Morphogenic regulator combinations for improved plant transformation

Abstract

Combinations of 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

TITLE: MORPHOGENIC REGULATOR COMBINATIONS FOR IMPROVEDPLANT TRANSFORMATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to provisional patent application U.S. Serial No. 63 / 606,743 filed December 6, 2023. 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.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on December 3, 2024, is named “P14584WOOO.xml” and is 62,544 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 especially 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, comprising introducing a first polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5,9, 23, 26, 27, 28, or 29 in a cereal plant cell (e.g., a maize or wheat plant cell); and culturing the cereal plant cell to produce the regenerable plant structure are provided.

[0005] Methods of producing a regenerable plant structure, comprising introducing a first polynucleotide encoding a first 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: 5 or 29, in a cereal plant cell (e.g., a maize or wheat plant cell); and culturing the cereal plant cell to produce the regenerable plant structure are provided.

[0006] Also provided are cereal plant cells (e.g., a maize or wheat plant cell) comprising a polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29, wherein expression of the first and second polypeptides increases proliferation, somatic embryogenesis, and / or regeneration capacity of the cereal plant cell.

[0007] Also provided are recombinant polynucleotides comprising a first polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell and the second polynucleotide is operably linked to a second heterologous promoter functional in a plant cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows a phylogenetic analysis of AP2 transcription factors. The amino acid sequences of 52 morphogenes from 8 plant species of diverse taxon in higher plants are analyzed. The dashed line surrounds a single and distinct clade including PLT3b, PLT3, PLT5, and PLT7 from different species.

[0009] FIGS. 2A-2B show somatic embryogenesis by top morphoregulator combinations in maize recalcitrant genotype GIDA8989. Somatic embryo induction and mScarlet expression before the selection step are shown of immature embryos transformed with pIN5093 (2A,panels A-B), pIN6289 (2A, panels C-D), pIN6291 (2B, panels E-F), and pIN6296 (2B, panels G-H).

[0010] FIG. 3 shows plant regeneration by top morphoregulator combinations in maize recalcitrant genotype GIDA8989 in panels A and B. Plant regeneration from the various construct treatments including those shown in FIGS. 2A and 2B (pIN5093, pIN6298, pIN6291, and pIN6296). Controls include single morphogene (pIN6293), no morphogene (pIN6294), process constructs (pIN2855), and mockoff - no Agrobacterium infection and no selection (OFF). Top mophoregulator combinations show better plant regeneration than controls.

[0011] FIG. 4 shows somatic embryogenesis response of GIPI9660 by the end of Resting stage. Somatic embryos with kinetin 0.1-0.2 mg / L show higher yield and faster growth than embryos with no kinetin treatment.DETAILED DESCRIPTION

[0012] 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 necessarily defines the other, as well as necessarily 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.

[0013] 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 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 the polynucleotide or polypeptide.

[0014] 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).

[0015] As used herein, the phrase “cereal plant” refers to a plant cultivated for its edible grain, including, but not limited to, rice, wheat, rye, oats, barley, sorghum, millet, and maize.

[0016] 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.

[0017] The term “genome” refers to the entire complement of genetic material (genes and noncoding sequences) that is present in an organism or cell, including both nuclear and organellar DNA.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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 a 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).

[0022] 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 plantcell; 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.

[0023] 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.

[0024] As used herein, the phrase “somatic embryo induction frequency” refers to the percentage of immature embryos producing somatic embryos.

[0025] 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 noncoding 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.

[0026] 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.

[0027] 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 a first polynucleotide encoding a WUSCHEL or WUSCHEL-related homeobox (WUS / WOX) polypeptide and a second polynucleotide encoding a Plethora 5, Plethora 7, Plethora 3b (PLT5, PLT7, PLT3b), BABY BOOM 1 (BBM1), Leafy Cotyledon 1 (LEC1), RWP-RK DOMAINCONTAINING PROTEIN 4 (RKD4), GROWTH-REGULATING FACTOR 4-GRF-INTERACTING FACTOR 1 (GRF4-GIF1) polypeptide in the plant cell. In certain embodiments, a third polynucleotide encoding a BABY BOOM 2 (BBM2) polypeptide is also introduced in the plant cell. In certain embodiments, the WUS / WOX polypeptide comprises a Miscanthus lutarioriparius WUS2 (M1WUS2) polypeptide of SEQ ID NO: 6 or an allelic variant thereof. In certain embodiments, the WUS / WOX polypeptide comprises a Sorghum bicolor W0X5 (SbWOX5) polypeptide of SEQ ID NO: 7 or an allelic variant thereof. In certain embodiments, the WUS / WOX polypeptide comprises a Setaria italica W0X4 (SiW0X4) polypeptide of SEQ ID NO: 8 or an allelic variant thereof. In certain embodiments, the WUS / WOX polypeptide comprises a Dichanthelium oligosanthes W0X2 (DoWOX2) polypeptide of SEQ ID NO: 10 or an allelic variant thereof. In certain embodiments the PLT5 polypeptide comprises a Zea mays PLT5 (ZmPLT5) polypeptide of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments the PLT7 polypeptide comprises a Zea mays PLT7 (ZmPLT7) polypeptide of SEQ ID NO: 2 or an allelic variant thereof. In certain embodiments the PLT3b polypeptide comprises a Zea mays PLT3b (ZmPLT3b) polypeptide of SEQ ID NO: 26 or an allelic variant thereof. In certain embodiments the PLT3b polypeptide comprises a Sorghum bicolor PLT3b (SbPLT3b) polypeptide of SEQ ID NO: 27 or an allelic variant thereof. In certain embodiments the PLT3b polypeptide comprises an Oryza sativa PLT3b (OsPLT3b) polypeptide of SEQ ID NO: 28 or an allelic variant thereof. In certain embodiments the BBM1 polypeptide comprises a Zea mays BBM1 (ZmBBMl) polypeptide of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments the LEC1 polypeptide comprises a Zea mays LEC1 (ZmLECl) polypeptide of SEQ ID NO: 4 or an allelic variant thereof. In certain embodiments the RKD4 polypeptide comprises a Zea mays RKD4 (ZmRKD4) polypeptide of SEQ ID NO: 5 or an allelic variant thereof. In certain embodiments the RKD4 polypeptide comprises a Zea mays RKD4 (ZmRKD4) polypeptide of SEQ ID NO: 29 or an allelic variant thereof. In certain embodiments the GRF4-GIF polypeptide comprises a Zea mays GRF4-GIF (ZmGRF4-GIF) polypeptide of SEQ ID NO: 9 or an allelic variant thereof. In certain embodiments the GRF4-GIF polypeptide comprises a Triticum aestivum GRF4-GIF (TaGRF4- GIF) polypeptide of SEQ ID NO: 23 or an allelic variant thereof. In certain embodiments, the BBM2 polypeptide comprises a Setaria italica BBM2 (SiBBM2) polypeptide of SEQ ID NO: 21 or an allelic variant thereof.

