stunt virus. (FBNSV; see e.g., GenBank Reference Sequences: GQ150780, GQ150781, GQ150782, GQ150778, GQ150779, GQ150783, GQ150784, GQ150785), parsley severe stunt associated virus (PSSaV; see e.g, GenBank Reference Sequences: MK039128, MK039129, MK039130. MK039132, MK039133, MK039134, MK039135), sophora yellow’ stunt virus (SYSV; see e., GenBank Reference Sequences: MH048843. MH048844, MH048846, MH048845, MH048842, MH048847. MH048848, MH048849), subterranean clover stunt virus (SCSV; see e.g., GenBank Reference Sequences: MK035728, MK035729, MK035730, MK035731, MK035732, MK035733, MK035734, MK035735), and cow vetch latent virus (CVLV; see e.g., GenBank Reference Sequences: MF535447, MF535448, MF535449, MF535450. MF535451, MF535452, MF535453, MF535454).

[0031] The phrase “Homolog} ’-Directed Repair (HDR)"’ refers to the repair of one or more double-stranded breaks in DNA using homologous recombination with a donor template (repair template). In molecular biology applications, HDR mechanisms can be used to facilitate site¬ specific gene / genome editing; for example, a double stranded break can be induced in a target sequence by introducing into a cell a nuclease targeting the sequence (such as a Cas nuclease), and a repair template containing a desired nucleotide sequence can be integrated or “knocked-in" via HDR at the location of the double-stranded break. In some embodiments, the repair template disclosed herein is a donor template for HDR.Docket No. P15048WO00

[0032] The terms “increase"’ or “decrease” refer to a positive (increase) or negative (decrease) difference relative to a reference value, such as a control. The difference can be a qualitative or quantitative. In some examples, the difference is statistically significant (e.g., P-Value less than 0.05 or 0.01). In some examples, the difference is an increase relative to a control of at least 5%, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least al least 80%, al least 90%, al least 100%, at least 150%, al least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, or greater than 500%. In some examples, the difference is a decrease relative to a control of at least 5%, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.

[0033] The terms “isolated"’ or “purified” refer to biological components (such as a nucleic acid, protein, or cell) that have been substantially separated from other biological components m the environment in which the component occurs, e.g., separated from other chromosomal and extra-chromosomal DNA and RNA, proteins and / or cells. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0034] Absolute purity or isolation is not required, it is intended as a relative term. Thus, for example, a purified / isolated protein, nucleic acid, or cell preparation is one in which the protein, nucleic acid, or cell is more enriched than the protein, nucleic acid, or cell is in its initial environment. In one example, a preparation is purified / isolated such that the protein, nucleic acid, or cell represents at least 50% of the total content of the preparation. A substantially purified protein or nucleic acid is at least 60%, at least 70 %, at least 80%, at least 90?l>, at least 95%, at least 98%, or at least 99% pure. " Ihus, in one specific, non-limiting example, a substantially purified protein or nucleic acid is 90% free of other components.

[0035] The phrase “modified plant” refers to a plant that includes artificial genetic modification A modified plant is not naturally occurring. In some examples, a modified plant is a genome- edited plant (e.g., by CRISPR-based editing). In other examples, a modified plant is a transgenic plant (a plant that includes a transgene).

[0036] The phrase “operably linked” refers to a nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably finked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frameDocket No. P15048WO00

[0037] The term “producing" in relation to a nucleic acid refers to generating copies of the nucleic acid. Producing includes, for example, amplifying, replicating, or multiplying a nucleic acid. In some examples, a nucleic acid is produced in a plant host cell, such as a soybean cell.

[0038] The term “promoter” refers to a nucleic acid control sequence that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor). In some examples, the vectors provided herein include an RNA polIII promoter (e.g., U6) or an RNA pol II promoter, (e.g.. a ubiquitin promoter, Cauliflower Mosaic Virus (CaMV) 35S promoter. RUBISCO promoter, or combinations thereof).

[0039] The term “recombinant” refers to a nucleic acid or protein that has a sequence made by an artificial combination of two otherwise separated segments of sequence (e.g., a “chimeric” sequence). This artificial combination can be accomplished by chemical synthesis or by manipulation of isolated segments of nucleic acids, for example, by standard molecular biology techniques (e.g., cloning).

[0040] The term “regulatory element” refers to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements include those that direct constitutive expression of a nucleotide sequence m many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.

[0041] The phrase “repair template” refers to a nucleic acid fragment used to modify a target nucleic acid via homology-dependent repair (HDR). HDR is a mechanism Eukaryotic cells use to repair double-stranded DNA breaks. Thus, the term “repair” does not refer to repairing function of a target, but rather repair of a double-stranded break induced in a target nucleic acid (for example, by a nuclease). In general, a repair template includes an insert sequence (e.g.. a sequence to introduce specific mutations, insertions, or deletions into the target) flanked by sequence homologous to the target nucleic acid. The repair template is incorporated into the target through HDR resulting in modification of the target nucleic acid. A DNA repair template can be single stranded or double stranded. In some examples, the DNA repair template is single stranded.Docket No. P15048WO00

[0042] The term “replicon’" refers to a nucleic acid molecule that replicates as a unit.

[0043] The term “reporter” refers to a protein whose expression is linked to the expression of a gene of interest. Exemplary reporter proteins include fluorescent proteins and chemiluminescent molecules, such as infrared -fluorescent proteins (IFPs), GFP, YFP, RFP, CFP, rnRFPl, mCherry, mOrange, DsRed, tdTomato, mKO, tagRFP, EGFP, mEGFP, mOrange2, mScarlet, maple, tagRFP-T, firefly luciferase, Renilla luciferase, and click beetle luciferase (see, e.g., US Pat. Pub. No 2010 / 0122355). In some examples, the reporter protein is positioned downstream of and in frame with a gene of interest, such that the reporter protein is co-expressed with the gene of interest.

[0044] The term “sequence identity” refers to the degree of similarity between amino acid or nucleic acid sequences. Sequence identity is frequently measured in terms of percentage identity (or percent identity'); the higher the percentage, the more similar the two sequences are. Homologs of a polypeptide (or nucleotide sequence) will possess a relatively high degree of sequence identity when aligned using standard methods.

[0045] Methods of alignment of sequences for comparison have been described The NCBI Basic Local Alignment Search Tool (BLAST) tool is often used and is available from several sources, including the National Center for Biotechnology Information (blast ncbi.nlm.nih.gov / Blast.cgi). Various types of BLAST are available, for example, blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website and other resources. In some examples, percent sequence identity’ is determined by using BLAST with default parameters.

[0046] The term “transformed” refers to a transformed cell into which an exogenous nucleic acid molecule has been introduced by a molecular biology7technique. As used herein, the temi transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including chemical methods e.g., calcium-phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), lipofection, nucleofection, receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope / capsid-DNA complexes), Agrobacten w?-mediated transformation, biolistics (particle gun accelerator or gene gun), or other transduction and / or transfection methods.

[0047] The term “transgene” refers to anucleic acid that is artificially introduced into an organism in which the nucleic acid does not naturally occur.

[0048] The term “vector” refers to a nucleic acid molecule that can be introduced into a host cell (for example, by transformation), thereby producing a transformed host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replicationDocket No. P15048WO00Recombinant DNA vectors are vectors containing recombinant DNA. A vector can also include one or more selectable marker genes and other genetic elements. Often vectors are plasmids, however, they can also be viral vectors, cosmids, or artificial chromosomes. In some examples, the vector is a transfer DNA (T-DNA) vector suitable for Agrobacterium-mediated transformation or a vector suitable for biolistics.

[0049] To the extent to which any of tire preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

[0050] Disclosed herein are nucleic acids including a recombinant Geminivirus-based replicon. The replicon includes a first transfer DNA (T-DNA) comprising one or more bacterial DNA transfer elements which are operably linked to: (i) DNA encoding a plant geminivirus coat protein (CP) and a plant geminivirus replication initiator protein (Rep); and (ii) a first geminivirus Rep recognition site (RRS) DNA encoding one or more gene editing reagents which can modify’ genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS (Fig. 2). In some embodiments, the T-DN A further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS (Fig. 4).

[0051] When delivered by a plant transforming bacterium to a plant part such as a shoot apical meristem, the T-DNA is expressed in the LI epidermal layer of cells (Fig. 1). Expression of the T-DNA results in the formation of a Gemini like particle comprising CP and the replicon with editing reagents (Figs. 3 and 5). This particle is naturally able to move through the LI cell layer into the subepidermal mesophyll L2 cell layer of the shoot apical meristem. There, the particle will dismantle in the L2 cells and the replicon will be transcribed. The resulting production of gene editing reagents will modify the plant genome.

[0052] The coat protein (CP) of gemini viruses is involved m viral movement from cell to cell and systemic spread in the plant (Wartig et al. (1997) Virology 228, 132-140 (1997); Liu et al. (1998) J. Gen. Virol. 79. 2265-2274 and Unseld et al. (2004) Virology 318. 90-101). CP will package the replicated replicons and move them from LI to L2 cell layers in the meristem.

[0053] Geminivirus CP proteins and polynucleotides encoding the same can be obtained either from geminiviruses that infect monocot plants (e.g., maize streak virus (MSV), wheat dwarf virus (WDV)) or from geminiviruses that infect dicot plants (e.g.. tobacco yellow dwarf virus, bean yellow dwarf virus (BeYDV), tomato golden mosaic virus (TGMV), Cabbage leaf curl virusDocket No. P15048WO00(CaLCuV), Sri Lankan cassava mosaic vims (SLCMV), tomato leaf curl virus (ToLCV), beet curly top virus (BCTV). tomato pseudo-curly top virus (TPCTV)). In certain embodiments, the geminivirus CP protein used in a monocot plant cell is obtained from a geminivirus that infects monocot plants. In certain embodiments, the geminivirus CP protein used in a dicot plant cell is obtained from a geminivirus that infects dicot plants. Useful geminivirus CP proteins include the proteins set forth in Table 1 below. In certain embodiments, the geminivirus CP protein encoded by the vectors provided herein can comprise an amino acid sequence with at least 50%, 60%, 70%.80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. 17. 18. 29. 43.51, 61, 73, 81, 91, 101, or 111. In certain embodiments, the geminivirus CP protein encoded by the vectors provided herein is encoded by a nucleic acid comprising a polynucleotide sequence with at least 50%, 60%, 70%. 80%. 85%. 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. 16, 28, 42, 50, 60, 72, 80, 90, 100, or 110.

[0054] Rep is a highly conserved protein in sequence, position, and function, while its structure and folding patterns present slight variability among different genera of Gemini viruses. The Rep protein acts as a rolling circle initiator to catalyze a site-specific cleavage and rejoining reaction in a conserved hairpin loop in the viral replication origin. Rep, with a size of approximately 360 amino acids, transcribes from a bidirectional core promoter located in the Rep recognition site (RRS) except for Mastre-, Capula, Becurto, and Grablo-virus Rep (C1: C2), which is a spliced version of Cl and C2 open reading frames (ORFs) (Hanley-Bowdoin et al., 1999, 2013; Varsani et al., 2017). In addition to Rep. Mastre-, Capula-, Becurto-. and Grablo-virus also code for replication-associated protein A (RepA'Cl) with sizes varying from 260 to 300 amino acids, transcribed from ORF Cl (Hofer et al., 1992; Collin et al., 1996; Gutierrez et al., 2004; Varsani et al., 2017). As used herein, the term Rep includes Rep, C1: C2, RepA / Cl, ALI, AC1, and Cl. In certain embodiments, the geminivirus Rep protein encoded by the vectors provided herein can have an ammo acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, 22, 34. 36, 45, 55, 57, 65, 67, 69, 75, 83, 85, 95, 105, 115, or 117. In certain embodiments, the geminivirus Rep protein encoded by7the vectors provided herein is encoded by a nucleic acid comprising a polynucleotide sequence with at least 50%, 60%, 70%. 80%. 85%. 90%. 95%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 7, 21, 33, 35.44, 54, 56, 64, 66, 68, 74, 82, 84, 94. 104, 114, or 116.

