Morphoregulators for maize transformation

Abstract

Morphoregulator polypeptides that can be used to improve the capacity of the plant cells to regenerate embryogenic plant tissues, plant organs, and whole plants are disclosed. Also disclosed are plant cells comprising the polypeptides and related methods for improving the capacity of the plant cells to regenerate embryogenic plant tissues, plant organs, and whole plants.

Description

INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 TITLE: MORPHOREGULATORS FOR MAIZE TRANSFORMATION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to provisional patent application U.S. Serial No. 63 / 661,751, filed June 19, 2024. The provisional patent application is herein incorporated by reference in its entirety, including without limitation, the specification, claims, and abstract, as well as any figures, tables, appendices, or drawings thereof. 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 June 13, 2025, is named P14795WO00_SequenceListing.xml and is 70,562 bytes in size. BACKGROUND

[0003] Plant morphoregulators, also named morphogenic genes or developmental genes, have been used to facilitate plant transformation and transformation of transformation-recalcitrant plant germplasm. Maize WUSCHEL (WUS) and BABY BOOM (BBM) genes have been used to stimulate somatic embryogenesis. Such somatic embryogenesis promotes formation of direct embryogenic callus from which new shoots can be generated, making some previously recalcitrant tissues accessible to transformation. Although embryogenic enhancers have been tried and work in certain combinations (e.g., ZmBBM2 and ZmWUS2), they often have limited effectiveness on some lines or cytotoxicity that requires sophisticated deployment. A need exists for additional morphogenic genes and morphogenic gene combinations to expand the range of germplasm accessible for transformation. SUMMARY

[0004] Methods of producing a regenerable plant structure, the method comprising introducing one or more polynucleotides encoding at least one morphoregulatory polypeptide of SEQ ID NO: 1 - 11, and / or an allelic variant thereof in a monocot plant cell; and culturing the monocot plant cell to produce the regenerable plant structure are provided. In certain embodiments, the one or more polynucleotides encode at least one additional morphoregulatory polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 least 99.5% sequence identity across the entire length of SEQ ID NO: 12, 13, 14, or 28 in the monocot plant cell.

[0005] Also provided are monocot plant cells comprising a heterologous promoter which is operably linked to a polynucleotide encoding a polypeptide of SEQ ID NO: 1 -11, and / or an allelic variant thereof, wherein expression of the polypeptide increases proliferation, somatic embryogenesis, and / or regeneration capacity of the monocot plant cell.

[0006] Also provided are recombinant polynucleotides comprising one or more polynucleotides encoding at least one morphoregulatory polypeptide of SEQ ID NO: 1 - 11, and / or an allelic variant thereof, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell. Vectors comprising the recombinant polynucleotide are also provided. DESCRIPTION OF THE DRAWINGS

[0007] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0008] The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the embodiments of the present disclosure and together with the description, serve to explain the principles of the disclosure. In the drawings:

[0009] FIG.1 shows a binary vector pG3R-WOY-SI containing pAXIG::WUS2, pLTP::BBM, pBdEF1a::YFP-NLS pZmUBI::GUS and pSbAHAS::ZmHRA

[0010] FIG.2 shows a maize B104 zygotic embryo transformed with pG3R-WOY-SI (FIG.1), seven days after transformation and stained for GUS expression.

[0011] FIG.3 shows the outline of the scRNA-seq strategy. Briefly, immature B104 embryos were transformed with a binary vector pG3R-WOY-SI (FIG. 1). Seven days post transformation, transgenic cells started somatic embryo development. In order to obtain the high-resolution single cell RNA sequencing (scRNA-seq) dataset, about 200 immature zygotic embryos expressing most YFP-positive somatic embryos were selected and used for enzymatic removal of the cell walls. In total 100,000 transgenic protoplasts were sorted using Fluorescence-Assisted Cell Sorting. The resulting population of cells was used for single-cell partitioning using 10x Genomics technology, followed by short-read mRNA-sequencing.

[0012] FIG.4 shows an scRNA-seq dataset Uniform Manifold Approximation and Projection (UMAP) representation. Transcript profiles of 6830 high-quality transformed cells after cell cycle regression were clustered and visualized through UMAP.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0013] FIG. 5 shows pG3P-RUBY-GUS-AG vector map. The pG3P-RUBY-GUS-AG destination vector was used for the induced somatic embryogenesis screen.

[0014] FIG. 6 shows morphoregulatory genes that induced somatic embryogensis. Immature zygotic maize embryos from three different cobs were transformed with the candidate morphoregulatory genes and imaged at different days after transformation (DAT). The candidate number, transcription family and gene identifier are given, next to the percentage of explants that show RUBY-expressing somatic embryos. Pictures of explants transformed with the candidates are shown, next to pictures of explants transformed with the negative control (tdTomato) or the positive control (BBM / WUS2) from the same experiment.

[0015] FIG. 7A and B show Morphoregulator 17 (SEQ ID NO: 1) induced somatic embryogenesis. Immature zygotic maize embryos from three different cobs were transformed with the morphoregulator 17 and imaged at 13 days after transformation (DAT). FIG. 7A. DAT, transcription family and gene identifier are given. Representative pictures of explants transformed with the candidate are shown, next to representative pictures of explants transformed with the negative control (tdTomato) or the positive control (BBM / WUS) from the same experiment. FIG.7B, 21 random explants are shown for morphoregulator 17, with 14 negative control (tdTomato) explants and 14 positive control (BBM / WUS) explants, next to the percentage of explants that were scored to show RUBY-expressing somatic embryos.

[0016] FIG.8 shows Morphoregulator 47 (SEQ ID NO: 7) increases the number of transgenic regenerants. Immature zygotic maize embryos from three different cobs were transformed with the morphoregulator 47 and imaged 29 days after transformation (DAT). Overview images are shown of the explants transformed with the negative control (tdTomato), the morphoregulator 47 or the positive control (BBM / WUS). The number of explants forming at least one RUBY- positive shoot is given. DETAILED DESCRIPTION

[0017] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5’ to 3’ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one defines the other, as well as defines the exact complements, as is known to one of ordinary skill in the art. Where a term is provided in the singular, the inventors also contemplate embodiments described by the plural of that term.

[0018] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism. In certain embodiments, an allelic variant of a polynucleotide or polypeptide can have at leastINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of the polynucleotide or polypeptide.

[0019] 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).

[0020] As used herein, the phrase “embryo productivity” refers to an assigned number that reflects how many somatic embryos develop on each immature embryo that is embryogenic, wherein the assigned number is determined by a subjective somatic embryogenesis score from 0 to 4 and wherein a higher score indicates an increase in somatic embryo production.

[0021] The term “genome” refers to the entire complement of genetic material (genes and non- coding sequences) that is present in an organism or cell, including both nuclear and organellar DNA.

[0022] As used herein, “heterologous” refers to a polynucleotide or peptide sequence located in, e.g., a genome or a vector, in a context other than that in which the sequence occurs in nature. For example, a promoter that is operably linked to a gene other than the gene that the promoter is operably linked to in nature is a heterologous promoter.

[0023] The phrase “improved plant cell regenerative potential” as used herein refers to the ability of a given plant cell to form a somatic embryo, embryogenic callus, a somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots in comparison to a control plant cell.

[0024] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0025] As used herein, the term “introduced” means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods. Thus, “introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct / expression construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into aINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., nuclear chromosome, plasmid, plastid, chloroplast, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0026] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.

[0027] As used herein, the phrase “plant cell” can refer either a plant cell having a plant cell wall or to a plant cell protoplast lacking a plant cell wall.

[0028] As used herein, the phrase “somatic embryo induction frequency” refers to the percentage of immature embryos producing somatic embryos.

[0029] As used herein, the phrase “target gene” can refer to a gene located in the genome that is to be modified by genome editing systems provided herein. Embodiments of target genes include (protein-)coding sequence, non-coding sequence, and combinations of coding and non- coding sequences. Target gene edits include nucleotide substitutions, insertions, and / or deletions in one or more elements of a gene that include a transcriptional enhancer or promoter, a 5’ or 3’ untranslated region, a mature or precursor RNA coding sequence, an intron, a splice donor and / or acceptor, a protein coding sequence, a polyadenylation site, and / or a transcriptional terminator.

[0030] To the extent to which any of the 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.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0031] Plant cells and related systems, methods, and compositions that provide for improved plant cell regenerative potential in comparison to control plant cells are provided herein. In certain embodiments, improved plant cell regenerative potential is provided by introducing one or more polynucleotides encoding a homeodomain-leucine zipper (HD-Zip) polypeptide, a growth-regulating factor (GRF) polypeptide, a DNA binding with one finger (DOF) polypeptide, a WKRY polypeptide, a basic leucine zipper (bZIP) polypeptide, a MYB polypeptide, an AP2 polypeptide, or a basic helix-loop-helix (bHLH) polypeptide. In certain embodiments, at least one additional polynucleotide encoding a BABY BOOM (BBM) polypeptide (e.g. SEQ ID NO: 12 or an allelic variant thereof), a WUSCHEL (WUS) polypeptide (e.g. SEQ ID NO: 13 or an allelic variant thereof), or another morphoregulatory polypeptide (e.g. SEQ ID NO: 14 or an allelic variant thereof), is also introduced in the plant cell.

[0032] In certain embodiments, the HD-Zip polypeptide comprises a Zea Mays hb77 (Zmhb77) polypeptide of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments, the GRF polypeptide comprises a Zea Mays GRFTF4 (ZmGRFTF4) polypeptide of SEQ ID NO: 2 or an allelic variant thereof and / or a Zea Mays GRFTF5 (ZmGRFTF5) polypeptide of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments, the DOF polypeptide comprises a Zea Mays DOF11 (ZmDOF11) polypeptide of SEQ ID NO: 4 or an allelic variant thereof and / or a Zea Mays DOF22 (ZmDOF22) polypeptide of SEQ ID NO: 5 or an allelic variant thereof. In certain embodiments, the WRKY polypeptide comprises a Zea Mays WRKY53 (ZmWRKY53) polypeptide of SEQ ID NO: 6 or an allelic variant thereof. In certain embodiments, the bZIP polypeptide comprises a Zea Mays bZIP75 (ZmbZIP75) polypeptide of SEQ ID NO: 7 or an allelic variant thereof. In certain embodiments, the MYB polypeptide comprises a Zea Mays MYB31 (ZmMYB31) polypeptide of SEQ ID NO: 8 or an allelic variant thereof. In certain embodiments, the AP2 polypeptide comprises a Zea Mays HSCF1 (Zm HSCF1) polypeptide of SEQ ID NO: 9 or an allelic variant thereof. In certain embodiments, the bHLH polypeptide comprises a Zea Mays bHLH160 (ZmbHLH160) polypeptide of SEQ ID NO: 10 or an allelic variant thereof and / or a Zea Mays bHLH48 (ZmbHLH48) polypeptide of SEQ ID NO: 11 or an allelic variant thereof. In certain embodiments the BBM polypeptide comprises a Zea mays BBM (ZmBBM) polypeptide of SEQ ID NO: 12 or an allelic variant thereof. In certain embodiments the WUS2 polypeptide comprises a Zea mays WUS2 (ZmWUS2) polypeptide of SEQ ID NO: 13 or an allelic variant thereof.