[0028] Embodiments of the present disclosure are directed to combinations of morphoregulators. In certain embodiments, the first polynucleotide encodes a first (WUS / WOX) polypeptide comprising the amino acid of SEQ ID NO: 6, 7, 8, or 10 or allelicvariants thereof. In certain embodiments, the second polynucleotide encodes a second (PLT5, PLT7, PLT3b, BBM1, LEC1, RKD4, GRF4-GIF1) polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, 29, or allelic variants thereof. In certain embodiments, the first polynucleotide encodes the first polypeptide which comprises the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, 29 or an allelic variant thereof. In certain embodiments, the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5 or an allelic variant thereof. In certain embodiments, the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 23 or an allelic variant thereof. In certain embodiments, a third polynucleotide encoding a third 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: 21 is also introduced into a plant cell.

[0029] In certain embodiments, the first polynucleotide encoding the first 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: 11, 12, 13, 14, or 25. In certain embodiments, the second polynucleotide encoding the second 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, 16, 17, 18 ,19, 20, 24, 31, 32, or 33. In embodiments comprising a third polynucleotide encoding a third polypeptide, the third polynucleotide can have 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: 22.

[0030] In certain embodiments, improved plant cell regenerative potential is provided by introducing a polynucleotide encoding a RKD4 polypeptide in the plant cell. In certain embodiments the RKD4 polypeptide comprises a Zea mays RKD4 (ZmRKD4) polypeptide of SEQ ID NO: 5, 29, or an allelic variant thereof.

[0031] In certain embodiments, the first and second polynucleotides encoding the first and second polypeptides are 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 certainembodiments, the first and second polynucleotides encoding the first and second polypeptides are introduced in plant cells are obtained from or located in any plant part or tissue or callus. In certain embodiments, the first and second polynucleotides encoding the first and second polypeptides are introduced in plant cells obtained from or located in a plant tissue, a cultured plant tissue explant, whole 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 first and second polynucleotides encoding the first and second polypeptides are introduced in a plant cell is derived from the LI or L2 layer of an immature or mature embryo of a monocot plant (e.g., maize, wheat, sorghum, or rice).

[0032] In certain embodiments, the first and second polynucleotides encoding the first and second polypeptides are 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 LI or L2 layer of an immature or mature embryo of a monocot plant (e.g., maize, wheat, sorghum, or rice).

[0033] In certain embodiments, the plant cells where the first and second polynucleotides encoding the first and second polypeptides are introduced, as well as the related methods, systems, or compositions provided herein can include plant cells obtained from or located inany 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 cere ale), sorghum (Sorghum bicolor), sugarcanes (Saccharum spp.) or wheat (Tritium aestivum). In certain embodiments, the plant cell is a cereal plant cell. In certain embodiments, the plant cell is a maize plant cell or a wheat plant cell.

[0034] 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 protoplastto 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 use 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.

[0035] In certain embodiments, the plant cells where the first and second polynucleotides encoding the first and second polypeptides 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).

[0036] In certain embodiments, the media can comprise a cytokinin and / or L-cysteine. In certain embodiments, the cytokinin comprises kinetin. In some embodiments, the inclusion of a cytokinin and / or L-cysteine promotes somatic embryogenesis of the plant cell. In certain embodiments, the media comprising cytokinin and / or L-cysteine is an early somatic embryogenesis media, including, for example, a coculture media (e.g., for coculture of explants with Agrobacterium) or a resting media used after coculture. See, e.g., Sidek et al., 2022, AIMS AgrFood 7: 536-552 and Frame et al., 2002, Plant Physiol, 129: 13-22. In certain embodiments, the cytokinin (e.g, kinetin) is included in the media in a concentration of from about 0.01 mg / L to about 0.2 mg / L, from about 0.05 mg / L to about 0.15 mg / L, from about 0.075 mg / L to about0.125 mg / L, from about 0.1 mg / L to about 0.15 mg / L, from about 0.1 mg / L to about 0.2 mg / L, from about 0.125 mg / L to about 0.175 mg / L, or any range therein. In certain embodiments, the L-cysteine is included in the media (e.g., a coculture or resting media) in a concentration of from about 150 mg / L to about 450 mg / L, from about 200 mg / L to about 400 mg / L, from about 300 mg / L to about 400 mg / L, from about 200 mg / L to about 400 mg / L, from about 250 mg / L to about 350 mg / L, from about 275 mg / L to about 325 mg / L, or any range therein.

[0037] 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) a first polynucleotide encoding a first WUS / WOX polypeptide; (b) a second polynucleotide encoding a second PLT5, PLT7, PLT3b, BBM1, LEC1, RKD4, GRF4-GIF1 polypeptide; and optionally (c) 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 first and second polynucleotides encoding the first and second polypeptides 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 cell 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.

[0038] In the systems and methods provided herein, the first and second polynucleotides encoding the first and second 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 first and second polynucleotides encoding the first and second polypeptides are introduced simultaneously. In other embodiments, the genome editing system is introduced after the first and second polynucleotides encoding the first and second polypeptides. In other embodiments, the genome editing system is introduced before the first and second polynucleotides encodingthe first and second polypeptides. In summary, the genome editing system can be provided to a plant cell either previous to, concurrently with, or subsequent to introducing the first and second polynucleotides encoding the first and second polypeptides to the plant cell.

[0039] Plant cells having improved plant cell regenerative potential conferred by expression of a first and second 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 first and second polynucleotides encoding the first and second polypeptides versus control plant cells without the first and / or second polynucleotides. In certain embodiments, expression of the first and second polypeptides results in an increased somatic embryo induction frequency or embryo productivity relative to a control plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides. 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 cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides. 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, 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.

[0040] In certain embodiments, an attribute of tissues selected for introduction of the first and second polynucleotides can be the presence of dividing cells and the ability to grow in tissueculture 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 Lbrz 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 first and second polynucleotides encoding the first and second polypeptides. 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 polynucleotides 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 polynucleotides 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 polynucleotides and optionally a genome editing system can be identified and / or selected via a positive growth selection based on the ability of those plant 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 polynucleotides. 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, embodimentswhere 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.

[0041] In some embodiments, methods provided herein can include the additional step of growing or regenerating a plant from a plant cell comprising the first and second polynucleotides encoding the first and second polypeptides 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 temperature 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 seed 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 cropsor 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.

[0042] The first and second polypeptides can be provided to a cell (e.g, a plant cell or plant protoplast) by any suitable technique. In certain embodiments, the first and second polypeptides are provided by directly contacting a plant cell with the first and second polypeptides or the first and second polynucleotide that encode the first and second polypeptides. In certain embodiments, the first and second polypeptides are provided by transporting the first and second polypeptides or a first and second polynucleotide that encodes first and second polypeptides into a plant cell or plant protoplast using a chemical, enzymatic, or physical agent. In certain embodiments, the first and second 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 first and second polynucleotides encoding the first and second polypeptides; see, e.g., Broothaerts etal. (2005) Nature, 433:629 - 633. In an embodiment, the first and second polypeptides are provided by transcription in a plant cell or plant protoplast of a DNA that encodes the first and second 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 first and second polypeptides. In certain embodiments, the first and second polypeptides are provided to the plant cell or plant protoplast as first and second polynucleotides that encodes the first and second 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.