[0055] The C3 protein (also named AL3, AC3, and REn) interacts with Rep and acts as a replication enhancer during geminivirus DNA accumulation. In certain embodiments, the geminivirus C3 protein can have an amino acid sequence w ith at least 50%, 60%, 70%, 80%, 85%, 90%. 95%, 98%, 99%. or 100% sequence identity to SEQ ID NO: 10, 24, 47, 77. 87, 97, or 107,Docket No. P15048WO00

[0056] The Rep recognition site (RRS) is a non-coding region encoded by all geminiviruses, which contains cis-acting regulatory elements for gene expression and replication, RRS includes intergenic regions (IR) and long intergenic regions (LIR). RRS includes a predicted hairpin structure containing the conserved (among most geminiviruses) nonanucleotide sequence (TAATATTAC) as part of the loop, and small repeated sequences, known as “herons, ’’ which are sequence-specific binding sites for Rep. Together, the iterons and hairpin form the origin of replication (oii). Geminiv irus replication is initiated by a Rep protein binding to RRS sequence on a circular dsDNA genome. However, if the geminivirus genome is linearized and contains flanking RRS sequences (also referred to as an LSL vector), Rep proteins bind to the RRS sequences and release circularized, single - stranded geminiviral replicons (GVRs). Replicons can then be used as a template for repiicase-mediated genome amplification. Consequently, any sequence present inside the flanking RRSs will be present in the replicon. In certain embodiments, the geminivirus RRS can have a nucleic acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 11, 25, 26, 37-40, 48, 58, 70, 78, 88, 98, 108, or 1 18.

[0057] Table 1 below' provides SEQ ID NO for the geminivirus genomes, proteins, and nucleic acids disclosed herein for geminiviruses SbMMV, TYLCV, CpYDV, MSV, WDV, TGMV, CaLCuV. SLCMV, ToLCV, and BeYDV. However, any geminivirus genomes can be used to construct the nucleic acid constructs described herein. Example Geminivirus replicons that can be adapted for use in the vectors described herein include Geminivirus replicons disclosed in U. S Patent No. 11,384,360, issued July 12, 2022; European Patent No. EP 2,436,769, granted April 1, 2015; and U. S. Patent No. 8,513,397, issued August 20, 2013, each of which are incorporated herein by reference.

[0058] Table 1: Geminivirus Protein and DNA sequencesSEQ ID NO. Source / Name (Database Accession)1 SbMMV Genome (NCBI: NC 014140.1)2 SbMMV Coat Protein coding sequence3 SbMMV Coat Protein4 SbMMV Pre-coat coding sequence5 SbMMV Pre-coat Protein6 SbMMV Replication Associated Protein (Rep) alternative coding sequence 7 SbMMV Rep reference coding sequence8 SbMMV Rep Protein9 SbMMV Replication Enhancer (REn) coding sequence10 SbMMV REn Protein11 SbMMV Intergenic region12 SbMMV replicon 5’ backbone sequence13 SbMMV replicon 3’ backbone sequenceDocket No. P15048WO00SbMMV pIN4614 SbMMV repliconTYLCV Genome (GenBank: X15656.1)TYLCV Coat Protein coding sequenceTYLCV Coat Protein variantTYLCV Coat Protein referenceTYLCV V2 protein coding sequenceTYLCV V2 ProteinTYLCV Rep coding sequenceTYLCV Rep ProteinTYLCV REn coding sequenceTYLCV REn ProteinTYLCV Intergenic regionTYLCV Intergenic regionCpYDV Genome (GenBank: DQ458791.1)Cp YDV Coat Protein coding sequenceCpYDV Coat ProteinCpYDV Movement Protein coding sequenceCpYDV Movement ProteinCpYDV Rep Reverse coding reverse completementCpYDV Rep (Cl) coding sequenceCpYDV Rep (Cl) ProteinCpYDV Rep (C1: C2) coding sequenceCpYDV Rep (C1: C2) ProteinCpYDV Intergenic region (long)CpYDV Intergenic region (upstream long)CpYDV Intergenic. region (downstream long)CpYDV Intergenic region (short)SbBMV Genome (NCBI: NC 038463.1)SbBMV Coat Protein coding sequenceSbBMV Coat ProteinSbBMV Rep coding sequenceSbBMV Rep ProteinSbBMV REn coding sequenceSbBMV REn ProteinSbBMV Intergenic. regionMSV Genome (NCBI: NC 075135.1)MSV Coat Protein coding sequenceMSV Coat ProteinMSV Movement Protein coding sequenceMSV Movement ProteinMSV Rep (Cl) coding sequenceMSV Rep (Cl) ProteinMSV Rep (C1: C2) coding sequenceMSV Rep (C1: C2) ProteinDocket No. P15048WO00MSV Intergenic regionWDV Genome (GenBank: X02869.1)WDV Coat Protein coding sequenceWDV Coat ProteinWDV Movement Protein coding sequenceWDV Movement ProteinWDV Rep variant coding sequenceWDV Rep variant ProteinW’DV Rep (Cl) coding sequenceWDV Rep (Cl) ProteinWDV Rep (Cl: C2) coding sequenceWDV Rep (C 1: C2) ProteinWDV Intergenic RegionTGMV Genome (GenBank: K02029.1)TGMV Coat Protein coding sequenceTGMV Coat ProteinTGMV Rep coding sequenceTGMV Rep ProteinTGMV REn coding sequenceTGMV REn ProteinTGMV Intergenic regionCaLCuV Genome (GenBank: U655292)CaLCuV Coat Protein coding sequenceCaLCuV Coat ProteinCaLCuV pCPCbLCVA.007 Rep coding sequenceCaLCuV pCPCbLCVA.007 Rep ProteinCaLCuV Rep reference coding sequenceCaLCuV Rep ProteinCaLCuV REn coding sequenceCaLCuV REn ProteinCaLCuV Intergenic regionSLCMV Genome (GenBank: AJ314737.1)SLCMV Coat Protein coding sequenceSLCMV Coat ProteinSLCMV V2 protein coding sequenceSLCMV V2 ProteinSLCMV Rep (Cl) coding sequenceSLCMV Rep (Cl) ProteinSLCMV REn coding sequenceSLCMV REn ProteinSLCMV Intergenic regionToLCV Genome (NCBI: NC 003896.1)ToLCV Coat Protein coding sequenceToLCV Coat ProteinDocket No. P15048WO00102 ToLCV Pre-coat coding sequence103 ToLCV Pre-coat Protein104 ToLCV Rep (Cl) coding sequence105 ToLCV Rep (Cl) Protein106 ToLCV REn coding sequence107 ToLCV REn Protein108 ToLCV Intergenic region109 BeYDV Genome (GenBank: Y11023)110 BeYDV Coat Protein coding sequence111 BeYDV Coat Protein112 BeYDV V2 protein coding sequence113 BeYDV V2 Protein114 BeYDV Rep (Cl) coding sequence115 BeYDV Rep (C 1) Protein116 BeYDV Rep (C1: C2) coding sequence117 BeYDV Rep (CT. C2) Protein118 BeYDV Intergenic region

[0059] Disclosed herein are nucleic acids including a recombinant Nanovirus-based replicon The replicon includes a first transfer DNA (T-DNA) comprising one or more bacterial DNA transfer elements which are operably linked to: (i) DNA encoding a plant nanovirus coat protein (CP) and a plant nanovirus replication initiator protein (Rep); and (ii) a first nanovirus Rep recognition site (RRS) DNA encoding one or more gene editing reagents which can modify genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS (Fig. 2). In some embodiments, the T-DNA further comprises (iii) DNA encoding a nanovirus Rep / M-Rep protein and a third geminivims RRS, wherein the DNA encoding the nanovirus Rep / M-Rep protein is located between the first and third RRS or between the second and third RRS (Fig.5).

[0060] When delivered by a plant transforming bacterium to a plant past such as a shoot apical meristem, the T-DNA is expressed in the LI epidermal layer of cells (Fig.1). Expression of the T-DNA results in the formation of a Nanoviral like particle comprising CP and the replicon with editing reagents (Figs. 3 and 5), This particle is naturally able to move through the LI cell layer into the subepidermal mesophyll I., 2 cell layer of the shoot apical meristem. There, the particle will dismantle in the L2 cells and the replicon will be transcribed. The resulting production of gene editing reagents w ill modify the plant genome.

[0061] The coat protein (CP) of nanoviruses is a structural protein involved in virion formation (encapsidation). The movement protein (MP) of nanoviruses are involved in viral movement from cell to cell and systemic spread in the plant (Wartig et al. (1997) Virology 228. 132-140 (1997); Liu et al. (1998) J. Gen. Virol. 79.2265-2274 and Unseld et al. (2004) Virology 318.90-101). CP will package the replicated replicons and move them from LI to L2 cell layers in the meristem. Nanovirus CP proteins and polynucleotides encoding the same can be obtained either from nanoviruses that infect monocot plants (e.g., abaca bunchy top vims (ABTV), cardamom bushy dw-arf virus (CdBDV). banana bunchy top virusDocket No. P15048WO00(BBTV)) or from nanoviruses that infect dicot plants (e.g., milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow7leaf virus (FBYLV), black medic leaf roil virus (BMLRV), faba bean necrotic yellow’s virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV), subterranean clover stunt virus (SCSV), and cow vetch latent virus (CVLV)). In certain embodiments, the nanovirus CP protein used in a monocot plant cell is obtained from a nanovirus that infects monocot plants. In certain embodiments, the nanovirus CP protein used in a dicot plant cell is obtained from a nanovirus that infects dicot plants. Useful nanovirus CP proteins include the proteins set forth in Table 2 below. In certain embodiments, the nanovirus CP protein encoded by the vectors provided herein can comprise an amino acid sequence with at least 50%. 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 120, 130. 140. 150, 160, 170, 180. 190, 200, 210, 220, 230, 240. 250, or 260. In certain embodiments, the nanovirus CP protein encoded by the vectors provided herein is encoded by a nucleic acid comprising a polynucleotide sequence with at least 50%. 60%, 70%, 80%. 85%, 90%. 95%, 98%, 99%. or 100% sequence identity to SEQ ID NO: 119, 129, 139, 149, 159. 169, 179, 189, 199, 209, 219, 229, 239. 249, or 259.

[0062] In certain embodiments, the nanovirus MP protein encoded by the vectors provided herein can comprise an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%. 90%, 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 31, 53, 63, 126, 136, 146, 156, 166, 176. 186. 196, 206, 216, 226, 236.246, 256, or 266. In certain embodiments, the nanovirus MP protein encoded by the vectors provided herein is encoded by a nucleic acid comprising a polynucleotide sequence with at least 50?4», 60%, 70%, 80%.85%, 90%, 95%, 98%. 99%. or 100% sequence identity to SEQ ID NO: 30, 52, 62, 125, 135. 145, 155.165, 175, 185, 195, 205, 215, 225, 235, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, or 265.