[0033] In certain embodiments, one of more of the genes encoding peptides set forth in SEQ ID NO: 1-11 and / or an allelic variant thereof is provided with an additional morphoregulatoryINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 gene. In some embodiments, the additional morphoregulatory gene encodes a protein comprising SEQ ID NO: 12, 13, 14, 28, or an allelic variant thereof. In some embodiments, the polypeptide allelic variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 14, or 28. In some embodiments, the at least one additional morphoregulatory polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12, 13, 14, or 28. In certain embodiments, the additional morphoregulatory gene is a morphoregulatory gene disclosed in Patent Application Publication Nos. US20220135990 and 20230189734, which are each incorporated herein by reference in their entireties.

[0034] Embodiments of the present disclosure are directed to combinations of morphoregulators. In certain embodiments, the one or more polynucleotides encode any two, three, four, or more peptides selected from SEQ ID NO: 1 - 11, and / or an allelic variant thereof. In certain embodiments, the one or more polynucleotides encode any two, three, four, or more peptides selected from SEQ ID NO: 1 - 5, and / or an allelic variant thereof. In certain embodiments, the one or more polynucleotides encode any two, three, four, or more peptides selected from SEQ ID NO: 6 - 11, and / or an allelic variant thereof. Thus, any combination of morphoregulators is encompassed by this disclosure. For example, in certain embodiments, the one or more polynucleotides encode at least SEQ ID NO: 1 or an allelic variant thereof and SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; SEQ ID NO: 9 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof. In certain embodiments, the one or more polynucleotides encode at least SEQ ID NO: 9 or an allelic variant thereof and SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof. In certain embodiments, the polynucleotides or combinations of polynucleotides include additional polynucleotides which encode at least one additional morphoregulatory polypeptide selected from SEQ ID NO: 12, 13, 14, and / or 28.

[0035] In certain embodiments, the polynucleotide encoding the polypeptide is introduced in isolated plant cells or plant protoplasts (i.e., are not located in undissociated or intact plant tissues, plant parts, or whole plants). In certain embodiments, the polynucleotide encoding the polypeptide is introduced in plant cells obtained from or located in any plant part or tissue or callus. In certain embodiments, the polynucleotide encoding the polypeptide is introduced in plant cells obtained from or located in a plant tissue, a cultured plant tissue explant, wholeINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 plant, intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, seedling, whole seed, halved seed or other seed fragment, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, embryogenic callus, callus, or plant cell suspension. In certain embodiments, the polynucleotide encoding the polypeptide is introduced in a plant cell is derived from the L1 or L2 layer of an immature or mature embryo of a monocot plant (e.g., maize, wheat, sorghum, or rice).

[0036] In certain embodiments, the polynucleotide encoding the polypeptide is introduced in plant cells that are located in undissociated or intact plant tissues, plant parts, plant explants, or whole plants. In certain embodiments, the plant cell can be located in an intact nodal bud, a cultured plant tissue explant, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, seedling, whole seed, halved seed or other seed fragment, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, or callus. In certain embodiments, the explants used include immature embryos. Immature embryos (e.g., immature maize embryos) include 1.8-2.2 mm embryos, 1-7 mm embryos, and 3-7 mm embryos. In certain embodiments, the aforementioned embryos are obtained from mature ear-derived seed, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, tassels, immature ears, and silks. In various aspects, the plant-derived explant includes immature embryos, 1.8-2.2 mm embryos, 1-7 mm embryos, and 3.5-7 mm embryos. In an aspect, the embryos used in the disclosed methods can be derived from mature ear-derived seed, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, tassel, immature ear, or silks. In certain embodiments, the plant cell is a pluripotent plant cell (e.g., a stem cell or meristem cell). In certain embodiments, the plant cell is located within the L1 or L2 layer of an immature or mature embryo of a monocot plant (e.g., maize, wheat, sorghum, or rice).

[0037] In certain embodiments, the plant cells where the polynucleotide encoding the polypeptide is introduced, as well as the related methods, systems, or compositions provided herein can include plant cells obtained from or located in any monocot plant species of interest, for example, row crop plants and turf grasses. In certain non-limiting embodiments, the plant cells are obtained from or located in barley (Hordeum vulgare), maize (Zea mays L.), millets (Setaria spp, Echinochloa spp, Eleusine spp, Panicum spp., Pennisetum spp.), oats (Avena sativa), oil palm (Ellis quineensis), rice (Oryza sativa L.), rye (Secale cereale), sorghum (Sorghum bicolor), sugarcanes (Saccharum spp.) or wheat (Tritium aestivum). In certainINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 embodiments, the plant cell is a monocot plant cell. In certain embodiments, the plant cell is a maize plant cell, wheat plant cell, or sorghum plant cell.

[0038] In certain embodiments, the plant cells can comprise haploid, diploid, or polyploid plant cells or plant protoplasts, for example, those obtained from a haploid, diploid, or polyploid plant, plant part or tissue, or callus. In certain embodiments, plant cells in culture (or the regenerated plant, progeny seed, and progeny plant) are haploid or can be induced to become haploid; techniques for making and using haploid plants and plant cells are known in the art, see, e.g., methods for generating haploids in Arabidopsis thaliana by crossing of a wild-type strain to a haploid-inducing strain that expresses altered forms of the centromere-specific histone CENH3, as described by Maruthachalam and Chan in “How to make haploid Arabidopsis thaliana”, protocol available at www[dot]openwetware[dot]org / images / d / d3 / Haploid_Arabidopsis_protocol[dot]pdf; (Ravi et al. (2014) Nature Communications, 5:5334, doi: 10.1038 / ncomms6334). Haploids can also be obtained in a wide variety of monocot plants (e.g., maize, wheat, rice, sorghum, barley) by crossing a plant comprising a mutated CENH3 gene with a wildtype diploid plant to generate haploid progeny as disclosed in US Patent No. 9,215,849, which is incorporated herein by reference in its entirety. Haploid-inducing maize lines that can be used to obtain haploid maize plants and / or cells include Stock 6, MHI (Moldovian Haploid Inducer), indeterminate gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, and well as transgenic haploid inducer lines disclosed in US Patent No.9,677,082, which is incorporated herein by reference in its entirety. 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, megagametophyte, and microspores. In certain embodiments where the plant cell or plant protoplast is haploid, the genetic complement can be doubled by chromosome doubling (e.g., by spontaneous chromosomal doubling by meiotic non-reduction, or by using a chromosome doubling agent such as colchicine, oryzalin, trifluralin, pronamide, nitrous oxide gas, anti-microtubule herbicides, anti-microtubule agents, and mitotic inhibitors) in the plant cell or plant protoplast to produce a doubled haploid plant cell or plant protoplast wherein the complement of genes or alleles is homozygous; yet other embodiments include regeneration of a doubled haploid plant from the doubled haploid plant cell or plant protoplast. Another embodiment is related to a hybrid plant having at least one parent plant that is a doubled haploid plant provided by this approach. Production of doubled haploid plants provides homozygosity in one generation, instead of requiring several generations of self-crossing to obtain homozygous plants. The useINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 of doubled haploids is advantageous in any situation where there is a desire to establish genetic purity (i.e. homozygosity) in the least possible time. Doubled haploid production can be particularly advantageous 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.

[0039] In certain embodiments, the plant cells where the polynucleotide encoding the polypeptide are introduced can be plant cells that are (a) encapsulated or enclosed in or attached to a polymer (e.g., pectin, agarose, or other polysaccharide) or other support (solid or semi- solid surfaces or matrices, or particles or nanoparticles); (b) encapsulated or enclosed in or attached to a vesicle or liposome or other fluid compartment; or (c) not encapsulated or enclosed or attached. In certain embodiments, the plant cells can be in liquid or suspension culture, or cultured in or on semi-solid or solid media, or in a combination of liquid and solid or semi-solid media (e.g., plant cells or protoplasts cultured on solid medium with a liquid medium overlay, or plant cells or protoplasts attached to solid beads or a matrix and grown with a liquid medium). In certain embodiments, the plant cells encapsulated in a polymer (e.g., pectin, agarose, or other polysaccharide) or other encapsulating material, enclosed in a vesicle or liposome, suspended in a mixed-phase medium (such as an emulsion or reverse emulsion), or embedded in or attached to a matrix or other solid support (e.g., beads or microbeads, membranes, or solid surfaces).

[0040] In a related embodiment, the disclosure provides arrangements of plant cells having improved plant cell regenerative potential in the systems, methods, and compositions described herein, such as arrangements of plant cells convenient for screening purposes or for high- throughput and / or multiplex transformation or gene editing experiments. In an embodiment, the disclosure provides an arrangement of multiple plant cells comprising a polynucleotide encoding at least one of a hb77, GRFTF4, GRFTF5, DOF11, DOF22, WRKY53, bZIP75, MYB31, HSCF1 , bHLH160, and / or bHLH48 polypeptide; optionally at least one additional polynucleotide encoding a BBM, WUS2, and / or other morphoregulatory polypeptide; and optionally a genome editing system. In another embodiment, the disclosure provides an array including a plurality of containers, each including at least one plant cell or plant protoplast having improved plant cell regenerative potential. In an embodiment, the disclosure provides arrangements of plant cells having the polynucleotide encoding the polypeptide and optionally the genome editing system, wherein the plant cells are in an arrayed format, for example, in multi-well plates, encapsulated or enclosed in vesicles, liposomes, or droplets (useful, (e.g., in a microfluidics device), or attached discretely to a matrix or to discrete particles or beads; a specific embodiment is such an arrangement of multiple plant cells having improved plant cellINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 regenerative potential provided in an arrayed format, further including a genome editing system (e.g., an RNA-guided nuclease, at least one guide RNA, and optionally a donor DNA template), which may be different for at least some locations on the array or even for each location on the array, and optionally at least one chemical, enzymatic, or physical delivery agent.