[0043] Transient expression of the first and second polypeptides can be achieved by a variety of techniques. Certain embodiments are useful in effectuating transient expression of the first and second polypeptides without remnants or selective genetic markers occurring in progeny. In certain embodiments, the first and second 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 semipurified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, the first and second polypeptides are targeted to the plant cell or cell nucleus in a manner that ensures transient expression (e.g., by methods adapted fromGao et al. 2016; or Li et al. 2009). In certain embodiments, the first and second polypeptides are delivered into the plant cell by delivery of the polypeptides themselves in the absence of any polynucleotides that encode the polypeptides. In certain embodiments, the first and second 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 first and second 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 first and second polypeptides are delivered into the plant cell by delivery of first and second polynucleotides that encodes the first and second 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 first and second polypeptides are delivered into the plant cell by delivery of first and second polynucleotides that encode the first and second polypeptides. In certain embodiments, the first and second polynucleotides that encode the first and second 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 first and second polynucleotides that encode the first and second 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 be 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) singlestranded 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 first or second 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.

[0044] Transient expression of the first and second 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 first and second 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 transientincrease in the expression of first and second 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 first and second 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 first and second 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)-based and / or any hybridization-based assay. Useful methods for quantitating proteins include immunoassays (e.g., ELISAs, RIAs) and / or mass spectrometry -based methods.

[0045] 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 Cas 12a nuclease, a nCasl2a nickase, a Casl2d (CasY), a Casl2e (CasX), a Casl2b (C2cl), a Casl2c (C2c3), a Casl2i, a Casl2j, a Casl2L, a Cas 14, 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).

[0046] 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. Cpfl endonuclease andcorresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 Al . 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 Al (published as WO 2016 / 007347 and claiming priority to US Provisional Patent Application 62 / 023,246). 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, Casl2c, Casl2i, 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 Cas 12a, Cas 12b, and Casl2e. All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.

[0047] 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 Cpfl-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 orsgRNAs 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 Casl2a proteins. In some instances, Casl2a can also recognize a 5’-CTA PAM motif. Other examples of potential Cast 2a 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). Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 Al, which is incorporated herein by reference for its disclosure of DNA encoding Cpfl endonucleases and guide RNAs and PAM sites.

[0048] 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 etal. {2013) Science, 339:819-823; Ran etal. (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 Cpfl 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 basepairing) 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 Al, 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 Al, the entire specification of which is incorporated herein by reference.

[0049] 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. For a review of the construction and use of ZFNs in plants and other organisms, see, e.g., Umov 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 multifmger 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 maybe 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.

[0050] Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a nonspecific 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. Set. USA, 108:2623 - 2628 and Mahfouz (2011) GM Crops, 2:99 - 103.

[0051] 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.

[0052] 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 polypeptide 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).

[0053] 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.

[0054] 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 (PL TP), 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-gly cerate 3 -kinase, chloroplastic-like protein, chlorophyll a-b binding protein 7, chloroplastic-like protein, ultraviolet-B-repressible protein, 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 can comprise a barley heat shock promoter (see, e.g., Harrington et al., Plant Methods (2020) 16: 137). In some embodiments, the barley heat shock promoter comprises a polynucleotide 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: 34.

[0055] 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, Ferre-D'Amare 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 Patent5,641,876, 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 cellspecific 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.

[0056] 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) consist 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 tr7 3' 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 saliva), disclosed in US Patent Application Publication 2002 / 0192813 Al, incorporated herein by reference.

[0057] Polynucleotides encoding morphogenic regulatory proteins used in expression vectors or polynucleotides provided herein include polynucleotides obtained from the source organism (e.g., genomic or cDNAs) and wholly synthetic polynucleotides. Wholly synthetic genes can be obtained by “back translation” or “reverse translation” (e.g., using a protein sequence and a codon usage table from a plant or cereal plant including maize or wheat to generate a DNA sequence) of the morphogenic polypeptides (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 23, 26, 27, 28, 29, or an allelic variant thereof). Reverse translation or back translation programsthat will accept a polypeptide sequence and a codon bias table as input to generate a polynucleotide sequence encoding the polypeptide include the Reverse Translate function on the world wide web internet site “bioinformatics.org / sms2 / rev_trans.html” (Stothard P (2000) Biotechniques 28: 1102-1104) and the “EMBOSS Backtranseq” function on the world wide web internet site “ebi.ac.uk / Tools / st / emboss_backtranseq / ” (Madeira et al. Nucleic Acids Research, 30 Jun 2019, 47(W1):W636-W641 DOI: 10.1093 / nar / gkz268). Methods for synthesizing genes suitable for expression in plants and in cereal plants including maize which are described in at least U.S. Pat. Nos. 5380831, 5500365, and 5689052, each incorporated herein by reference in their entireties, can be adapted for use in synthesizing genes encoding any of the morphogenic proteins described herein.

[0058] 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 Z>eta-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 com, 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

[0059] The following numbered embodiments also form part of the present disclosure:

[0060] 1. A method of producing a regenerable plant structure, the method comprising: introducing a first polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29 in a cereal plant cell; and culturing the cereal plant cell to produce the regenerable plant structure.

[0061] 2 A method of producing a regenerable plant structure, the method comprising: introducing a first polynucleotide encoding a first 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: 5 or 29, in a cereal plant cell; and culturing the cereal plant cell to produce the regenerable plant structure.

[0062] 3. The method of embodiment 1 or 2, wherein the cereal plant cell is a maize or wheat plant cell.

[0063] 4. The method of any one of embodiments 1-3, wherein the cereal plant cell is in an explant comprising an immature embryo or embryogenic callus.

[0064] 5 The method of any one of embodiments 1-4, wherein the first polynucleotide encoding the first 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: 11, 12, 13, 14, or 25.

[0065] 6. The method of any one of embodiments 1-5, wherein the second polynucleotide encoding the second 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, 16, 17, 18, 19, 20, 24, 30, 31, 32, or 33.

[0066] 7 The method of any one of embodiments 1-6, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell and the second polynucleotide is operably linked to a second heterologous promoter functional in a plant cell.

[0067] 8. The method of embodiment 7, wherein the first and / or second heterologous promoter comprises a barley heat shock promoter, optionally wherein the barley heat shock promoter is a promoter of the barley HvHSP17 gene or optionally wherein the barley heatshock promoter comprises a polynucleotide 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: 34.

[0068] 9. The method of any one of embodiments 1-8, wherein the first and second polynucleotides are stably incorporated into the genome of the cereal plant cell.

[0069] 10. The method of any one of embodiments 1-9, wherein the first and second polynucleotides are transiently expressed in the cereal plant cell.

[0070] 11. The method of embodiment 10, wherein the first and / or second polynucleotide are flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are lox sites recognized by a ere recombinase or FRT sites recognized by a FLP recombinase.

[0071] 12. The method of embodiment 11, further comprising providing the site-specific recombinase to the cultured plant cells after introduction of the first and / or second polynucleotide and / or after introduction of the genome editing system.

[0072] 13. The method of any one of embodiments 1-12, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, 29, or an allelic variant thereof.

[0073] 14. The method of any one of embodiments 1-12, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5 or an allelic variant thereof.

[0074] 15. The method of any one of embodiments 1-12, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 23 or an allelic variant thereof.

[0075] 16. The method of embodiment 15, further comprising introducing a third polynucleotide encoding a third 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: 21 in the cereal plant cell.

[0076] 17. The method of any one of embodiments 1-16, wherein the cereal plant cell comprises a regeneration-recalcitrant germplasm.

[0077] 18. The method of any one of embodiments 1-17, wherein the regenerable plant structure comprises a somatic embryo, embryogenic callus, somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots.

