Embryogenic factors for cellular reprogramming of plant cells

Abstract

By plant cells are treated with cell reprogramming factors to change cell fate and development. Embryogenic factor genes and / or morphological development genes of induced embryos are used as cell reprogramming factors, and the polynucleotides comprising polypeptides or encoding gene products are used to generate double haploid or haploid plants from gametes. By contacting the embryogenic factor gene products and / or morphological development gene polypeptides of the induced embryogenic reorganization of the isolated cell with the purification of exogenous source to process maize microspores, embryogenesis is caused. When transforming with the gene construct comprising regulatory elements and structural genes, the gamete development of maize plants is an embryoid, and the regulatory elements and structural genes can play a role in a cascade mode to change the cell fate of plant cells. Embryogenic factor proteins and / or morphological development proteins expressed from gene constructs are used for non-original treatment methods and for plant in situ cell reprogramming.

Description

Technical Field

[0001] The present disclosure relates to the field of plant molecular biology, and more particularly, the present disclosure relates to rapid and efficient methods for producing non-transformed plants and transformed plants.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 835,500 (filed April 18, 2019) and U.S. Provisional Patent Application No. 62 / 947,786 (filed December 13, 2019), both of which are hereby incorporated by reference in their entirety.

[0004] References to electronically submitted sequence listings

[0005] An official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing, with the file name "20200417_8046-WO-PCT_ST25", created on April 17, 2020, and having a size of 1,984,864 bytes, and submitted simultaneously with this specification. The sequence listing contained in the ASCII formatted file is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0006] Plant breeding programs identify new varieties by screening many plants to identify individuals with desired characteristics. Typically, large numbers of generations from crosses are grown and evaluated, ideally across many years and multiple environments, to select plants with the most desirable characteristics.

[0007] Typical breeding methods make two kinds of parental plants hybridize, and progeny 1 hybrid (F1 hybrid) is the first hybrid generation.When two kinds of parental strains (typically inbred strains) from different heterozygous groups are hybridized, the hybrid vigor in commercial F1 hybrid is observed.Hybrid vigor (function of improvement or increase of any biological quality produced owing to combining the genetic contribution of its parents) is important for commercial maize seed production.The improvement of commercial hybrid performance needs to continue to develop new inbred parent system.

[0008] The maize inbred line development method can use the double haploid production of the maternal parent (producing female characteristics), wherein after the female ear (fertilized with pollen from so-called "haploid induction" line) of the plant produced from the first generation of hybridization is fertilized, the maternal haploid embryo is selected. Contrary to the copy of the inheritance of both maternal and paternal genomes, the pollen of the haploid induction strain is used to pollinate the female flower, causing the ovule level containing only the haploid maternal genome to rise, thus producing the maternal haploid embryo. The ovule in the female flower is the product of meiosis, and each maternal ovule is a unique meiosis recombinant haploid genome, thereby allowing the use of the in vitro tissue culture method including the chromosome doubling process to separate and process immature maternal haploid embryos, so that the rapid generation of the maternal double haploid recombinant population becomes possible. Many maize maternal haploid embryos produced by fertilizing target plants with pollen from a maize haploid inducer line cannot be regenerated into fertile, doubled haploid plants, and few, if any, in vitro tissue culture and plantlet regeneration methods will produce multiple fertile plants from a single haploid embryo. Therefore, there is a need for improved methods of producing doubled haploid plants from maternal gamete doubled haploids in maize.

[0009] Most maize inbreds are recalcitrant to microspore isolation, in vitro tissue culture and plantlet regeneration methods to produce paternal (male-producing) gamete dihaploids. Therefore, there is a need for methods to produce dihaploid plants from paternal gamete dihaploids in maize.

[0010] Thus, plant breeders would also benefit from methods for developing populations of recombinant inbred lines that do not require extensive pollination control methods or the extended time required to propagate self-fertilizing lines to an isogenic state. Summary of the invention

[0011] In one aspect, a method for generating plant embryos is provided, the method comprising (a) obtaining embryogenic cells by providing a cell reprogramming agent to a plant cell to obtain an embryogenic cell fate in the plant cell, the cell reprogramming agent being a cell reprogramming polypeptide selected from the group consisting of (i) a morphological development gene polypeptide; or (ii) an embryogenic factor polypeptide; or (iii) an inactivated CRISPR-Cas nuclease translation fusion polypeptide, a combination of (i) and (ii); a combination of (i) and (iii); a combination of (ii) and (iii); and (b) producing the plant embryo from the plant cell.

[0012] In one aspect, the plant cell is a gamete cell. In one aspect, the gamete cell is a maternal gamete cell. In another aspect, the gamete cell is a paternal gamete cell.

[0013] In one aspect, the cell reprogramming agent is not produced by a recombinant DNA construct stably integrated in a gamete cell. In another aspect, the cell reprogramming agent is produced by a recombinant DNA construct stably integrated in a gamete cell.

[0014] In one aspect, the morphological development gene polypeptide is selected from the group consisting of (i) WUS / WOX homeobox polypeptide; (ii) Babyboom (BBM) polypeptide or ovule development protein 2 (ODP2) polypeptide; (iii) LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii).

[0015] In one aspect, the cell reprogramming polypeptide further comprises a cell penetrating peptide (CPP).

[0016] In one aspect, the cell reprogramming polypeptide further comprises a secretion signal peptide (SSP).

[0017] In one aspect, the cell reprogramming factors are present in tissue culture medium.

[0018] In one aspect, the method comprises co-culturing the microspores with an embryogenesis inducing suspension feeder cell culture, wherein the embryogenesis inducing suspension feeder cell culture expresses an embryogenesis inducing polypeptide, or co-culturing the microspores with the cell reprogramming factors in the culture medium.

[0019] In one aspect, the embryogenic factor polypeptide is selected from the group consisting of (i) transcription factor bHLH30-like polypeptides; (ii) hybrid D-type cyclin polypeptides; (iii) mitogen-activated kinase protein polypeptides; (iv) plant lipid transfer polypeptides; (v) cyclin delta-2 polypeptides; (vi) oberon-like protein polypeptides;

[0020] (vii) polynucleotide adenylyltransferase polypeptides; (viii) GATA zinc finger polypeptides; (ix) homeobox-leucine zipper polypeptides; (x) hydrolase polypeptides; (xi) telomerase reverse transcriptase polypeptides; (xii) zinc finger polypeptides; (xiii) GRAS family transcription factor polypeptides; (xiv) mlo defense gene homolog polypeptides; (xv) 3-ketoacyl-CoA synthase 11-like polypeptides; (xvi) plant sulfate peptide polypeptides; and

[0021] (i) and combinations comprising any two or more embryogenic factor polypeptides simultaneously.

[0022] In one aspect, the cell reprogramming polypeptide is a cell cycle regulator polypeptide selected from the group of cell cycle regulators consisting of cyclin dependent kinases (CDKs).

[0023] In one aspect, the cell reprogramming polypeptide is selected from the group consisting of: (i) embryogenic factor polypeptides and (ii) cell cycle regulatory factor polypeptides; (iii) morphological development gene polypeptides; (iv) a combination of (i) and (ii); a combination of (ii) and (iii); or a combination of (i), (ii) and (iii).

[0024] In one aspect, the method further comprises culturing haploid plant embryos. In one aspect, the method comprises contacting haploid plant embryos with a chromosome doubling agent for a period of time sufficient to produce doubled haploid plant embryos. In one aspect, the method wherein the microspores are obtained from maize, rice, sorghum, Brassica, soybean, wheat, and cotton.

[0025] In one aspect, a method for generating haploid plant embryos is provided, the method comprising (a) providing a plant comprising an expression cassette, wherein the expression cassette comprises a haploid-inducing expression cassette, wherein the expression cassette comprises an egg cell-preferring regulatory element operably linked to a polynucleotide encoding a morphological development gene and an embryogenic factor and / or a cell cycle regulatory factor polypeptide; (b) hybridizing the plant of (a) as a female parent with a male parent expressing a color marker; (c) isolating female haploid embryos; (d) contacting the haploid embryos with a chromosome doubling agent; and (e) producing doubled haploid plants from the doubled haploids.

[0026] In one aspect, a method for improving haploid induction of a maize haploid inducer is provided, the method comprising (a) providing an expression cassette for a haploid inducer plant, wherein the expression cassette comprises a haploid induction expression cassette, wherein the expression cassette comprises an egg cell-preferred or pollen cell-preferred regulatory element operably linked to a polynucleotide encoding a morphological development gene and / or an embryogenic factor and / or a cell cycle regulatory factor polypeptide; (b) crossing the plant of (a) with a female; (c) isolating haploid embryos from the female parent; (d) contacting the haploid embryos with a chromosome doubling agent; and (e) producing a doubled haploid plant from the doubled haploid.

[0027] In one aspect, a method for generating haploid plant embryos is provided, the method comprising (a) providing a plant comprising an expression cassette, wherein the expression cassette comprises a dCas9 cell reprogramming expression cassette, wherein the expression cassette comprises an egg cell-preferred or pollen cell-preferred regulatory element operably linked to a polynucleotide encoding an inactivated Cas9 translational fusion polypeptide; (b) crossing the plant of (a) as a female parent with a male parent expressing a color marker; (c) isolating female haploid embryos; (d) contacting the haploid embryos with a chromosome doubling agent; and (e) generating doubled haploid plants from the doubled haploids.

[0028] In one aspect, a method for generating haploid plant embryos is provided, the method comprising (a) providing a plant comprising an expression cassette, wherein the expression cassette comprises a tapetum cell-preferred regulatory element operably linked to a polynucleotide encoding a cell reprogramming polypeptide; (b) crossing the plant of (a) with a wild-type inbred plant to provide an F1 hybrid; (c) recovering embryogenic microspores from the F1 hybrid of (b); and (d) producing the haploid plant embryos from the embryogenic microspores.

[0029] In one aspect, the cell reprogramming polypeptide is a morphological development polypeptide. In one aspect, the cell reprogramming polypeptide is an embryogenic factor polypeptide. In one aspect, the cell reprogramming polypeptide is a cell cycle regulator polypeptide.

[0030] In one aspect, the cell reprogramming polypeptide is selected from the group consisting of: (i) embryogenic factor polypeptides and (ii) cell cycle regulatory factor polypeptides; and (iii) morphological development gene polypeptides; (iv) a combination of (i) and (ii); a combination of (ii) and (iii); or a combination of (i), (ii) and (iii).

[0031] In one aspect, the morphological development polypeptide is selected from the group consisting of (i) a WUS / WOX homeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide; (iii) a LEC1 polypeptide.

[0032] In one aspect, the method further comprises modifying genomic DNA by a site-specific nuclease. In one aspect, the expression cassette further comprises a polynucleotide encoding a site-specific nuclease. In one aspect, the site-specific nuclease is selected from the group consisting of zinc finger nucleases, meganucleases, TALENs, and CRISPR-Cas endonucleases. In one aspect, the CRISPR-Cas nuclease is aCas9, Cpf1, or Casα nuclease. In one aspect, during microspore embryogenesis, modification of genomic DNA is performed by a Cas endonuclease. In one aspect, the modification of DNA is an insertion, deletion, or substitution mutation. In one aspect, the Cas endonuclease is expressed from an expression cassette, and the Cas endonuclease further comprises a cell-penetrating peptide. In one aspect, the method further comprises providing a guide RNA expressed from the expression cassette. In one aspect, the modification of DNA is performed by exogenously providing a guide RNA and a Cas endonuclease as a ribonucleoprotein complex to the embryogenic microspore. In one aspect, the plant is homozygous for the expression cassette.

[0033] On the one hand, the expression cassette further comprises a signal peptide. On the one hand, the expression cassette further comprises a cell penetrating peptide (CPP). On the one hand, the method further comprises contacting the haploid plant embryo with a chromosome doubling agent for a period of time sufficient to produce a double haploid plant embryo. On the one hand, the plant is maize, rice, sorghum, Brassica, soybean, wheat or cotton. On the one hand, the method further comprises regenerating a double haploid plant from the double haploid plant embryo.

[0034] In one aspect, a method for generating a double haploid plant is provided, the method comprising (a) providing a plant comprising an expression cassette, wherein the expression cassette comprises an endosperm cell-preferred regulatory element operably linked to a polynucleotide encoding an embryogenesis-inducing polypeptide; (b) hybridizing the plant of (a) with a wild-type F1 hybrid; (c) recovering haploid embryos from the hybridization of (b); (d) contacting the haploid embryos with a chromosome doubling agent for a period of time sufficient to produce double haploid embryos; and (e) regenerating the double haploid plants from the double haploid embryos of (d). In one aspect, the embryogenesis-inducing polypeptide is a morphological development polypeptide. In one aspect, the morphological development polypeptide is selected from the group consisting of (i) WUS / WOX homeobox polypeptides; (ii) Babyboom (BBM) polypeptides or ovule development protein 2 (ODP2) polypeptides; (iii) LEC1 polypeptides; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii). In one aspect, the expression cassette further comprises a polynucleotide encoding a gene editing nuclease. On the one hand, the method further comprises modifying genomic DNA by site-specific nuclease. On the one hand, the expression cassette further comprises a polynucleotide encoding a site-specific nuclease. On the one hand, the site-specific nuclease is selected from the group consisting of zinc finger nuclease, meganuclease, TALEN and CRISPR-Cas endonuclease. On the one hand, the CRISPR-Cas nuclease is Cas9 or Cpf1 nuclease. On the one hand, during haploid embryonic embryogenesis, modification of genomic DNA is performed by Cas endonuclease. On the one hand, the modification of DNA is insertion, deletion or substitution mutation. On the one hand, the Cas endonuclease is expressed from the expression cassette, and the Cas endonuclease further comprises a cell penetrating peptide. On the one hand, the method further comprises providing a guide RNA expressed from the expression cassette. On the one hand, the modification of DNA is performed by exogenously providing guide RNA and Cas endonuclease as a ribonucleoprotein complex to the haploid embryo of the embryogenesis. On the one hand, the plant is homozygous for the expression cassette. On the one hand, the expression cassette further comprises a signal peptide. On the one hand, the expression cassette further comprises a cell penetrating peptide (CPP). On the one hand, the expression cassette further comprises a color-marked or fluorescently-labeled polynucleotide encoding an adjustable element. On the one hand, reclaiming the haploid embryo comprises screening for the presence or absence of the color-marked, fluorescently-labeled or adjustable element. On the one hand, the screening occurs in cell viability and cell sorting microfluidic devices for automatic fluorescence detection, to identify, sort and select the haploid embryo comprising the expression cassette and the haploid embryo not comprising the expression cassette.