[0063] Rep (M-Rep) is a highly conserved protein in sequence, position, and function, while its structure and folding patterns present slight variability among different genera of Nanoviruses. The Rep protein acts as a rolling circle initiator to catal ze a site-specific cleavage and rejoining reaction in a conserved hairpin loop in the viral replication origin. Rep. with a size of approximately 290 amino acids, transcribes from a promoter located in the Common region (CR). Rep coding region is preceded by a promoter sequence with a TATA box and the polyadenylatioti signal and is followed by a poly adenylation signal, this leads to transcription of the replication origin and synthesis of a terminally redundant mRNA that is capable of folding into extended secondary structures, and may serve to regulate the expression of the encoded Rep protein (Grigoras el al.. 2008). As used herein, the term Rep includes Rep and M-Rep. In certain embodiments, the nanoviridae Rep protein encoded by the vectors provided herein can have an amino acid sequence with at least 50%, 60%, 70%, 80%. 85%, 90%, 95%, 98%. 99%. or 100% sequence identity to SEQ ID NO: 122, 132. 142. 152, 162. 172, 182, 192, 202. 212, 222, 232. 242, 252, or 262. In certain embodiments, the nanoviridae Rep protein encoded by the vectors provided herein is encoded by a nucleic acid comprising a polynucleotide sequence with at least 50%. 60%, 70%, 80%, 85%, 90%. 95%. 98%.Docket No. P15048WO0099%, or 100% sequence identity to SEQ ID NO: 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, or 261.

[0064] The Clink protein promotes cell c cle progression and enhances viral DNA replication. In certain embodiments, the nanoviridae Clink protein can have an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 124, 134, 144, 154, 164, 174, 184, 194, 204. 214, 224, 234, 244, 254, 264.[0065| Non-coding Regions (NCR) are non-coding regions encoded by all nanoviruses, which contains cis-acting regulatory elements for gene expression and replication. NCRs include two common regions (CR) which are the common stem-loop region (CR-SL) and the second common region (CR-II) or(CR-M) for nanoviruses and babuviruses respectivel. The CR-SL includes a predicted hairpin structure containing the conserved nucleotide sequence (TATTATTAC) or (TAGTATT C) for babuviruses or nanoviruses, respectively. The CR-SL also encompasses short repeated sequences (iterons) that are presumed to be binding sites for Rep protein (Herrera-Valencia et al., 2006). Together, the iterons and hairpin form the origin of replication (ori). Nanoviridae replication is initiated by a Rep protein binding to these iteron sequences on a circular dsDNA genome.

[0066] Table 2 below provides SEQ ID NO for the nanoviridae genomes and proteins disclosed herein for nanoviruses ABTV, CdBDV, BBTV, MDV, MVCDV, PYSV, FBYLV, BMLRV, FBNYV, PNYDV, FBNSV, PSSaV, SYSV, SCSV, CVLV. However, any nanovirus genomes can be used to construct the nucleic acid constructs described herein.

[0067] Table 2: Nanovirus Protein and DNA sequencesSEQ ID NO. Source / Name (Database Accession)119 ABTV Coat Protein (DNA-S)120 ABTV Coat Protein (CP)121 ABTV Replication Initiator Protein (DNA-R)122 ABTV Replication Initiator Protein (Rep)123 ABTV7Cell Cycle Regulation (DNA-C)124 ABTV Cell Cycle Regulation Protein (Clink)125 ABTV Movement Protein (DNA-M)126 ABTV Movement Protein (MP)127 ABTV Nuclear Shuttle Protein (DNA-N)128 ABTV Nuclear Shuttle Protein (N SP)129 CdBDV Coat Protein (DNA-S)130 CdBDV’ Coal Protein (CP)131 CdBDV Replication Initiator Protein (DNA-R)132 CdBDV Replication Initiator Protein (Rep)133 CdBDV Cell Cycle Regulation (DNA-C)134 CdBDV Cell Cycle Regulation Protein (Clink)135 CdBDV Movement Protein (DNA-M)136 CdBDV Movement Protein (MP)137 CdBDV Nuclear Shuttle Protein (DNA-N)Docket No. P15048WO00CdBDV Nuclear Shutle Protein (NSP)BBTV Coat Protein (DNA-S)BBTV Coat Protein (CP)BBTV Replication Initiator Protein (DNA-R)BBTV Replication Initiator Protein (Rep)BBTV Cell Cycle Regulation (DNA-C)BBTV Cell Cycle Regulation Protein (Clink)BBTV Movement Protein (DNA-M)BBTV Movement Protein (MP)BBTV Nuclear Shuttle Protein (DNA-N)BBTV Nuclear Shuttle Protein (NSP)MDV Coat Protein (DNA-S)MDV Coat Protein (CP)MDV Replication Initiator Protein (DNA-R)MDV Replication Initiator Protein (Rep)MDV Cell Cycle Regulation (DNA-C)MDV Cell Cycle Regulation Protein (Clink)MDV Movement Protein (DNA-M)MDV Movement Protein (MP)MDV Nuclear Shuttle Protein (DNA-N)MDV Nuclear Shuttle Protein (NSP)MVCDV Coat Protein (DNA-S)MVCDV Coat Protein (CP)MVCDV Replication Initiator Protein (DNA-R)MVCDV Replication Initiator Protein (Rep)MVCDV Cell Cycle Regulation (DNA-C)MVCDV Cell Cycle Regulation Protein (Clink)MVCDV Movement Protein (DNA-M)MVCDV Movement Protein (MP)MVCDV Nuclear Shuttle Protein (DNA-N)MVCDV Nuclear Shuttle Protein (NSP)PYSV Coat Protein (DNA-S)PYSV Coat Protein (CP)PYSV Replication Initiator Protein (DNA-R)PYSV Replication Initiator Protein (Rep)PYSV Cell Cycle Regulation (DNA-C)PYSV Cell Cycle Regulation Protein (Clink)PYSV Movement Protein (DNA-M)PYSV Movement Protein (MP)PYSV Nuclear Shuttle Protein (DNA-N)PYSV Nuclear Shuttle Protein (NSP)FBYLV Coat Protein (DNA-S)FBYLV Coat Protein (CP)FBYLV Replication Initiator Protein (DNA-R)Docket No. P15048WO00FBYLV Replication Initiator Protein (Rep)FBYLV Cell Cycle Regulation (DNA-C)FBYLV Cell Cycle Regulation Protein (Clink)FBYLV Movement Protein (DNA-M)FBYLV Movement Protein (MP)FBYLV Nuclear Shuttle Protein (DNA-N)FBYLV Nuclear Shuttle Protein (NSP)BMLRV Coat Protein (DNA-S)BMLRV Coat Protein (CP)BMLRV Replication Initiator Protein (DNA-R)BMLRV Replication Initiator Protein (Rep)BMLRV Cell Cycle Regulation (DNA-C)BMLRV Cell Cycle Regulation Protein (Clink)BMLRV Movement Protein (DNA-M)BMLRV Movement Protein (MP)BMLRV Nuclear Shuttle Protein (DNA-N)BMLRV Nuclear Shuttle Protein (NSP)FBNYV Coat Protein (DNA-S)FBNYV Coat Protein (CP)FBN YV Replication Initiator Protein (DNA-R)FBNYV Replication Initiator Protein (Rep)FBNYV Cell Cycle Regulation (DNA-C)FBNYV Cell Cycle Regulation Protein (Clink)FBNYV Movement Protein (DNA-M)FBNYV Movement Protein (MP)FBNYV Nuclear Shuttle Protein (DNA-N)FBNYV Nuclear Shuttle Protein (NSP)PNYDV Coat Protein (DNA-S)PNYDV Coat Protein (CP)PNYDV Replication Initiator Protein (DNA-R)PNYDV Replication Initiator Protein (Rep)PNYDV Cell Cycle Regulation (DNA-C)PNYDV Cell Cycle Regulation Protein (Clink)PNYDV Movement Protein (DNA-M)PNYDV Movement Protein (NIP)PNYDV Nuclear Shuttle Protein (DNA-N)PNYDV Nuclear Shuttle Protein (NSP)FBNSV Coat Protein (DN A-S)FBNSV Coat Protein (CP)FBNSV Replication Initiator Protein (DNA-R)FBNSV Replication Initiator Protein (Rep)FBNSV Cell Cycle Regulation (DNA-C)FBNSV Cell Cycle Regulation Protein (Clink)FBNSV Movement Protein (DNA-M)Docket No. P15048WO00FBNSV Movement Protein (MP)FBNSV Nuclear Shuttle Protein (DNA-N)FBNSV Nuclear Shuttle Protein (NSP)PSSaV Coat Protein (DNA-S)PSSaV Coat Protein (CP)PSSaV Replication Initiator Protein (DNA-R)PSSaV Replication Initiator Protein (Rep)PSSaV Cell Cycle Regulation (DNA-C)PSSaV Cell Cycle Regulation Protein (Clink)PSSaV Movement Protein (DNA-M)PSSaV Movement Protein (MP)PSSaV Nuclear Shuttle Protein (DNA-N)PSSaV Nuclear Shuttle Protein (NSP)SYSV Coat Protein (DNA-S)SYSV Coat Protein (CP)SYSV Replication Initiator Protein (DNA-R)SYSV Replication Initiator Protein (Rep)SYSV Cell Cycle Regulation (DNA-C)SYSV Cell Cycle Regulation Protein (Clink)SYSV Movement Protein (DNA-M)SYSV Movement Protein (MP)SYSV Nuclear Shuttle Protein (DNA-N)SYSV Nuclear Shuttle Protein (NSP)SCSV Coat Protein (DNA-S)SC SV Coat Protein (CP)SCSV Replication Initiator Protein (DNA-R)SCSV Replication Initiator Protein (Rep)SCSV Cell Cycle Regulation (DNA-C)SCSV Cell Cycle Regulation Protein (Clink)SCSV Movement Protein (DNA-M)SCSV Movement Protein (MP)SCSV Nuclear Shuttle Protein (DNA-N)SCSV Nuclear Shuttle Protein (NSP)CVLV Coat Protein (DNA-S)CVLV Coat Protein (CP)CVLV Replication Initiator Protein (DNA-R)CVLV Replication Initiator Protein (Rep)CVLV Cell Cvcie Regulation (DNA-C)CVLV Cell Cycle Regulation Protein (Clink)CVLV Movement Protein (DNA-M)CVLV Movement Protein (MP)CVLV Nuclear Shuttle Protein (DNA-N)CVLV Nuclear Shuttle Protein (N SP)Docket No. P15048WO00

[0068] The T-DNA described herein can include nucleic acids encoding gene editing reagents such as CRISPR (Cas9) CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.