[0078] 19. The method of any one of embodiments 1-18, wherein the introducing comprises bacterial-mediated transformation or biolistic-mediated transformation.

[0079] 20. The method of any one of embodiments 1-19, wherein the cereal plant cell is cultured in a media comprising a cytokinin.

[0080] 21. The method of embodiment 20, wherein the cytokinin comprises kinetin.

[0081] 22. The method of embodiment 20 or 21, wherein the cytokinin is included in the media in a concentration of from about 0.01 mg / L to about 0.2 mg / L or from about 0. Img / L to about 0.15 mg / L.

[0082] 23. The method of any one of embodiments 1-21, wherein the cereal plant cell is cultured in a media comprising L-cysteine.

[0083] 24. The method of embodiment 23, wherein the L-cysteine is included in the media in a concentration of from about 150 mg / L to about 450 mg / L or from about 300 mg / L to about 400 mg / L.

[0084] 25. The method of any one of embodiments 1-24, wherein expression of the first and second polypeptides result in an increased somatic embryo induction frequency or increased embryo productivity relative to a control cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

[0085] 26. The method of embodiment 25, 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 cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

[0086] 27. The method of embodiment 25 or 26, 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.

[0087] 28. The method of any one of embodiments 1-27, further comprising introducing a genome editing system in the cereal plant cell.

[0088] 29. The method of any one of embodiments 1-28, 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.

[0089] 30. The method of any one of embodiments 1-29, 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.

[0090] 31. A cereal plant cell comprising a polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29 wherein expression of the first and second polypeptides increases proliferation, somatic embryogenesis, and / or regeneration capacity of the cereal plant cell.

[0091] 32. A cereal plant cell comprising a first polynucleotide encoding a first 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: 5 or 29.

[0092] 33. The cereal plant cell of embodiment 31 or 32, wherein the cereal plant cell is a maize or wheat plant cell.

[0093] 34. The cereal plant cell of any one of embodiments 31-33, wherein the first polynucleotide encoding the first 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: SEQ ID NO: 11, 12, 13, 14, or 25.

[0094] 35. The cereal plant cell of any one of embodiments 31-34, wherein the second polynucleotide encoding the second 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, 16, 17, 18, 19, 20, 24, 30, 31, 32, or 33.

[0095] 36. The cereal plant cell of any one of embodiments 31-35, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell andthe second polynucleotides is operably linked to a second heterologous promoter functional in a plant cell.

[0096] 37. The cereal plant cell of embodiment 36, wherein the first and / or second heterologous promoter comprises a barley heat shock promoter, optionally wherein the barley heat shock promoter is a promoter of the barley HvHSP17 gene or optionally wherein the barley heat shock promoter comprises a polynucleotide 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: 34.

[0097] 38. The cereal plant cell of any one of embodiments 31-37, wherein the first and second polynucleotides are stably incorporated into the genome of the cereal plant cell.

[0098] 39. The cereal plant cell of any one of embodiments 31-38, wherein the first and second polynucleotides are transiently expressed in the cereal plant cell.

[0099] 40. The cereal plant cell of any one of embodiments 31-39, wherein the first and / or second polynucleotide are flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are lox sites recognized by a ere recombinase or FRT sites recognized by a FLP recombinase.

[0100] 41. The cereal plant cell of any one of embodiments 31-40, wherein the first and second polynucleotides comprise an mRNA.

[0101] 42. The cereal plant cell of any one of embodiments 31-41, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, 29, or an allelic variant thereof.

[0102] 43. The cereal plant cell of any one of embodiments 31-42, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5 or an allelic variant thereof.

[0103] 44. The cereal plant cell of any one of embodiments 31-43, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 23 or an allelic variant thereof.

[0104] 45. The cereal plant cell of any one of embodiments 31-44, further comprising a third polynucleotide encoding a third polypeptide having at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity across the entire length of SEQ ID NO: 21.

[0105] 46. The cereal plant cell of any one of embodiments 31-37, wherein the cereal plant cell comprises a regeneration-recalcitrant germplasm.

[0106] 47. The cereal plant cell of any one of embodiments 31-46, wherein expression of the first and second polypeptides results in an increased somatic embryo induction frequency or increased embryo productivity relative to a control cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

[0107] 48. The cereal plant cell of embodiment 47, 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 cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

[0108] 49. The cereal plant cell of embodiment 47 or 48, 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.

[0109] 50. The cereal plant cell of any one of embodiments 31-49, further comprising a genome editing system.

[0110] 51. The cereal plant cell of embodiment 50, 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.[OHl] 52. The cereal plant cell of embodiment 51, 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.

[0112] 53. A cereal plant, tissue, organ, callus, or cell culture comprising the cereal plant cell of any one of embodiments 31-52.

[0113] 54. A method for producing a cereal plant, the method comprising: regenerating a cereal plant from the cereal plant cell of any one of embodiments 31-53.

[0114] 55. The method of any one of embodiments 31-54, wherein the regenerating step comprises culturing the cereal plant cell in a media comprising a cytokinin.

[0115] 56. The method of embodiment 55, wherein the cytokinin comprises kinetin.

[0116] 57. The method of embodiment 55 or 56, wherein the cytokinin is included in the media in a concentration of from about 0.01 mg / L to about 0.2 mg / L or from about 0. Img / L to about 0.15 mg / L.

[0117] 58. The method of any one of embodiments 31-57, wherein the regenerating step comprises culturing the cereal plant cell in a media comprising L-cysteine.

[0118] 59. The method of embodiment 58, wherein the L-cysteine is included in the media in a concentration of from about 150 mg / L to about 450 mg / L or from about 300 mg / L to about 400 mg / L.

[0119] 60. The method of any one of embodiments 54-59, further comprising selecting a progeny of the cereal plant that lacks the first and second polynucleotides.

[0120] 61. The method of any one of embodiments 54-60, wherein the cereal plant comprises an inserted transgene, a target gene edit, or a genome edit.

[0121] 62. A recombinant polynucleotide comprising: a first polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, , 26, 27, 28, or 29, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell and the second polynucleotide is operably linked to a second heterologous promoter functional in a plant cell.

[0122] 63. The recombinant polynucleotide of embodiment 62, wherein the first polynucleotide 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: 11, 12, 13, 14, or 25 and the second polynucleotide 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, 16, 17, 18, 19, 20, 24, 26, 27, 28, or 29.

[0123] 64. The recombinant polynucleotide of embodiment 62 or 63, wherein the first polynucleotide encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10, or an allelic variant thereof.

[0124] 65. The recombinant polynucleotide of any one of embodiments 62-64, wherein the second polynucleotide encodes a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, 29, or an allelic variant thereof.

[0125] 66. The recombinant polynucleotide of any one of embodiments 62-65, wherein the first and / or second polynucleotide are flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are lox sites recognized by a ere recombinase or FRT sites recognized by a FLP recombinase.