[0035] On the one hand, an embryogenic microspore is provided, the embryogenic microspore comprises an increased amount of an embryogenic inducing polypeptide compared to a control microspore, wherein the polypeptide is not produced in the microspore. On the one hand, an embryoid or embryogenic tissue produced from an embryogenic microspore is provided. On the one hand, an embryogenic microspore is provided, the embryogenic microspore comprises a heterologous cell reprogramming agent, wherein the heterologous cell reprogramming agent is not produced in the microspore. On the one hand, the cell reprogramming agent is selected from the group consisting of: (i) embryogenic inducing polypeptide; or (ii) embryogenic inducing compound; or (iii) a combination of (i) and (ii). On the one hand, the embryogenic inducing polypeptide is selected from the group consisting of (i) WUS / WOX homeobox polypeptide; (ii) Babyboom (BBM) polypeptide or ovule development protein 2 (ODP2) polypeptide; (iii) LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii). In one aspect, the embryogenic inducing compound is hemin or a kinase inhibitor or a combination thereof. In one aspect, the embryogenic microspore is capable of producing a haploid embryo. In one aspect, the embryogenic microspore is a maize embryogenic microspore. In one aspect, the embryogenic microspore is from rice, sorghum, Brassica, soybean, wheat or cotton. In one aspect, a plant cell comprising an expression cassette is provided, wherein the expression cassette comprises a tapetum cell preference regulatory element, the tapetum cell preference regulatory element is operably linked to a polynucleotide encoding an embryogenic inducing polypeptide, and wherein the embryogenic inducing polypeptide can be secreted or transported into the microspore. In one aspect, the embryogenic inducing polypeptide comprises a cell penetrating peptide. In one aspect, the embryogenesis-inducing polypeptide is a morphological development polypeptide selected from the group consisting of: (i) WUS / WOX homeobox polypeptides; (ii) Babyboom (BBM) polypeptides or ovule development protein 2 (ODP2) polypeptides; (iii) LEC1 polypeptides; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii). In one aspect, a plant cell comprising an expression cassette is provided, wherein the expression cassette comprises an endosperm cell-preferred regulatory element, the endosperm cell-preferred regulatory element is operably linked to a polynucleotide encoding an embryogenesis-inducing polypeptide, and wherein the embryogenesis-inducing polypeptide is produced in endosperm cells, peri-embryonic regions (ESRs), basal endosperm transfer layers (BETLs), or a combination thereof, and can be secreted or transported into embryonic cells. In one aspect, a plant cell population comprising the plant cell and the embryonic cell is provided, wherein the embryonic cell comprises a secreted or transported embryogenesis-inducing polypeptide.In one aspect, the embryogenesis-inducing polypeptide is a morphological developmental polypeptide selected from the group consisting of: (i) a WUS / WOX homeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide; (iii) a LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii).

[0036] 1. A method for producing a non-transgenic doubled haploid plant, the method comprising:

[0037] (a) obtaining embryogenic microspores by providing the microspores with a cell reprogramming agent selected from the group consisting of:

[0038] i) cell reprogramming polypeptide;

[0039] ii) cell reprogramming polynucleotides;

[0040] iii) a combination of i) and ii);

[0041] (b) producing haploid plant embryos from the embryogenic microspores;

[0042] (c) contacting the haploid embryo with a chromosome doubling agent for a period of time sufficient to produce doubled haploid embryos; and

[0043] (d) regenerating a doubled haploid plant from the doubled haploid embryo of (c).

[0044] 2. The method of claim 1, wherein the cell reprogramming polypeptide is selected from the group consisting of:

[0045] (a) Morphological developmental polypeptides;

[0046] (b) embryogenic factor polypeptides;

[0047] (c) synthetic transcription factor polypeptides;

[0048] (d) a combination of (a) and (b);

[0049] (e) a combination of (a) and (c);

[0050] (f) a combination of (b) and (c); and

[0051] (g) A combination of (a), (b) and (c).

[0052] 3. The method of claim 2, wherein the morphogenesis polypeptide is selected from the group consisting of:

[0053] (a) functional WUS / WOX polypeptides;

[0054] (b) Babyboom (BBM) polypeptide or ovule development protein 2 (ODP2) polypeptide;

[0055] (c) LEC1 polypeptide;

[0056] (d) a combination of (a) and (b);

[0057] (e) a combination of (a) and (c); and

[0058] (f) A combination of (b) and (c).

[0059] 4. The method of claim 2, wherein the embryogenic factor polypeptide comprises:

[0060] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 17;

[0061] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 18;

[0062] (c) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 19;

[0063] (d) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 20;

[0064] (e) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 21;

[0065] (f) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 22;

[0066] (g) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:23;

[0067] (h) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:24;

[0068] 5. The method of claim 2, wherein the synthetic transcription factor polypeptide is selected from the group consisting of:

[0069] (a) dCas9-translation fusion protein;

[0070] (b) dCasα-translation fusion protein; and

[0071] (c) A combination of (a) and (b).

[0072] 6 . The method of claim 1 , wherein providing the cell reprogramming polypeptide is performed by contacting the microspores with the cell reprogramming polypeptide.

[0073] 7. The method of any one of claims 1, 2 or 6, wherein the cell reprogramming polypeptide further comprises a cell penetrating peptide (CPP).

[0074] 8. The method of claim 7, wherein the cell penetrating peptide (CPP) is selected from the group consisting of:

[0075] (a) Protein transduction domain CPP,

[0076] (b) amphipathic peptide CPP,

[0077] (c) a synthetic cationic polyhistidine polypeptide CPP,

[0078] (d) synthetic cationic polylysine polypeptide CPP,

[0079] (e) a synthetic cationic polyarginine polypeptide CPP,

[0080] (f) Dendritic polycationic CPP,

[0081] (g) vascular endothelial cadherin CPP,

[0082] (h) transportan CPP,

[0083] (i) Monomer or dimer of HIV-1 TAT CPP,

[0084] (j) penetratin CPP,

[0085] (k) a synthetic cationic homoarginine oligopeptide CPP, and

[0086] (l) γ-zein protein CPP.

[0087] 9. The method of claim 1, further comprising isolating and culturing the doubled haploid embryos.

[0088] 10. A doubled haploid plant produced by the method of claim 1 or 9.

[0089] 11. The method of claim 5, wherein the cell reprogramming polypeptide comprises a dCas9-translation fusion protein having cell reprogramming activity, the method comprising:

[0090] (a) introducing into a cell a polypeptide comprising an amino acid sequence selected from the group consisting of: a dCas9-chromatin modifying translational fusion protein comprising:

[0091] i) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:78;

[0092] ii) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95% or 98%) identical to SEQ ID NO:79;

[0093] iii) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95% or 98%) identical to SEQ ID NO:80;

[0094] iv) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95% or 98%) identical to SEQ ID NO:81;

[0095] v) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:82;

[0096] (b) introducing the polypeptide into a cell in combination with a polypeptide comprising an amino acid sequence selected from the group consisting of: a translational fusion protein comprising a dCas9-transcriptional activator fusion protein comprising:

[0097] i) inactivated Spy Cas9 (polynucleotide SEQ ID NO: 83 encoding polypeptide SEQ ID NO: 84). and

[0098] ii) the transcriptional activator domain of the Arabidopsis CBF1 protein (polynucleotide SEQ ID NO: 85 encoding the polypeptide SEQ ID NO: 86), or

[0099] iii) a transcriptional activator domain of the VP64 domain (polynucleotide SEQ ID NO: 244 encoding the polypeptide SEQ ID NO: 245);

[0100] (c) introducing the polypeptide into a cell in combination with a polypeptide comprising an amino acid sequence selected from the group consisting of: a translational fusion protein comprising a dCas9-transcriptional repressor fusion protein comprising:

[0101] i) a transcriptional repressor motif as shown in Table 16;

[0102] ii) a transcriptional repressor domain as shown in Table 17.

[0103] 12. The method of claim 11, wherein the dCas9-translation fusion protein is combined with at least one guide RNA (gRNA) selected from the group consisting of:

[0104] (a) a gRNA having sequence homology to a locus encoding a functional WUS / WOX polypeptide;

[0105] (b) a gRNA having sequence homology to a locus encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide;

[0106] (c) a gRNA having sequence homology to a gene locus encoding a LEC1 polypeptide;

[0107] (d) a gRNA having sequence homology to a locus encoding an embryogenic factor polypeptide;

[0108] (e) a gRNA having sequence homology to a gene locus encoding a C2H2-type zinc finger protein;

[0109] (f) a gRNA having sequence homology to a gene locus encoding a MADS-box transcription factor protein;

[0110] (g) a gRNA having sequence homology to a gene locus encoding a polycomb group (PcG) protein or a subunit thereof; and

[0111] (h) a gRNA having sequence homology to a gene locus encoding a CHD3 chromatin remodeling factor or a subunit thereof, thereby forming a ribonucleoprotein complex comprising a protein (polynucleotides encoding polypeptides 17 to 32) that promotes cell reprogramming.

[0112] 13. The method of claim 12, wherein the locus encoding the embryogenic factor polypeptide is a nucleotide sequence selected from the group consisting of:

[0113] (a) at least one of SEQ ID NOs: 1-16;

[0114] (b) a nucleotide sequence that is at least 95% identical to at least one of SEQ ID NOs: 1-16; and

[0115] (c) a nucleotide sequence that is at least 70% identical to at least one of SEQ ID NOs: 1-16.

[0116] 14. The method of claim 12, wherein designing a gRNA for a gene locus comprises:

[0117] (a) selecting DNA sequences flanking the locus encoding the gRNA target site, including proximal regulatory regions up to 10,000 nucleotides upstream or downstream of the locus, or

[0118] (b) selecting DNA sequences flanking the locus encoding the gRNA target site, including cis-regulatory elements greater than 10,000 nucleotides upstream or downstream of the locus, or

[0119] (c) The method of (a) and (b) and (c), or any combination thereof.

[0120] 15. The method of claims 11-12, wherein a polynucleotide is used to introduce a dCas9 translational fusion polypeptide into a cell, comprising:

[0121] (a) attaching a double-stranded or single-stranded DNA polynucleotide containing a dCas9-translational fusion protein expression cassette and a guide RNA expression cassette to particles for introduction and delivery into cells;

[0122] (b) attaching a double-stranded or single-stranded DNA polynucleotide containing a dCas9-translation fusion protein expression cassette and at least one single gRNA as an RNA molecule to particles for introduction and delivery into cells;

[0123] (c) attaching an RNA polynucleotide containing a dCas9 translational fusion protein and a double-stranded or single-stranded DNA polynucleotide containing a guide RNA expression cassette to particles for introduction and delivery into cells;

[0124] (d) Attaching an RNA polynucleotide containing a dCas9 translational fusion protein and at least one single gRNA as an RNA molecule to particles for introduction and delivery into cells.

[0125] 16. The method of claims 11-12, wherein the dCas9 translational fusion protein is introduced into a plant cell using a polypeptide, comprising:

[0126] (a) attaching a polypeptide containing a dCas9 translational fusion protein and a double-stranded or single-stranded DNA polynucleotide containing a guide RNA expression cassette to particles for introduction and delivery into cells;

[0127] (b) Attaching a polypeptide containing a dCas9 translational fusion protein and at least one single gRNA as an RNA molecule to particles for introduction and delivery into cells.

[0128] 17. The method of claim 15, wherein the dCas9 translational fusion protein is fused to a cell penetrating peptide, the cell penetrating peptide comprising:

[0129] (a) Zea mays (Z. mays) knotted1 CPP (SEQ ID NO: 49),

[0130] (b) Saccharomyces pombe TP10 CPP (SEQ ID NO: 51),

[0131] (c) Candida albicans Zebra CPP (SEQ ID NO: 53),

[0132] (d) PEP1 CPP (SEQ ID NO: 55),

[0133] (e) HIV-1 TAT CPP (SEQ ID NO: 57), and

[0134] (f) γ-zein cell penetrating peptide (SEQ ID NO: 59).

[0135] 18. The method of claims 11-17, wherein the method comprises exposing a plant cell to two or more dCas9 translational fusion proteins complexed with one or more gRNAs to obtain a plant from the treated cell.

[0136] 19. A method for producing a non-transgenic doubled haploid plant, the method comprising:

[0137] (a) expressing a tapetum cell-preferred regulatory element operably linked to a polynucleotide encoding a cell reprogramming polypeptide,

[0138] (b) secreting the expressed cell reprogramming polypeptide and transporting the polypeptide into microspores,

[0139] (c) obtaining embryogenic microspores responsive to the cell reprogramming polypeptide,

[0140] (d) producing said haploid plant embryos from said embryogenic microspores,

[0141] (e) contacting the haploid embryo with a chromosome doubling agent for a period of time sufficient to produce doubled haploid embryos; and

[0142] (f) regenerating the doubled haploid plant from the doubled haploid embryo of (e).

[0143] 20. The plant cell of claim 19, wherein the cell reprogramming polypeptide comprises a cell penetrating peptide.

[0144] 21. The plant cell of claim 19, wherein the cell reprogramming polypeptide is a morphological development polypeptide selected from the group consisting of:

[0145] (g) WUS / WOX homeobox polypeptide;

[0146] (h) Babyboom (BBM) polypeptide or ovule development protein 2 (ODP2) polypeptide;

[0147] (i) LEC1 polypeptide;

[0148] (j) a combination of (a) and (b); and

[0149] (k) A combination of (a) and (c).

[0150] 22. The plant cell of claim 19, wherein the embryogenic factor polypeptide comprises:

[0151] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 17;

[0152] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 18;

[0153] (c) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 19;

[0154] (d) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 20;

[0155] (e) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 21;

[0156] (f) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 22;

[0157] (g) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:23;

[0158] (h) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:24;

[0159] 23. The method of claim 20, wherein the cell reprogramming polypeptide comprises a secretion signal peptide.

[0160] 24. The method of claim 20, wherein the cell reprogramming polypeptide comprises a cell reprogramming-glucocorticoid receptor translational fusion polypeptide.

[0161] 25. The method of claim 24, wherein the fused cell reprogramming-glucocorticoid receptor polypeptide is conditionally localized to the nucleus by external application of a ligand to an in vitro tissue culture medium, thereby controlling the protein activity.

[0162] 26. The method of claim 2, wherein one or more cell reprogramming polypeptides and one or more cell cycle regulatory proteins are provided to the cells simultaneously.

[0163] 27. The method of claim 26, wherein the cell cycle regulatory protein is a cyclin-dependent kinase selected from the group consisting of:

[0164] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 114;

[0165] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 115;

[0166] (c) A combination of (a) and (b).

[0167] 28. A method for obtaining a genome-modified doubled haploid plant, the method comprising:

[0168] (a) obtaining a first plant having genomic DNA of a maize plant to be edited, the first plant being transformed with an expression cassette comprising:

[0169] i. Morphological development genes, or

[0170] ii. Embryogenic factors, or

[0171] iii. Genome modification components, or

[0172] iv. CRE excision component, or

[0173] v. (i) and (ii) and (iii) and (iv), or any combination thereof;

[0174] (b) obtaining a second plant, wherein the second plant comprises a marker gene;

[0175] (c) pollinating the first plant with pollen from the second plant;

[0176] (d) selecting at least one haploid offspring produced by the pollination step (c), wherein the haploid offspring comprises the genome of the first plant but does not comprise the genome of the second plant, and the genome of the first plant has been modified by the gene editing component.