[0041] In the systems and methods provided herein, the polynucleotides encoding the polypeptides and a genome editing system can be introduced in the plant cell in any temporal order. In certain embodiments, the genome editing system and the polynucleotides encoding the polypeptides are introduced simultaneously. In other embodiments, the genome editing system is introduced after the polynucleotides encoding the polypeptides. In other embodiments, the genome editing system is introduced before the polynucleotides encoding the polypeptides. In summary, the genome editing system can be provided to a plant cell either previous to, concurrently with, or subsequent to introducing the polynucleotides encoding the polypeptides to the plant cell.

[0042] Plant cells having improved plant cell regenerative potential conferred by expression of a polypeptide are provided herein. Also provided by the disclosure are compositions derived from or grown from the plant cell or plant protoplast having improved plant cell regenerative potential, provided by the systems and methods disclosed herein; such compositions include multiple protoplasts or cells, callus, a somatic embryo, a somatic meristem, embryogenic callus, or a regenerated plant grown from the plant cell or plant protoplast having improved plant cell regenerative potential. Improved plant cell regenerative potential in plant cells can be assessed by a variety of techniques. In certain embodiments, such techniques can compare the numbers and / or amount of regenerable plant structures (e.g., immature embryos, somatic embryos, embryogenic calli, somatic meristems, organogenic calli, shoots, or shoots further comprising roots) formed and / or recovered from a given number of plant cells comprising the polynucleotide encoding the polypeptide versus control plant cells without the polynucleotide. In certain embodiments, expression of the polypeptide results in an increased somatic embryo induction frequency or embryo productivity relative to a control plant cell lacking the polynucleotide encoding the polypeptide. In certain embodiments, the somatic embryo induction frequency is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In certain embodiments, somatic embryo induction frequency is increased at least 2-fold, at least 5-fold, at least 10-fold, or at least 20- fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide. In certain embodiments, the embryo productivity is at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6,INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, or at least about 2.5. In certain embodiments, the embryo productivity is from about 1 to about 3 or from about 1.5 to about 2.5. When both somatic embryo induction frequency and embryo productivity are high, the transformation rate is high and the culture is robust.

[0043] In certain embodiments, an attribute of tissues selected for introduction of the polynucleotide can be the presence of dividing cells and the ability to grow in tissue culture media. These tissues include, but are not limited to, dividing cells from young maize leaf, meristems and scutellar tissue from about 8 or 10 to about 12 or 14 days after pollination (DAP) embryos. The isolation of maize embryos has been described in several publications (Brettschneider, Becker, and Lörz 1997; Leduc et al. 1996; Frame et al. 2011; K. Wang and Frame 2009). In certain embodiments, basal leaf tissues (e.g., leaf tissues located about 0 to 3 cm from the ligule of a maize plant; Kirienko, Luo, and Sylvester 2012) are selected for introduction of the polynucleotides. In certain embodiments, such increases in numbers and / or amounts of regenerable plant structures can be observed in about 1, 2, or 3 to about 7, 10, 14, 30, or 60 days following the introduction of a polynucleotide encoding the polypeptide. Methods for obtaining regenerable plant structures and regenerating plants from the plant cells provided herein can be adapted from methods disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, single plant cells subjected to the introduction of the polynucleotide will give rise to single regenerable plant structures. In certain embodiments, the single regenerable plant cell structure can form from a single cell on, or within, an explant that has been subjected to the introduction of the polynucleotides and optionally subjected to treatment with a genome editing system. In certain embodiments, initiation or formation of the single plant cell regenerable structure can occur where single- cell-derived cell or tissue proliferation (e.g., growth of callus, non-differentiated callus, embryogenic callus and organogenic callus) occurring before initiation of the regenerable plant structure is reduced or absent. In certain embodiments, regenerable plant structures from plant cells subjected to the introduction of the polynucleotide and optionally a genome editing system can be form the regenerable plant structure in the absence of exogenous cytokinin or with levels of cytokinin that are lower than those required to initiate formation of the regenerable structure from a control cell. In certain embodiments, regenerable plant structures from plant cells subjected to the introduction of the polynucleotide and optionally a genome editing system can be identified and / or selected via a positive growth selection based on the ability of those plantINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 cells to initiate and / or form the regenerable plant structures more rapidly than adjacent plant cells that have not been subjected to the introduction of the polynucleotide. In certain embodiments, such positive growth selection can obviate or reduce the need to use a traditional negative selection system where an antibiotic or herbicide is used to inhibit growth of adjacent, non-transformed cells that do not contain a gene that confers resistance to the antibiotic or herbicide. Nonetheless, embodiments where a selectable marker gene conferring resistance to an antibiotic, herbicide, or other agent can be introduced into the plant cell at least temporarily during initiation and / or formation of the regenerable plant cell structures to facilitate identification and recovery.

[0044] In some embodiments, methods provided herein can include the additional step of growing or regenerating a plant from a plant cell comprising the polynucleotide encoding the polypeptide or from a regenerable plant structure obtained from that plant cell. In certain embodiments, the plant can further comprise an inserted transgene, a target gene edit, or genome edit as provided by the methods and compositions disclosed herein. In certain embodiments, callus is produced from the plant cell, and plantlets and plants produced from such callus. In other embodiments, whole seedlings or plants are grown directly from the plant cell without a callus stage. Thus, additional related aspects are directed to whole seedlings and plants grown or regenerated from the plant cell or plant protoplast having a target gene edit or genome edit, as well as the seeds of such plants. In certain embodiments wherein the plant cell or plant protoplast is subjected to genetic or epigenetic modification (for example, stable or transient expression of a transgene, gene silencing, epigenetic silencing, or genome editing by means of, e.g., an RNA-guided nuclease), the grown or regenerated plant exhibits a phenotype associated with the genetic or epigenetic modification. In certain embodiments, the grown or regenerated plant includes in its genome two or more genetic or epigenetic modifications that in combination provide at least one phenotype of interest. In certain embodiments, a heterogeneous population of plant cells having a target gene edit or genome edit, at least some of which include at least one genetic or epigenetic modification, is provided by the method; related aspects include a plant having a phenotype of interest associated with the genetic or epigenetic modification, provided by either regeneration of a plant having the phenotype of interest from a plant cell or plant protoplast selected from the heterogeneous population of plant cells having a target gene or genome edit, or by selection of a plant having the phenotype of interest from a heterogeneous population of plants grown or regenerated from the population of plant cells having a target gene edit or genome edit. Examples of phenotypes of interest include herbicide resistance, improved tolerance of abiotic stress (e.g., tolerance of temperatureINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 extremes, drought, or salt) or biotic stress (e.g., resistance to nematode, 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). In an embodiment, a heterogeneous population of plant cells having a target gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) is exposed to conditions permitting expression of the phenotype of interest; e.g., selection for herbicide resistance can include exposing the population of plant cells having a target gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) 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. Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can be adapted from published procedures (Roest and Gilissen, Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran and Smith, Crop Sci. 30(6):1328-1337; Ikeuchi et al., Development, 2016, 143: 1442-1451). Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can also be adapted from US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. Also provided are heterogeneous populations, arrays, or libraries of such plants, succeeding generations or seeds of such plants grown or regenerated from the plant cells or plant protoplasts, having a target gene edit or genome edit, parts of the plants (including plant parts used in grafting as scions or rootstocks), or products (e.g., fruits or other edible plant parts, cleaned grains or seeds, edible oils, flours or starches, proteins, and other processed products) made from the plants or their seeds. Embodiments include plants grown or regenerated from the plant cells having a target gene edit or genome edit, wherein the plants contain cells or tissues that do not have a genetic or epigenetic modification, e.g., grafted plants in which the scion or rootstock contains a genetic or epigenetic modification, or chimeric plants in which some but not all cells or tissues contain a genetic or epigenetic modification. Grafted plants can be grafts between the same or different (generally related) species. Additional related aspects include a hybrid plant provided by crossing a first plant grown or regenerated from a plant cell or plant protoplast having a target gene edit or genome edit and having at least one genetic or epigenetic modification, with a second plant, wherein the hybrid plant contains the genetic or epigenetic modification; also contemplated is seed produced by the hybrid plant. Also envisioned as related aspects are progeny seed and progeny plants, including hybrid seedINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 and hybrid plants, having the regenerated plant as a parent or ancestor. The plant cells and derivative plants and seeds disclosed herein can be used for various purposes useful to the consumer or grower. The intact plant itself may be desirable, e.g., plants grown as cover crops or as ornamentals. In other embodiments, processed products are made from the plant or its seeds, such as extracted proteins, oils, sugars, and starches, fermentation products, animal feed or human food, wood and wood products, pharmaceuticals, and various industrial products.

[0045] The polypeptides can be provided to a cell (e.g., a plant cell or plant protoplast) by any suitable technique. In certain embodiments, the polypeptides are provided by directly contacting a plant cell with the polypeptide or the polynucleotide that encodes the polypeptide. In certain embodiments, the polypeptides are provided by transporting the polypeptides or a polynucleotide that encodes the polypeptide into a plant cell or plant protoplast using a chemical, enzymatic, or physical agent. In certain embodiments, the polypeptides are provided by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of a plant cell or plant protoplast with polynucleotides encoding the polypeptides; see, e.g., Broothaerts et al. (2005) Nature, 433:629 - 633. In an embodiment, the polypeptides are provided by transcription in a plant cell or plant protoplast of a DNA that encodes the polypeptides and is stably integrated in the genome of the plant cell or is provided to the plant cell or plant protoplast in the form of a plasmid or expression vector (e.g., a viral vector) that encodes the polypeptides. In certain embodiments, the polypeptides are provided to the plant cell or plant protoplast as polynucleotides that encode the polypeptides, e.g., in the form of an RNA (e.g., mRNA or RNA containing an internal ribosome entry site (IRES)) encoding the polypeptides. A genome editing system can also be introduced into the plant cells by similar techniques.