[0069] CRISPR-type genome editing can be adapted for use in the plant cells and methods provided herein in several ways. CRISPR elements, e.g.. gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or guide RNAs in combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are provided directly to the eukaryotic cell (eg., soybean plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi -purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, soybean plants or soybean plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Type II Cas nuclease such as Cas9), a Type V Cas nuclease such as a Casl2a, CasI2b, Casl2c, Casl2f, Casl2i, Casl2j, Casl2L, or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA or scoutRNA) to form an RNA-guided endonuclease / guide RN A complex which can specifically bind sequences in the gDNA target site that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g.. 5 -NGG are typically targeted for design of crRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequences include 5 -NGG (Streptococcus pyogenes'), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), 5’-NNGRRT or 5’-NNGRR (Staphylococcus aureus Cas9. SaCas9). and 5'-NNNGATT (Neisseria meningitidis). T-nch PAM sites (e.g., 5’-TTN or 5 -TTTV, where “V’?is A. C, or G) are typically targeted for design of crRNAs or sgRNAs used with Type V (e.g., Cas 12a, Cast 2f, Casl2i, or Cas 12j) proteins. Tn certain embodiments provided herein, a miniature Type V Cas endonuclease, which include Cas 12f, Cas 12i, Cas 12j, and vanants thereof, is used. Miniature Type V Cas endonucleases are disclosed in Nugyen et al., doi.org / 10.1016. / j.sbi.2022.102466 as well as in US20210395784AI, US20210254038A1, W02024091775, and US 11649444B 1, each of which are incorporated herein by reference in thereDocket No. P15048WO00entireties. In some instances, Cas t 2a can also recognize a 5 -CTA PAM motif. Other examples of potential Type V Cas nuclease sites (e.g. Casl2a, Casl2f. Casl2i, or Casl2j) PAM sequences include TIN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN. CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Cpfl endonuclease and corresponding guide R As and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 Al, which is incorporated herein by reference for its disclosure of DNA encoding Type V Cas endonucleases and guide RNAs and PAM sites. Engineered endonucleases with altered or eliminated PAM recognition sites can also be used.

[0070] Identification of a suitable PAM site is only one factor in selecting an efficacious gRNA. Different guides can also have different cutting efficiencies and specificities, which depend on a number of factors. In some embodiments, identification of effective gRN A target sequences m the genome is approached through a combination of in silico selection and experimental evaluation.

[0071] In some cases, a target sequence that perfectly hybridizes with the gRNA spacer sequence occurs only once in a given plant genome. In some embodiments, the genome comprises additional sequences that imperfectly hybridize with the gRNA spacer sequence, for example, sequences having one or more mismatches (e.g., 1, 2, 3, 4, or 5 mismatches) and / or bulges, relative to the gRNA spacer sequence. In some embodiments, the genome comprises sequences that hybridize the gRNA spacer sequence that are adjacent to a PAM sequence having at least one mismatch relative to the canonical PAM sequence. Such genomic sequences (e.g., target sequences that imperfectly hybridize the gRNA spacer sequence and / or target sequences comprising a non-canonical PAM sequences) are called off-target sites. A favorable off-target profile is typically one that minimizes or eliminates the number of off-target sites and / or the frequency of cutting at these sites.

[0072] The nuclease efficiency and occurrence of off-target activity' for a given gRNA / endonuclease combination can be influenced by a number of factors including similanties and dissimilarities between the target site and various off-target sites, as well as the particular endonuclease used. For example, the ability of a given gRNA to promote cleavage at a target sequence in a genomic DNA molecul e may relate to the accessibility of the target sequence, which depends on one or more factors that include the chromatin structure of the genomic DNA molecule and / or proximity to transcription factor binding sites. For example, target sequences located within a region of the genomic DNA molecule having a high condensed chromatin structure are less accessible than target sequences located within a region of the genomic DNA molecule having anDocket No. P15048WO00open chromatin structure. As a further example, target sequences proximal to a region of the genomic DNA molecule bound by a transcription factor or other regulatory protein may be less accessible than target sequences proximal a region of the genomic DNA molecule that is unbound by regulatory proteins. Moreover, the cell state and type of cell may influence the accessibility' of target sequences, for example, by influencing the chromatin structure of genomic DNA.

[0073] In certain embodiments, zinc finger nucleases or zinc finger nickases can also be used in the methods provided herein. Zinc-finger nucleases are site-specific endonucleases comprising two protein domains: a DNA-binding domain, comprising a plurality of individual zinc finger repeats that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain that comprises a nuclease domain (ty pically Fokl). The cleavage domain dimerizes m order to cleave DNA; therefore, a pair of ZFNs are required to target non-palindromic target polynucleotides. The zinc finger binding domains of the zinc finger nuclease or nickase provide specificity and can be engineered to specifically recognize any' desired target DNA sequence. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc “fingers” each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity' of the zinc finger binding domain. One approach, termed “modular assembly ”, relies on the functional autonomy' of individual zinc fingers with DNA. In this approach, a given sequence is targeted by identifying zinc fingers for each component triplet m the sequence and linking them into a multifinger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger ammo acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. Exemplary' selection methods (e.g, phage display and yeast two-hybrid systems) can be adapted for use in the methods described herein. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DN. Thus, cleavage by Fokl as part of a ZFN requires two adjacent andDocket No. P15048WO00independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites.

[0074] Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly- conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to anon-specific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl, can be conveniently used.

[0075] Also disclosed are methods of producing or expressing a nucleic acid in a plant cell (including protoplasts, or plant cells located in an intact plant, plant part, or plant tissue), including transforming (introducing) a nucleic acid or vector including a geminivirus replicon into a plant cell, thereby generating a transformed plant ceil. In some examples, the geminivirus replicon includes an expression cassette. In some examples, the geminivirus replicon includes an expression cassette used to express an exogenous gene in a host plant (e.g., soybean plant). Nucleic acid production or expression in the transformed plant cell can be stable or transient. In some examples, the nucleic acid is produced or expressed transiently. In some examples, the plant cell is a soybean cell (Glycine max)

[0076] Also disclosed are methods of modifying genetic material of a plant cell (including protoplasts, or plant cells located in an intact plant, plant part, or plant tissue), including transforming the plant cell with a nucleic acid or vector including ageminivirus replicon, thereby-generating a transformed plant cell. In some examples, the geminivirus replicon includes a repair template. In some examples, transforming the plant cell with the nucleic acid or vector disclosed herein produces a transformed plant cell containing an induced genetic modification (e.g., an insertion, deletion, or substitution). In some embodiments, the modified genetic material is genomic DNA In some embodiments, modifying the genetic material of the plant cell includesDocket No. P15048WO00introducing an insertion, deletion, or substitution into a target nucleic acid. In some embodiments, modifying the genetic material is facilitated by a homology -dependent repair (HDR) mechanism. In some examples, the plant cell is a soybean cell (Glycine max).

[0077] In some examples, the method of modifying genetic material of a plant cell includes transforming the plant cell with a nucleic acid or vector including a geminivirus replicon including a repair template, as disclosed herein. In some examples, the method further includes introducing a nuclease, for example, a nuclease capable of inducing a double-stranded DNA break that can be repaired via HDR with the repair template Thus, in some embodiments, the repair template and nuclease target the same gene or loci. In some examples, the method further includes introducing a guide RNA (gRN A), for example, to guide a Cas nuclease to a particular target gene or loci. The nucleic acid or vector including the geminivirus replicon, the nuclease, and / or the gRNA can all be included on the same vector, or different vectors. In some examples, a host plant (e.g.. a soybean plant) is engineered to express an inducible nuclease and / or gRNA, which is induced following transformation with the nucleic acid or vector including the geminivirus replicon. While the nucleic acid or vector encoding the geminivirus replicon, the nuclease, and / or the gRNA. need not be simultaneously introduced, in some examples they are introduced in the plant cell simultaneously or at substantially the same time. In some examples, the nucleic acid or vector including the geminivirus replicon also encodes a Cas nuclease and a gRNA. In some embodiments, the methods further include transforming the plant cell with a vir plasmid, or other replicon containing the vir genes from Agrobacterium.

[0078] Methods for transformation include any suitable method of introducing a nucleic acid or vector into a host cell. In some embodiments, a plant cell is transformed w-ith a nucleic acid or vector disclosed herein by bacterial-mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp.. Phyllobacterium sp.) transformation. In some embodiments, the plant cell is transformed GaAgrobacierium-mediated transformation In certain embodiments, nucleic acids or vectors described herein are introduced into plant cells by microprojectile bombardment or transfection.

[0079] In some examples, production or expression of an exogenous nucleic acid (e.g., repair template or expression cassette) achieved with the geminivirus replicon disclosed herein is significantly higher than production or expression using other geminivirus replicons (e.g., SbBMV or BeYDV). In some examples, production or expression is at least 25% higher, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%. at least 250%, at least 275%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, atDocket No. P15048WO00least 800%, at least 900%, at least 1000% higher, or more, relative to other geminivirus replicons (e.g., SbBMV or BeYDV). in some examples, production or expression is at least two-fold greater than production or expression achieved using other geminivirus replicons (e.g., SbBMV or BeYDV), such as at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least flfold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, or more, than production or expression achieved using other geminivirus replicons (e.g., SbBMV or BeYDV).

[0080] In some embodiments, the plant cell (including protoplasts) is an isolated plant cell (e.g., a plant cell isolated from a whole plant or plant part or tissue, or a plant cell in suspension or plate culture). In some examples, the isolated plant cell is obtained or isolated from a whole plant, or plant part or tissue, for example (and without limitation), an intact nodal bud, a shoot apex or shoot apical meristem, a root apex or root apical meristem, lateral meristem, intercalary meristem, a seedling (e.g, a germinating seed or small seedling or a larger seedling with one or more true leaves), a whole seed (e.g., an intact seed, or a seed with part or all of its seed coat removed or treated to make permeable), a halved seed or other seed fragment, an embryo (e.g.. a mature dissected zygotic embryo, a developing embryo, a dry or rehydrated or freshly excised embryo), or callus.

[0081] In some embodiments, the plant cell is not an isolated plant cell, for example, the plant cell is located in an intact or growing plant or in a plant part or tissue. In certain embodiments the plant cell is located in a meristem (e.g., a SAM comprising LI and L2 layers) of a wliole plant or plant part. In such examples, the method can be performed in situ or in planta.

[0082] Generally, transformed plant cells (including protoplasts) are capable of division and further differentiation. In some examples, a transformed plant cell is regenerated to a whole plant (e.g., a transformed soybean cell is regenerated to a soybean plant). In some examples, the methods further include one or more steps of growing or regenerating a plant from a transformed plant cell, for example, a transformed plant cell including, producing or expressing a nucleic acid (e.g., a nucleic acid including a geminivirus replicon), or including an induced genetic modification as disclosed herein, thereby generating a modified plant. In such examples, the grown or regenerated plant contains at least some cells or tissues producing or expressing the transformed nucleic acid or including the induced genetic modification. In some examples, a callus is produced from the transformed plant cell, and plantlets and plants are produced from the callus. In other examples, whole seedlings or plants are grown directly from die transformed plant cell without a callus stage. Thus, additional aspects of the disclosure are directed to whole seedlings and plants grown or regenerated from transformed plant cells produced by the methodsDocket No. P15048WO00disclosed herein, as well as the seeds of such plants. In some examples, the grown or regenerated plant exhibits a phenotype associated with expression of the nucleic acid or the induced genetic modification. Non-limiting phenotypes include herbicide resistance, improved tolerance of abiotic stress (e.g., tolerance of temperature extremes, drought, or salt) or biotic stress (e. g._ resistance to bacterial or fungal pathogens), improved utilization of nutrients or water, modified lipid, carbohydrate, or protein composition, improved flavor or appearance, improved storage characteristics (e.g.. resistance to bruising, browning, or softening), increased yield, altered morphology (e.g., floral architecture or color, plant height, branching, root structure), or expression of a selectable marker.