[0177] 29. The method of claim 28, wherein the gene editing component uses a DNA modifying enzyme, which is a site-directed nuclease selected from the group consisting of: a meganuclease (MN), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas9 nuclease, a Casα nuclease, a Cpf1 nuclease, a dCas9-FokI, a dCpf1-FokI, a chimeric Cas9-cytidine deaminase, a chimeric Cas9 adenine deaminase, a chimeric FEN1-Fok1, a Mega-TAL, a nickase Cas9 (nCas9), a chimeric dCas9-non-FokI nuclease, and a dCpf1-non-FokI nuclease.

[0178] 30. The method of claim 28, wherein the marker gene is selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, C1, CRC, R-nj, R1-scm and anthocyanin pigments.

[0179] 31. The method of claim 28, wherein the edited haploid offspring are treated with a chromosome doubling agent to produce edited doubled haploid offspring.

[0180] 32. The method of claim 31, wherein the chromosome doubling agent is colchicine, nafate, dithipyr, trifluralin, or another known anti-microtubule agent.

[0181] 33. The method of claim 32, further comprising isolating and culturing the doubled haploid plant embryos.

[0182] 34. The method of claim 29, wherein at least one guide RNA molecule is provided to a ribonucleoprotein complex comprising a DNA modifying enzyme.

[0183] 35. A method for producing a doubled haploid plant, the method comprising:

[0184] (a) providing genetic chromosome doubling proteins to haploid cells;

[0185] (b) contacting the haploid cell with a genetic chromosome doubling protein for a period of time sufficient to produce a doubled haploid cell;

[0186] (c) obtaining double haploid cells;

[0187] (d) Regeneration of doubled haploid plants from the doubled haploid cells of (c).

[0188] 36. The method of claim 35, wherein the genetic chromosome doubling agent is a genetic chromosome doubling polypeptide.

[0189] 37. The method of claim 36, wherein the genetic chromosome doubling polypeptide is selected from the group consisting of:

[0190] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 141;

[0191] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 197;

[0192] (c) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 198;

[0193] (d) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 199;

[0194] (e) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 200;

[0195] (f) a combination of (a) and (b), or

[0196] (g) a combination of (a) and (c), or

[0197] (h) a combination of (a) and (d), or

[0198] (i) A combination of (a) and (e).

[0199] 38. The method of claim 36, wherein treating the haploid cells comprises:

[0200] (a) treating haploid cells with a solution containing Agrobacterium expressing a polypeptide for doubling genetic chromosomes;

[0201] (b) translocating the genetic chromosome doubling polypeptide from the Agrobacterium into a plant cell;

[0202] (c) recovering the plant cells from the Agrobacterium, thereby completing the treatment;

[0203] (d) Obtaining double haploid offspring.

[0204] 39. The method of claim 36, wherein the genetic chromosome duplication comprises a protein that causes mitotic spindle instability.

[0205] 40. The method of claim 39, wherein the instability-causing protein interferes with the segregation of daughter chromatids during mitosis.

[0206] 41. The method of claim 40, wherein the interfering activity impairs microtubule polymerization.

[0207] 42. The method of claim 41, wherein microtubule polymerization is interfered with by providing tubulin.

[0208] 43. The method of claim 42, wherein the tubulin provided has an altered activity.

[0209] 44. The method of claim 43, wherein the altered activity that interferes with microtubule polymerization impairs α-β-tubulin heterodimer activity.

[0210] 45. The method of claim 44, wherein the activity of the tubulin subunit is impaired using a mutated subunit comprising a loss of:

[0211] (a) provides the C-terminal domain of the subunit of the T7 loop responsible for nucleotide hydrolysis;

[0212] (b) the N-terminal domain of the subunit that provides the nucleotide binding site;

[0213] (c) H8 helix;

[0214] (d) T5 loop;

[0215] (e) B9 tablet;

[0216] (f) H7 helix;

[0217] (g) GDP (β-tubulin); or

[0218] (h) A combination of any two or more of (a) to (g).

[0219] 46. ​​The method of claim 45, wherein the mutated subunit impairs the guanine nucleotide binding site and / or GTPase activity.

[0220] 47. The method of claim 46, wherein the tubulin conserved site [SAG]-GGTG-[SA]-G (SEQ ID NO: 423) is mutated.

[0221] 48. The method of claim 36, wherein the genetic chromosome doubling agent comprises degrading mitotic cycle proteins, resulting in an intranuclear cycle comprising G1-S-G2 phases.

[0222] 49. The method of claim 48, wherein promoting endoreduplication comprises providing a cell cycle switch protein to the haploid cell.

[0223] 50. The method of claim 49, wherein the cell cycle switch protein is selected from the group comprising:

[0224] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 197;

[0225] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 198;

[0226] (c) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 199;

[0227] (d) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 200;

[0228] 51. The method of claim 28, wherein the plant comprises a heterologous polynucleotide operably linked to a promoter, wherein the polynucleotide encodes one or more polypeptides that cause a plant egg cell to become embryogenic when the polynucleotide is expressed in a plant cell.

[0229] 52. The method of claim 51, wherein the heterologous polynucleotide is stably incorporated into the genome of the plant.

[0230] 53. The method of claim 52, wherein the plant has increased levels of a polypeptide selected from the group consisting of:

[0231] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 111;

[0232] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:21;

[0233] (c) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 112;

[0234] (d) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 113;

[0235] (e) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 114;

[0236] (f) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 115;

[0237] (g) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 116;

[0238] (h) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 117;

[0239] (i) A combination of (d) and (h).

[0240] 54. The method of claim 53, wherein the plant has elevated levels of reprogramming in the egg cell promoting embryogenic cell fate in response to the polypeptide.

[0241] 55. The method of claim 54, wherein the reprogramming renders the oocyte parthenogenetic, and wherein the parthenogenetic oocyte contains only the maternal genome (1n).

[0242] 56. The method of claim 55, wherein the plant has a phenotype comprising an elevated level of maternal haploid induction.

[0243] 57. The method of claim 51, wherein the promoter is a tissue-preferred promoter.

[0244] 58. The method of claim 57, wherein the tissue-preferred promoter causes the one or more polypeptides to be expressed in the egg cell.

[0245] 59. The method of claim 28, wherein the cloned doubled haploid plants are propagated from the genomically modified haploid embryos using a method comprising:

[0246] (a) contacting the genome-modified cell with a bacterial strain comprising a T-DNA containing a morphogenetic gene expression cassette;

[0247] (b) eliciting a growth response in a second genome-modified cell; and

[0248] (c) regenerating a cloned genome-modified plant from the second genome-modified plant cell;

[0249] (d) contacting the cloned genome-modified plant with a chromosome doubling agent for a period of time sufficient to produce doubled haploid embryos; and

[0250] (e) regenerating the cloned genome-modified plant.

[0251] 60. A method for improving the haploid induction ability of a maize haploid inducer line, the method comprising:

[0252] (a) obtaining a maize plant having a known haploid induction capability transformed with a haploid induction expression cassette, the haploid induction expression cassette comprising:

[0253] i. known morphogenesis genes, or

[0254] ii embryogenic factors, or

[0255] iii. a combination of (i) and (ii);

[0256] (b) obtaining a donor plant, wherein the donor plant provides a donor ear;

[0257] (c) pollinating the ears of the donor ear with pollen from the transformed haploid inducer;

[0258] (d) selecting at least one haploid offspring produced by the pollination step (c), wherein the haploid offspring comprises the genome of the donor plant but does not comprise the genome of the transformed haploid inducer plant;

[0259] (e) contacting the haploid embryo with a chromosome doubling agent for a period of time sufficient to produce doubled haploid embryos; and

[0260] (f) regenerating the doubled haploid plant from the doubled haploid embryo of (e).

[0261] 61. The method of claim 59, wherein the transformable maize plant is selected from and / or derived from the group comprising lines Stock6, RWK, RWZ, UH400.

[0262] 62. A method for producing haploid plant embryos, the method comprising the steps of:

[0263] (a) providing microspores or pollen containing a cell reprogramming agent;

[0264] (b) pollinating embryo sac cells, particularly egg cells, of a plant from which haploid embryos are to be prepared using the microspores or pollen;

[0265] (c) allowing the microspore or pollen to release the cell reprogramming agent in or near the embryo sac cell, particularly the egg cell, to improve the method for obtaining haploid plant embryos.

[0266] 63. The method of claim 2, wherein the cell reprogramming polypeptide comprises a catalytically inactive Casα (dCasα) endonuclease, the endonuclease comprising a translational fusion protein having cell reprogramming activity with an amino acid sequence at least 85% to 100% identical to one of SEQ ID NOs: 219-226, the method comprising:

[0267] (a) introducing into a cell a polypeptide comprising an amino acid sequence selected from the group consisting of: a dCasα chromatin-modifying translational fusion protein comprising:

[0268] i) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:67;

[0269] ii) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95% or 98%) identical to SEQ ID NO:68;

[0270] iii) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:69;

[0271] iv) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95% or 98%) identical to SEQ ID NO:70;

[0272] v) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:71;

[0273] vi) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:72;

[0274] (b) introducing the polypeptide into a cell in combination with a polypeptide comprising an amino acid sequence selected from the group consisting of: a translational fusion protein comprising a dCasα-transcriptional activator fusion protein comprising:

[0275] i) an inactivated Casα as shown in Table 14, and;

[0276] ii) the transcriptional activator domain of the Arabidopsis CBF1 protein (polynucleotide SEQ ID NO: 85 encoding the polypeptide SEQ ID NO: 86), or

[0277] iii) a transcriptional activator domain of the VP64 domain (polynucleotide SEQ ID NO: 244 encoding the polypeptide SEQ ID NO: 245);

[0278] (c) introducing the polypeptide into a cell in combination with a polypeptide comprising an amino acid sequence selected from the group consisting of: a translational fusion protein comprising a dCasα transcriptional repressor fusion protein consisting of:

[0279] i) a transcriptional repressor motif as shown in Table 16;

[0280] ii) a transcriptional repressor domain as shown in Table 17.

[0281] 64. Combining the protein of claim 63 with at least one guide RNA (gRNA) to create a ribonucleoprotein complex comprising

[0282] (a) designing a gRNA sequence having homology to a sequence having identity to a locus encoding a WUS protein, wherein the WUS protein promotes cell reprogramming;

[0283] (b) designing a gRNA sequence that has homology to a sequence that is identical to a locus encoding a BBM protein that promotes cell reprogramming;

[0284] (c) designing a gRNA sequence having homology to a sequence having identity to a locus encoding a LEC1 protein, wherein the LEC1 protein promotes cell reprogramming;

[0285] (d) designing a gRNA sequence having homology to a sequence identical to a gene locus encoding an embryogenic factor protein (polynucleotides SEQ ID NOs: 1 to 16 encoding polypeptides SEQ ID NOs: 17 to 32), wherein the embryogenic factor protein promotes cell reprogramming;

[0286] (e) designing a gRNA sequence that has homology to a sequence that is identical to a gene locus encoding a C2H2-type zinc finger protein that represses a cell reprogramming factor;

[0287] (f) designing a gRNA sequence that has homology to a sequence that is identical to a locus encoding a MADS-box transcription factor protein that represses a cellular reprogramming factor;

[0288] (g) designing a gRNA sequence that has homology to a sequence that is identical to a locus encoding a polycomb group (PcG) protein or a subunit thereof, wherein the polycomb group (PcG) protein or a subunit thereof represses cellular reprogramming;

[0289] (h) Designing a gRNA sequence that has homology to a sequence that is identical to a locus encoding a CHD3 chromatin remodeling factor or a subunit thereof, wherein the CHD3 chromatin remodeling factor or a subunit thereof represses cellular reprogramming.

[0290] 65. The method of claim 64, wherein designing a gRNA for a locus comprises:

[0291] (a) selecting DNA sequences flanking the locus encoding the gRNA target site, including proximal regulatory regions up to 10,000 nucleotides upstream or downstream of the locus, or

[0292] (b) selecting DNA sequences flanking the locus encoding the gRNA target site, including cis-regulatory elements greater than 10,000 nucleotides upstream or downstream of the locus, or

[0293] (c) The method of (a) and (b) and (c), or any combination thereof.

[0294] 66. The method of claim 63, wherein a polynucleotide is used to introduce a dCasα translational fusion polypeptide into a cell, comprising:

[0295] (a) attaching a double-stranded or single-stranded DNA polynucleotide containing a dCasα translational fusion protein expression cassette and a guide RNA expression cassette to a particle for introduction and delivery into a cell, or

[0296] (b) attaching a double-stranded or single-stranded DNA polynucleotide containing a dCasα translational fusion protein expression cassette and at least one single gRNA as an RNA molecule to particles for introduction and delivery into cells, or

[0297] (c) attaching an RNA polynucleotide containing a dCasα translational fusion protein and a double-stranded or single-stranded DNA polynucleotide containing a guide RNA expression cassette to particles for introduction and delivery into cells, or

[0298] (d) Attaching an RNA polynucleotide containing a dCasα translational fusion protein and at least one single gRNA as an RNA molecule to particles for introduction and delivery into cells.

[0299] 67. The method of claim 63, wherein the dCasα translational fusion protein is introduced into a plant cell using a polypeptide, comprising:

[0300] (a) attaching a polypeptide containing a dCasα translational fusion protein and a double-stranded or single-stranded DNA polynucleotide containing a guide RNA expression cassette to particles for introduction and delivery into cells;

[0301] (b) Attaching a polypeptide containing a dCasα translational fusion protein and at least one single gRNA as an RNA molecule to particles for introduction and delivery into cells.

[0302] 68. The method of claim 67, wherein the dCasα translational fusion protein is fused to a cell penetrating peptide comprising:

[0303] (a) maize knotted1 CPP (SEQ ID NO: 49),

[0304] (b) Saccharomyces pombe TP10CPP (SEQ ID NO: 51),

[0305] (c) Candida albicans Zebra CPP (SEQ ID NO: 53),

[0306] (d) PEP1 CPP (SEQ ID NO: 55),

[0307] (e) HIV-1 TAT CPP (SEQ ID NO: 57), and

[0308] (f) γ-zein cell penetrating peptide (SEQ ID NO: 59).

[0309] 69. The method of claims 63-69, wherein the treating comprises exposing the plant cells to two or more dCasα and / or dCas9 translational fusion proteins complexed with one or more gRNAs to obtain plants from the treated cells.

[0310] 70. A method for obtaining a transgenic plant, the method comprising:

[0311] (a) transforming a plant cell with an expression cassette comprising at least one polynucleotide encoding an embryogenic factor polypeptide to obtain a transformed plant tissue;

[0312] (b) regenerating a transformed plant from cells of a tissue contacted by a protein expressed by the polynucleotide; and

[0313] (c) selecting genetically engineered plants in which the genomic modification is present.