[0046] Transient expression of the polypeptides can be achieved by a variety of techniques. Certain embodiments are useful in effectuating transient expression of the polypeptides without remnants or selective genetic markers occurring in progeny. In certain embodiments, the polypeptides are provided directly to the 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, the polypeptides are targeted to the plant cell or cell nucleus in a manner that ensures transient expression (e.g., by methods adapted from Gao et al. 2016; or Li et al. 2009). In certain embodiments, the polypeptides are delivered into the plant cell by delivery of the polypeptides themselves in theINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 absence of any polynucleotides that encode the polypeptides. In certain embodiments, the polypeptides can be produced in a heterologous system, purified and delivered to plant cells by particle bombardment (e.g., by methods adapted from Martin-Ortigosa and Wang 2014). In embodiments where the polypeptides are delivered in the absence of any encoding polynucleotides, the delivered polypeptide is expected to degrade over time in the absence of ongoing expression from any introduced encoding polynucleotides to result in transient expression. In certain embodiments, the polypeptides are delivered into the plant cell by delivery of polynucleotides that encode the polypeptides. In certain embodiments, the polypeptides can be encoded on a bacterial plasmid and delivered to plant tissue by particle bombardment (e.g., by methods adapted from Hamada et al. 2018; or Kirienko, Luo, and Sylvester 2012). In certain embodiments, the polypeptides can be encoded on a T-DNA and transiently transferred to plant cells using Agrobacterium (e.g., by methods adapted from Leonelli et al. 2016; or Wu et al. 2014). In certain embodiments, the polypeptides can be encoded in a viral genome and delivered to plants (e.g., by methods adapted from Honig et al. 2015). In certain embodiments, the polypeptides can be encoded in mRNA or an RNA comprising an IRES and delivered to target plant cells. In certain embodiments, the polypeptides are delivered into the plant cell by delivery of polynucleotides that encode the polypeptides. In certain embodiments, the polynucleotides that encode the polypeptides are not integrated into a plant cell genome (e.g., as a polynucleotide lacking sequences that provide for integration, by agroinfiltration on an integration deficient T-DNA vector or system, or in a viral vector), is not operably linked to polynucleotides which provide for autonomous replication, and / or only provided with factors (e.g., viral replication proteins) that provide for autonomous replication. Suitable techniques for transient expression including biolistic and other delivery of polynucleotides, agroinfiltration, and use of viral vectors disclosed by Canto, 2016 and others can be adapted for transient expression of the polypeptides provided herein. In certain embodiments, the polynucleotides that encode the polypeptides are integrated into a plant cell genome (e.g., a nuclear or plastid genome) and transient expression of the polypeptides are effectuated by excision of the polynucleotides and / or regulated expression of the polypeptides. Excision of a polynucleotide encoding the polypeptide can be provided by use of site-specific recombination systems (e.g., Cre-Lox, FLP-FRT). Excision of a polynucleotide encoding one or more of the polypeptide(s) (e.g., morphogenic polypeptides and / or nucleases) can also be provided by use of autoexcision methods, including those described by International Publication No. WO 2023 / 201186, which is herein incorporated by reference in its entirety and specifically with respect to such disclosure. Regulated expression of the polypeptide can beINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 effectuated by methods including: (i) operable linkage of the polynucleotide encoding the polypeptide to a developmentally-regulated, de-repressable, and / or inducible promoter; and / or (ii) introduction of a polynucleotide (e.g., dsRNA or amiRNA) that can induce siRNA- mediated inhibition of the polypeptide. Suitable site-specific recombination systems as well as developmentally-regulated, de-repressable, and / or inducible promoters include those disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. Polynucleotides that can be used to effectuate transient expression of the polypeptides include: (a) double-stranded RNA; (b) single-stranded RNA; (c) chemically modified RNA; (d) double-stranded DNA; (e) single-stranded DNA; (f) chemically modified DNA; or (g) a combination of (a) - (f). Certain embodiments of the polynucleotides further include additional nucleotide sequences that provide useful functionality; non-limiting examples of such additional nucleotide sequences include an aptamer or riboswitch sequence, nucleotide sequence that provides secondary structure such as stem-loops or that provides a sequence-specific site for an enzyme (e.g., a sequence-specific recombinase or endonuclease site), T-DNA (e.g., DNA sequence encoding a polypeptide is enclosed between left and right T-DNA borders from Agrobacterium spp. or from other bacteria that infect or induce tumors in plants), a DNA nuclear-targeting sequence, a regulatory sequence such as a promoter sequence, and a transcript-stabilizing or -destabilizing sequence. Certain embodiments of the polynucleotides include those wherein the polynucleotide is complexed with, or covalently or non-covalently bound to, a non-nucleic acid element, e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a cell- penetrating or pore-forming peptide, a polymer, a detectable label, a quantum dot, or a particulate or nanoparticulate.

[0047] Transient expression of the polypeptides can be for a period of time and / or in an amount sufficient to result in improved regenerative potential in comparison to a control plant cell. In certain embodiments, the transient increase in the expression of the polypeptides is for a period of about 1, 2, 4, 8, 12, 16, 20, 24, 30, or 36 hours to about 72, 96, 120, 144, 168, 192, 276, or 336 hours. In certain embodiments, the transient increase in the expression of polypeptides is for a period of about 2, 4, 8, 12, or 16 hours to about 18, 20, 24, 30 or 36 hours. In certain embodiments, the transient increase in the expression of polypeptides is for a period of about 18, 20, 24, 30 or 36 hours to about 60, 80, 100, 120, 168, or 192 hours. Such transient increases in expression of polypeptides can be measured by methods whereby accumulated gene products including mRNAs and / or proteins are measured. Useful methods of measuring mRNAs include quantitative reverse transcriptase Polymerase Chain Reaction (qRT-PCR)-INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 based and / or any hybridization-based assay. Useful methods for quantitating proteins include immunoassays (e.g., ELISAs, RIAs) and / or mass spectrometry-based methods.

[0048] Genome editing systems of use in the methods provided herein include molecules capable of introducing a double-strand break (“DSB”) or single-strand break (“SSB”) at a specific site or sequence in a double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA or donor or other DNA template polynucleotides. Examples of such gene editing molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type nuclease system, a type II Cas nuclease, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Cas12a nuclease, a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b (C2c1), a Cas12c (C2c3), a Cas12i, a Cas12j, a Cas12L, a Cas14, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (including introduction of a DSB or SSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for use with an RNA-guided nuclease, or a DNA encoding a gRNA for use with an RNA-guided nuclease; (d) donor DNA template polynucleotides suitable for insertion at a break in genomic DNA by homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ); and (e) other DNA templates (e.g., dsDNA, ssDNA, or combinations thereof) suitable for insertion at a break in genomic DNA (e.g., by non-homologous end joining (NHEJ).

[0049] CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016 / 0138008A1 and US2015 / 0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 A1. Plant RNA promoters for expressing CRISPR guide RNA and plant codon- optimized CRISPR Cas9 endonuclease are disclosed in International Patent Application PCT / US2015 / 018104 (published as WO 2015 / 131101 and claiming priority to US Provisional Patent Application 61 / 945,700). Methods of using CRISPR technology for genome editing in plants are disclosed in US Patent Application Publications US 2015 / 0082478A1 and US 2015 / 0059010A1 and in International Patent Application PCT / US2015 / 038767 A1 (published as WO 2016 / 007347 and claiming priority to US Provisional Patent Application 62 / 023,246).INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 In certain embodiments, an RNA-guided endonuclease that leaves a blunt end following cleavage of the target site is used. Blunt-end cutting RNA-guided endonucleases include Cas9, Cas12c, Cas12i, and Cas 12h (Yan et al., 2019). In certain embodiments, an RNA-guided endonuclease that leaves a staggered single stranded DNA overhanging end following cleavage of the target site following cleavage of the target site is used. Staggered-end cutting RNA- guided endonucleases include Cas12a, Cas12b, and Cas12e. All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.

[0050] 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 (e.g., 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, plants or plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpf1-type 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) to form an RNA-guided endonuclease / guide RNA 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-rich 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 Cas12a proteins. In some instances, Cas12a can also recognize a 5’-CTA PAM motif. Other examples of potential Cas12a PAM sequences include TTN, 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). Cpf1INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 A1, which is incorporated herein by reference for its disclosure of DNA encoding Cpf1 endonucleases and guide RNAs and PAM sites.

[0051] For the purposes of gene editing, CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281 – 2308. At least 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpf1 at least 16 nucleotides of gRNA sequence are needed to achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence were reported necessary for efficient DNA cleavage in vitro; see Zetsche et al. (2015) Cell, 163:759 - 771. In practice, guide RNA sequences are generally designed to have a length of 17 - 24 nucleotides (frequently 19, 20, or 21 nucleotides) and exact complementarity (i.e., perfect base-pairing) to the targeted gene or nucleic acid sequence; guide RNAs having less than 100% complementarity to the target sequence can be used (e.g., a gRNA with a length of 20 nucleotides and 1 - 4 mismatches to the target sequence) but can increase the potential for off- target effects. The design of effective guide RNAs for use in plant genome editing is disclosed in US Patent Application Publication 2015 / 0082478 A1, the entire specification of which is incorporated herein by reference. Efficient gene editing has been achieved using a chimeric “single guide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing); see, for example, Cong et al. (2013) Science, 339:819 - 823; Xing et al. (2014) BMC Plant Biol., 14:327 - 340. Chemically modified sgRNAs have been demonstrated to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985 - 991. The design of effective gRNAs for use in plant genome editing is disclosed in US Patent Application Publication 2015 / 0082478 A1, the entire specification of which is incorporated herein by reference.

[0052] Other nucleases capable of effecting site-specific modification of a target nucleotide sequence in the systems, methods, and compositions provided herein include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL-effector nucleases or TALENs), Argonaute proteins, and a meganuclease or engineered meganuclease. Zinc finger nucleases (ZFNs) are engineered proteins comprising a zinc finger DNA-binding domain fused to a nucleic acid cleavage domain, e.g., a nuclease. The zinc finger binding domains provide specificity and can be engineered to specifically recognize any desired target DNA sequence.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 For a review of the construction and use of ZFNs in plants and other organisms, see, e.g., Urnov et al. (2010) Nature Rev. Genet., 11:636 - 646. 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 in 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 amino 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. See, e.g., US Patents 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) are well known and described in the literature. In addition, enhancement of binding specificity for zinc finger binding domains has been described in US Patent 6,794,136, incorporated herein by reference in its entirety. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. Examples of linker sequences are publicly known, e.g., see US Patents 6,479,626; 6,903,185; and 7,153,949, incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl as part of a ZFN requires two adjacent and independent 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. Fokl variants with enhanced activities have been described; see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96 - 107.

[0053] Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate theINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 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 a non- 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. For a description of the use of TALENs in plants, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623 - 2628 and Mahfouz (2011) GM Crops, 2:99 - 103.