[0083] The methods can include a selection step of selecting transformed plant cells (or seedlings or plants grown or regenerated therefrom) with a desired phenotype, for example, transformed plant cells (or seedlings or plants) can be exposed to conditions permitting expression of a phenotype of interest; e.g., selection for herbicide resistance can include exposing the population of plant cells (or seedlings or plants) to an amount of herbicide or other substance that inhibits growth or is toxic, allowing identification and selection of those resistant plant cells (or seedlings or plants) that survive treatment. Plant cells (or seedlings or plants grown or regenerated therefrom) can be selected based on manifestation of a desired phenotype. Such plants can be selected, for example, for further analysis or plant breeding.

[0084] The plant cell can be haploid, diploid, or polyploid. In some examples, the plant cell is haploid or can be induced to become haploid. Examples of haploid cells include but are not limited to plant cells obtained from haploid plants and plant cells obtained from reproductive tissues, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, and microspores. In some examples where the plant cell is haploid, the method of modify ing the genetic material of the plant cell can further include a step of chromosome doubling (e.g., by spontaneous chromosomal doubling by meiotic non-reduction, or by using a chromosome doubling agent such as colchicine, oryzalin, or trifluralin) to produce a doubled haploid plant cell that is homozygous for the induced genetic modification. Thus, aspects of the disclosure are related to haploid plant cells having the altered target nucleotide sequence as well as a doubled haploid plant cells or a doubled haploid plant that is homozygous for an induced genetic modification. Another aspect of the disclosure is related to a hybrid plant having at least one parent plant that is a doubled haploid plant provided by the method. Production of doubled haploid plants by these methods provides homozygosity in one generation, instead of requiring several generations of self-crossing to obtain homozygous plants; this may be particularlyDocket No. P15048WO00advantageous in slow-growing plants, such as fruit and other trees, or for producing hybrid plants that are offspring of at least one doubled-haploid plant.

[0085] The plant cell can be obtained from a dicot or a monocot plant. Non -limiting pl nts include row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses. Specific, non¬ limiting examples of commercially important plants include: alfalfa (Medicago saliva'), almonds (Prunus dulcis), apples (Malus x domestica), apricots Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibiricd), asparagus {Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape, (Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucus carota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppers and other capsicum peppers (Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C. baccatum). chrysanthemums (Chrysanthemum spp.), coconut (Cocos nucifera), coffee (Coffiea spp. including Coffiea arabica and Coffiea canephor a), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata), cucumber (Cucumis salivas), currants and gooseberries (Ribes spp.). eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp ), flax (Linum usitatissumum L ), geraniums (Pelargonium spp.), grapefruit (Citrus parodist), grapes (Vitus spp.) including wine grapes (Vitus vinifer ), guava (Psidium guajav ), irises (Iris spp,), lemon (Citrus limori), lettuce (Lactuca saliva), limes (Citrus spp.), maize (Zea mays L.), mango (Mangifera indica), mangosteen (Garcinia mangostana), melon (Cucumis meld), millets (Setaria spp., Echinochloa spp., Eleusine spp, Panicum spp., Pemisetum spp.), oats (Avena saliva), oil palm (Ellis quineensis), olive (Olea europaea), onion (Allium cepa), orange (Citrus sinensis), papaya (Carica papaya), peaches and nectarines (Prunus persica), pear (Pyrus spp.). pea (Pisa sativum), peanut (Arachis hypogaea), peonies (Paeonta spp.), petunias (Petunia spp.), pineapple (Ananas comosus), plantains (Musa spp.), plum (Prunus domestica), poinsettia (Euphorbia pulcherrima), Polish canola (Brassica rapa), poplar Populus spp.), potato (Solanum tuberosum), pumpkin (Cucurbita pepo), rice (Oryza sativa L.), roses (Rosa spp.), rubber Hevea brasiliensis). rye (Secale cereale). safflower (Carthainus tinctorius L), sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycine max), squash (Cucurbita pepo), strawberries (Fragaria, spp. Fragaria x ananas set), sugar beet Beta vulgaris), sugarcanes (Saccharum spp.), sunflower (Helianthus annus), sweet potato (Ipomoea batatas), tangerine Citrus tangerina), tea (Camellia sinensis), tobacco (Nicotiana labacum L.), tomato (Lycopersicon esculentum), tulips (Tulipa spp.), turnip (Brassica rapa rapa), walnuts (Juglans spp. I...), watermelon (Citrulus lanatus).Docket No. P15048WO00wheat (Triticum aestivum), and yams (Discorea spp ). In a non-limiting example, the plant cell is a soybean cell {Glycine max}.

[0086] In some examples, the plant cell is obtained from a crop plant characterized as being of or derived from an ‘'elite” germplasm or genetic background, for example, from an inbred crop plant that is an elite strain of germplasm, or from a hy brid crop plant that is the progeny of at least one elite strain of germplasm (e.g., progeny of an inbred male parent of a first elite strain and an inbred female parent of a second elite strain). As used herein, an “elite” strain or line of a crop plant is one that has resulted from usually multiple rounds of breeding and selection for superior performance, e.g., superior yield or other agronomic trait. As used herein, “line” or “strain” includes plants that share identical parentage and are generally inbred to some degree, and which are generally homozygous at most genetic loci. Plants of a given line or strain exhibit a consistent and predictable phenotype and agronomic performance. A plant is “homozygous” when it has only one type of allele at a given locus, e.g., a diploid plant with two identical copies of an allele at a given locus.

[0087] The following numbered embodiments also form part of the present disclosure:

[0088] 1. A method for modifying genomic DNA of a plant comprising:(a) contacting a whole plant or part thereof with a bacterial plant transformation culture comprising a plant transforming bacterium containing a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to:(i) DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP); and a plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep); and(ii) a first geminivirus Rep recognition site (RRS) or a first nanovirus Rep recognition site (RRS), DN A encoding one or more gene editing reagents which can modify genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS; and(b) selecting a propagule of the plant comprising the modification of the genomic DNA.2. The method of embodiment 1, wherein the first transfer DNA comprising DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP) and a plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep) further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.Docket No. P15048WO003. The method of embodiments 1 or 2, wherein the bacterial plant transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a geminivirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRSs of the second transfer DNA.4. The method of embodiments 1 or 2, wherein the bacterial plant transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a nanovirus RRS, DNA encoding a nanovirus Rep protein, and another nanovirus RRS, wherein the DNA encoding the nanovirus Rep protein is located between the nanovirus RRSs of the second transfer DNA.5. The method of any one of embodiments 1 to 4, wherein the first transfer DNA further comprises a nanovirus Cell Cycle Regulation Protein (Clink).6. The method of embodiment 5, wherein the Clink protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 124, 134, 144, 154, 164, 174, 184. 194, 204, 214. 224, 234, 244, 254, 264.7. The method of any one of claims 1 to 6, wherein the first transfer DNA further comprises a geminivirus or nanovirus Movement Protein Movement Protein (MP).8. The method of embodiment 7, w'herein the MP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%. 85%, 90%, 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 31. 53, 63, 126. 136, 146, 156, 166, 176. 186, 196, 206. 216, 226, 236. 246. 256, or 266.9. The method of any one of embodiments 1 to 8, wherein the geminivirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 3, 17, 18. 29, 43, 51, 61, 73, 81, 91, 101, or 111.10. The method of any one of embodiments 1 to 9, wherein the geminivirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%. 85'%, 90%, 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 8, 22, 34, 36, 45, 55, 57, 65. 67, 69, 75, 83. 85, 95, 105. 115, or 117.11. The method of any one of embodiments 2 to 10, w'herein the geminivirus C3 protein has an amino acid sequence with at least 50%, 60%, 70%, 80%. 85%, 90%, 95%, 98%. 99%. or 100% sequence identity’ to SEQ ID NO: 10, 24. 47, 77, 87, 97. or 107.Docket No. P15048WO0012. The method of any one of embodiments 1 to 11, wherein the geminivirus RRS has a nucleic acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11, 25, 26, 37-40, 48, 58, 70, 78, 88, 98, 108, or 118.13. The method of any one of embodiments 1 to 12, wherein the geminivirus coat protein. Rep protein, Rep / C3 protein and RRSs are obtained from the same geminivirus genus or from the same geminivirus species.14. The method of any one of embodiments 1 to 13, wherein the geminivirus Rep and Rep / C3 proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein.15. The method of any one of embodiments 1 to 14, wherein the nanovirus Rep proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein.16. The method of any one of embodiments 1 to 15, wherein the nanovirus CP and Rep proteins are obtained from the same nano virus genus or from the same nanovirus species.17. The method of any one of embodiments 1 to 16, wherein the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV), cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV),18. The method of any one of embodiments 1 to 16, wherein the plant is a dicot and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roll virus (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV). subterranean clover stunt virus (SCSV), or cow vetch latent virus (CVLV).19. The method of any one of embodiments 1 to 18, wherein the RRS is a Nanovirus common region.20. The method of embodiment 19, wherein the Nanovirus common region comprises the nucleic acid sequence TATTATTAC or TAGTATTAC.21. The method of any one of embodiments 1 to 20, wherein the nanovirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%. 98%, 99%, or 100% sequence identity to SEQ ID NO: 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 260.Docket No. P15048WO0022. The method of any one of embodiments 1 to 21, wherein the nanovirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122, 132, 142, 152. 162, 172, 182, 192. 202, 212, 222, 232. 242. 252, or 262.23. The method of any one of embodiments 1 to 22, wherein the gene editing reagents comprise a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.24. The method of any one of embodiments 1 to 23, wherein the geminivims CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein to form a gemini-like viral particle (GLVP).25. The method of any one of embodiments 1 to 23. wherein the nanovirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein to form a nanovirus-like viral particle.26. The method of any one of embodiments 1 to 25, wherein the plant transforming bacterium is:(i) an Agrobacterium sp. and the transfer DNA is an Agrobacterium T-DNA; or(ii) a Rhizobium sp., Sinorhizobium sp.. Mesorhizobiutn sp., Bradyrhizobium sp., Azobacter sp., or Phvllobacterium sp.27. The method of any one of embodiments 1 to 26. wherein the part of the plant contacted by the bacterial transformation culture is a plant part containing a meristem.28. The method of embodiment 27, wherein the meristem is a shoot apical meristem or an intercalary meristem of the plant.29. The method of embodiment 27, wherein the meristem is a vegetative meristem or a floral meristem.30. The method of any one of embodiments 27 to 29, wherein the LI layer of the meristem is contacted by the bacterial transformation culture.31. The method of any one of embodiments 27 to 30, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture.Docket No. P15048WO0032. The method of any one of embodiments 27 to 30, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture and one or more gene editing reagents and / or a GLVP or nanovirus-like particle is detectable in the L2 layer of the meristem.33. The method of any one of embodiments 27, or 30 to 32, wherein the meristem is a vegetative apical meristem.34. The method of any one of embodiments 1 to 16 or 19 to 33, wherein the plant is a dicot plant and the geminivirus is: (i) a Begomovirus and the RRS is a Begomovirus common region; (ii) a Curiovirus or a Topocuvirus and the RRRS is a Curtovirus or a Topocuvirus intergenic region; or (iii) a Mastrevirus and the RRS is a Mastrevirus Large Intergenic region (LIR),35. The method of embodiment 34. wherein the Begomovirus is a soybean mild mottle virus, cabbage leaf curl virus (CaLCuV), tomato golden mosaic virus (TGMV). tomato yellow leaf curl (TYLCuV), soybean chlorotic blotch virus (SCBV), or soybean blistering mosaic virus (SBMV).36. The method of any one of embodiments 1 to 16 or 19 to 33, wherein the plant is a monocot plant and the geminivirus is a Mastrevirus.37. The method of embodiment 36. wherein the Mastrevirus is a Maize Streak Virus, Tobacco yellow dwarf virus (TYDV), or bean yellow dwarf virus (BeYDV).38. The method of any one of embodiments 1 to 37. wherein the gene editing reagent(s) comprise: (i) an RNA directed DNA endonuclease and a guide RNA; (ii) a zinc finger nuclease: (ii) a transcription activator-like effector nuclease (TALEN); or (iv) a meganuclease.39. The method of any one of embodiments 1 to 38, wherein the propagule is an embryo, pollen, seed, or seedling.40. A non-human cell comprising first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to:(i) DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP); anda plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep); and(ii) a first geminivirus large intergenic region (RRS), DNA encoding one or more gene editing reagents, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS.Docket No. P15048WO0041. The cell of embodiment 40, wherein the first transfer DNA further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS,42. The cell of embodiments 40 or 41, further comprising a second transfer DNA comprising a geminivirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRSs of the second transfer DNA.43. The cell of embodiments 40 or 41, further comprising a second transfer DNA comprising a nanovirus RRS, DNA encoding a nanovirus Rep protein, and another nanovirus RRS, wherein the DNA encoding the nano virus Rep protein is located between the nanovirus RRSs of the second transfer DNA.44. The cell of any one of embodiments 40 to 43, wherein the first transfer DNA further comprises a nanovirus Cell Cycle Regulation Protein (Clink)45. The cell of embodiment 44. wherein the Clink protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%. 98%, 99%, or 100% sequence identity to SEQ ID NO: 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224. 234, 244, 254. 264.46. The cell of any one of embodiments 40 to 45, wherein the first transfer DNA further comprises a geminivirus or nanovirus Movement Protein Movement Protein (MP).47. The cell of embodiment 46. wherein the MP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 31, 53, 63, 126, 136, 146, 156. 166, 176, 186, 196, 206. 216, 226, 236, 246. 256, or 266.48. The cell of any one of embodiments 40 to 47, wherein the geminivirus CP protein has an amino acid sequence with at least 50%, 60%, 70%. 80%, 85%, 90%. 95%. 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 17, 18, 29, 43, 51, 61, 73, 81, 91, 101, or 111.49. The cell of any one of embodiments 40 to 48, w herein the geminivirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 8, 22, 34. 36, 45, 55, 57, 65, 67, 69, 75, 83, 85, 95, 105, 115, or 117.Docket No. P15048WO0050. The cell of any one of embodiments 40 to 49, wherein the geminivirus C3 protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, 24, 47, 77, 87, 97, or 107.51. The cell of any one of embodiments 40 to 50, wherein the geminivirus RRS has a nucleic acid sequence with at least 50%. 60%, 70%, 80%, 85%, 90%. 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 11, 25, 26, 37-40, 48, 58, 70, 78. 88, 98, 108, or 118.52. Tire cell of any one of embodiments 40 to 51, wherein the nanovirus Rep proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein.53. The cell of any one of embodiments 40 to 52, wherein the nanovirus CP and Rep protein are obtained from the same nanovirus genus or from the same nanovirus species.54. The cell of any one of embodiments 40 to 53, wherein the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV), cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV).55. The cell of any one of embodiments 40 to 53, wherein the plant is a dicot and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roil virus (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV), subterranean clover stunt virus (SCSV), or cow vetch latent virus (CVLV).56. The cell of any one of embodiments 40 to 55, wherein the RRS is a Nanovirus common region.57. The cell of embodiment 56, wherein the Nanovims common region comprises the nucleic acid sequence TATTATTAC or TAGTATTAC.58. The cell of any one of embodiments 40 to 57, wherein the nanovirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 120, 130, 140, 150, 160. 170. 180, 190, 200, 210, 220, 230, 240, 250. or 26059. The cell of any one of embodiments 40 to 58, wherein the nanovirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122, 132. 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, or 262.Docket No. P15048WO0060. The cell of any one of embodiments 40 to 59, wherein the gene editing reagents comprise a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.61. The cell of any one of embodiments 40 to 60, wherein the cell is a bacterial cell optionally selected from the group consisting of and E.coli, Agrobacterium sp., a Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. cell.62. A bacterial culture, comprising a plurality of cells of embodiment 61.63. The bacterial culture of any one of embodiments 40 to 62, wherein the bacterial culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a geminivinis RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRS of the second transfer DNA.64. A plant gene editing system comprising a whole plant wherein a part of the plant comprising a meristem is contacted with a bacterial transformation culture comprising a plant transforming bacterium containing a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to:(i) DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP); anda plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep); and(li) a first geminivirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS.65. The system of embodiment 64, wherein the first transfer DNA further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.66. The system of embodiments 64 or 65, wherein the bacterial transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a geminivirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNADocket No. P15048WO00encoding the geminivirus Rep / C3 protein is located between the geminivirus RRS of the second transfer DNA.67. The system of any one of embodiments 64 to 66, wherein the bacterial plant transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a nanovirus RRS, DNA encoding a nanovirus Rep protein, and another nanovirus RRS, wherein the DNA encoding the nanovirus Rep protein is located between the nanovirus RRSs of the second transfer DNA.68. Tire system of any one of embodiments 64 to 67, wherein the first transfer DNA further comprises a nanovirus Cell Cycle Regulation Protein (Clink).69. The system of embodiment 68, wherein the Clink protein has an amino acid sequence with at least 50%, 60%. 70%. 80%, 85%. 90%. 95%. 98%, 99%, or 100% sequence identity to SEQ ID NO: 124.134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264.70. The system of any one of embodiments 64 to 69, wherein the first transfer DNA further comprises a geminivirus or nanovirus Movement Protein Movement Protein (MP).71. The system of embodiment 70, wherein the MP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31, 53, 63, 126. 136, 146, 156, 166, 176, 186, 196, 206. 216, 226, 236. 246, 256, or 266.72. The system of any one of embodiments 64 to 71, wherein the geminivirus CP protein has an amino acid sequence with at least 50%. 60%, 70%, 80%. 85%. 90%, 95%, 98%. 99%. or 100% sequence identity’ to SEQ ID NO: 3, 17, 18, 29. 43, 51, 61, 73. 81. 91, 101. or 111.73. The system of any one of embodiments 64 to 72, wherein the geminivirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%. 80%. 85%, 90%, 95%. 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, 22. 34. 36, 45, 55. 57. 65, 67, 69, 75. 83. 85. 95, 105. 1 15. or 117.74. The system of any one of embodiments 64 to 73. wherein the geminivirus C3 protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. 24, 47, 77, 87, 97, or 107.75. The system of any one of embodiments 64 to 74, wherein the geminivirus RRS has a nucleic acid sequence with at least 50%, 60%. 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11, 25, 26, 37-40, 48, 58, 70, 78, 88, 98, 108, or 118.Docket No. P15048WO0076. The system of any one of embodiments 64 to 75, wherein the nanovirus Rep proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein.77. The system of any one of embodiments 64 to 76, wherein the nanovirus CP and Rep protein are obtained from the same nanovirus genus or from the same nanovirus species.78. The system of any one of embodiments 64 to 77, wherein the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV). cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV).79. 'The system of any one of embodiments 64 to 77, wherein the plant is a dicot and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV). pea yellow stunt v irus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roll virus (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV). parsley severe stunt associated virus (PSSaV). sophora yellow stunt virus (SY SV), subterranean clover stunt virus (SCSV), or cow vetch latent virus (CVLV).80. The system of any one of embodiments 64 to 79, wherein the RRS is a Nanovirus common region.81. The system of embodiment 80, wherein the Nanovirus common region comprises the nucleic acid sequence TATTATTAC or TAGTATTAC.82. The system of any one of embodiments 64 to 81, wherein the nanovirus CP protein has an amino acid sequence with at least 50%, 60%, 70%. 80%, 85%, 90%, 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 26083. The system of any one of embodiments 64 to 82. wherein the nanovirus Rep protein has an amino acid sequence wdth at least 50%, 60%, 70%. 80%, 85%, 90%, 95%. 98%, 99%, or 100% sequence identity to SEQ ID NO: 122, 132. 142, 152, 162. 172, 182, 192. 202, 212. 222, 232, 242, 252, or 262.84. The system of any one of embodiments 64 to 83. wherein the gene editing reagents comprise a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.Docket No. P15048WO0085. The system of any one of embodiments 64 to 84, wherein the geminivirus coat protein, Rep protein, Rep / C3 protein and RRSs are obtained from the same geminivirus genus or from the same geminivirus species.86. The system of any one of embodiments 64 to 85, wherein the Geminivirus Rep and Rep / c3 proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein.87. The system of any one of embodiments 64 to 86, wherein the geminivirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein to form a gemini-like viral particle (GLVP).88. The system of any one of embodiments 64 to 86, wherein the nanovirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein to form a nanovirus-like viral particle.89. The system of any one of embodiments 64 to 88. wherein the plant transforming bacterium is:(i) an Agrobacterium sp. and the transfer DNA is an Agrobacterium T-DNA; or(ii) a Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., or Phyllobacterium sp.90. The system of any one of embodiments 64 to 89. wherein the part of the plant contacted by the bacterial transformation culture is a plant part containing a meristem.91. The system of embodiment 90, wherein the meristem is a shoot apical meristem or an intercalary meristem of the plant.92. The system of embodiment 90, wherein the meristem is a vegetative meristem or a floral meristem.93. The system of any one of embodiments 90 to 92. wherein an LI layer of the meristem is contacted by the bacterial transformation culture.94. The system of any one of embodiments 90 to 93. wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture.Docket No. P15048WG0095. The system of any one of embodiments 90 to 94, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture and one or more of gene editing reagent and / or a GLVP or nanovirus-like particle is detectable in the L2 layer of the meristem.96. The system of any one of embodiments 64 to 95, wherein the plant is a dicot plant and the geminivirus is: (i) a Begomovirus and the RRS is a Begomovirus common region; (ii) a Curtovirus or a Topocuvirus and the RRRS is a Curtovirus or a Topocuvirus intergenic region; or (iii) a Mastrevirus and the RRS is a Mastrevirus Large Intergenic region (LIR)97. The system of embodiment 96, wherein the Begomovirus is a soybean mild mottle virus, cabbage leaf curl virus (CaLCuV), tomato golden mosaic vims (TGMV), tomato yellow leaf curl (TYLCuV), soybean chlorotic blotch virus (SCBV), or soybean blistering mosaic virus (SBMV).98. The system of any one of embodiments 66 to 95, wherein the plant is a monocot plant and the geminivirus is a Mastrevirus.99. The system of embodiment 98, wherein the Mastrevirus is a Maize Streak Virus. Tobacco yellow dwarf virus (TYDV), or bean yellow dwarf virus (BeYDV).100 The system of any one of embodiments 64 to 99. wherein the gene editing reagent(s) comprise: (i) an RNA directed DN A endonuclease and a guide RNA; (ii) a zinc finger nuclease; (ii) a transcription activator-like effector nuclease (TALEN); or (iv) a meganuclease.101. The system of any one of embodiments 64 to 100, wherein a DNA insertion, deletion, and / or substitution is effected in the genomic DNA of an L2 layer meristem cell by the gene editing reagents.102. The system of any one of embodiments 64 to 101, wherein a DNA insertion, deletion, and / or substitution effected in the genomic DNA by the gene editing reagents is recoverable in a plant propagule obtained from the whole plant.103 The system of embodiment 102. wherein the plant propagule is a seed, embryo, or pollen.EXAMPLES[0089| The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.Docket No. P15048WO00Example 1: Transformation[0090| The nucleic acid construct described herein can be utilized to stably or preferably transiently transform plant cells In stable transformation, the nucleic acid molecule described herein is integrated into the plant genome, and as such it represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the cell transformed but not integrated into the genome, and as such represents a transient trait. Agrobacterium-mediated gene transfer is described in Klee, H. J. et al. (1987); Annu Rev Plant Physiol 38, 467-486; Klee, H. J. and Rogers, S. G (1989); Cell Culture and Somatic Cell Genetics of Plants, Vol 6, Molecular Biology of Plant Nuclear Genes, pp. 2-25, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, Cal.; and Gatenby, A. A. (1989); Regulation and Expression of Plant Genes in Microorganisms, pp. 93-112, Plant Biotechnology, S. Kung and C. J. Arntzen, eds., Butterworth Publishers, Boston, Mass.