[0314] 71. The method of claim 71, wherein the embryogenic factor polypeptide comprises:

[0315] (a) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 17;

[0316] (b) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 18;

[0317] (c) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 19;

[0318] (d) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 20;

[0319] (e) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 21;

[0320] (f) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO: 22;

[0321] (g) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:23;

[0322] (h) an amino acid sequence that is at least 70% (e.g., at least 80%, 90%, 95%, or 98%) identical to SEQ ID NO:24;

[0323] 72. A method for locus-specific gene regulation of a target gene, the method comprising:

[0324] (a) providing a strand-specific RNA transcript to a cell, wherein the strand-specific RNA transcript contains a sequence homologous to a DNA regulatory element;

[0325] (b) binding the strand-specific RNA transcript to an RNA binding protein; and (c) switching the gene regulatory expression state, wherein the DNA regulatory element can function as an enhancer or a repressor depending on the direction of transcription;

[0326] (d) improving cellular reprogramming of the treated cells;

[0327] (e) regenerating a plant from the treated cells.

[0328] 73. A method for improving cellular reprogramming in a plant cell, the method comprising providing to the cell an RNA molecule that binds to a protein, thereby reducing the enzymatic activity of the protein.

[0329] 74. The method of claim 75, wherein the RNA molecule is a non-coding RNA.

[0330] 75. The method of claim 76, wherein the non-coding RNA has sequence homology with a response element.

[0331] 76. The method of claim 76, wherein the response element is a trithorax response element

[0332] 77. The method of claim 76, wherein the RNA molecule can be

[0333] 78. The method of claim 76, wherein the RNA molecule is transcribed in a strand-specific manner.

[0334] 79. The method of claim 75, wherein the protein is a subunit of a Polycomb group complex.

[0335] 80. The method of claim 81, wherein the reduction in polycomb group complex activity results in reduced gene silencing.

[0336] 9.) The method of claim 8, wherein the reduction in gene silencing is caused by the loss of a silencing histone modification, such as the loss of trimethylation of lysine 27 on histone 3 (H3K27me3).

[0337] 81. A method for obtaining a doubled haploid plant, the method comprising:

[0338] (a) obtaining a first plant transformed with an expression cassette comprising:

[0339] i. Morphological development genes, or

[0340] ii embryogenic factors, or

[0341] iii. (i) and (ii), or any combination thereof;

[0342] (b) obtaining a second plant, wherein the second plant comprises a marker gene;

[0343] (c) pollinating the first plant with pollen from the second plant;

[0344] (d) selecting at least one haploid offspring produced by the pollination step (c), wherein the haploid offspring comprises the genome of the first plant but does not comprise the genome of the second plant.

[0345] 82. The method of claim 81, wherein the marker gene is selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, C1, CRC, R-nj, R1-scm and anthocyanin pigments.

[0346] 83. The method of claim 81, wherein the haploid offspring are treated with a chromosome doubling agent to produce edited doubled haploid offspring.

[0347] 84. The method of claim 83, wherein the chromosome doubling agent is colchicine, nafate, dithipyr, trifluralin, or another known anti-microtubule agent.

[0348] 85. The method of claim 83, further comprising isolating and culturing the doubled haploid plant embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0349] Figure 1 Shown the relative efficacy of somatic embryogenesis induction after 14 days. The relative efficacy of somatic embryogenesis is shown as relative to control plasmid infection, and after infection with experimental plasmid, the percentage change (%; y axis) of the response embryo that shows somatic embryogenesis development on the 14th day. Shown for each in embryogenic factor 1-16, the relative efficacy of somatic embryogenesis.

[0350] Figure 2A Shown is the efficacy of somatic embryogenesis of immature maize embryos in response to plasmids co-expressing the DZ470 cyclin gene and a cyclin-dependent kinase gene (CDKA1 or CDKA3) in combination with the WUSCHEL gene (% response; y-axis). Figure 2B Shown is the efficacy of somatic embryogenesis of immature maize embryos in response to plasmids co-expressing the DZ470 cyclin gene and a cyclin-dependent kinase gene (CDKA1 or CDKA3) in combination with the ODP2 / BBM gene (% response; y-axis).

[0351] Figure 3It is a schematic diagram of improved microspore embryogenesis using cell reprogramming treatment (which activates microspore embryogenesis of male-producing characteristics). The microspore generation step leading to microspore development is shown. The method disclosed herein describes a method for providing a cell reprogramming agent to plant cells to change cell fate, such as first by applying low temperature stress pretreatment to the male tassel, wherein the cell fate is no longer able to develop microgametogenesis, and secondly, by applying cell reprogramming treatment. Cell reprogramming treatment leads to improved microspore embryogenesis response of producing male characteristics, characterized in that cells proliferate into multicellular structures, followed by embryo-like structures, followed by microspore-derived embryoids, and then microspore-derived plants after embryoid germination and regeneration. The method disclosed herein can use chromosome doubling treatment, such as chromosome doubling treatment can be applied to microspore-derived plantlets to obtain microspore-derived double haploid plants.

[0352] Figure 4A-4E The relative efficacy of various DNA particle bombardment treatments compared to untreated controls (NTC) is shown. Figure 4A Shown are the rates of embryo-like structure (ELS) productivity in response to particle bombardment with expression vectors containing: i) WUS2 and ODP2 / BBM polynucleotides (labeled DG, (SEQ ID NO: 210)), ii) dCas9-CBF1A polynucleotide without gRNA (labeled dCas9, (SEQ ID NO: 85)), iii) gRNA molecules targeting WUS and BBM genomic loci (without dCas9-CBF1A polynucleotide) (labeled gRNA, PHP89615 (SEQ ID NO: 201), PHP89613 (SEQ ID NO: 203), PHP89611 (SEQ ID NO: 205), and RV038531 (SEQ ID NO: 206)), iv) a combination of gRNA molecules targeting WUS and BBM genomic loci and dCas9-CBF1A expression plasmid (labeled RNP), and v) unlabeled gold particles (labeled gold). All the above treatments were normalized to untreated control (NTC) cells. The hatched and solid bars represent two (2) replicates of the experiment. Figure 4B and Figure 4C Images of developing embryoid bodies are shown in response to: iv) RNP treatment targeting the WUS and BBM genomic loci ( Figure 4B and Figure 4C 5 mm and 3 mm scale bars, respectively). Figure 4D Microspore embryo-like structures (ELS) in response to iii) gRNA treatment using freshly isolated microspores are shown (line 1 = 0.9 mm). Figure 4EEmbryoids developing in response to iii) gRNA treatment using 14-day microspore cultures are shown (0.5 mm scale bar).

[0353] Figure 5A-5D Embryoid development in response to dCas9-HAT1 RNP treatment targeting WUS and BBM genomic loci is shown. Cellular reprogramming response of microspore-derived cells is shown. ( Figure 5A )(4 mm scale bar) No-bombardment control treatment 14 days after bombardment, ( Figure 5B ) (6 mm scale bar) dCas9-HAT1 RNP processing 14 days after bombardment, ( Figure 5C ) No-bombardment control treatment 28 days after bombardment (imaged in 100 mm diameter dishes), and ( Figure 5D ) dCas9-HAT1 RNP treatment 28 days after bombardment (imaged in a 100 mm diameter dish).

[0354] Fig. 6A The use of a truncated BBM (BBM) operably linked to an egg cell promoter (ZmRKD2 egg cell promoter or egg cell Pv-egg cell PRO (TR1) egg cell promoter) was shown. 404 ) genes (respectively, producing ZmRKD2pro::BBM 404 Expression cassette or Pv-oocyte PRO(TR1)pro::BBM 404 Schematic representation of the method for maternal haploid induction using expression cassettes (SEQ ID NO: 119 and SEQ ID NO: 120, respectively). Figure 6B Shows the use of ZmRKD2pro::BBM 404 or Pv-EGG CELL PRO(TR1)pro::BBM 404 Percentage of CFP-negative embryos lacking the paternal genome after transformation with the expression cassette (% paternal genome deletion; y-axis).

[0355] Figure 7 A schematic diagram of simultaneous haploid induction and genome modification using color-coded nonhaploid inducers as pollen donors is shown.

[0356] Figure 8A-8F Shows from Figure 7 Photographic images of diploid and haploid embryos for two (2) independent events isolated from the haploid induced cross described in . Fig. 8A and Figure 8B Shown are CFP and RFP expression in diploid and haploid embryos from two (2) independent events under bright field illumination. Figure 8C and Fig.8DShown are CFP and RFP expression in diploid and haploid embryos from two (2) independent events under fluorescent illumination. Fig. 8E and Figure 8F Shown is the expression of CFP and RFP in diploid and haploid embryos from two (2) independent events under fluorescent illumination. Figure 8C and Fig.8D In , haploid detection was demonstrated by identifying two (2) independent events of embryos lacking CFP. Fig. 8E and Figure 8F Haploids with putative CRE excision events are shown in Figure 5 by identifying CFP-deficient and RFP-deficient embryos from two (2) independent events. DETAILED DESCRIPTION

[0357] The disclosure herein will be described more fully hereinafter with reference to the accompanying drawings, in which some but not all possible aspects are shown. Indeed, the disclosure can be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure can satisfy applicable legal requirements.

[0358] Those skilled in the art to which the disclosed methods and compositions relate will consider that many modifications and other aspects disclosed herein have the benefit of the teachings presented in the following description and the associated drawings. Therefore, it should be understood that the disclosure is not limited to the specific aspects disclosed, and modifications and other aspects are intended to be included within the scope of the appended claims. Although specific terms are employed herein, these terms are used only in a general and descriptive sense and not for purposes of limitation.

[0359] It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. As used in the specification and claims, the term "comprising" may include aspects of "consisting of". Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. In this specification and the claims that follow, reference will be made to several terms defined herein.

[0360] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a protein" includes one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless expressly stated otherwise.

[0361] All patents, publications, and patent applications mentioned in this specification are indicative of the levels of those skilled in the art to which the disclosure pertains. All patents, publications, and patent applications are incorporated herein by reference in their entirety to the same extent as if each individual patent, publication, or patent application was expressly and individually indicated to be incorporated by reference in its entirety.

[0362] In plants, germline cells (germline) provide transgenerational inheritance of genetic information in each offspring by producing spore mother cells during sporogenesis. For example, sporogenesis provides megaspore mother cells that develop female gametes, egg cells and central cells that produce embryos and endosperm, respectively; or microspore mother cells that develop male gametes, producing four haploid microspores, each of which further develops into mature pollen grains. A key aspect of the unique role of germline cells is to provide genetic information that future offspring receive, with half of the genetic contribution coming from female gametes and half of the genetic contribution coming from male gametes. The egg cell is fertilized with a sperm cell to form a diploid zygote, while a second sperm cell fuses with the two polar nuclei of the central cell to form a triploid endosperm. The endosperm is the terminal nutritional tissue of the embryo, but does not contribute to the germline. After fertilization, the zygote produces an embryo, a process known as zygotic embryogenesis, which is a characteristic of sexual reproduction. The newly formed embryo undergoes an embryogenic developmental program comprising an underlying regulatory program influenced by genetic determinants and epigenetic reprogramming, leading from the embryogenic cell state to the acquisition of one or more differentiated cell fates, ultimately giving rise to the plant with all its differentiated tissues.

[0363] The method disclosed herein can change such developmental programs of the above-mentioned plant sexual reproduction. Such methods are valuable as plant reproduction methods for agricultural purposes. For example, the mechanism of changing the sporophyte fate to reproductive fate or returning the obtained cell state to the embryogenic cell state (e.g., germline or pluripotent cell state) from the differentiated cell state is particularly interesting. The disclosure provides methods using the molecular mechanisms under the cell reprogramming technology, which can be used for agricultural purposes and crop improvement.

[0364] Cell reprogramming refers to a method of providing a stimulus to change the cell fate of a treated cell. Cell reprogramming generally involves reverting a differentiated, more specialized cell to an induced pluripotent stem cell state. Such methods may also include transdifferentiation, defined as converting a cell other than a stem cell into a second cell type.

[0365] As used herein, "reprogramming" ("reprogram" or "reprograming" or "reprogramed") is the process of making mature specialized cells return or sensitize to induced pluripotent stem cells or to further develop into the embryonic / embryogenic state of embryonic or embryonic-like structures. In the cell colony "reprogrammed", it is expected that not all cells are "reprogrammed" to the same degree or to the same embryonic / embryogenic state. It is generally expected that the mixing of cells in the various states of reprogramming or the mosaic of cells. Compared with the cells not yet exposed to the method and composition provided herein, it is expected that the method and composition provided herein can increase the ratio or percentage of cells reprogrammed and in the desired embryonic / embryogenic state. Reprogramming also refers to the reconstruction of germ cell development. When embryogenesis inducing polypeptide and / or small molecule compounds contact plant cells so that plant cell embryogenesis, reprogramming can occur. The method disclosed herein provides contacting haploid plant cells with embryogenesis inducing agents (such as, for example, polypeptides and / or small molecule compounds) to reprogram cell fate and make cells become embryogenic. Alternatively, a polynucleotide encoding an embryogenesis-inducing polypeptide may be introduced and expressed in a plant cell, wherein the embryogenesis-inducing polypeptide affects surrounding / neighboring cells, thereby causing those surrounding / neighboring cells to become embryogenic. Cells may be reprogrammed in situ or ex situ.

[0366] As used herein, "cell reprogramming factors" or "embryogenesis inducers" include but are not limited to small molecules, compounds, embryogenic factor gene products, and morphological developmental gene embryogenic induction gene products that function independently or synergistically (including, for example, microspore embryogenic induction) in cell fate reprogramming. When cells are contacted with small molecules, it is believed that these reprogramming molecules activate the expression of endogenous genes in cells that trigger embryogenic responses in contacted cells.

[0367] As used herein, a "cell reprogramming treatment" is any treatment disclosed herein that elicits an embryogenic response in a contacted cell.

[0368] Treatment with a cell reprogramming agent (embryogenesis inducing polypeptide or embryogenesis inducing compound) or cell reprogramming treatment of plant cells within a tissue of an organism prior to cell isolation or cell extraction for experiments and / or measurements being performed in an external environment is referred to as "in situ" treatment or treatment method.

[0369] Cell reprogramming affects cell fate, and may cause various types of cell fate changes.A kind of cell fate is to become omnipotent cells, characterized in that cells of sporophyte and extraembryonic cells (such as endosperm cells in plants) can be formed.Another kind of cell fate is to become pluripotent cells, characterized in that cells of all cell types (excluding extraembryonic cells) comprising sporophyte can be produced.Embryogenic cells that can directly organogenesis can be considered as pluripotent.Another kind of cell fate is characterized in that it becomes pluripotent cells, which is defined as cells that can develop into more than one cell type but more limited than pluripotent cells (such as plant cells that experience indirect organogenesis).Reprogramming can also refer to the epigenetic marker characteristics of cell states that eliminate differentiation or are more specialized, and rebuild the epigenetic marker characteristics of embryogenic cell states.

[0370] The present disclosure includes methods for cell reprogramming using embryogenic factors as cell reprogramming agents to produce non-transgenic or transgenic plants. These embryogenic factors can be used in combination with morphogenic genes.

[0371] The disclosure also includes a method for carrying out cell reprogramming to produce non-transgenic plants or transgenic plants by treating cells with heterologous proteins as cell reprogramming agents, wherein the cell reprogramming agent includes a heterologous protein compounded with a guide RNA, which can change the gene regulation of the endogenous locus encoding embryogenic factors. The heterologous protein used as a cell reprogramming agent can also be combined with a gRNA for recruiting the heterologous protein / gRNA complex to the locus encoding the morphogenesis gene, for recruiting the heterologous protein / gRNA complex to the endogenous locus encoding the embryogenic factor.