[0054] Argonautes are proteins that can function as sequence-specific endonucleases by binding a polynucleotide (e.g., a single-stranded DNA or single-stranded RNA) that includes sequence complementary to a target nucleotide sequence) that guides the Argonaut to the target nucleotide sequence and effects site-specific alteration of the target nucleotide sequence; see, e.g., US Patent Application Publication 2015 / 0089681, incorporated herein by reference in its entirety.

[0055] In related embodiments, zinc finger nucleases, TALENs, and Argonautes are used in conjunction with other functional domains. For example, the nuclease activity of these nucleic acid targeting systems can be altered so that the enzyme binds to but does not cleave the DNA. Examples of functional domains include transposase domains, integrase domains, recombinase domains, resolvase domains, invertase domains, protease domains, DNA methyltransferase domains, DNA hydroxylmethylase domains, DNA demethylase domains, histone acetylase domains, histone deacetylase domains, nuclease domains, repressor domains, activator domains, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domains, cellular uptake activity associated domains, nucleic acid binding domains, antibody presentation domains, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferases, histone demethylases, histone kinases, histone phosphatases, histone ribosylases, histone deribosylases, histone ubiquitinases, histone deubiquitinases, histone biotinases and histone tail proteases. Non-limiting examples of functional domains include a transcriptional activation domain, a transcription repression domain, and an SHH1, SUVH2, or SUVH9 polypeptideINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 capable of reducing expression of a target nucleotide sequence via epigenetic modification; see, e.g., US Patent Application Publication 2016 / 0017348, incorporated herein by reference in its entirety. Genomic DNA may also be modified via base editing using a fusion between a catalytically inactive Cas9 (dCas9) fused to a cytidine deaminase which converts cytosine (C) to uridine (U), thereby effecting a C to T substitution; see Komor et al. (2016) Nature, 533:420 - 424. In other embodiments, adenine base editors (ABEs) can be used to convert A / T base pairs to G / C base pairs in genomic DNA (Gaudelli et al., 2017).

[0056] Other genome altering reagents used in plant cells and methods provided herein include transgenes or vectors comprising the same. Such transgenes can confer useful traits that include herbicide tolerance, pest tolerance (e.g., tolerance to insects, nematodes, or plant pathogenic fungi and bacteria), improved yield, increased and / or qualitatively improved oil, starch, and protein content, improved abiotic stress tolerance (e.g., improved or enhanced water use efficiency or drought tolerance, osmotic stress tolerance, high salinity stress tolerance, heat stress tolerance, enhanced cold tolerance, including cold germination tolerance), and the like. Such transgenes include both transgenes that confer the trait by expression of an exogenous protein as well as transgenes that confer the trait by inhibiting expression of endogenous plant genes (e.g., by inducing an siRNA response which inhibits expression of the endogenous plant genes). Transgenes that can provide such traits are disclosed in US Patent Application Publication Nos. 20170121722 and 20170275636, which are each incorporated herein by reference in their entireties and specifically with respect to such disclosures.

[0057] In some embodiments, one or more polynucleotides or vectors driving expression of one or more polynucleotides encoding any of the polypeptides and / or genome editing systems are introduced into a plant cell. In certain embodiments, a polynucleotide vector comprises a regulatory element such as a promoter operably linked to one or more polynucleotides encoding the polypeptide or genome editing system. In such embodiments, expression of these polynucleotides can be controlled by selection of the appropriate promoter, particularly promoters functional in a plant cell; useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter). Developmentally regulated promoters that can be used include Phospholipid Transfer Protein (PLTP), fructose- 1,6-bisphosphatase protein, NAD(P)-binding Rossmann-Fold protein, adipocyte plasma membrane-associated protein-like protein, Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratory reduction 6 protein, D-glycerate 3-kinase, chloroplastic-like protein, chlorophyll a-b binding protein 7, chloroplastic-like protein, ultraviolet-B-repressible protein,INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 Soul heme-binding family protein, Photosystem I reaction center subunit psi-N protein, and short-chain dehydrogenase / reductase protein that are disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, the promoter is operably linked to nucleotide sequences encoding multiple guide RNAs, wherein the sequences encoding guide RNAs are separated by a cleavage site such as a nucleotide sequence encoding a microRNA recognition / cleavage site or a self-cleaving ribozyme (see, e.g., Ferré-D'Amaré and Scott (2014) Cold Spring Harbor Perspectives Biol., 2:a003574). In certain embodiments, the promoter is an RNA polymerase III promoter operably linked to a nucleotide sequence encoding one or more guide RNAs. In certain embodiments, the promoter operably linked to one or more polynucleotides is a constitutive promoter that drives gene expression in plant cells. In certain embodiments, the promoter drives gene expression in the nucleus or in an organelle such as a chloroplast or mitochondrion. Examples of constitutive promoters include a CaMV 35S promoter as disclosed in US Patents 5,858,742 and 5,322,938, a rice actin promoter as disclosed in US Patent 5,641,876, a maize chloroplast aldolase promoter as disclosed in US Patent 7,151,204, and the nopaline synthase (NOS) and octopine synthase (OCS) promoters from Agrobacterium tumefaciens. In certain embodiments, the promoter operably linked to one or more polynucleotides encoding elements of a genome-editing system is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PPDK) promoter, which is active in photosynthetic tissues. Other contemplated promoters include cell-specific or tissue-specific or developmentally regulated promoters, for example, a promoter that limits the expression of the nucleic acid targeting system to germline or reproductive cells (e.g., promoters of genes encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in germline or reproductive cells). In certain embodiments, the genome alteration is limited only to those cells from which DNA is inherited in subsequent generations, which is advantageous where it is desirable that expression of the genome-editing system be limited in order to avoid genotoxicity or other unwanted effects. All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.

[0058] Expression vectors or polynucleotides provided herein may contain a DNA segment near the 3′ end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA, and may also support promoter activity. Such a 3’ element is commonly referred to as a “3′-untranslated region” or “3′-UTR” or a “polyadenylation signal.” In some cases, plant gene-based 3’ elements (or terminators) consistINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 of both the 3’-UTR and downstream non-transcribed sequence (Nuccio et al., 2015). Useful 3′ elements include: Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, and tr73′ elements disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference, and 3′ elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose-1,6- biphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza sativa), disclosed in US Patent Application Publication 2002 / 0192813 A1, incorporated herein by reference.

[0059] In certain embodiments, a vector or polynucleotide comprising an expression cassette includes additional components, e.g., a polynucleotide encoding a drug resistance or herbicide gene or a polynucleotide encoding a detectable marker such as green fluorescent protein (GFP) or beta-glucuronidase (gus) to allow convenient screening or selection of cells expressing the vector or polynucleotide. Selectable markers include genes that confer resistance to herbicidal compounds, such as glyphosate, sulfonylureas, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Since the expression of WUS / WOX genes can accelerate somatic embryogenesis and embryo maturation, selectable marker genes, selective agents, and conditions can be adjusted to minimize formation of un-edited or untransformed regenerable plant structures (e.g., “escapes”). Such selectable marker genes and selective agents include the maize HRA gene (Lee et al., 1988, EMBO J 7:1241-1248) which confers resistance to sulfonylureas and imidazolinones, the CP4 gene that confers resistance to glyphosate (US Reissue Patent RE039247, specifically incorporated herein by reference in its entirety and with respect to such genes and related selection methods), the GAT gene which confers resistance to glyphosate (Castle et al., 2004, Science 304:1151-1154), genes that confer resistance to spectinomycin such as the aadA gene (Svab et al., 1990, Plant Mol Biol.14:197- 205) and the bar gene that confers resistance to glufosinate ammonium (White et al., 1990, Nucl. Acids Res.25:1062), and PAT (or moPAT for corn, see Rasco-Gaunt et al., 2003, Plant Cell Rep.21:569-76; also see Sivamani et al., 2019) and the PMI gene that permits growth on mannose-containing medium (Negrotto et al., 2000, Plant Cell Rep.22:684-690). Embodiments

[0060] The following numbered embodiments also form part of the present disclosure:

[0061] 1. A method of producing a regenerable plant structure, the method comprising: introducing one or more polynucleotides encoding at least one morphoregulatory polypeptide of SEQ ID NO: 1 - 11, and / or an allelic variant thereof in a monocot plant cell; and culturing the monocot plant cell to produce the regenerable plant structure.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0062] 2. The method of embodiment 1, wherein the allelic variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 10, or 11.

[0063] 3. The method of any one of embodiments 1-2, wherein the one or more polynucleotides encodes at least SEQ ID NO: 1 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; SEQ ID NO: 9 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof.

[0064] 4. The method of any one of embodiments 1-3, wherein the one or more polynucleotides encodes at least SEQ ID NO: 9 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8, and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof.

[0065] 5. The method of claim any one of embodiments 1-4, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; and / or SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

[0066] 6. The method of any one of embodiments 1-5, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; and / or SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

[0067] 7. The method of any one of embodiments 1-6, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: SEQ ID NO: 6 or an allelic variant thereof; SEQ ID NO: 7 or an allelic variant thereof; SEQ ID NO: 9 or an allelic variant thereof; or SEQ ID NO: 11 and / or an allelic variant thereof.

[0068] 8. The method of any one of embodiments 1-7, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: SEQ ID NO: 3 or an allelic variant thereof; and / or SEQ ID NO: 8 or an allelic variant thereof.

[0069] 9. The method of any one of embodiments 1-8, wherein the one or more polynucleotides encodes any two peptides selected from SEQ ID NO: 1 – 11, and / or an allelic variant thereof.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0070] 10. The method of claim any one of embodiments 1-9, wherein the one or more polynucleotides encodes any two peptides selected from SEQ ID NO: 1 – 5, and / or an allelic variant thereof.

[0071] 11. The method of any one of embodiments 1-10, wherein the one or more polynucleotides encodes any two peptides selected from SEQ ID NO: 6 - 11, and / or an allelic variant thereof.

[0072] 12. The method of any one of embodiments 1-11, wherein the one or more polynucleotides encode at least one additional morphoregulatory polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12, 13, 14, or 28 in the monocot plant cell.

[0073] 13. The method of any one of embodiments 1-12, wherein the monocot plant cell is a maize, wheat, or sorghum plant cell.

[0074] 14. The method of any one of embodiments 1-13, wherein the monocot plant cell is in an explant comprising an immature embryo or embryogenic callus.

[0075] 15. The method of any one of embodiments 1-14, wherein the polynucleotide encoding the polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 15-24, or 25.

[0076] 16. The method of any one of embodiments 12-15, wherein the polynucleotide encoding the additional polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 26 or27.