[0091] The Agrobacterium-mediated system includes the use of plasmid vectors that contain defined DNA segments which integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary' depending upon the plant species and the Agrobacterium delivery' system. A widely used approach is the leaf-disc procedure, which can be performed with any tissue explant that provides a good source for initiation of whole-plant differentiation (Horsch, R. B et al. (1988). " Leaf disc transformation." Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). A supplementary approach employs the Agrobacterium delivery' system in combination with vacuum infiltration. The Agrobacterium system is especially useful in the creation of transgenic dicotyledonous plants. There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues Additional direct DNA transfer techniques include glass or silicone carbide whiskers (see, for example, Dunwell, Methods Mol Biol. 1999; 111:3 75-82)

[0092] Following stable transformation, plant propagation then occurs. The most common method of plant propagation is by seed. The disadvantage of regeneration by seed propagation, however, is the lack of uniformity in the crop due to heterozy gosity, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. In other words, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the regeneration be effected such that the regenerated plant has identical traits andDocket No. P15048WO00characteristics to those of the parent transgenic plant. The preferred method of regenerating a transformed plant is by micropropagation, which provides a rapid, consistent reproduction of the transformed plants.

[0093] Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. The micropropagation process involves four basic stages: stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one. the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the newly grown tissue samples are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that they can continue to grow in the natural environment.

[0094] Transient transformation of, for example, leaf cells, meristematic cells, or the whole plant is also envisaged herein. Transient transformation can be effected by any of the direct DNA transfer methods described above or by mechanical or vector mediated viral infection using the plant viruses derived plasmid described herein. Thus, the present disclosure provides a gemini virus based nucleic acid construct which spreads systemically throughout the host plant and yet does not induce symptoms thereinReferencesIlyina T. V., Koonin E. V. (1992). Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria. eucaryotes and archaebacteria. Nucleic Acid Res 20, 3279-3285. doi: 10.1093 / nar / 20.13.3279.Koonin E. V., Ilyina T. V. (1992). Gemimvirus replication proteins are related to prokary otic plasmid rolling circle DNA replication initiator proteins. J. Gen. Virol. 73, 2763–2766. doi: 10.1099 / 0022-1317-73-10-2763.Oshima K., Kakizawa S., Nishigawa H., Kuboyama T., Miyata S.-I., Ugaki M., et al. (2001). A plasmid of phytoplasma encodes a unique replication protein having both plasmid-and virus-like domains: clue to viral ancestry or result of virus / plasmid recombination? Virology 285, 270–277. doi: 10.1006 / viro.2001.0938.Docket No. P15048WO00Krupovic M., Ravantti J. J., Bamford D. H. (2009). Geminiviruses: a tale of a plasmid becoming a virus. BMC Evol. Biol. 9, 112–111. doi: 10.1186 / 1471-2148-9-112.Hanley-Bowdoin L., Settlage S. B., Orozco B. M., Nagar S., Robertson D. (1999).Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit. Rev. Plant Set. 18, 71-106Varsani A., Roumagnac P.. Fuchs M., Navas-Castillo J.. Moriones E., Idris A., et al. (2017). Capulavirus and Grablovirus: two new generain the family Gemini viridae. Arch. Virol. 162, 1819-1831. doi: 10.1007 / s00705-017-3268-6.Hofer J., Dekker E. L., Reynolds H. V., Woolston C. J., Cox B. S., Mullineaux P. M. (1992). Coordinate regulation of replication and virion sense gene expression in wheat dwarf virus. Plant Cell 4, 213-223. doi: 10.1105 / tpc.4.2.213.Collin S., Fernández-lobato M., Gooding P. S., Mullineaux P. M., Fenoll C. (1996) The two nonstructural proteins from wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins. Virology 219, 324-329. doi: 10.1006 / viro.1996.0256. Gutierrez C. Ramirez-Parra E., Castellano M. M., Sanz-Burgos A. P.. Luque A., Missich R. (2004). Geminivirus DNA replication and cell cycle interactions. Vet. Microbiol. 98, 111-119. doi: 10.1016 / j. vetmic.2003.10.012.Varsani A., Roumagnac P., Fuchs M., Navas-Castillo J., Moriones E., Idris A., et al. (2017). Capulavirus and Grablovirus: two new genera in the family Geminiviridae. Arch. Virol. 162, 1819-1831. doi: 10.1007 / s00705-017-3268-6.

Claims

Docket No. P15048WO00We claim:

1. A method for modifying genomic DNA of a plant comprising:(a) contacting a whole plant or part thereof with a bacterial plant transformation culture comprising a plant transforming bacterium containing a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to:(i) DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP): anda plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep); and(ii) a first geminivirus Rep recognition site (RRS) or a first nanovirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents which can modify genomic DNA of the plant, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS; and(b) selecting a propagule of the plant comprising the modification of the genomic DNA.

2. The method of claim 1. wherein the first transfer DNA comprising DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP) and a plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep) further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS. wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.

3. The method of claim 1. wherein the bacterial plant transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a geminivirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRSs of the second transfer DNA.

4. The method of claim 1, wherein the bacterial plant transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a nanovirus RRS, DNA encoding a nanovirus Rep protein, and another nanovirus RRS, -wherein the DNA encoding the nanovirus Rep protein is located between the nanovirus RRSs of the second transfer DNA.Docket No. P15048WO005. The method of claim 1. wherein the first transfer DNA further comprises a nanovirus Cell Cycle Regulation Protein (Clink).

6. The method of claim 5, wherein the Clink protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

124. 134, 144, 154, 164.

174. 184, 194. 204, 214, 224, 234.

244. 254, 264.

7. The method of claim 1, wherein the first transfer DNA further comprises a geminivirus or nanovirus Movement Protein Movement Protein (MP).

8. The method of claim 7, wherein the MP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31, 53, 63, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, or 266.

9. The method of claim 1, wherein the geminivirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 17, 18, 29, 43, 51, 61, 73, 81, 91, 101, or 111.

10. The method of claim 1, wherein the geminivirus Rep protein has an amino acid sequence with at least 50%. 60%, 70%, 80%. 85%, 90%, 95%, 98%. 99%, or 100% sequence identity to SEQ ID NO: 8, 22, 34, 36, 45, 55, 57, 65, 67, 69, 75, 83, 85, 95, 105, 115, or 117.

11. The method of claim 1, wherein the geminivirus C3 protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 24, 47, 77, 87, 97, or 107.

12. The method of claim 1, wherein the geminivirus RRS has a nucleic acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

11.

25. 26, 37-40, 48, 58, 70, 78, 88, 98, 108. or 118.

13. The method of claim 1. wherein the geminivirus coat protein, Rep protein, Rep / C3 protein and RRSs are obtained from the same geminivirus genus or from the same geminivirus species.Docket No. P15048WO0014. The method of claim 1. wherein the geminivirus Rep and Rep / C3 proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein15. The method of claim 1, wherein the nano virus Rep proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein.

16. The method of claim 1. wherein the nanovirus CP and Rep proteins are obtained from the same nanovirus genus or from the same nanovirus species.

17. The method of claim 1, wherein the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV), cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV).

18. The method of claim 1. wherein the plant is a di cot and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roll virus (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV). faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV), subterranean clover stunt virus (SCSV), or cow vetch latent virus (CVLV).

19. The method of claim 1. wherein the RRS is a Nanovirus common region.

20. The method of claim 19, wherein the Nanovirus common region comprises the nucleic acid sequence TATTATTAC or TAGTAITAC.

21. The method of claim 1, wherein the nanovirus CP protein has an amino acid sequence with at least 50%, 60%. 70%, 80%, 85%. 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 260.Docket No. P15048WO0022. The method of claim 1. wherein the nanovirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122, 132, 142, 152. 162, 172, 182, 192. 202, 212, 222. 232, 242, 252, or 262.

23. The method of claim 1, wherein the gene editing reagents comprise a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.

24. The method of claim 1, wherein the geminivirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein to form a gemini-like viral particle (GLVP).

25. The method of claim 1, wherein the nanovirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein to form a nanovirus-like viral particle.

26. The method of any one of claims 1 to 25. wherein the plant transforming bacterium is:(i) an Agrobacterium sp. and the transfer DNA is an Agrobacterium T-DNA; or (ii) a Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., or Phyllobacterium sp.

27. The method of any one of claims 1 to 25, wherein the part of the plant contacted by the bacterial transformation culture is a plant part containing a meristem.

28. The method of claim 27, wherein the meristem is a shoot apical meristem or an intercalary meristem of the plant.

29. The method of claim 27, wherein the meristem is a vegetative meristem or a floral meristem.

30. The method of claim 27, wherein the LI layer of the meristem is contacted by the bacterial transformation culture.Docket No. P15048WO0031. The method of claim 27, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture.

32. The method of any one of claims 27 to 30, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture and one or more gene editing reagents and' or a GLVP or nanovirus-like particle is detectable in the L2 layer of the meristem.

33. The method of claim 27, wherein the meristem is a vegetative apical meristem.

34. The method of any one of claims 1 to 16 or 19 to 25, wherein the plant is a dicot plant and the geminivirus is: (i) a Begomovirus and the RRS is a Begomovirus common region; (ii) a Curtovirus or a Topocuvirus and the RRRS is a Curtovirus or a Topocuvirus intergenic region; or (iii) a Mastrevirus and the RRS is a Mastrevirus Large Intergenic region (LIR).

35. The method of claim 34, wherein the Begomovirus is a soybean mild mottle virus, cabbage leaf curl virus (CaLCuV), tomato golden mosaic vims (TGMV), tomato yellow leaf curl (TYLCuV), soybean chlorotic blotch virus (SCBV), or soybean blistering mosaic vims (SBMV).

36. The method of any one of claims 1 to 16 or 19 to 25, wherein the plant is a monocot plant and the geminivirus is a Mastrevirus.

37. The method of claim 36, wherein the Mastrevirus is a Maize Streak Virus, Tobacco yellow dwarf virus (TYDV), or bean yellow dwarf virus (BeYDV).

38. The method of any one of claims 1 to 22 or 23 to 25, wherein the gene editing reagent(s) comprise: (i) an RNA directed DNA endonuclease and a guide RNA; (ii) a zinc finger nuclease; (ii) a transcription activator-like effector nuclease (TALEN); or (iv) a meganuclease.