[0372] The present disclosure provides methods for producing transgenic plants by rapidly inducing somatic embryogenesis in wild-type cells using embryogenic factors alone or in combination with morphogenic genes. These methods can be used in plant breeding programs, and in particular crop plant breeding programs.

[0373] The present disclosure also provides methods for producing non-transgenic microspore-derived doubled haploid populations by co-culturing microspores with purified proteins, such as embryogenic factor gene product proteins, with or without chemical treatment.

[0374] The present disclosure also provides methods for improving productivity of maize maize doubled haploid systems, specifically by improving the regeneration of haploid plants per sampled haploid embryo.

[0375] As used herein, "asexual reproduction" means reproduction without the fusion of gametes.

[0376] As used herein, "central cell" means the female gamete that produces the endosperm.

[0377] As used herein, "egg cell" means the female gamete that gives rise to an embryo.

[0378] As used herein, "megaspore mother cell" means a cell that develops into a female gametophyte, also known as a megaspore mother cell or a functional megaspore (FMS).

[0379] As used herein, "microspore mother cell" means a cell that develops into a male gametophyte, also known as a microsporocyte.

[0380] As used herein, "gametogenesis" means the development of the gametophyte from a spore.

[0381] As used herein, "parthenogenesis" means the formation of an embryo from an unfertilized egg cell.

[0382] As used herein, "pseudofertilization" means the fertilization-dependent formation of the endosperm from a central cell.

[0383] As used herein, "sexual reproduction" means a mode of reproduction in which female (egg) and male (sperm) gametes fuse to form a zygote.

[0384] As used herein, "somatic embryogenesis" means the formation of embryos from spore somatic cells in the absence of gamete and seed formation.

[0385] As used herein, "sporogenesis" means the formation of spores from a spore mother cell.

[0386] As used herein, "spore mother cell" means the first cell of the reproductive lineage, formed from the sporophyte cells in the female and male reproductive tissues of plants.

[0387] As used herein, "vegetative propagation" means a form of propagation in which new plants are formed without the formation of embryos.

[0388] As used herein, the term "embryo" means embryos and their progeny, immature and mature embryos, immature zygotic embryos, zygotic embryos, somatic embryos, embryonic callus, and embryos derived from mature ear-derived seeds. The embryo is a structure capable of germinating to form a plant.

[0389] As used herein, "haploid" means a plant or plant cell that has a single set of chromosomes (genome) and a reduced chromosome number (n) equal to the number of chromosomes in the gametes.

[0390] As used herein, the term "1n" or "1n cell" means a cell containing a single set of chromosomes, typically a product of meiosis. Examples of 1n cells include gametes, such as sperm cells, egg cells, or tissues derived from gametes by mitosis, such as 1n embryos or 1n plants. In maize, where plants are typically diploid and gametes are haploid, embryos or plants derived from such gametes are referred to as haploid embryos and haploid plants.

[0391] As used herein, "diploid" means a plant or plant cell that has two sets (genomes) of chromosomes, and the number of chromosomes (2n) is equal to the number of chromosomes in the zygote.

[0392] As used herein, the term "2n" or "2n cell" means a cell containing two sets of chromosomes. Examples of 2n cells include a zygote, an embryo produced by mitosis of a zygote, or a plant produced by germination of a 2n embryo.

[0393] As used herein, "haploid plant" means a plant having a single set of chromosomes (genome) and a reduced chromosome number (n) equal to the number of chromosomes in the gametes.

[0394] As used herein, the term "diploid plant" means a plant having two sets (genomes) of chromosomes, and the number of chromosomes (2n) is equal to the number of chromosomes in the zygote.

[0395] As used herein, "doubled haploid" means that a haploid cell is a cell with a single genome (male or female).

[0396] As used herein, "anther" is the part of the stamen that contains a microsporangium attached to a filament. In angiosperms (flowering plants), the microsporangium produces a microsporocyte (also called a microspore mother cell), which then produces four microspores by meiosis. The microspores divide by mitosis to produce pollen grains.

[0397] As used herein, "locule" is the compartment within the anther that contains the male gametes during microgametogenesis.

[0398] The term "microgametogenesis" is the process in plant reproduction in which microgametophytes (referred to herein as "microspores") develop into three-celled pollen grains.

[0399] As used herein, "microsporangium" or plural "microsporangia" is a sporangium that produces spores that give rise to male gametophytes. In nearly all land plants, the sporangium is the site of meiosis and produces genetically distinct haploid spores.

[0400] The term "microspore embryogenesis" means the activation of embryogenesis in microspores that causes or induces the microspores to be in an embryogenic state with male-producing characteristics.

[0401] The term "microspore-derived embryo" or "microspore-derived embryoid" means one or more cells derived from a microspore that have the cell fate and developmental characteristics of a cell undergoing embryogenesis. An embryo or embryoid is an organized structure capable of germination in vitro to produce a plantlet.

[0402] The term "androgenic" refers to the induction of androgenesis, in which the embryo contains only the paternal chromosomes of haploid or diploid cells (parthenogenesis).

[0403] As used herein, a "haploid" plant has a single set of chromosomes (genome), and the reduced chromosome number (n) in a haploid plant is equal to the number of chromosomes in the gametes.

[0404] As used herein, a "diploid" plant has two sets (genomes) of chromosomes, and the number of chromosomes (2n) is equal to the number of chromosomes in the zygote.

[0405] As used herein, "doubled haploid" or "doubled haploid plant or cell" is developed by doubling the haploid chromosome set. Plants or seeds obtained from doubled haploid plants (self-fertilized for any number of generations) can still be identified as doubled haploid plants. Doubled haploid plants are considered to be homozygous plants. If the plant is fertile, it is doubled haploid, even if the entire vegetative part of the plant is not composed of cells with doubled chromosome sets. For example, if the plant contains viable gametes, it is considered to be a doubled haploid plant even if it is mosaic.

[0406] As used herein, a "doubled haploid embryo" is an embryo having one or more cells that contain two homozygous sets of chromosomes that can subsequently grow into a doubled haploid plant.

[0407] As used herein, the term "clone" means a plurality of propagated plant cells or plants that are genetically, epigenetically and morphologically identical.

[0408] As used herein, the term "gamete" means a reproductive cell produced by meiosis, such as a sperm cell, an egg cell, or an ovule cell.

[0409] As used herein, the term "haploid embryo" means a gamete-derived somatic cell structure.

[0410] As used herein, the term "somatic structure" means a tissue, an organ, or an organism.

[0411] As used herein, the term "somatic cell" is a non-gametic cell. Somatic cells, tissues or plants can be haploid, diploid, triploid, tetraploid, hexaploid, etc. A whole set of chromosomes is referred to as 1n (haploid), wherein the number of chromosomes found in a single set of chromosomes is referred to as the haploid number (x). For example, in the diploid plant maize (Zea mays), 2n=2x=20 total chromosomes, and in the diploid rice Asian cultivated rice (Oryza sativa), 2n=2x=24 total chromosomes. In triploid plants such as bananas, 2n=3x=33 total chromosomes. In the hexaploid wheat common wheat (Triticum aestivum), 2n=6x=42. Ploidy levels may also vary between cultivars within the same species, for example in Saccharum officinarum where 2n=10x=80 chromosomes, but commercial sugarcane varieties range from 100 to 130 chromosomes.

[0412] As used herein, the term "culture medium" includes compounds in liquid, gaseous or solid state.

[0413] As used herein, the term "selectable marker" means a transgene that, when expressed in a transformed / transfected cell, confers resistance to a selective agent such as antibiotics, herbicides, and other compounds that are toxic to non-transformed / non-transfected cells.

[0414] As used herein, the term "EAR" means "ethylene response element binding factor-associated amphipathic repressor motif", which has a general consensus sequence of LLxLxL, DNLxxP, LxLxPP, R / KLFGV or TLLLFR as a transcriptional repression signal within a transcription factor. Adding an EAR-type repressor element to a DNA binding protein (such as a transcription factor, dCAS9 or LEXA (as an example)) confers a transcriptional repression function to the fusion protein (Kagale, S., and Rozwadowski, K. 2010. Plant Signaling and Behavior 5: 691-694).

[0415] As used herein, the term "transcription factor" means a protein that controls the transcription rate of a specific gene by binding to the DNA sequence of the promoter and up-regulating or down-regulating expression. Examples of transcription factors (also morphogenic genes) include members of the AP2 / EREBP family (including BBM (ODP2), plethora and aintegumenta subfamilies, CAAT-box binding proteins (such as LEC1 and HAP3) and members of the MYB, bHLH, NAC, MADS, bZIP and WRKY families.

[0416] As used herein, the term "synthetic transcription factor" refers to a molecule comprising at least two domains, a recognition domain and a regulatory domain that do not occur naturally in nature.

[0417] As used herein, the term "expression cassette" means a unique component of a vector DNA consisting of coding and non-coding sequences including 5' and 3' regulatory sequences that control expression in transformed / transfected cells.

[0418] As used herein, the term "coding sequence" means the portion of a DNA sequence defined by the start and stop codons that encode the amino acids of a protein.

[0419] As used herein, the term "non-coding sequence" means a portion of a DNA sequence that is transcribed to produce messenger RNA but does not encode amino acids for a protein, such as a 5' untranslated region, introns, and a 3' untranslated region. Non-coding sequences may also refer to RNA molecules, such as microRNAs, interfering RNAs, or RNA hairpins, which when expressed can downregulate the expression of endogenous genes or other transgenics.

[0420] As used herein, the term "regulatory sequence" means a segment of a nucleic acid molecule that is capable of increasing or decreasing gene expression. Regulatory sequences include promoters, terminators, enhancer elements, silencing elements, 5'UTR and 3'UTR (untranslated regions).

[0421] As used herein, the term "transfer cassette" means a T-DNA comprising one or more expression cassettes flanked by a right border and a left border.

[0422] As used herein, the term "T-DNA" means the portion of a Ti plasmid that is inserted into the genome of a host plant cell.

[0423] As used herein, the term "embryogenic factor" means a gene that enhances cell reprogramming when expressed, and the gene can promote the formation of the improvement of somatic cell-derived structures. More precisely, the ectopic expression of embryogenic factors stimulates the de novo formation of organogenesis structures (such as structures from embryogenic callus), which can improve the formation of embryos. The de novo embryogenic formation of this stimulation occurs in cells expressing embryogenic factors or in neighboring cells. The embryogenic factor gene can be a transcription factor that regulates the expression of other genes, or affects the gene of hormone levels in plant tissues, or affects the gene of the enzyme that affects cell reprogramming in plant cells, any of which can stimulate the change of embryogenesis. As used herein, the term "embryogenic factor" means an embryogenic factor gene and / or a protein that serves as a cell reprogramming agent expressed by an embryogenic factor gene.

[0424] Embryogenic factors are involved in plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, initiation of somatic embryogenesis, acceleration of somatic embryo maturation, initiation and / or development of apical meristem, initiation and / or development of shoot meristem, or a combination thereof.

[0425] Identification of embryogenic factors and / or methods for altering the regulation of genes encoding embryogenic factors that result in the induction of cellular reprogramming, particularly in cells of recalcitrant cell types or recalcitrant species or recalcitrant genotypes, are useful techniques for plant breeding methods. The present methods can be used in plant breeding methods (including plant transformation methods, doubled haploid plant production methods, or a combination thereof) by using embryogenic factors as cellular reprogramming agents, and using methods that include treating cells with heterologous proteins to modulate endogenous loci encoding embryogenic factors in the genome of the cells.

[0426] The embryogenic factors disclosed herein are directed to stimulating in vitro embryogenesis, which is characterized by the development of embryos outside the body of a plant, such as stimulating in vitro embryogenesis from microspores contacted with the embryogenic factor.

[0427] In one aspect, the embryogenic factors disclosed herein relate to stimulating in vivo embryogenesis, characterized in that embryos develop in situ, such as stimulating in vivo embryogenesis from an egg cell contacted with the embryogenic factors. In one aspect, the embryogenic factors disclosed herein relate to stimulating embryogenesis in cells from other plant tissues, including but not limited to protoplasts, leaves, hypocotyls, or any other plant explants that can be used in sporophytic embryogenesis methods.

[0428] In one aspect, the present disclosure provides a method for producing a haploid plant, the method comprising expressing an embryogenic factor in an egg cell of a sexually reproducing plant, resulting in an increase in the percentage of offspring containing half the number of chromosomes as compared to the parent.

[0429] In one aspect, the present disclosure provides a method for producing haploid plants, the method comprising (a) infecting plant cells with a bacterial strain containing a plasmid, the plasmid comprising a transfer DNA containing a degenerate Cas9 fusion protein gene and a guide RNA having sequence identity with a locus encoding an embryogenic factor gene, and (b) expressing a degenerate Cas9 fusion protein in a cell to stimulate cell reprogramming, resulting in obtaining an embryogenic cell fate, and (c) regenerating plants from treated cells. In one aspect, the treated cell is a sporophyte cell having the same number of chromosomes as the parent. In one aspect, the treated cell is a gametophyte cell having half the number of chromosomes as the parent.

[0430] In one aspect, the present disclosure provides a method for producing plants using asexual reproduction. Asexual reproduction is a mode of reproduction in flowering plants characterized by the development of diploid cells in an embryo sac into embryos without fertilization. Parthenogenesis is a form of asexual reproduction and, in a broader sense, can include the formation of de novo embryos from haploid gametophytic cells (e.g., egg cells produced by megasporogenesis, or microspores produced by microsporogenesis).

[0431] In one aspect, the present disclosure provides a method of (a) infecting a plant cell with a bacterial strain containing a plasmid comprising a transfer DNA containing an embryogenic factor gene operably linked to a regulatory element active before or during megasporogenesis, and (b) regenerating progeny in the absence of sexual fertilization in an egg cell to produce apomictic clones.

[0432] Particularly for plant cells and maize microspores, the present disclosure provides methods for improving the developmental fate of cell reprogramming toward embryogenesis, including improving the stress adaptation process caused by cell separation and separation techniques. On the one hand, a method for inhibiting the development of protoplasts in microspores into starch plastids, or dedifferentiating starch plastids into protoplasts or promoting autophagy in maize microspores is provided.

[0433] The present disclosure provides an efficient and effective method for producing a population of recombinant inbred lines, including but not limited to a method for initiating embryogenesis in a plant cell so as to enable the production of a double haploid recombinant population. The present disclosure also provides a method for achieving cell reprogramming and embryogenic growth stimulation in non-transformed cells, and particularly in gametes or haploid cells, during gamete or haploid cell development. The present disclosure provides a method for promoting microspore embryogenesis in a cell, tissue or organ of a plant by contacting a cell, tissue or organ with a cell reprogramming factor (including an embryogenic factor) that can reprogram cells, tissues or organs (wherein embryogenesis is induced in cells, tissues or organs), the cell reprogramming factor being such as, for example, an embryogenic inducing compound.