[0077] 17. The method of any one of embodiments 1-16, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

[0078] 18. The method of any one of embodiments 1-17, wherein the polynucleotide is stably incorporated into the genome of the monocot plant cell.

[0079] 19. The method of any one of embodiments 1-18, wherein the polynucleotide is transiently expressed in the monocot plant cell.

[0080] 20. The method of any one of embodiments 1-19, wherein the polynucleotide is flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are loxP sites recognized by a cre recombinase or FRT sites recognized by a FLP recombinase.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0081] 21. The method of embodiment 20, further comprising providing the site-specific recombinase to the cultured plant cells after introduction of the polynucleotide and / or after introduction of the genome editing system.

[0082] 22. The method of any one of embodiments 1-21, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 6, 8, 9, and / or an allelic variant thereof.

[0083] 23. The method of any one of embodiments 1-22, wherein the monocot plant cell comprises a regeneration-recalcitrant germplasm.

[0084] 24. The method of any one of embodiments 1-23, wherein the regenerable plant structure comprises a somatic embryo, embryogenic callus, somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots.

[0085] 25. The method of any one of embodiments 1-24, wherein the introducing comprises bacterial-mediated transformation or biolistic-mediated transformation.

[0086] 26. The method of any one of embodiments 1-25, wherein expression of the polypeptide results in an increased somatic embryo induction frequency or increased embryo productivity relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0087] 27. The method of any one of embodiments 1-26, wherein the percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker is increased by at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0088] 28. The method of any one of embodiments 1-27, wherein the percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker is increased at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10- fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0089] 29. The method of embodiment 27 or 28, wherein the plant transformation marker comprises a non-destructive marker comprising a fluorescent protein, an anthocyanin biosynthetic protein, or CYP76AD1, DODA, and Glucosyltransferase proteins.

[0090] 30. The method of any one of embodiments 1-29, wherein the somatic embryo induction frequency is increased at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, optionally wherein the somatic embryo induction frequency is increased at least 2-fold, at least 5-fold, at least 10-fold, or atINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0091] 31. The method of any one of embodiments 1-30, wherein the embryo productivity is increased to an assigned embryo productivity score of at least about 1, at least about 1.5, at least about 2, or at least about 2.5 somatic embryos per embryogenic immature embryo, optionally wherein the embryo productivity is increased to an assigned embryo productivity score of about 1 to about 3 or from about 1.5 to about 2.5 somatic embryos per embryogenic immature embryo.

[0092] 32. The method of any one of embodiments 1-31, further comprising introducing a genome editing system in the monocot plant cell.

[0093] 33. The method of embodiment 32, wherein the genome editing system comprises a CRISPR-based system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system, and optionally a donor template polynucleotide.

[0094] 34. The method of embodiment 33, wherein the CRISPR-based system comprises (i) an RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease; and (ii) a guide RNA or a polynucleotide encoding the gRNA.

[0095] 35. A monocot plant cell comprising a heterologous promoter which is operably linked to a polynucleotide encoding a polypeptide of SEQ ID NO: 1 -11, and / or an allelic variant thereof, wherein expression of the polypeptide increases proliferation, somatic embryogenesis, and / or regeneration capacity of the monocot plant cell.

[0096] 36. The monocot plant cell of embodiment 35, wherein the allelic variant has least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 10, or 11.

[0097] 37. The monocot plant cell of any one of embodiments 35-36, wherein the polynucleotide encodes at least SEQ ID NO: 1 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; SEQ ID NO: 9 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof.

[0098] 38. The monocot plant cell of any one of embodiments 35-37, wherein the polynucleotide encodes at least SEQ ID NO: 9 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0099] 39. The monocot plant cell of any one of embodiments 35-38, further comprising at least one additional polynucleotide encoding an additional polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12,13, 14, or 28 in the monocot plant cell.

[0100] 40. The monocot plant cell of any one of embodiments 35-39, wherein the polynucleotide encodes a polypeptide of SEQ ID NO: 6, 7, 9, 11 and / or an allelic variant thereof, and wherein the plant cell further comprises an additional heterologous promoter which is operably linked to an additional polynucleotide encoding an additional polypeptide of SEQ ID NO: 13 or an allelic variant thereof.

[0101] 41. The monocot plant cell of any one of embodiments 35-40, wherein the polynucleotide encodes a polypeptide of SEQ ID NO: 3, 8, and / or an allelic variant thereof, and wherein the plant cell further comprises an additional heterologous promoter which is operably linked to an additional polynucleotide encoding an additional polypeptide of SEQ ID NO: 12 or an allelic variant thereof.

[0102] 42. The monocot plant cell of any one of embodiments 35-41, wherein the monocot plant cell is a maize, wheat, or sorghum plant cell.

[0103] 43. The monocot plant cell of any one of embodiments 35-42, wherein the polynucleotide encoding the polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 15-24, or 25.

[0104] 44. The monocot plant cell of any one of embodiments 39-43, wherein the additional polynucleotide encoding the additional polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 26 or 27.

[0105] 45. The monocot plant cell of any one of embodiments 35-44, wherein the polynucleotide is stably incorporated into the genome of the monocot plant cell.

[0106] 46. The monocot plant cell of any one of embodiments 35-45, wherein the polynucleotide is transiently expressed in the monocot plant cell.

[0107] 47. The monocot plant cell of any one of embodiments 35-46, wherein the polynucleotide is flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are loxP sites recognized by a cre recombinase or FRT sites recognized by a FLP recombinase.

[0108] 48. The monocot plant cell of any one of embodiments 35-47, wherein the polynucleotide comprises an mRNA.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0109] 49. The monocot plant cell of any one of embodiments 35-48, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 6, 8, 9, or an allelic variant thereof.

[0110] 50. The monocot plant cell of any one of embodiments 35-49, wherein the monocot plant cell comprises a regeneration-recalcitrant germplasm.

[0111] 51. The monocot plant cell of any one of embodiments 35-50, wherein expression of the polypeptide results in at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% increase in percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0112] 52. The monocot plant cell of any one of embodiments 35-51, wherein the percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker is increased at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0113] 53. The monocot plant cell of any one of embodiments 51 or 52, wherein the plant transformation marker comprises a non-destructive marker comprising a fluorescent protein, an anthocyanin biosynthetic protein, or CYP76AD1, DODA, and Glucosyltransferase proteins.

[0114] 54. The monocot plant cell of any one of embodiments 35-53, wherein expression of the polypeptide results in an increased somatic embryo induction frequency or embryo productivity relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0115] 55. The monocot plant cell of any one of embodiments 35-54, wherein the somatic embryo induction frequency is increased at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, optionally wherein the somatic embryo induction frequency is increased at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

[0116] 56. The monocot plant cell of any one of embodiments 35-55, wherein the embryo productivity is increased to an assigned embryo productivity score of at least about 1, at least about 1.5, at least about 2, or at least about 2.5 somatic embryos per embryogenic immature embryo, optionally wherein the embryo productivity is increased to an assigned embryoINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 productivity score of about 1 to about 3 or from about 1.5 to about 2.5 somatic embryos per embryogenic immature embryo.

[0117] 57. The monocot plant cell of any one of embodiments 35-57, further comprising a genome editing system.

[0118] 58. The monocot plant cell of embodiment 57, wherein the genome editing system comprises a CRISPR-based system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system, and optionally a donor template polynucleotide.

[0119] 59. The monocot plant cell of embodiment 58, wherein the CRISPR-based system comprises (i) a RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease; and (ii) a guide RNA or a polynucleotide encoding the gRNA.

[0120] 60. A monocot plant, tissue, organ, callus, or cell culture comprising the monocot plant cell of any one of embodiments 35-59.

[0121] 61. A method for producing a monocot plant, the method comprising: regenerating a monocot plant from the monocot plant cell of any one of embodiments 35-60.

[0122] 62. The method of embodiment 61, further comprising selecting a progeny of the monocot plant that lacks the polynucleotide.

[0123] 63. The method of embodiment 61 or 62, wherein the monocot plant comprises an inserted transgene, a target gene edit, or a genome edit.

[0124] 64. The monocot plant produced by the method of any one of embodiments 61-63.

[0125] 65. A recombinant polynucleotide comprising: one or more polynucleotides encoding at least one morphoregulatory polypeptide of SEQ ID NO: 1 - 11, and / or an allelic variant thereof, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

[0126] 66. The recombinant polynucleotide of embodiment 65, wherein the allelic variant has least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 10, or 11.

[0127] 67. The recombinant polynucleotide of any one of embodiments 65-66, wherein the one or more polynucleotides encodes at least SEQ ID NO: 1 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; SEQ ID NO: 9 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof.

[0128] 68. The recombinant polynucleotide of any one of embodiments 65-67, wherein the one or more polynucleotides encodes at least SEQ ID NO: 9 or an allelic variant thereof and:INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or SEQ ID NO: 10, 11, and / or an allelic variant thereof.

[0129] 69. The recombinant polynucleotide of any one of embodiments 65-68, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

[0130] 70. The recombinant polynucleotide of any one of embodiments 65-69, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: SEQ ID NO: 2, 3, and / or an allelic variant thereof; SEQ ID NO: 4, 5, and / or an allelic variant thereof; SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

[0131] 71. The recombinant polynucleotide of any one of embodiments 65-70, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: SEQ ID NO: 6 or an allelic variant thereof; SEQ ID NO: 7 or an allelic variant thereof; SEQ ID NO: 9 or an allelic variant thereof; or SEQ ID NO: 11 or an allelic variant thereof.

[0132] 72. The recombinant polynucleotide of any one of embodiments 65-71, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: SEQ ID NO: 3 or an allelic variant thereof; or SEQ ID NO: 8 or an allelic variant thereof.

[0133] 73. The recombinant polynucleotide of any one of embodiments 65-72, wherein the one or more polynucleotides encode at least one additional morphoregulatory polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12,13, 14, or 28 in the monocot plant cell.

[0134] 74. The recombinant polynucleotide of any one of embodiments 65-73, wherein the polynucleotide encoding the polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 15-24, or 25.

[0135] 75. The recombinant polynucleotide of any one of embodiments 73-74, wherein the polynucleotide encoding the additional polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 26 or 27.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00

[0136] 76. The recombinant polynucleotide of any one of embodiments 65-75, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 6, 8, 9, and / or an allelic variant thereof.

[0137] 77. The recombinant polynucleotide of any one of embodiments 65-76, wherein the polynucleotide is flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are loxP sites recognized by a cre recombinase or FRT sites recognized by a FLP recombinase.