39. The method of any one of claims 1 to 25, wherein the propagule is an embryo, pollen, seed, or seedling.

40. A non-human cell comprising first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to:Docket No. P15048WO00(i) DNA encoding a plant gemini virus coat protein (CP) or a plant nanovirus coat protein (CP): anda plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep); and(ii) a first geminivirus large intergenic region (RRS), DNA encoding one or more gene editing reagents, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS.

41. The cell of claim 40, wherein the first transfer DNA further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.

42. The cell of claim 40, further comprising a second transfer DNA comprising a geminivirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRSs of the second transfer DNA.

43. The cell of claim 40, further comprising a second transfer DNA comprising a nanovirus RRS. DNA encoding a nanovirus Rep protein, and another nanovirus RRS. wherein the DNA encoding the nanovirus Rep protein is located between the nanovirus RRSs of the second transfer DNA.

44. The cell of claim 40, wherein the first transfer DNA further comprises a nanovirus Cell Cycle Regulation Protein (Clink).

45. The cell of claim 44, wherein the Clink protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264.

46. The cell of any one of claims 40 to 45, wherein the first transfer DNA further comprises a geminivirus or nanovirus Movement Protein Movement Protein (MP).Docket No. P15048WO0047. The cell of claim 46, wherein the MP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%. or 100% sequence identity to SEQ ID NO: 31, 53, 63, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, or 266.

48. The cell of any one of claims 40 to 45, wherein the gemmivirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity' to SEQ ID NO: 3, 17, 18, 29, 43, 51, 61, 73, 81, 91, 101, or 111.

49. The cell of any one of claims 40 to 45, wherein the gemimvirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%. 80%, 85%, 90%. 95%, 98 %, 99%, or 100% sequence identity to SEQ ID NO: 8, 22, 34, 36, 45, 55, 57, 65, 67, 69, 75.

83.

85. 95, 105, 115. or 117.

50. The cell of any one of claims 40 to 45, wherein the gemmivirus C3 protein has an amino acid sequence with at least 50%, 60%. 70%. 80%, 85%. 90%. 95%, 98%. 99%. or 100% sequence identity to SEQ ID NO: 10, 24, 47, 77, 87.

97. or 107.

51. The cell of any one of claims 40 to 45, wherein the gemimvirus RRS has a nucleic acid sequence with alleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11, 25, 26, 37-40. 48, 58, 70, 78, 88, 98, 108. or 118.

52. The cell of any one of claims 40 to 45, wherein the nanovirus Rep proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein.

53. The cell of any one of claims 40 to 45, wherein the nanovirus CP and Rep protein are obtained from the same nanovirus genus or from the same nanovirus species.

54. The cell of any one of claims 40 to 45. wherein the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV), cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV).

55. The cell of any one of claims 40 to 45. wherein the plant is a dicot and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stuntDocket No. P15048WO00virus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roll virus (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV), subterranean clover stunt virus (SCSV), or cow vetch latent virus (CVLV).

56. The cell of any one of claims 40 to 45, wherein the RRS is a Nanovirus common region.

57. The cell of claim 56, wherein the Nanovirus common region comprises the nucleic acid sequence TATTATTAC or TAGTATTAC.

58. The cell of any one of claims 40 to 45, wherein the nanovirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 260.

59. The cell of any one of claims 40 to 45, wherein the nanovirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, or 262.

60. The cell of any one of claims 40 to 45, wherein the gene editing reagents comprise a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.

61. The cell of any one of claims 40 to 45, wherein the cell is a bacterial cell optionally selected from the group consisting of and E.coli. Agrobacterium sp., & Rhizobium sp., Sinorhizobium sp., Mesorhizobium. sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. cell.

62. A bacterial culture, comprising a plurality' of cells of claim 61.

63. The bacterial culture of any one of claims 40 to 62, wherein the bacterial culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a geminivirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS,Docket No. P15048WO00wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRS of the second transfer DNA.

64. A plant gene editing system comprising a whole plant wherein a part of the plant comprising a meristem is contacted with a bacterial transformation culture comprising a plant transforming bacterium containing a first transfer DNA comprising one or more bacterial DNA transfer elements which are operably linked to:(i) DNA encoding a plant geminivirus coat protein (CP) or a plant nanovirus coat protein (CP); anda plant geminivirus replication initiator protein (Rep) or a plant nanovirus replication initiator protein (Rep); and(ii) a first geminivirus Rep recognition site (RRS), DNA encoding one or more gene editing reagents, and a second RRS, wherein the DNA encoding the gene editing reagents is located between the first and the second RRS.

65. The system of claim 64, wherein the first transfer DNA further comprises (iii) DNA encoding a geminivirus Rep / C3 protein and a third geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the first and third RRS or between the second and third RRS.

66. The system of claim 64. wherein the bacterial transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a gemimvirus RRS, DNA encoding a geminivirus Rep / C3 protein, and another geminivirus RRS, wherein the DNA encoding the geminivirus Rep / C3 protein is located between the geminivirus RRS of the second transfer DNA.

67. The system of claim 64, wherein the bacterial plant transformation culture further comprises a plant transforming bacterium containing a second transfer DNA comprising a nanovirus RRS, DNA encoding a nanovirus Rep protein, and another nanovirus RRS, wherein the DNA encoding the nanovirus Rep protein is located between the nanovirus RRSs of the second transfer DNA.

68. The system of claim 64, wherein the first transfer DNA further comprises a nanovirus Cell Cycle Regulation Protein (Clink).Docket No. P15048WO0069. The system of claim 68, wherein the Clink protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 26470. The system of any one of claims 64 to 69, wherein the first transfer DNA further comprises a geminivirus or nanovirus Movement Protein Movement Protein (MP).

71. The system of claim 70, wherein the MP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31, 53, 63, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, or 266.

72. The system of any one of claims 64 to 69, wherein the geminivirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, 17, 18, 29, 43, 51, 61, 73, 81, 91, 101, or 111.

73. The system of any one of claims 64 to 69, wherein the geminivirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8, 22, 34, 36, 45, 55, 57, 65, 67, 69, 75. 83, 85, 95, 105, 115, or 117.

74. The system of any one of claims 64 to 69, wherein the geminivirus C3 protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, 24, 47, 77, 87, 97. or 107.

75. The system of any one of claims 64 to 69, wherein the geminivirus RRS has a nucleic acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11, 25, 26, 37-40, 48, 58, 70, 78, 88, 98, 108, or 118.

76. The system of any one of claims 64 to 69, wherein the nanovirus Rep proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein.Docket No. P15048WO0077. The system of any one of claims 64 to 69, wherein the nanovirus CP and Rep protein are obtained from the same nanovirus genus or from the same nanovirus species.

78. The system of any one of claims 64 to 69, wherein the plant is a monocot plant and the nanovirus is an abaca bunchy top virus (ABTV), cardamom bushy dwarf virus (CdBDV), or banana bunchy top virus (BBTV).

79. The system of any one of claims 64 to 69, wherein the plant is a dicot and the nanovirus is a milk vetch dwarf virus (MDV), milk vetch chlorotic dwarf virus (MVCDV), pea yellow stunt virus (PYSV), faba bean yellow leaf virus (FBYLV), black medic leaf roll vims (BMLRV), faba bean necrotic yellows virus (FBNYV), pea necrotic yellow dwarf virus (PNYDV), faba bean necrotic stunt virus (FBNSV), parsley severe stunt associated virus (PSSaV), sophora yellow stunt virus (SYSV). subterranean clover stunt vims (SCSV), or cow- vetch latent vims (CVLV).

80. The system of any one of claims 64 to 69, wherein the RRS is a Nanovirus common region.

81. The system of claim 80, wherein the Nanovirus common region comprises the nucleic acid sequence TATTATTAC or TAGTATTAC.

82. The system of any one of claims 64 to 69, wherein the nanovirus CP protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

120. 130, 140, 150, 160, 170, 180, 190. 200, 210, 220, 230, 240.

250. or 260.

83. The system of any one of claims 64 to 69, wherein the nanovirus Rep protein has an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, or 262.

84. The system of any one of claims 64 to 69, wherein the gene editing reagents comprise a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to a gene of interest in the DNA of the plant.Docket No. P15048WO0085. The system of any one of claims 64 to 69, wherein the geminivirus coat protein, Rep protein, Rep / C3 protein and RRSs are obtained from the same geminivirus genus or from the same geminivirus species.

86. The system of any one of claims 64 to 69, wherein the Geminivirus Rep and Rep / c3 proteins can replicate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein.

87. The system of any one of claims 64 to 69, wherein the geminivirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep / C3 protein to form a gemini-like viral particle (GLVP).

88. The system of any one of claims 64 to 69, wherein the nanovirus CP can encapsidate circular DNA comprising an RRS and DNA encoding the gene editing reagents or an RRS and DNA encoding the Rep protein to form a nanovirus-like viral particle.

89. The system of any one of claims 64 to 69, wherein the plant transforming bacterium is:(i) an Agrobacterium, sp. and the transfer DNA is an Agrobacterium T-DNA; or (ii) aRhizobium sp., Sinorhizobium sp., Mesorhizobium sp.. Bradyrhizobium sp., Azobacter sp., o Phyllobacterium sp.

90. The system of any one of claims 64 to 69, wherein the part of the plant contacted by the bacterial transformation culture is a plant part containing a meristem.

91. The system of claim 90, wherein the meristem is a shoot apical meristem or an intercalary meristem of the plant.

92. The system of claim 90, wherein the meristem is a vegetative meristem or a floral meristem.

93. The system of claim 90, wherein an LI layer of the meristem is contacted by the bacterial transformation culture.Docket No. P15048WO0094. The system of claim 90, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture.

95. The system of claim 90 to 94, wherein the L2 layer of the meristem is not directly contacted by the bacterial transformation culture and one or more of gene editing reagent and or a GLVP or nanovirus-like particle is detectable in the L2 layer of the meristem.

96. The system of any one of claims 64 to 69, wherein the plant is a dicot plant and the geminivirus is: (i) a Begomovirus and the RRS is a Begomovirus common region; (ii) a Curtovirus or a Topocuvirus and the RRRS is a Curtovirus or a Topocuvirus intergenic region; or (iii) a Mastrevirus and the RRS is a Mastrevirus Large Intergenic region (LIR)97. The system of claim 96, wherein the Begomovirus is a soybean mild mottle virus, cabbage leaf curl virus (CaLCuV), tomato golden mosaic virus (TGMV), tomato yellow leaf curl (TYLCuV), soybean chlorotic blotch virus (SCBV), or soybean blistering mosaic virus (SBMV).

98. The system of any one of claims 64 to 69, wherein the plant is a monocot plant and the geminivirus is a Mastrevirus.

99. The system of claim 98, wherein the Mastrevirus is a Maize Streak Virus, Tobacco yellow dwarf virus (TYDV), or bean yellow dwarf virus (BeYDV).

100. The system of any one of claims 64 to 69, wherein the gene editing reagent(s) comprise: (i) an RNA directed DNA endonuclease and a guide RNA; ( ii ) a zinc finger nuclease; (ii) a transcription activator-like effector nuclease (TALEN); or (iv) a meganuclease.

101. The system of any one of claims 64 to 69, wherein a DNA insertion, deletion, and / or substitution is effected in the genomic DNA of an L2 layer meristem cell by the gene editing reagents.

102. The system of any one of claims 64 to 69, wherein a DNA insertion, deletion, and / or substitution effected in the genomic DNA by the gene editing reagents is recoverable in a plant propagule obtained from the whole plant.Docket No. P15048WO00103. The system of claim 102, wherein the plant propagule is a seed, embryo, or pollen.

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