[0434] The disclosure provides a method for reprogramming microspores by co-cultivating microspores with purified protein such as embryogenic factor gene products. On the other hand, a method for reprogramming microspores by co-cultivating microspores in the presence of cells expressing embryogenic factor gene products is provided. The time period of co-cultivating (contacting) with embryogenic induction cell reprogramming agents will change according to the intractability of processed microspores. For example, on the one hand, microspore embryogenesis is proved by the existence of multicellular structure (MCS) in sporopollenin coating and / or the rupture of microspore exine and / or the existence of embryonic structure (ELS). On the one hand, microspores are co-cultivated with embryogenic induction cell reprogramming agents until some characteristics are observed, such as MCS and / or ELS. Alternatively, other phenotypic and / or genotypic markers are also used to determine processed microspore embryogenesis state or cell reprogramming state. Typically, less than 1 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 In some embodiments, the present invention provides a method for co-cultivating the microspores of the cultured microspores for 60 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, 80 days, The present disclosure also provides methods for generating a microspore-derived doubled haploid population by promoting microspore embryogenesis from a tissue or organ of a progeny plant (e.g., a first generation F1 hybrid or alternatively in a later progeny or backcross progeny) generated from a genetic cross of two different strains under hemizygous transgenic conditions using the methods described herein.

[0435] The present disclosure provides methods for promoting microspore embryogenesis from tissues or organs of first generation F1 hybrids derived by transforming the F1 embryos themselves into the F1 hybrids directly regenerated under hemizygous transgenic conditions for the purpose of generating a microspore-derived doubled haploid population. In additional aspects, the generated and / or treated microspores and / or microspore-derived cells are contacted with a chromosome doubling agent to promote diploidization of microspore-derived embryoids.

[0436] Embryogenic factors that can be used in combination with morphological development genes are related to plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, the initiation of somatic embryogenesis, the acceleration of somatic embryo maturation, the initiation and / or development of apical meristems, the initiation and / or development of shoot meristems, or a combination thereof, to improve cell reprogramming. When embryogenic factors are co-expressed with morphological development genes, an improved method for obtaining plants is provided.

[0437] The present disclosure provides a method for improving cell reprogramming and generating transgenic events, the method comprising (a) infecting plant cells with a bacterial strain containing a plasmid, the plasmid comprising a transfer DNA containing an embryogenic factor gene and / or a morphogenic gene and (b) regenerating a transgenic event. The embryogenic factor gene is selected from any one of the embryogenic factor genes disclosed herein. The morphogenic gene is selected from (i) a nucleotide sequence encoding a functional WUS / WOX polypeptide, or (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or (iii) a combination of (i) and (ii).

[0438] The present disclosure provides methods for promoting microspore embryogenesis in cells, tissues or organs of plants by contacting the cells, tissues or organs with cell reprogramming factors (including embryogenic factors) capable of reprogramming the cells, tissues or organs (wherein embryogenesis is induced in the cells, tissues or organs), such as, for example, embryogenic factors and / or embryogenic inducing compounds that induce protein products of exogenous morphogenetic developmental genes.

[0439] Also provided is a method for generating a microspore-derived doubled haploid population in non-transformed cells, and particularly in gametes or haploid cells, during gamete or haploid cell development by ectopically expressing a fusion protein gene product of an embryogenic factor that induces embryogenesis and a translocation signal fusion protein gene product and / or a fusion protein gene product of an embryogenic inducing morphological development gene and a translocation signal fusion protein gene product in plant tissues or organs, thereby achieving cell reprogramming and embryogenic growth stimulation. On the other hand, the disclosure provides during gamete or haploid cell development, in non-transformed cells, and particularly in gametes or haploid cells, a method for producing a double haploid population derived from microspores in plant tissues or organs by the following method: using an embryogenic factor gene that induces embryogenesis operably connected to a translocation signal peptide and a fluorescent protein and / or an embryogenic morphological development gene that is operably connected to a translocation signal peptide and a fluorescent protein and selecting based on the presence or absence of an embryogenic factor gene / translocation signal peptide / fluorescent protein fusion and / or a morphological development gene / translocation signal peptide / fluorescent protein fusion that induces embryogenesis, thereby achieving cell reprogramming and embryogenic growth stimulation. The disclosure provides a method for reprogramming microspores by co-culturing microspores with purified proteins such as embryogenic factor gene products and / or morphological development gene embryogenic induction gene products. On the other hand, a method for reprogramming microspores by co-culturing microspores in the presence of cells expressing embryogenic factor gene products and / or morphological development gene embryogenic induction gene products is provided.

[0440] The present disclosure provides a method for cloning and propagating a plantlet derived from a cell of a maternal haploid embryo, wherein the maternal haploid embryo is produced by ectopically expressing an embryogenic factor with or without a translocation signal peptide and / or a morphological development gene with or without a translocation signal in a plant tissue or organ. A method for cloning and propagating a plurality of gene-edited plantlets derived from a cell of a maternal haploid embryo is also provided, wherein the maternal haploid embryo is produced by ectopically expressing an embryogenic factor with or without a translocation signal peptide in a plant tissue or organ, and / or ectopically expressing a morphological development gene with or without a translocation signal peptide, which is fused to a gene product of a nuclease gene with or without a translocation signal peptide.

[0441] The present disclosure also provides a method for promoting embryogenesis and gene editing in endosperm cells using a transformed haploid inducer system in maternally derived haploid embryo cells, wherein the transformed haploid inducer system expresses a gene product of an embryogenic factor with or without a translocation signal and a nuclease gene with or without a fertilization translocation signal peptide and / or a morphological development gene with or without a translocation signal peptide and a nuclease gene with or without a fertilization translocation signal peptide. In a further aspect, the treated maternal haploid embryo and / or embryo-derived cell is contacted with a chromosome doubling agent to promote diploidization and regeneration of maternally derived somatic embryos.

[0442] As used herein, the term "morphogenic gene" or "morphogenic development gene" means a gene that stimulates the formation of somatic derived structures that can produce plants when ectopically expressed. More precisely, the ectopic expression of morphogenic genes stimulates the de novo formation of somatic embryos or organogenesis structures (e.g., shoot meristems) that can produce plants. This stimulated de novo formation occurs in cells expressing morphogenic genes or in neighboring cells. Morphogenic genes can be transcription factors that regulate the expression of other genes, or genes that affect hormone levels in plant tissues, both of which can stimulate morphological changes. Morphogenic genes can be stably incorporated into the genome of a plant or can be transiently expressed. As used herein, the term "morphogenic factor" means a morphogenic gene and / or a protein expressed by a morphogenic gene.

[0443] Morphogenic genes related to plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, regeneration, initiation of somatic embryogenesis, accelerated somatic embryo maturation, initiation and / or development of apical meristem, initiation and / or development of shoot meristem, initiation and / or development of shoots, or combinations thereof (e.g., WUS / WOX genes (WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, or WOX9), see U.S. Patents 7,348,468 and 7,256,322 and U.S. Patent Application Publications 2017 / 0121722 and 2007 / 0271628; Laux et al. (1996) Development 122:87-96; and Mayer et al. (1998) Cell 95:805-815; van der Graaff et al., 2009, Genome Biology [Genome Biology] 10: 248; Dolzblasz et al. 2016. Mol. Plant [Molecular Plant] 19: 1028-39) can be used in the methods of the present disclosure. It is expected that the regulation of WUS / WOX can regulate plant and / or plant tissue phenotypes, including plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, regeneration, initiation of somatic embryogenesis, acceleration of somatic embryo maturation, initiation and / or development of apical meristem, initiation and / or development of shoot meristem, initiation and / or development of shoots, or a combination thereof. Expression of Arabidopsis WUS can induce stem cells in vegetative tissues, which can differentiate into somatic embryos (Zuo, et al. (2002) Plant J [Plant Journal] 30: 349-359). Also of interest in this regard are the MYB118 gene (see U.S. Pat. No. 7,148,402), the MYB115 gene (see Wang et al. (2008) Cell Research 224-235), the BABYBOOM gene (BBM; see Boutilier et al. (2002) Plant Cell 14: 1737-1749), or the CLAVATA gene (see, e.g., U.S. Pat. No. 7,179,963). Morphogenic genes useful in the present disclosure include, but are not limited to, functional WUS / WOX genes.

[0444] The morphogenic polynucleotide sequences and amino acid sequences of WUS / WOX homeobox polypeptides can be used in the disclosed methods. As defined herein, a "functional WUS / WOX nucleotide" or "functional WUS / WOX gene" is any polynucleotide encoding a protein containing a homeobox DNA binding domain, a WUS box, and an EAR repressor domain (Ikeda et al., 2009 Plant Cell [Plant Cell] 21: 3493-3505). As demonstrated by Rodriguez et al., 2016 PNAS www.pnas.org / cgi / doi / 10.1073 / pnas.1607673113, removal of the dimerization sequence left behind the homeobox DNA binding domain, the WUS box, and the EAR repressor domain produces a functional WUS / WOX polypeptide. The WUSCHEL protein (hereafter referred to as WUS) plays a key role in the initiation and maintenance of the apical meristem, which contains a pool of pluripotent stem cells (Endrizzi et al., (1996) Plant Journal 10:967-979; Laux et al., (1996) Development 122:87-96; and Mayer et al., (1998) Cell 95:805-815). Arabidopsis mutants of the WUS gene contain stem cells that are misspecified and appear to undergo differentiation. WUS encodes a homeodomain protein that may function as a transcriptional regulator (Mayer et al., (1998) Cell 95:805-815). The stem cell population of the Arabidopsis shoot meristem is thought to be maintained by a regulatory loop between the CLAVATA (CLV) gene, which promotes organ initiation, and the WUS gene, which is required for stem cell identity, wherein the CLV gene represses WUS at the transcriptional level, and WUS expression is sufficient to induce meristem cell identity and expression of the stem cell marker CLV3 (Brand et al., (2000) Science 289:617-619; Schoof et al., (2000) Cell 100:635-644). Constitutive expression of WUS in Arabidopsis has been shown to result in adventitious shoot proliferation in leaves (in situ) (Laux, T., Talk Presented at the XVI International Botanical Congress Meeting, August 1-7, 1999, St. Louis, Mo.).

[0445] In one aspect, the functional WUS / WOX homeobox polypeptides useful in the methods of the present disclosure are WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX5A or WOX9 polypeptides (see, U.S. Pat. Nos. 7,348,468 and 7,256,322 and U.S. Patent Application Publication Nos. 2017 / 0121722 and 2007 / 0271628, which are incorporated herein by reference in their entirety and van der Graaff et al., 2009, Genome Biology 10: 248). Functional WUS / WOX homeobox polypeptides useful in the methods of the present disclosure may be obtained or derived from any plant. Functional WUS / WOX nucleotides encoding proteins containing a homeobox DNA binding domain, a WUS box and an EAR repressor domain useful in the methods of the present disclosure are listed in Table 1 below.

[0446] Table 1.

[0447]

[0448]

[0449]

[0450] Other morphogenic genes that can be used in the present disclosure include, but are not limited to, LEC1 (U.S. Patent No. 6,825,397, incorporated herein by reference in its entirety, Lotan et al., 1998, Cell 193: 1195-1205), LEC2 (Stone et al., 2008, PNAS 105: 3151-3156; Belide et al., 2013, Plant Cell Tiss. Organ Cult 113: 543-553), KN1 / STM (Sinha et al., 1993. Genes Dev 7: 787-795), the IPT gene of Agrobacterium (Ebinuma and Komamine, 2001, In vitro Cell. Dev Biol-Plant 37: 103-113), MONOPTEROS-δ (Ckurshumova et al., 2014, New Phytol. 204:556-566), the Agrobacterium AV-6b gene (Wabiko and Minemura 1996, Plant Physiol. 112:939-951), a combination of the Agrobacterium IAA-h and IAA-m genes (Endo et al., 2002, Plant Cell Rep. 20:923-928), the Arabidopsis SERK gene (Hecht et al., 2001, Plant Physiol. 127:803-816), the Arabidopsis AGL15 gene (Harding et al., 2003, Plant Physiol. 128:804-816), the Arabidopsis thaliana AGL16 gene (Harding et al., 2003, Plant Physiol. 129:811-813), the Arabidopsis thaliana AGL17 gene (Harding et al., 2003, Plant Physiol. 130:813-814), the Arabidopsis thaliana AGL18 gene (Harding et al., 2003, Plant Physiol. 131:814-815), the Arabidopsis thaliana AGL19 gene (Harding et al., 2003, Plant Physiol. 132:815-816), the Arabidopsis thaliana AGL17 gene (Harding et al., 2003, Plant Physiol. Physiol. [Plant Physiology] 133: 653-663), FUSCA gene (Castle and Meinke, Plant Cell [Plant Cell] 6: 25-41) and PICKLE gene (Ogas et al., 1999, PNAS [Proceedings of the National Academy of Sciences of the United States of America] 96: 13839-13844).

[0451] The disclosure also includes plants obtained by any method or composition disclosed herein. The disclosure also includes seeds from plants obtained by any method or composition disclosed herein. As used herein, the term "plant" refers to whole plants, plant organs (such as leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny thereof. As used herein, the term "plant" refers to whole plants, plant organs (such as leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (such as callus, undifferentiated callus, immature and mature embryos, immature zygotic embryos, immature cotyledons, hypocotyls, suspension culture cells, protoplasts, leaves, leaf cells, root cells, phloem cells and pollen). Plant cell includes but is not limited to from following cell: seed, suspension culture, explant, immature embryo, embryo, zygotic embryo, somatic embryo, embryogenic callus, meristem, somatic meristem, organogenic callus, protoplast, embryo derived from mature ear derived seed, leaf base, leaf from mature plant, leaf tip, immature inflorescence, tassel, immature ear, long whisker, cotyledon, immature cotyledon, meristem region, callus, cell from leaf, cell from stem, cell from root, cell from bud, gametophyte, sporophyte, pollen, microspore, multicellular structure (MCS) and embryo-like structure (ELS). Plant part comprises differentiation and undifferentiated tissue, includes but is not limited to root, stem, bud, leaf, pollen, seed, tumor tissue and various forms of culture in the cell (for example single cell, protoplast, embryo and callus). Plant tissue can be in plant or in plant organ, tissue or cell culture. Grain means mature seeds produced by commercial growers for purposes other than cultivation or propagation of species. Progeny, variants and mutants of regenerated plants are also included within the scope of the present disclosure, as long as these progeny, variants and mutants are derived from regenerated plants made using the methods and compositions disclosed herein and / or contain introduced polynucleotides disclosed herein.

[0452] As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant that contains a heterologous polynucleotide within its genome. Typically, the heterologous polynucleotide is stably integrated into the transgenic or transformed plant genome so that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. It should be understood that, as used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of heterologous nucleic acids, including those transgenics that were initially so altered and those generated from the initial transgene by sexual hybridization or asexual propagation. A transgenic plant is defined as a mature fertile plant containing a transgene.