[0138] 78. A vector comprising the recombinant polynucleotide of any one of embodiments 65-77. EXAMPLES Example 1. Single cell-RNA sequencing of induced somatic embryogenesis in maize

[0139] Somatic embryogenesis is a process that allows a single somatic adult cell to regenerate by asexually forming an embryo and is used for the generation of transgenic and gene edited plants. In monocots, it is known that the co-expression of two transcription factors, OVULE DEVELOPMENT PROTEIN2 / BABY BOOM (ODP2 / BBM, SEQ ID NO: 12) and WUSCHEL2 (WUS2, SEQ ID NO: 13) can trigger the direct formation of somatic embryos without the intermediary callus phase (Lowe et al., 2018, doi: 10.1007 / s11627-018-9905-2). Here, we transformed immature embryos of the model maize inbred line B104 with a binary vector pG3R-WOY-SI containing pAXIG::WUS2, pLTP::BBM, pBdEF1a::YFP-NLS pZmUBI::GUS and pSbAHAS::ZmHRA as part of the T-DNA (FIG. 1). This binary vector was introduced in the Agrobacterium tumefaciens strain EHA105 recA- containing the ternary vector pVS1-VIR2 to increase transformation efficiency. Seven days post transformation, transgenic cells started somatic embryo development (FIG. 2). In order to obtain the high- resolution single cell RNA sequencing (scRNA-seq) dataset, about 200 immature zygotic embryos expressing most YFP-positive somatic embryos were selected and used for enzymatic removal of the cell walls. In total 100,000 transgenic protoplasts were sorted using Fluorescence-Assisted Cell Sorting. By limiting the time of cell wall removal, the outer cell layers were enriched and sort time was limited. Including an additional dye, 4',6-diamidino-2- phenylindole (DAPI) excluded the sorting of dead cells. The resulting population of cells was used for single-cell partitioning using 10x Genomics technology, followed by short-read mRNA-sequencing (FIG.3).

[0140] After mapping reads to the Zm-B73-REFERENCE-NAM-5.0 genome, a total of 18,979 individual cells were identified. Initial filtering on the number of genes per cell, number ofINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 Unique Molecular Identifiers (UMI) per cell, and maximal mitochondrial and chloroplast gene percentage per cell resulted in 12,016 higher-quality cells. To further enhance data quality, a cluster of cells identified as background noise (free-floating mRNA from lysed or dead cells) by the 10x Genomics processing pipeline was removed. This step resulted in 10,834 higher- quality cells. Additionally, only cells expressing at least one transgene (ODP2 / BBM, WUS2, GUS, YFP or HRA) were selected. The stringent selection process resulted in a dataset consisting of 31,635 genes from 6830 high-quality transformed cells. Finally, cell cycle regression was performed to reduce clustering based on cell cycle-related genes. Upon data processing cells were divided into 17 unique clusters and dimensional reduction was performed using Uniform Manifold Approximation and Projection (UMAP) for visualization (FIG.4). Table 1. Overview of identified morphoregulatory genesExample 2. Identification of somatic embryogenesis related genes in literature.

[0141] To collect known SE-related genes, a text mining (TM) approach on scientific literature was performed (Vandepoele and Lukicheva, unpublished). This involved processing abstracts and full-text articles to extract gene-trait-species "triples," associating a trait with a gene in a species. Each triple is characterized by the number of times (evidences) it was found in the literature. Due to limited molecular information for maize, triples from Arabidopsis via orthology transfer were included. Relevant traits such as embryo, regeneration, meristem, and development were focused on. 73 relevant triples were manually verified to evaluate theirINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 quality, identifying 38 correct genes and 10 as a threshold on minimum number of evidences per triple. This threshold was applied to generate a final set of maize triples and those inferred from Arabidopsis through orthology. The resulting triples were used to discover embryogenesis-related gene regulatory networks (GRNs) using the MINI-EX tool (Example 3). The 38 manually curated genes were further used to assess the TF prioritization performance and prioritize candidate regulators in the identified GRNs. Example 3. Prioritization of candidate regulators using cell-type-specific gene regulatory networks.

[0142] To establish cell-type-specific gene regulatory networks (GRNs), the MINI-EX tool (Ferrari et al., 2022, doi: 10.1016 / j.molp.2022.10.016) was utilized. MINI-EX initially constructs a co-expression network on the full dataset and subsequently infers cluster-specific networks by retaining only transcription factors (TFs) and their target genes that are differently expressed within a given cluster. The MINI-EX output consists of a ranked list of regulons, where a regulon is defined as a TF and the target genes it regulates at the cluster level. Regulons are ranked both globally and at the cluster level based on their network properties (e.g., network centrality, betweenness, etc.) and their relevance to user-defined terms of interest. Clusters involved in the SE process were identified by analyzing the expression of SE-related genes retrieved from literature and TF families known to be involved in that process within the differentially expressed genes of each cluster. Clusters 0, 5, 8, 11, 12 and, 15 were focused on as relevant. Next, TFs ranked by MINI-EX in the top 50 of these relevant clusters were selected. To select cluster-specific TFs, their expression profiles were analyzed in the scRNA-seq dataset through feature plots. This approach narrowed the candidate list to 60 TFs. This candidate list included Zm00001eb006480 (SEQ ID NO: 28), and Zm00001eb389400 (SEQ ID NO.9), an AP2 family gene. Zm00001eb099390 (SEQ ID NO: 14), a basic helix-loop-helix (bHLH) TF, was also found in the candidate list. Finally, BABY BOOM itself was obtained (Zm00001eb144510 (SEQ ID NO: 12); BBM is also known as ODP2 or ereb53). Example 4. A rapid screen for somatic embryogenesis using the RUBY reporter.

[0143] Evaluation proceeded with candidate genes from the MINI-EX analyses. Coding sequences were retrieved from B73, BsaI and AarI restriction sites removed and local codon optimization was performed when needed for gene synthesis. Finally, flanking overhangs that are compatible with the Golden Gate cloning method were introduced. All candidates were cloned under control of the ZmUBI1 promoter and terminator in the destination vector pG3P- RUBY-GUS-AG, containing the non-invasive betalain marker pPvUBI::RUBY (He et al.,INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 2020, doi: 10.1038 / s41438-020-00390-1) and the reporter pOsACT::GUS (Figure 5). The resulting expression vectors were transformed into Agrobacterium tumefaciens strain EHA105 recA- pVS1-VIR2.

[0144] Maize B104 immature embryo transformation was performed essentially as described (Aesaert et al., 2021). Briefly, wild-type B104 maize cobs were harvested 11-12 days after pollination and refrigerated overnight before the Agrobacterium-mediated transformation by co-cultivation. Three days post-transformation (DAT), embryos were transferred to resting media supplemented with cefotaxime and vancomycin effective against Agrobacterium. At nine DAT, embryos were transferred to a selection medium containing phosphinothricin (PTT). At DAT 16, embryos were moved to maturation medium to further promote development into plants. The potential formation of induced somatic embryos, increased formation of callus and / or transgenic shoots and roots was monitored based on the betalain pigment appearing in transgenic tissue. As a comparison, the fluorescent protein tdTomato driven by the ZmUBI1 promoter was used as a negative control, while pNOS::WUS2 and pZmUBI1::BBM together were used as a positive control.

[0145] This screen identified 11 morphoregulators (Table 1). Morphoregulators 17, 20, 25, 33, 44, 45, 53, 60, 62, and 63 showed increased number of developing structures expressing RUBY (FIG. 6, FIG. 7, & Table 2) and / or an increased number of explants with RUBY-expressing induced somatic embryos (FIG. 6, FIG. 7, & Table 3) compared to the negative control (tdTomato). Morphoregulators 47 and 63 showed an increased number of explants with one or more shoots expressing RUBY (FIG.8, Table 4). These candidates have not previously been directly proven to work as morphoregulators. Table 2.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00Table 3.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00Table 4.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 Example 5. Transformation Efficiency Trials

[0146] To assess transformation efficiency of the candidate morphoregulators alone and in combination, explants of corn genotype GIDA8989 (recalcitrant line) were transformed using Agrobacterium-mediated transformation.

[0147] The candidate morphoregulators were first individually overexpressed to evaluate transformation efficiency when used alone. These results are shown in Table 5. The candidates were overexpressed using the ZmUBI1 promoter as indicated. Explants not treated with a morphoregulator were used as a negative control. Table 5.

[0148] Candidate morphoregulator combinations were then evaluated in a dual overexpression format with the second overexpressed gene being SiBBM2, MlWUS2.2, or a second candidate morphoregulator. These results are shown in Table 6. The candidates were overexpressed usingINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 either the ZmUBI1 promoter or the nopaline synthase (NOS) promoter, as indicated. Explants not treated with any morphoregulators were used as a negative control. Table 6.

Claims

INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 CLAIMS 1. A method of producing a regenerable plant structure, the method comprising: (i) introducing one or more polynucleotides encoding at least one morphoregulatory polypeptide of SEQ ID NO: 1 - 11, and / or an allelic variant thereof in a monocot plant cell; and (ii) culturing the monocot plant cell to produce the regenerable plant structure.

2. The method of claim 1, wherein the allelic variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 10, or 11.

3. The method of claim 1, wherein the one or more polynucleotides encodes at least SEQ ID NO: 1 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; (iv) SEQ ID NO: 9 and / or an allelic variant thereof; or (v) SEQ ID NO: 10, 11, and / or an allelic variant thereof.

4. The method of claim 1, wherein the one or more polynucleotides encodes at least SEQ ID NO: 9 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 10, 11, and / or an allelic variant thereof.

5. The method of claim 1, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 6. The method of claim 1, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

7. The method of claim 1, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: (i) SEQ ID NO: 6 or an allelic variant thereof; (ii) SEQ ID NO: 7 or an allelic variant thereof; (iii) SEQ ID NO: 9 or an allelic variant thereof; or (iv) SEQ ID NO: 11 or an allelic variant thereof.

8. The method of claim 1, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: (i) SEQ ID NO: 3 or an allelic variant thereof; or (ii) SEQ ID NO: 8 or an allelic variant thereof.

9. The method of claim 1, wherein the one or more polynucleotides encodes any two peptides selected from SEQ ID NO: 1 - 11, and / or an allelic variant thereof.

10. The method of claim 1, wherein the one or more polynucleotides encodes any two peptides selected from SEQ ID NO: 1 - 5, and / or an allelic variant thereof.