[0126] 67. A vector comprising the recombinant polynucleotide of any one of embodiments 62-66.EXAMPLESExample 1

[0127] This example describes the methods used in the experiments of Examples 2 to 3.Explants

[0128] Immature embryos of maize recalcitrant lines SITU1978, GIDA8989, and BEDB2121 were isolated 9-13 days after pollination.Transformation

[0129] Agrobacterium-mediated transformation was performed essentially by conventional methods (see US5591616, US7939328) using DL-phosphinothricin (PPT) or Imazapyr as a selection agent with modifications for either rapid protocol or standard protocol. For the rapid protocol, the transformation steps included Embryo Inoculation (1 day), Coculture (3-4 days), Rest (7-14 days), Shoot Induction (14 days), Shoot Elongation (7 days), Rooting (14-21 days), and Plantlet transfer to soil. For the standard protocol, the transformation steps included, Embryo Inoculation (1 day), Coculture (3-4 days), Rest (7-days), Selection 1 (7 days), Selection 2 (14 days), Selection 3 (7 days), Pre-Regeneration (5-7 days), Regeneration (14 days), and Shoot Growth (1-3 weeks), and Plantlet transfer to soil.Vectors

[0130] Super-Binary T-DNA vectors or ternary vectors with the afore mentioned relevant expression cassettes between left and right borders were used in the experiments.Transformation efficiency

[0131] Transformation efficiency was calculated as the number of transgenic plants over total number of explants (immature embryos) to start with. Each transgenic plant was confirmed by qPCR confirmation of integration of one or more than one transgene.

[0132] Table 1 lists plasmids and their components used in transformation experiments.Table 1Example 2

[0133] Immature zygotic explants of maize genotypes SITU1978 and GIDA8989 were transformed with Agrobacterium carrying one of plasmids pIN4471, pIN2541, or pIN5093 as indicated. pIN2542 is an empty control construct that does not contain a morphogene expression cassette. pIN4471 also contains Cre-LoxP excision system to remove morphoregulator gene before the end of Rest culture stage.

[0134] Results are shown in Table 2. This example illustrates that coexpression of some of genes such as MlWUS2-SiBBM2, promoted transformation efficiency as compared with empty control (pIN2541) in recalcitrant maize lines GIDA8989 and SITU1978.Table 2. Morphoregulator-enhanced maize transformationna: not applicableExample 3

[0135] This example illustrates that combinations of morphoregulators enhance maize transformation.

[0136] Immature zygotic explants of maize genotypes BEDB2121 and GIDA8989 were transformed with Agrobacterium carrying one of plasmids pIN3759, pIN2403, a combination of pIN3759 and pIN2403, or a combination of pIN3759 and pIN2402. pIN3759 contains M1WUS2 expression cassette alone whereas pIN2402 contains ZmGRF / GIF chimera cassette alone. Both constructs carry PAT gene a selectable marker but do not carry Cre-LoxP excision system.

[0137] Results are shown in Table 3. Combinations of pIN3759 and pIN2403 or pIN2402 showed increased transformation efficiency (TE%) as compared to any of the constructs alone. Table 3na: not applicableExample 4

[0138] Immature zygotic explants of wheat genotype InteGrain were transformed with Agrobacterium carrying one of plasmids pIN2854, pIN5093, or a combination of pIN2854 and pIN5093. pIN2854 contains GRF4-GIF1 expression cassette alone. pIN5093 contains the WUS2-BBM2 expression casette. pIN2854 + pIN5093 indicates the two Agrobacterium strains with each carrying one of these constructs were mixed together and used to infect wheat explants. Results are shown in Table 4. Combinations of pIN2854 and pIN5093 increased wheat 1 & 2 copy number ratios.Table 4na: not applicable

[0139] Examples 3 and 4 demonstrate that coexpression of MlWUS2-ZmGRF4 / GIFl (Zm: Zea mays') or MlWUS2.2-SiBBM2-TaGRF4 / GIF 1 induced higher somatic embryogenesis and transformation efficiencies than expression of ZmGRF4-GlFl or TaGRF4 / GIFl alone in maize and wheat, respectively.Example 5. Phylogenetic analysis of AP2 transcription factor family members including clade classifications.

[0140] The AP2 transcription factor family is a large group of transcription factors that regulate plant growth and development. Three members of this transcription factor family, maize Plethora 5 (PLT5, SEQ ID NOs: 1 and 15), Plethora 7 (PLT7, SEQ ID NOs: 2 and 16), and Plethora 3b (PLT3b, also named AINTEGUMENTA-LIKE 5 (AIL5), or AP2-60, SEQ ID NOs: 26-28 and 30-32), belong to the same clade as the PLT3 / 5 / 7 / clade (dashed box in FIG. 1) within the AP2 super family. This PLT3 / 5 / 7 clade is separate from and distinctly different from the clade containing BBM and PLT1 / 2. FIG. 1 illustrates the evolutionary relationship between the BBM clade, the PLT3 / 5 / 7 clade, and other clades across diverse plant species including both dicots and monocots. Although these genes share evolutionary origins, they have distinct functions and gene expression patterns. BBM is primarily involved in inducing somatic embryogenesis especially early somatic embryogenesis, while PLT3b, PLT3, PLT5, and PLT7 focus on meristem maintenance and stem cell regulation. In addition, BBM and PLT3 / 5 / 7 clades share less than 40% overall amino acid sequence homology. This evidence suggests the sequence and functional divergence between maize BBM and PLT3b / PLT3 / PLT5 / PLT7 proteins.Example 6. Co-expression of two morphoregulators improved somatic embryogenesis and transformation of a maize recalcitrant genotype.

[0141] This example illustrates that morphoregulators improved somatic embryogenesis, transformation and / or gene editing via Agrobacterium-mediated transformation and describes the methods used in the experiments of Examples 6-9.

[0142] Explants

[0143] Immature embryos of GIDA8989, GIPI9660, GILO8924, BEDB2121, GIQA9901, GIDA9910 were isolated 9-13 days after pollination.

[0144] Transformation

[0145] Agr()bacterium-mQ(T\a.iQ transformation was performed essentially by conventional methods (see US Patent No. 5,591,616 and US Patent No. 7,939,328, which are herein incorporated by reference in their entirety), using DL-phosphinothricin (PPT) as a selection agent. The transformation steps were Embryo Inoculation (1 day), Coculture (1 days), Resting (13-14 days), Shoot Induction / S el ection (14 days), Shoot El ongation / S el ection (14 days), Rooting (14 days), followed by transplant to soil. Medium compositions used in these experiments are listed in Table 5.

[0146] Two days before infection, 10 pl glycerol stock of Agrobacterium strain carrying a morphoregulator gene-containing plasmid from an ultra-freezer was inoculated into a 50 ml falcon tube that contains 10 ml YEP liquid medium (10 g / L Yeast Extract, 10 g / L Peptone, 5 g / L NaCl, 50 mg / L), placed on an orbital shaker set at 225 rpm, and incubated overnight at 28 °C. In the morning of the following day, the 10 ml overnight Agrobacterium culture was poured into 40 ml YEP liquid medium in a 250 ml flask and incubated on the same shaker. About 16 hours before infection, the Agrobacterium cell suspensions were spun-down in a centrifuge (4000 rpm, 15 min, 4 °C) and resuspended in a 250 ml flask containing 50 ml of AB minimum medium (Table 5) amended with 200 pM acetosyringone and 50 mg / L spectinomycin and shaken overnight. Early in the morning on the day of infection, the Agrobacterium culture was spun down (4000 rpm, 15 min, 4 °C) again. The supernatant was removed and the Agrobacterium infection medium with acetosyringone was added. The Agrobacterium cells were resuspended and the optical density (O.D. 660 nm) was adjusted to about 0.25 to 1.0.Table 5. Medium compositions for Agrobacterium-mediated transformation of maize (per liter)