[0453] A transgenic "event" is produced by transforming a plant cell with a heterologous DNA construct comprising a nucleic acid expression cassette comprising a gene of interest; regenerating a population of plants resulting from the insertion of the transferred gene into the genome of the plant; and selecting plants characterized by insertion at a specific genomic location. An event is phenotypically characterized by the expression of the inserted gene. At the genetic level, an event is part of the genetic makeup of a plant. The term "event" also refers to progeny resulting from a sexual cross between a transformant and another plant, wherein the progeny comprises the heterologous DNA.

[0454] Composition and method of the present disclosure can be applied to plant species of wide range, including dicotyledon and monocotyledon.Representative examples of plants that can be processed according to method disclosed herein include but are not limited to wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, barley, oat, rice, maize, triticale, sorghum, rye, millet, flax, sugarcane, banana, cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus (Eucalyptus), wheat grass (wheat grass), turf grass, alfalfa, clover, soybean, peanut, citrus, papaya, Setaria sp, cocoa, cucumber, apple, Capsicum (Capsicum), bamboo, melon, ornamental plants (including commercial garden and bulb flower species), fruit trees, vegetable species, Brassica species and interspecific hybrids.In a preferred embodiment, composition and method of the present disclosure are applied to maize plants.

[0455] The methods disclosed herein involve introducing a polypeptide, a polynucleotide (i.e., DNA or RNA) or a nucleotide construct (i.e., DNA or RNA) into a plant. As used herein, "introducing" is intended to refer to presenting a polynucleotide, a polypeptide or a nucleotide construct to a plant in a manner that allows the polynucleotide, a polypeptide or a nucleotide construct to enter the interior of a plant cell. The methods disclosed herein do not depend on a particular method for introducing a polynucleotide, a polypeptide or a nucleotide construct into a plant, as long as the polynucleotide, a polypeptide or a nucleotide construct is able to enter the interior of at least one cell of the plant. Methods for introducing a polynucleotide, a polypeptide or a nucleotide construct into a plant are known in the art, including but not limited to stable transformation methods, transient transformation methods, and virus-mediated methods.

[0456] As used herein, "stable transformation" is a transformation in which a polynucleotide or nucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that a polynucleotide or nucleotide construct is introduced into the plant and is not integrated into the genome of the plant, or a polypeptide is introduced into the plant. In addition, in certain embodiments, "transient" can mean that there is an embryogenesis inducing agent in a cell, wherein this agent has been exogenously applied or secreted from adjacent cells or produced from an extrachromosomal position (e.g., a plasmid or another independent replication source), or is not produced by a recombinant DNA construct that is stably integrated in the same cell.

[0457] As used herein, "contact", "contact with" or "contact with" means "direct contact" or "indirect contact". For example, a cell is placed under conditions where the cell can be in contact with any embryogenic inducing substance disclosed herein, including but not limited to embryogenic factors, morphological development genes, small molecules or doubling agents that induce embryogenesis. Such substances are allowed to be present in an environment (e.g., culture medium or expressed in cells or expressed in neighboring cells) in which the cell survives and can act on the cell. For example, a culture medium containing a doubling agent can be in direct contact with a haploid cell, or a culture medium containing a doubling agent can be separated from a haploid cell by filter paper, plant tissue or other cells, so that the doubling agent passes through the filter paper or the cell is transferred to the haploid cell.

[0458] As used herein, the term "biparental hybrid" is the cross fertilization of two genetically different plants to obtain the first hybrid generation of offspring and / or any subsequent successive hybrid generations. As used herein, biparental hybrids include offspring as progeny of any hybrid generation of offspring, including cross fertilization of offspring with one of its parental lines or individuals genetically similar to its parents to obtain a closer genetic identity with the parents, known as a "backcross," and / or any subsequent successive backcross generations.

[0459] The methods provided herein rely on the use of bacterial-mediated and / or biolistic-mediated gene transfer to produce regenerable plant cells. Bacterial strains that can be used in the methods of the present disclosure include, but are not limited to, Agrobacteria disarmament, Ochrobactrum bacteria, or Rhizobiaceae bacteria (U.S. Patent No. 9,365,859, which is incorporated herein by reference in its entirety). Standard protocols for particle bombardment (Finer and McMullen, 1991, In Vitro Cell Dev. Biol. -Plant 27: 175-182), Agrobacterium-mediated transformation (Jia et al., 2015, Int J. Mol. Sci. 16: 18552-18543; US 2017 / 0121722, incorporated herein by reference in its entirety), or Ochrobacterium-mediated transformation (US 2018 / 0216123, incorporated herein by reference in its entirety) can be used for the methods and compositions of the present disclosure. A variety of methods for introducing heterologous genes into plants are known and can be used to insert polynucleotides into plant hosts, including biological and physical plant transformation protocols. See, e.g., Miki et al., "Procedures for Introducing Foreign DNA into Plants," Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds. (CRC Press, Inc., Boca Raton, pp. 67-88 (1993). The method of choice varies with the host plant and includes chemical transfection methods such as calcium phosphate, microbial-mediated gene transfer such as Agrobacterium (Horsch, et al., (1985) Science 227:1229-31), Ochrobacterium (US2018 / 0216123), electroporation, microinjection, and biolistic bombardment. Expression cassettes and vectors for plant cell or tissue transformation and regeneration of transgenic plants, as well as in vitro culture methods are known and available. See, e.g., Gruber, et al., "Vectors for Plant Transformation [Vectors for Plant Transformation]," Methods in Plant Molecular Biology and Biotechnology [Methods in Plant Molecular Biology and Biotechnology], ibid., pp. 89-119.

[0460] Transformation protocols and protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocot or dicot). Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No. 5,563,055 and Zhao, et al., U.S. Pat. No. 5,981,840), Ochrobacterium-mediated transformation (US Pat. No. 5,981,840), and sphingobacterium-mediated transformation (US Pat. No. 5,981,840). 2018 / 0216123), direct gene transfer (Paszkowski, et al., (1984) EMBO J [Journal of the European Molecular Biology Association] 3: 2717-2722) and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., (1995), in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, Gamborg and Phillips, eds. (Springer-Verlag, Berlin; McCabe et al. (1988) Biotechnology [Biotechnology] 6: 923-926); and Lec1 transformation method (WO 00 / 28058). See also Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.Theoretical and Applied Genetics 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855, 5,322,783, and 5,324,646; Klein et al. (1988) Plant Genetics 10:117-130 (rice); Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, Chapman et al., eds. (Longman, New York, NY, 1986). =New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Ishida et al. (1996) Nature Biotechnology 14:745-750 (transformation via Agrobacterium tumefaciens tumefaciens), all of which are incorporated herein by reference in their entirety.Methods and compositions for rapid plant transformation are also found in 2017 / 0121722, which is incorporated herein by reference in its entirety. Vectors useful for plant transformation are found in U.S. Patent Application Serial No. 15 / 765,521, which is incorporated herein by reference in its entirety. .

[0461] Methods for harvesting tassels (including sterilization methods) and tassel pretreatment (e.g., temperature pretreatment) are known in the art and will vary depending on the intended use of the tassel. Specifically, before selecting tassels for microspore culture, the microspores must be brought to an appropriate stage, usually between the uninucleate stage and the binucleate stage. Typically, for tassels with anthers and microspores at the appropriate stage, the tassels are dissociated and each tassel is individually wrapped, for example, in aluminum foil.

[0462] Isolation of microspores typically occurs after pretreatment of the tassels in a reduced temperature environment to improve the response of male production characteristics. A commonly used technique is to place the foil-wrapped tassels at 10°C for 1 to 21 days. In addition, pre-culture of anthers can be performed in a mannitol solution (e.g., 0.3M liquid mannitol plus 50mg / L ascorbic acid) (U.S. Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, incorporated herein by reference in their entirety).

[0463] Before use, the tassels can be surface sterilized in a 40% Clorox (diluted 8.25% sodium hypochlorite v / v) solution (with two drops of Tween 80) for about fifteen minutes while gently agitating on a reciprocating shaker. The tassels are then rinsed three or more times in sterile water at room temperature and placed in a large petri dish and typically kept uncovered for 1-1.5 hours under sterile conditions to evaporate excess moisture before microspores are isolated. Another method of surface sterilization includes placing the spikelets dissociated from the tassel in a permeable basket, which is then immersed in a 40% Clorox (diluted 8.25% sodium hypochlorite v / v) solution (with two drops of Tween 80) for fifteen minutes and then rinsed as described above. The spikelets are placed in a large petri dish and typically kept uncovered for 1-1.5 hours to evaporate excess moisture before microspores are isolated.

[0464] Procedures for isolating maize anthers and spikelets include, but are not limited to, the glass rod immersion method (Pescitelli, et al., (1990) Plant Cell Rep. 8:628-31), the blending method, the blade tissue cutting method (see U.S. Patent 5,445,961, incorporated herein by reference in its entirety), the tissue homogenizer method (Gaillard, et al., (1991) Plant Cell Rep. 10:55-8), and the tissue grinder method (Mandaron et al., (1990) Theor Appl Genet 80:134-138).

[0465] After the microspores are separated from the surrounding somatic tissue, they are usually placed in fresh isolation medium immediately after the microspores are separated from any anther fragments. Many medium compositions are known in the art. A common method for separating microspores from anther fragments is to pass a blended microspore anther fragment slurry from the isolation procedure through a sieve (Pescitelli (1989) Plant Cell Rep. [Plant Cell Report] 7: 673-6, Gaillard, et al., (1991) and U.S. Patent 5,445,961, which are incorporated herein by reference in their entirety). Alternatively, the microspore anther fragment slurry is passed through several layers of cheesecloth or mesh filters (Coumans, (1989) Plant Cell Rep. [Plant Cell Report] 7: 618-21). Further separation can be performed using discontinuous density centrifugation methods or additional filtration methods, including but not limited to methods using sucrose or Percoll gradients (Coumans, (1989), Pescitelli et al., (1990)). Alternatively, a final high sucrose (0.44M) centrifugation method can be used to further separate the selection of cells captured at the 20%-30% interface of a Percoll gradient in the range of 20%-50% after centrifugation at 225g for 3 minutes (Gaillard, et al., (1991)). Further variations of separation methods are known in the art (Vergne et al., (1991) In: Negrutiu I. (ed.) BioMethods. Birkhauser, Basel, Boston, Bedinger and Edgerton, (1990) Plant Physiol. [Plant Physiology] 92: 474-9, Gaillard, et al., (1991)) and can be optimized as needed.

[0466] The specific culture medium used during isolation, for example, is typically composed of 6% sucrose, 50 mg / L ascorbic acid, 400 mg / L proline, 0.05 mg / L biotin and 10 mg / L nicotinic acid (see Petolino and Genovesi (1994) The Maize Handbook, Freeling, M., Walbot, V. (eds.) Springer-Verlag, New York). Various other media and solutions used to culture maize microspores are similar to those used for other cereal tissue culture procedures, and various modifications may be used (see Genovesi and Magill, (1982) Plant Cell Rep. 1:257-60, Martin and Widholm, (1996) Plant Cell Rep. 15:781-85, Magnard et al., (2000) Plant Mol Biol 44:559-74, Testillano et al., (2002) Int J Dev Biol 46:1035-47, Testillano et al., (2004) Chromosoma 112:342-9, Shariatpanahi et al., (2006) Plant Cell Plant Cell Rep 25:1294-9, Shim et al. (2006) Protoplasma 228:79-86, Soriano et al. (2008) Plant Cell Rep 27:805-11, Cistue et al. (2009) Plant Cell Rep 28:727-35, Jacquard et al. (2009) Planta 229:393-402, Jacquard et al. (2009) Plant Cell Rep 28:1329-39, Shim et al. (2009) Genome 52:166-74, Sanchez-Diaz et al. (2013) Plant Reprod 26:287-96). Common features of maize culture media generally include the use of N6, NLN or YP basal salts formulations with relatively high sugar concentrations (6%-12%), which may have components including triiodobenzoic acid, various plant hormones and / or proline.

[0467] The compositions and methods disclosed herein include producing double haploid plants from gametes by contacting plant cells with embryogenic factor gene products and / or morphological development gene embryogenic induction gene products, wherein the embryogenic factor gene products and / or morphological development gene embryogenic induction gene products can induce cell reprogramming and activate embryogenesis in cells. By contacting isolated microspores with embryogenic factor gene products and / or morphological development gene embryogenic induction gene products, a non-in situ cell reprogramming method for induction of male characteristics is provided.

[0468] Optionally, the ex situ methods of the present disclosure use isolated microspores co-cultured with suspended "feeder cells" expressing embryogenic factor gene products and / or morphogenic gene products to further promote cellular reprogramming to activate microspore embryogenesis.

[0469] Optionally, the ex situ cell reprogramming methods of the present disclosure can be combined and used with microspores isolated from plant tissue produced using the in situ cell reprogramming methods disclosed herein.

[0470] The present disclosure provides a method for reprogramming in situ cells for regenerating maternal haploid embryos by transforming a maize haploid inducer system to stably integrate and express a heterologous expression cassette, wherein the heterologous expression cassette encodes a morphological development polypeptide that stimulates somatic embryogenesis and also encodes a second component of a gene for gene editing purposes. Both components can include a fusion peptide using a secretion signal peptide that is operably connected to a promoter expressed in endosperm. Reporter genes or selectable marker genes can also be included in the expression cassette of the present disclosure. Examples of suitable reporter genes known in the art can be found, for example, in Jefferson et al., (1991) Plant Molecular Biology Manual, Gelvin et al., eds. (Kluwer Academic Publishers), pp. 1-33; DeWet et al., (1987) Mol. Cell. Biol. 7:725-737; Goff et al., (1990) EMBO J. 9:2517-2522; Kain et al., (1995) BioTechniniques 19:650-655; and Chiu et al., (1996) Current Biology 6:325-330, which are incorporated herein by reference in their entirety.

[0471] Selectable marker genes for selecting transformed cells or tissues may include genes that confer antibiotic resistance or herbicide resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al., (1983) EMBO J. [Journal of the European Molecular Biology Association] 2: 987-992); methotrexate (Herrera Estrella et al., (1983) Nature [Nature] 303: 209-213; Meijer et al., (1991) Plant Mol. Biol. [Plant Molecular Biology] 16: 807-820); hygromycin (Waldron et al., (1985) Plant Mol. Biol. [Plant Molecular Biology] 5: 103-108 and Zhijian et al., (1995) Plant Mol. Biol. [Plant Molecular Biology] 5: 104-108); Science 108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176); sulfonamides (Guerineau et al. (1990) Plant Mol. Biol. 7:171-176); Mol. Biol. [Plant Molecular Biology] 15: 127-36); bromoxynil (Stalker et al., (1988) Science [Science] 242: 419-423); glyphosate (Shaw et al., (1986) Science [Science] 233: 478-481 and U.S. Patent Application Serial Nos. 10 / 004,357 and 10 / 427,692); glufosinate (DeBlock et al., (1987) EMBO J. [Journal of the European Molecular Biology Association] 6: 2513-2518), which are incorporated herein by reference in their entirety.