11. The method of claim 1, wherein the one or more polynucleotides encodes any two peptides selected from SEQ ID NO: 6 - 11, and / or an allelic variant thereof.

12. The method of claim 1, wherein the one or more polynucleotides encode at least one additional morphoregulatory polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12, 13, 14, or 28 in the monocot plant cell.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 13. The method of claim 1, wherein the monocot plant cell is a maize, wheat, or sorghum plant cell.

14. The method of claim 1, wherein the monocot plant cell is in an explant comprising an immature embryo or embryogenic callus.

15. The method of claim 1, wherein the polynucleotide encoding the polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 15-24, or 25.

16. The method of claim 12, wherein the polynucleotide encoding the additional polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 26 or 27.

17. The method of claim 1, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

18. The method of claim 1, wherein the polynucleotide is stably incorporated into the genome of the monocot plant cell.

19. The method of claim 1, wherein the polynucleotide is transiently expressed in the monocot plant cell.

20. The method of claim 1, wherein the polynucleotide is flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are loxP sites recognized by a cre recombinase or FRT sites recognized by a FLP recombinase.

21. The method of claim 20, further comprising providing the site-specific recombinase to the cultured plant cells after introduction of the polynucleotide and / or after introduction of the genome editing system.

22. The method of claim 1, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 6, 8, 9, and / or an allelic variant thereof.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 23. The method of claim 1, wherein the monocot plant cell comprises a regeneration- recalcitrant germplasm.

24. The method of claim 1, wherein the regenerable plant structure comprises a somatic embryo, embryogenic callus, somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots.

25. The method of claim 1, wherein the introducing comprises bacterial-mediated transformation or biolistic-mediated transformation.

26. The method of claim 1, wherein expression of the polypeptide results in an increased somatic embryo induction frequency or increased embryo productivity relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

27. The method of claim 1, wherein the percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker is increased by at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

28. The method of claim 1, wherein the percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker is increased at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

29. The method of claim 27 or 28, wherein the plant transformation marker comprises a non-destructive marker comprising a fluorescent protein, an anthocyanin biosynthetic protein, or CYP76AD1, DODA, and Glucosyltransferase proteins.

30. The method of claim 1, wherein the somatic embryo induction frequency is increased at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, optionally wherein the somatic embryo induction frequency is increased at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 31. The method of claim 1, wherein the embryo productivity is increased to an assigned embryo productivity score of at least about 1, at least about 1.5, at least about 2, or at least about 2.5 somatic embryos per embryogenic immature embryo, optionally wherein the embryo productivity is increased to an assigned embryo productivity score of about 1 to about 3 or from about 1.5 to about 2.5 somatic embryos per embryogenic immature embryo.

32. The method of any one of claims 1-28, further comprising introducing a genome editing system in the monocot plant cell.

33. The method of claim 32, wherein the genome editing system comprises a CRISPR- based system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system, and optionally a donor template polynucleotide.

34. The method of claim 33, wherein the CRISPR-based system comprises (i) an RNA- guided nuclease or a polynucleotide encoding the RNA-guided nuclease; and (ii) a guide RNA or a polynucleotide encoding the gRNA.

35. A monocot plant cell comprising a heterologous promoter which is operably linked to a polynucleotide encoding a polypeptide of SEQ ID NO: 1 -11, and / or an allelic variant thereof, wherein expression of the polypeptide increases proliferation, somatic embryogenesis, and / or regeneration capacity of the monocot plant cell.

36. The monocot plant cell of claim 35, wherein the allelic variant has least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 10, or 11.

37. The monocot plant cell of claim 35, wherein the polynucleotide encodes at least SEQ ID NO: 1 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; (iv) SEQ ID NO: 9 and / or an allelic variant thereof; or (v) SEQ ID NO: 10, 11, and / or an allelic variant thereof.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 38. The monocot plant cell of claim 35, wherein the polynucleotide encodes at least SEQ ID NO: 9 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 10, 11, and / or an allelic variant thereof.

39. The monocot plant cell of claim 35, further comprising at least one additional polynucleotide encoding an additional polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12, 13, 14, or 28 in the monocot plant cell.

40. The monocot plant cell of claim 35, wherein the polynucleotide encodes a polypeptide of SEQ ID NO: 6, 7, 9, 11 and / or an allelic variant thereof, and wherein the plant cell further comprises an additional heterologous promoter which is operably linked to an additional polynucleotide encoding an additional polypeptide of SEQ ID NO: 13 or an allelic variant thereof.

41. The monocot plant cell of claim 35, wherein the polynucleotide encodes a polypeptide of SEQ ID NO: 3, 8, and / or an allelic variant thereof, and wherein the plant cell further comprises an additional heterologous promoter which is operably linked to an additional polynucleotide encoding an additional polypeptide of SEQ ID NO: 12 or an allelic variant thereof.

42. The monocot plant cell of claim 35, wherein the monocot plant cell is a maize, wheat, or sorghum plant cell.

43. The monocot plant cell of claim 35, wherein the polynucleotide encoding the polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 15-24, or 25.

44. The monocot plant cell of claim 39, wherein the additional polynucleotide encoding the additional polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at leastINTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 26 or 27.

45. The monocot plant cell of claim 35, wherein the polynucleotide is stably incorporated into the genome of the monocot plant cell.

46. The monocot plant cell of claim 35, wherein the polynucleotide is transiently expressed in the monocot plant cell.

47. The monocot plant cell of claim 35, wherein the polynucleotide is flanked by site- specific recombinase recognition sites, optionally wherein the site-specific recombination sites are loxP sites recognized by a cre recombinase or FRT sites recognized by a FLP recombinase.

48. The monocot plant cell of claim 35, wherein the polynucleotide comprises an mRNA.

49. The monocot plant cell of claim 35, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 6, 8, 9, or an allelic variant thereof.

50. The monocot plant cell of claim 35, wherein the monocot plant cell comprises a regeneration-recalcitrant germplasm.

51. The monocot plant cell of claim 35, wherein expression of the polypeptide results in at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% increase in percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

52. The monocot plant cell of claim 35, wherein the percentage of embryogenic explants, induced somatic embryos, and / or shoots expressing a plant transformation marker is increased at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 53. The monocot plant cell of claim 51 or 52, wherein the plant transformation marker comprises a non-destructive marker comprising a fluorescent protein, an anthocyanin biosynthetic protein, or CYP76AD1, DODA, and Glucosyltransferase proteins.

54. The monocot plant cell of claim 35, wherein expression of the polypeptide results in an increased somatic embryo induction frequency or embryo productivity relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

55. The monocot plant cell of claim 35, wherein the somatic embryo induction frequency is increased at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, optionally wherein the somatic embryo induction frequency is increased at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold relative to a control monocot plant cell lacking the polynucleotide encoding the polypeptide.

56. The monocot plant cell of claim 35, wherein the embryo productivity is increased to an assigned embryo productivity score of at least about 1, at least about 1.5, at least about 2, or at least about 2.5 somatic embryos per embryogenic immature embryo, optionally wherein the embryo productivity is increased to an assigned embryo productivity score of about 1 to about 3 or from about 1.5 to about 2.5 somatic embryos per embryogenic immature embryo.

57. The monocot plant cell of claim 35, further comprising a genome editing system.

58. The monocot plant cell of claim 57, wherein the genome editing system comprises a CRISPR-based system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease (ZFN) system, and optionally a donor template polynucleotide.

59. The monocot plant cell of claim 58, wherein the CRISPR-based system comprises (i) a RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease; and (ii) a guide RNA or a polynucleotide encoding the gRNA.

60. A monocot plant, tissue, organ, callus, or cell culture comprising the monocot plant cell of any one of claims 35-52 or 54-59.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 61. A method for producing a monocot plant, the method comprising: regenerating a monocot plant from the monocot plant cell of any one of claims 35-52 or 54-59.

62. The method of claim 61, further comprising selecting a progeny of the monocot plant that lacks the polynucleotide.

63. The method of claim 61, wherein the monocot plant comprises an inserted transgene, a target gene edit, or a genome edit.

64. The monocot plant produced by the method of claim 61.

65. A recombinant polynucleotide comprising: one or more polynucleotides encoding at least one morphoregulatory polypeptide of SEQ ID NO: 1 - 11, and / or an allelic variant thereof, wherein the polynucleotide is operably linked to a heterologous promoter functional in a plant cell.

66. The recombinant polynucleotide of claim 65, wherein the allelic variant has least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 1 - 10, or 11.

67. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encodes at least SEQ ID NO: 1 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; (iv) SEQ ID NO: 9 and / or an allelic variant thereof; or (v) SEQ ID NO: 10, 11, and / or an allelic variant thereof.

68. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encodes at least SEQ ID NO: 9 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof;INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 10, 11, and / or an allelic variant thereof.

69. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

70. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: (i) SEQ ID NO: 2, 3, and / or an allelic variant thereof; (ii) SEQ ID NO: 4, 5, and / or an allelic variant thereof; (iii) SEQ ID NO: 6, 7, 8 and / or an allelic variant thereof; or (iv) SEQ ID NO: 9, 10, 11, and / or an allelic variant thereof.

71. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encodes SEQ ID NO: 13 or an allelic variant thereof and: (i) SEQ ID NO: 6 or an allelic variant thereof; (ii) SEQ ID NO: 7 or an allelic variant thereof; (iii) SEQ ID NO: 9 or an allelic variant thereof; or (iv) SEQ ID NO: 11 or an allelic variant thereof.

72. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encodes SEQ ID NO: 12 or an allelic variant thereof and: (i) SEQ ID NO: 3 or an allelic variant thereof; or (ii) SEQ ID NO: 8 or an allelic variant thereof.

73. The recombinant polynucleotide of claim 65, wherein the one or more polynucleotides encode at least one additional morphoregulatory polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 12,13, 14, or 28in the monocot plant cell.INTERNATIONAL PATENT APPLICATION Docket No. P14795WO00 74. The recombinant polynucleotide of claim 65, wherein the polynucleotide encoding the polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 15-24, or 25.

75. The recombinant polynucleotide of claim 73, wherein the polynucleotide encoding the additional polypeptide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 26 or 27.

76. The recombinant polynucleotide of claim 65, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 3, 6, 8, 9, and / or an allelic variant thereof.

77. The recombinant polynucleotide of claim 65, wherein the polynucleotide is flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are loxP sites recognized by a cre recombinase or FRT sites recognized by a FLP recombinase.

78. A vector comprising the recombinant polynucleotide of any one of claims 65-77.

Images