[0147] Maize ears were surface sterilized for 20 min in 50 % (v / v) bleach (8.25 % sodium hypochlorite) plus 2 drops of Tween-20 followed by 3 rinses in sterile water. Immature embryos (IES) were isolated from ears and placed in 2 ml of the Agrobacterium infection medium with acetosyringone solution (ZM9271). The optimal size of the embryos varied based on the inbred, ranging from 1.5 to 2.0 mm. The infection solution was then drawn off and 1.8 ml of Agrobacterium suspension was added to the embryos and the tube vortexed for 5 sec. The microcentrifuge tube was allowed to stand for 5 min in the hood. The suspension mixture of Agrobacterium and embryos were poured onto Coculture medium (ZM9429). The Agrobacterium suspension was drawn off and the embryos were placed with scutella side up on the media. The plates were covered with lids and placed in a box with a lid and incubated in darkness at 21 °C for 1 day. Embryos were then transferred to Resting 1 medium (ZM9404) for 2-3 days followed Rest 2 medium (ZM9273) for 13-14 days at 28 °C in the dark before they were transferred to Maturation medium (ZM9174) amended with 5 mg / L or various levels of phosphinothricin (PPT), based on maize genotypes, at 28 °C in low light (around 50 mol / m2 / sec). Two weeks later, shoots were transferred to Regeneration medium (ZM9176) with 5 mg / L or various levels of PPT based on maize genotypes, at 28 °C in full light (150 mol / m2 / sec). Two weeks later, shoots were excised and transferred to Rooting medium (ZM9193) in full light. After 2-3 weeks plantlets with roots were transferred to greenhouse and analyzed using qPCR and nanopore assays.

[0148] To evaluate the impact of morphoregulators on promoting somatic embryogenesis, transformation, immature zygotic embryos of one of several recalcitrant corn genotypes, GIDA8989, GIPI9660, GILO8924, BEDB32121, GIQU9909, GIDA9910, GIBU9987, andGIKU8837, were transformed with Agrobacterium strain AGL1 carrying one of plasmids pIN5093, pIN6289, pIN6291, pIN6296, pIN6293, pIN6294, pIN6638, pIN6639, pIN6778, pIN6779, pIN6780, pIN6781, or pIN2855 as indicated.

[0149] Individual, regenerated plants were sampled by the end of Rooting medium and assayed by qPCR and nanopore assays as early detection of the transgenics. FIGS. 2A-2B and 3 illustrate somatic embryogenesis and plant regeneration responses from various top candidate combinations. Table 6 lists various combination and control constructs. Table 7 shows transformation of various recalcitrant maize genotypes using top combinations as compared to a control construct without morphogenes. These top morphogene combinations enabled many fold increases in transformation and / or editing frequencies. Transformation efficiencies of various combination constructs are listed in descending order: pIN5093, pIN95643, pIN6289, pIN6291, pIN6778, pIN6779, and pIN6296.

[0150] Vectors

[0151] Binary T-DNA vectors carrying the Cre-LoxP excision components in a ternary system with the following expression cassettes between left and right borders were used in the experiments (Table 6).Table 6

[0152] Co-expression of MlWUS2-SiBBM2 (pIN5093), or MlWUS2-ZmPLT5 (pIN6289) enabled transformation efficiencies of 18.9% or 16.7% and morphogene excision rates of 88.2% or 100%, respectively, in a recalcitrant maize genotype GIDA8989. Co-expression of ZmPLT5-SiBBM2 showed much lower transformation efficiency (5.6%) and morphogene excision efficiency (40%). However, all the above combination constructs showed better transformation and excision efficiencies than single WUS2 or no morphogene control constructs (Table 7).Table 7. Transformation and excision efficiencies by co-expression of two morphogenes.Example 7. Barley heat shock promoter enabled more efficient transformation and editing frequencies.

[0153] Immature zygotic embryo explants of corn genotypes GIDA8989, GIPI9660, and GILO8924 were transformed with AGL1 carrying one of plasmids pIN5643, pIN5958, or pIN2855 as indicated. Of these, pIN5643 and pIN5958 carry a gene editing unit.

[0154] Two weeks later, the cultured immature embryos were photographed and somatic embryogenesis responses were recorded. Explants infected with AGL1 harboring pIN5643 and pIN5958 that carry HvHSP17 and ZmHSP17 were treated for 3 hours at 38 °C or 2 hours at 42 °C, respectively, to induce Cre-LoxP excision of morphoregulators.

[0155] Explants were then sub-cultured and regenerated plants were analyzed for the presence of transgenes, morphogene excision and edits. In a side-by-side experiment comparing the two promoters driving Cre gene, ZmRAB17 desiccation promoter in construct pIN5094 induced a similar efficiency of morphogene excision (100%) but allowed much a lower transformation efficiency (8%) than the barely heat shock promoter in pIN5093 (85.7% excision and 18.7% transformation efficiency). In the following separate experiments comparing barley and maize heat shock promoters, the barley heat shock promoter (SEQ ID NO: 34) in pIN5643 enabled significantly higher efficiencies of transformation and high-quality editing than maize heat shock promoter in pIN5958 (Table 8).Table 8. Transformation and editing efficiencies by ZmRAB17, HvHSP17, or ZmHSP17 promoter.n.a.: not applicable due to the lack of editing cassetteExample 8. Use of kinetin in resting medium improved somatic embryogenesis, transformation frequency, and / or high-quality editing efficiency of various maize genotypes

[0156] Immature zygotic explants of corn genotypes GIDA8989, GIPI9660, GILO8924, and BEDB2121 were transformed with Agrobacterium strain AGL1 carrying one of plasmids pIN5093, pIN5643, or pIN2855 as indicated.

[0157] Expression of the fluorescent marker mScarlet indicated successful transformation at the time of transfer to the resting stage.

[0158] After one day co-culture, immature embryos were transferred to Rest 1 medium containing no or 0.1 mg / L kinetin for 1 to 3 days, followed by Rest 2 medium for 13-14 days (Table 5).

[0159] Afterwards, the cultured immature embryos were photographed and somatic embryogenesis responses recorded. Immature embryo explants were then sub-cultured until plants were recovered and analyzed by qPCR and AmSeq to determine presence of transgene, morphogene excision frequency, and high-quality editing efficiency. Kinetin at 0.1 mg / L in Rest 1 medium (Table 9) for 1, 2, or 3 days (depending on maize genotypes) or 0.15 mg / L in Rest 1 medium (Table 10) for 2 or 3 days enabled a significantly higher somatic embryogenesis responses, transformation efficiency, and / or high-quality editing efficiency than no kinetin control Rest medium (FIG. 4).Table 9. Use of kinetin in resting medium improved transformation and editing efficiencies of recalcitrant maize genotypes.Table 10. Optimum kinetin concentration in resting medium improved transformation and editing efficiencies of recalcitrant maize genotypes.n.a.: not applicable due to lack of editing cassette.Example 9. Use of L-cysteine in coculture medium improved somatic embryogenesis and transformation efficiency of various maize genotypes

[0160] In this experiment, the construct pIN5643 was used to test the effect of L-cysteine on recalcitrant genotypes for improved somatic embryogenesis (SE) which led to enhanced transformation and editing efficiencies.

[0161] Immature zygotic embryo explants of corn genotypes GIDA8989 and GIPI9660 were transformed with Agrobacterium strain AGL1 carrying plasmid pIN5643 or pIN2855.

[0162] Expression of the fluorescent marker mScarlet indicated successful transformation at the time of transfer to the resting stage.

[0163] Two weeks after the co-culture phase, the immature embryos producing new direct somatic embryos were photographed and somatic embryogenesis responses were recorded. L- cysteine at 300 mg / L enabled a drastic increase of transformation frequency and editing as compared with no or other concentrations of L-cysteine (Table 11).Table 11. L-cysteine improved transformation and editing efficiencies of recalcitrant maize genotypes.Example 10. Improved protocol and WUS2.2-RKD4 (pIN6291) enables to improve somatic embryogenesis and / or transformation efficiencies in extended maize genotypes.

[0164] Immature zygotic embryo explants of corn genotype GIPI9660, GIDA9924, GILO8924, GIDA9910 and ZEQU2124 were transformed with Agrobacterium strain AGL1 carrying plasmid pIN6291 or pIN6365, as indicated.

[0165] Two weeks later, somatic embryogenesis responses were recorded for the treated immature embryos. pIN629 l -transformed immature embryo explants had increased somatic embryogenesis, whereas pIN6365 had decreased somatic embryogenesis., (Table 12).

[0166] By the end of the transformation experiments, pIN6291 showed 8.1% transformation efficiency as compared to 0.0% with the empty control construct pIN6365 in the highly recalcitrant genotype ZEQU2124 (Table 13).Table 12. Somatic embryogenesis results comparing control to WUS2.2-RKD4 from two independent replicates.Table 13. Transformation efficiency results comparing control to WUS2.2-RKD4 in genotype ZEQU2124.Example 10. Improved biolistic-mediated transformation and gene editing of maize recalcitrant genotypes employing morphoregulators and ribonucleoproteins (RNP)

[0167] This example illustrates that the morphoregulator combination of RKD4+M1WUS2.2 improved ribonucleoprotein (RNP)-mediated transformation efficiency via particle bombardment. All constructs used in these experiments are listed in Table 6. All medium compositions for particle bombardment are listed in Table 14.Table 14. Medium compositions for particle bombardment of maize immature embryos.All ingredient amounts are per liter unless otherwise mentioned.

[0168] Immature embryos of preferred sizes, ranging from 1.5-2.2mm, more preferably 1.8- 2.2 mm, were isolated from corn ears and placed on IE isolation medium (ZM7044) with scutellum face up. The next day, embryos were moved to osmoticum media ZM7045 about four hours prior to bombardment.RNP was prepared by pipetting 4 l nuclease, 5.7 l crRNA, 4.9 pl IxPBS and 5.4ul nuclease free water into a 2 ml low-retention centrifuge tube and mixing well. Then, the followingreagents were pipetted into a 2 ml low retention centrifuge tube containing 25 pl of 0.6 pm gold particles (48 pg / pl stock): 20 pl RNP, 3.2 pl plasmid (250 ng / pl concentration) carrying morphoregulator gene with or without the selectable marker gene PAT, 4.9 pl duplexed oligos (500 ng / pl stock) and 8 pl TransIT-X2. This solution was mixed well, centrifuged for 30 sec at 8000rpm, followed by removal of the supernatant and resuspending in 400ul of water. For bombardment, 50 pl of the mixture produced above was loaded onto each microcarrier, airdried for 30-40 min, until a dry, yellow powder became visible. Particle bombardment was performed using a BioRad PDS-1000 / He device at 28 inches of Mercury chamber vacuum and using a 650 PSI rupture disc.Immature embryos of two regenerable corn genotypes GILO8924 and GIPI9660 were transformed with biolistic PDS-1000 / He device using RNP and construct pIN6291 carrying RKD4+M1WUS2.2 combination or empty control construct pIN2528 without morphogenes. The RNP complex contained CasCom plus gRNA for editing.

[0169] After particle bombardment, explants were left on the osmoticum media (ZM7045) overnight. Next day, explants were transferred to rest-2 media which either contained different levels of kinetin (ZM9437) or did not have kinetin (ZM9373) and were cultured for two weeks. Cultures were maintained in the dark up to this stage. Further, explants were moved to maturation media (ZM9174) for two weeks at 50 pmol / m2 / sec light with a 16 hour light / 8 hour dark photoperiod, and then transferred to regeneration media (ZM9177) for another two weeks at 150 pmol / m2 / sec light with a 16 hour light / 8 hour dark photoperiod. Finally, the isolated shoots were placed on Rooting media (ZM9193) for about two weeks at 150 pmol / m2 / sec light with a 16 hour light / 8 hour dark photoperiod and the rooted seedlings were sent to the greenhouse.Table 15. Plant regeneration and editing data with the use of RKD+WUS2.2 gene but without the use of kinetin in the media.

[0170] As seen in Table 15, for the genotype GIDA8924, the number of plants regenerated with the use of RKD+WUS2.2 genes was lower (42%) compared to no morpho genes (74%). However, the addition of kinetin in the media led to higher plant regeneration with 0.1 mg / L kinetin giving 57% and 0.15 mg / L kinetin giving 97% plant regeneration. A similar trend was reflected in the number of edited plants obtained with no morphogenes giving 14% editing efficiency, the use of a combination of morphogenes without kinetin giving 12% editing efficiency, the use of a combination of morphogenes with 0.1 mg / L kinetin yielding 21% editing efficiency, and the use of a combination of morphogenes with 0.15 mg / L kinetin giving 24% regeneration of edited shoots.In the genotype GIPI9660, the effect of combination of morphogenes and kinetin in the media was in the opposite direction. While no morphogenes gave 24% shoot regeneration, use of a combination of morphogenes led to 44% shoot regeneration. However, application of kinetin together with a combination of morphogenes led to lower shoot regeneration with 0.1 mg / Lkinetin leading to 18% and with 0.15 mg / L leading to 33% shoot regeneration. A similar trend was reflected in the regeneration of edited shoots for this genotype.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a regenerable plant structure, the method comprising: introducing a first polynucleotide encoding a first polypeptide having at least 90%, at least95%, 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29 in a cereal plant cell; and culturing the cereal plant cell to produce the regenerable plant structure.

2. A method of producing a regenerable plant structure, the method comprising: introducing a first polynucleotide encoding a first polypeptide having at least 90%, at least95%, 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: 5 or 29, in a cereal plant cell; and culturing the cereal plant cell to produce the regenerable plant structure.

3. The method of claim 1 or 2, wherein the cereal plant cell is a maize or wheat plant cell.

4. The method of claim 1 or 2, wherein the cereal plant cell is in an explant comprising an immature embryo or embryogenic callus.

5. The method of claim 1 or 2, wherein the first polynucleotide encoding the first 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: 11, 12, 13, 14, or 25.

6. The method of claim 1 or 2, wherein the second polynucleotide encoding the second 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, 16, 17, 18, 19, 20, 24, 30, 31, 32, or 33.

7. The method of claim 1 or 2, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell and the second polynucleotide is operably linked to a second heterologous promoter functional in a plant cell.

8. The method of claim 7, wherein the first and / or second heterologous promoter comprises a barley heat shock promoter having at least at least 95% sequence identity across the entire length of SEQ ID NO: 34.

9. The method of claim 1 or 2, wherein the first and second polynucleotides are stably incorporated into the genome of the cereal plant cell.

10. The method of claim 1 or 2, wherein the first and second polynucleotides are transiently expressed in the cereal plant cell.

11. The method of claim 10, wherein the first and / or second polynucleotide are flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are lox sites recognized by a ere recombinase or FRT sites recognized by a FLP recombinase.

12. The method of claim 11, further comprising providing the site-specific recombinase to the cultured plant cells after introduction of the first and / or second polynucleotide and / or after introduction of the genome editing system.

13. The method of claim 1 or 2, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29 or an allelic variant thereof.

14. The method of claim 1 or 2, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5 or an allelic variant thereof.

15. The method of claim 1 or 2, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 23 or an allelic variant thereof.

16. The method of claim 15, further comprising introducing a third polynucleotide encoding a third 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: 21 in the cereal plant cell.

17. The method of claim 1 or 2, wherein the cereal plant cell comprises a regenerationrecalcitrant germplasm.

18. The method of claim 1 or 2, wherein the regenerable plant structure comprises a somatic embryo, embryogenic callus, somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots.

19. The method of claim 1 or 2, wherein the introducing comprises bacterial -mediated transformation or biolistic-mediated transformation.

20. The method of claim 1 or 2, wherein the cereal plant cell is cultured in a media comprising a cytokinin.

21. The method of claim 20, wherein the cytokinin comprises kinetin.

22. The method of claim 20, wherein the cytokinin is included in the media in a concentration of from about 0.01 mg / L to about 0.2 mg / L or from about 0. Img / L to about 0.15 mg / L.

23. The method of claim 1 or 2, wherein the cereal plant cell is cultured in a media comprising L-cysteine.

24. The method of claim 23, wherein the L-cysteine is included in the media in a concentration of from about 150 mg / L to about 450 mg / L or from about 300 mg / L to about 400 mg / L.

25. The method of claim 1 or 2, wherein expression of the first and second polypeptides result in an increased somatic embryo induction frequency or increased embryo productivity relative to a control cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

26. The method of claim 25, 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 cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

27. The method of claim 25, 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.

28. The method of any one of claims 1 or 2, further comprising introducing a genome editing system in the cereal plant cell.

29. The method of claim 28, 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.

30. The method of claim 29, 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.

31. A cereal plant cell comprising a polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29, wherein expression of the first and second polypeptides increases proliferation, somatic embryogenesis, and / or regeneration capacity of the cereal plant cell.

32. A cereal plant cell comprising a first polynucleotide encoding a first 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: 5 or 29.

33. The cereal plant cell of claim 31 or 32, wherein the cereal plant cell is a maize or wheat plant cell.

34. The cereal plant cell of claim 31 or 32, wherein the first polynucleotide encoding the first 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: SEQ ID NO: 11, 12, 13, 14, or 25.

35. The cereal plant cell of claim 31 or 32, wherein the second polynucleotide encoding the second 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, 16, 17, 18, 19, 20, 24, 30, 31, 32, or 33.

36. The cereal plant cell of claim 31 or 32, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell and the second polynucleotides is operably linked to a second heterologous promoter functional in a plant cell.

37. The cereal plant cell of claim 36, wherein the first and / or second heterologous promoter comprises a barley heat shock promoter having at least at least 95% sequence identity across the entire length of SEQ ID NO: 34.

38. The cereal plant cell of claim 31 or 32, wherein the first and second polynucleotides are stably incorporated into the genome of the cereal plant cell.

39. The cereal plant cell of claim 31 or 32, wherein the first and second polynucleotides are transiently expressed in the cereal plant cell.

40. The cereal plant cell of claim 31 or 32, wherein the first and / or second polynucleotide are flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are lox sites recognized by a ere recombinase or FRT sites recognized by a FLP recombinase.

41. The cereal plant cell of claim 31 or 32, wherein the first and second polynucleotides comprise an mRNA.

42. The cereal plant cell of claim 31 or 32, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29 or an allelic variant thereof.

43. The cereal plant cell of claim 31 or 32, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5 or an allelic variant thereof.

44. The cereal plant cell of claim 31 or 32, wherein the first polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10 or an allelic variant thereof and the second polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 23 or an allelic variant thereof.

45. The cereal plant cell of claim 44, further comprising a third polynucleotide encoding a third 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: 21.

46. The cereal plant cell of claim 31 or 32, wherein the cereal plant cell comprises a regenerati on-recal citrant germpl asm .

47. The cereal plant cell of claim 31 or 32, wherein expression of the first and second polypeptides results in an increased somatic embryo induction frequency or increased embryo productivity relative to a control cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

48. The cereal plant cell of claim 47, 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 cereal plant cell lacking the first and / or second polynucleotides encoding the first and / or second polypeptides.

49. The cereal plant cell of claim 47, 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.

50. The cereal plant cell of claim 31 or 32, further comprising a genome editing system.

51. The cereal plant cell of claim 50, 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.

52. The cereal plant cell of claim 51, 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.

53. A cereal plant, tissue, organ, callus, or cell culture comprising the cereal plant cell of claim 31 or 32.

54. A method for producing a cereal plant, the method comprising: regenerating a cereal plant from the cereal plant cell of claim 31 or 32.

55. The method of claim 54, wherein the regenerating step comprises culturing the cereal plant cell in a media comprising a cytokinin.

56. The method of claim 55, wherein the cytokinin comprises kinetin.

57. The method of claim 55, wherein the cytokinin is included in the media in a concentration of from about 0.01 mg / L to about 0.2 mg / L or from about 0. Img / L to about 0.15 mg / L.

58. The method of claim 54, wherein the regenerating step comprises culturing the cereal plant cell in a media comprising L-cysteine.

59. The method of claim 58, wherein the L-cysteine is included in the media in a concentration of from about 150 mg / L to about 450 mg / L or from about 300 mg / L to about 400 mg / L.

60. The method of claim 54, further comprising selecting a progeny of the cereal plant that lacks the first and second polynucleotides.

61. The method of claim 60, wherein the cereal plant comprises an inserted transgene, a target gene edit, or a genome edit.

62. A recombinant polynucleotide comprising: a first polynucleotide encoding a first 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: 6, 7, 8, or 10 and a second polynucleotide encoding a second 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: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29, wherein the first polynucleotide is operably linked to a first heterologous promoter functional in a plant cell and the second polynucleotide is operably linked to a second heterologous promoter functional in a plant cell.

63. The recombinant polynucleotide of claim 62, wherein the first polynucleotide 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: 11, 12, 13, 14, or 25 and the second polynucleotide 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, 16, 17, 18, 19, 20, 24, 26, 27, 28, or 29.

64. The recombinant polynucleotide of claim 62, wherein the first polynucleotide encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 10, or an allelic variant thereof.

65. The recombinant polynucleotide of claim 62, wherein the second polynucleotide encodes a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 9, 23, 26, 27, 28, or 29, or an allelic variant thereof.

66. The recombinant polynucleotide of claim 62, wherein the first and / or second polynucleotide are flanked by site-specific recombinase recognition sites, optionally wherein the site-specific recombination sites are lox sites recognized by a ere recombinase or FRT sites recognized by a FLP recombinase.

67. A vector comprising the recombinant polynucleotide of any one of claims 62-66.

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