[0472] Other genes may be used in the expression cassettes of the present disclosure (also aiding in the recovery of transgenic events) and include, but are not limited to, GUS (β-glucuronidase; Jefferson, (1987) Plant Mol. Biol. Rep. 5:387), GFP (green fluorescent protein; Chalfie et al., (1994) Science 263:802), luciferase (Riggs et al., (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen et al., (1992) Methods Enzymol. 216:397-414), and a maize gene encoding anthocyanin production (Ludwig et al., (1990) Science 247:449), which are incorporated herein by reference in their entirety.

[0473] The method of the present disclosure also provides the following expression: a first component, which comprises a plurality of embryogenic factor genes and / or morphogenic genes operably linked to a promoter in an expression cassette, and a second component, which comprises genes that can be used for gene editing purposes, including but not limited to: Streptococcus pyogenes (CRISPR) CAS9 or other nuclease proteins (including but not limited to zinc finger nucleases, large-range nucleases or transcription activator-like effector nucleases). The first component is used in the transformed maize haploid inducer line to fertilize the female ear of the target plant, which is useful for improving double haploid production, while the second component can improve the regeneration of gene-edited maize double haploids.

[0474] The present disclosure also provides methods of contacting haploid cells with an amount of a chromosome doubling agent before, during, after, or overlapping with any part of the separation and embryogenesis induction methods for generating a doubled haploid population of paternal gametes (producing male characteristics) or maternal gametes (producing female characteristics).

[0475] For plant cells outside the tissue of an organism (e.g., extracted cells that have been isolated for experiments and / or measurements completed in an external environment), the use of cell reprogramming agents (embryogenesis-inducing polypeptides or embryogenesis-inducing compounds) or cell reprogramming treatments is referred to as "ex situ" treatments or treatment methods.

[0476] As used herein, "recombinant" means a cell or vector that has been modified by introducing heterologous nucleic acids or derived from cells that have been so modified. Thus, for example, a recombinant cell is a cell expressing a gene that is not found in a natural (non-recombinant) cell in the same form or position, or a cell expressing a natural gene in an expression pattern different from that of a natural (non-recombinant) cell, for example, due to deliberate human intervention, the natural gene is abnormally expressed, insufficiently expressed, reduced in expression, or not expressed at all. As used herein, the term "recombinant" does not encompass changes in cells or vectors caused by naturally occurring events (e.g., spontaneous mutations, natural transformation / transduction / transposition) (such as events that occur without deliberate human intervention).

[0477] As used herein, a "recombinant expression cassette" is a nucleic acid construct that is recombinantly or synthetically produced and has a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be integrated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed and a promoter.

[0478] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0479] As used herein, polypeptides useful in the methods of the present disclosure can be further engineered with cell penetrating peptides (referred to herein as "CPPs"). CPPs useful in the methods of the present invention are a class of short peptides that have translocation across cell membranes and act as nanocarriers for delivering proteins to plant cells. Exemplary CPP families include, but are not limited to, CPPs derived from protein transduction domains, amphipathic peptides, and synthetic cationic polypeptides (e.g., polylysine, polyhistidine, and polyarginine) or dendritic polycationic molecules. Exemplary CPPs useful in the methods of the present disclosure include, but are not limited to, monomers and dimers of peptide vascular endothelial cadherin CPPs, transporter CPPs, HIV-1 TAT basic domain CPPs, penetrant CPPs, synthetic cationic high arginine oligopeptide CPPs (see Eudes and Chugh. (2008) Plant Signal Behav. [Plant Signal Behavior] 3: 549-550) and gamma zein CPPs (see US8581036, incorporated herein by reference in its entirety). The present disclosure provides methods of using γ-zein CPP morphogenetic protein translational fusion proteins for contacting γ-zein-linked structures with plant cells and allowing uptake of the γ-zein-linked structures into plant cells to alter the cell fate of plant cells.

[0480] The term "regulatory element" refers to a nucleic acid molecule with gene regulatory activity, that is, a nucleic acid molecule that can affect the transcription and / or translation expression pattern of an operably linked transcribable polynucleotide. Therefore, the term "gene regulatory activity" refers to the ability to affect the expression of an operably linked transcribable polynucleotide molecule by affecting the transcription and / or translation of the operably linked transcribable polynucleotide molecule. Gene regulatory activity can be positive and / or negative, and the influence can be characterized by its following characteristics: time, space, development, tissue, environment, physiology, pathology, cell cycle and / or chemical response, and can also be characterized by quantitative indication or qualitative indication.

[0481] Cis-regulatory elements are regulatory elements that affect gene expression. Cis-regulatory elements are non-coding DNA regions that regulate the transcription of adjacent genes, usually DNA sequences near the genes they regulate. Cis-regulatory elements usually regulate gene transcription by encoding DNA sequences that confer binding to transcription factors.

[0482] As used herein, "promoter" is an exemplary regulatory element, and generally refers to a nucleotide sequence that can control the expression of a coding sequence or functional RNA. Generally speaking, the coding sequence is located at the 3' end of the promoter sequence. The promoter sequence comprises a proximal element and a more distal upstream element, the latter element being generally referred to as an enhancer. Therefore, an "enhancer" is a nucleotide sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. The promoter may all be derived from natural genes, or may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. It will be appreciated by those skilled in the art that different regulatory elements may guide the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions.

[0483] Plant promoters are promoters that can initiate transcription in plant cells. Exemplary plant promoters include, but are not limited to, promoters obtained from plants, plant viruses, and bacteria (such as Agrobacterium or Rhizobium) containing genes expressed in plant cells. Examples are promoters that preferentially initiate transcription in certain tissues (such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma). Such promoters are called "tissue preference" promoters. "Cell type" specific promoters mainly drive expression in certain cell types (e.g., vascular cells in roots or leaves) in one or more organs. "Inducible" or "regulatory" promoters refer to promoters under environmental control. Examples of environmental conditions that can be affected by inducible promoters include the presence of anaerobic conditions or illumination. Another type of promoter is a developmentally regulated promoter, such as a promoter that drives expression during pollen development. Tissue preference promoters, cell type specific promoters, developmentally regulated promoters, and inducible promoters are members of the "non-constitutive" promoter category. A "constitutive" promoter is a promoter that causes a nucleic acid fragment to be expressed in most cell types at most times under most environmental conditions and states of development or cellular differentiation.

[0484] "Translation leader sequence" refers to a nucleotide sequence located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the fully processed mRNA upstream of the translation initiation sequence. The translation leader sequence can affect a variety of parameters, including processing of the primary transcript to mRNA, mRNA stability and / or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster, (1995) Mol. Biotechnol [Molecular Biotechnology] 3: 225-236).

[0485] Promoters useful in the methods of the present disclosure include those regulating paternal and / or maternal embryogenesis. For paternal embryogenesis, exemplary promoters include tassel-preferred promoters, anther-preferred promoters, and tapetum-preferred promoters. Tissue-specific, tissue-preferred, or stage-specific regulatory elements further include anther-specific LAT52 (Twell, et al., (1989) Mol. Gen. Genet. [Contemporary Genetics] 217: 240-245), microspore-specific promoters (such as apg gene promoters) (Twell, et al., (1993) Sex. Plant Genet. [Sexual Plant Reproduction] 6: 217-224), tapetum-specific promoters (such as TA29 gene promoter (Mariani, et al., (1990) Nature 347: 737; U.S. Patent No. 6,372,967), stamen-specific promoters (such as MS26 gene promoter, MS44 gene promoter, MS45 gene promoter, 5126 gene promoter, BS7 gene promoter, PG47 gene promoter (U.S. Patent No. 5,412,085; U.S. Patent No. 5,545,546; Zheng et al., (1993) Plant J [Plant Journal] 3 (2): 261-271), SGB6 gene promoter (U.S. Patent No. 5,470,359), G9 gene promoter (U.S. Patent No. 5,8937,850; U.S. Patent No. 5,589,610), SB200 gene promoter (WO 2002 / 26789) etc. Tissue-preferred promoters active in cells of male reproductive organs can be particularly used in the methods of the present disclosure.

[0486] For maternal embryogenesis, exemplary promoters include seed-preferred promoters. "Seed-preferred" promoters include "seed-specific" promoters (promoters active during seed development, such as promoters of seed storage proteins) and "seed germination" promoters (promoters active during seed germination) (see Thompson et al. (1989) BioEssays [Biological Assays] 10: 108, which is incorporated herein by reference). Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced signal) promoter; cZ19B1 (maize 19 kDa zein) promoter; and milps (inositol-1-phosphate synthase) promoter (see WO 00 / 11177 and U.S. Pat. No. 6,225,529, which are incorporated herein by reference in their entirety). Other promoters that can be used in the methods of the present disclosure include, but are not limited to, endosperm-specific promoters, such as the gamma-zein promoter (Boronat et al. (1986) Plant Science 47: 95-102) and embryo-specific promoters, such as the globulin-1 promoter. For monocots, seed-specific promoters include, but are not limited to, the maize 15 kDa promoter, the 22 kDa zein promoter, the 27 kDa zein promoter, the gamma-zein promoter, the waxy promoter, the shrunken 1 promoter, the shrunken 2 promoter, the globulin 1 promoter, and the like. The seed-preferred promoters from the end1 and end2 genes (see WO 00 / 12733) can be used in the methods of the present disclosure. Additional seed-preferred promoters useful in the methods of the present disclosure include the oleosin promoter (WO 00 / 0028058), the lipid transfer protein (LTP) promoter (US Pat. No. 5,525,716), the Lec1 promoter, the Jipl promoter, and the milps3 promoter (see WO 02 / 42424).

[0487] As used herein, a "signal peptide" or "secretion signal peptide" sequence refers to a region of a protein that interacts with a protein transport system and translocates or targets a protein for delivery to a specific destination. Examples of signal peptides or secretion signal peptides that can be used in the methods of the present disclosure include, but are not limited to, signal peptides that target proteins to the extracellular matrix of plant cells, such as the Nicotiana plumbaginifolia elongated gene signal peptide (DeLoose, et al., (1991) Gene 99: 95-100); signal peptides that cause protein secretion, such as the PRIb signal peptide (Lind, et al., (1992) Plant Mol. Biol. 18: 47-53) or the barley alpha amylase (BAA) signal peptide (Rahmatullah, et al., (1989) Plant Mol. Biol. 12: 119).

[0488] Secretion signal peptides useful in the methods of the present disclosure include those containing domains found in the superfamily of bifunctional inhibitor / plant lipid transfer proteins / seed storage helical domain proteins (characteristically encoding eight conserved cysteine ​​residues important for secondary structure) (including but not limited to lipid transfer proteins such as LILY-LIM2 (Q43534), sorghum (XP_002445754), barley (BAK05897), rice-OSC4 (BAD09233), rice-MEN-8 (XP_006660357), and maize-MZm3-3 (NP_001105123), which can be used to engineer plant-specific proteins for male expression). Secretion signal peptides that target proteins from the endosperm to the embryo can be used to engineer translational fusion proteins for female expression that can be used in the methods of the present disclosure.

[0489] As used herein, "heterologous" refers to a nucleic acid that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene that is from a species different from the species from which the structural gene was derived or, if from the same species, one or both are substantially modified from their original form and / or genomic location.

[0490] The embryogenic factor and / or morphological development gene that can be used for the induction embryogenesis in the method of the present disclosure can be provided in the expression cassette for expressing in the target plant. The expression cassette can include 5' and 3' regulatory sequences that are operably connected to the embryogenic factor gene sequence and / or morphological development gene sequence of the induction embryogenesis disclosed herein. "Operably connected" is intended to represent the functional connection between two or more elements. For example, the operably connected between the target polynucleotide and the regulatory sequence (i.e., promoter) is a functional connection that allows the target polynucleotide to be expressed. The operably connected element can be continuous or non-continuous. When used to refer to the connection (fusion protein) of two protein coding regions, operably connected means that the coding region is in the same reading frame. The box can additionally include at least one other gene to be co-transformed into the organism. Alternatively, one or more embryogenic factor genes and / or one or more morphological development genes that induce embryogenesis can be provided on multiple expression cassettes. Such expression cassette has multiple restriction sites for inserting embryogenic factor gene sequences and / or morphological development gene sequences that induce embryogenesis, which will be located under the transcriptional regulation of the regulatory region (one or more promoters). The expression cassette may additionally contain a selectable marker gene.

[0491] As used herein, chimeric signal peptide-embryogenic factor gene fusions and / or chimeric signal peptide-morphogenic gene fusions can be further engineered with translocation or nuclear localization signal sequences at the C-terminus of the polypeptide to promote improved cell reprogramming efficiency and embryogenic induction. The disclosed methods provide gene constructs encoding WUSCHEL proteins fused to polypeptides derived from bacterial virulence proteins that confer in situ translocation of secreted proteins. Agrobacterium tumefaciens and Agrobacterium rhizogenes are examples of plant pathogens that can transfer plasmid-encoded bacterial genes located on transferred DNA (T-DNA) into plant cells in a manner that depends on translocation of bacterial virulence (Vir) proteins. The translocation of fusions between CRE recombinase and VIR protein polypeptides (specifically VirE2 or VirF peptide sequences) directly demonstrates the role of translocation of proteins into plant cells conferred by Vir peptides (Vergunst et al., (2000) Science 290: 979-82). In addition, the C-terminal 27 amino acids of the GALLS protein of Agrobacterium rhizogenes have been shown to play a role in protein transport and nuclear localization (Hodges et al., (2006) J. Bacteriol. 188: 8222-30). The use of peptides encoding translocation or nuclear localization signals is known in the art (see US 6800791, which is incorporated herein by reference in its entirety).

[0492] The expression cassettes useful in the methods of the present disclosure may contain polynucleotides encoding: Ms44 signal peptide-WUSCHEL fusions with translocation or nuclear localization signal sequences, or similar Ms44 signal peptide-ODP2 fusions with translocation fusion peptides, which can be further engineered with cell penetrating peptides (referred to herein as "CPPs"). CPPs useful in the methods of the present invention are a class of short peptides that have properties of translocation across cell membranes and act as nanocarriers for delivering proteins into plant cells. Exemplary CPP families include, but are not limited to, CPPs derived from protein transduction domains, amphipathic peptides, and synthetic cationic polypeptides (e.g., polylysine, polyhistidine, and polyarginine) or dendritic polycationic molecules. Exemplary CPPs that can be used in the methods of the present disclosure include, but are not limited to, peptide vascular endothelial cadherin CPP, transporter CPP, monomers and dimers of HIV-1 TAT basic domain Cpp, penetratin CPP, synthetic cationic homoarginine oligopeptide CPP (see Eudes and Chugh. (2008) Plant Signal Behav. [Plant Signal Behavior] 3: 549-550) and gamma zein CPP (see US 8581036, which is incorporated herein by reference in its entirety). The present disclosure provides methods for using gamma-zein CPP morphogenetic protein translation fusion proteins for contacting gamma-zein-linked structures with plant cells and allowing the gamma-zein-linked structures to be taken up into plant cells to change the cell fate of plant cells. Also provided are engineered embryogenesis-inducing morphogenetic proteins for use in the methods of the present disclosure, the engineered embryogenesis-inducing morphogenetic proteins comprising a CPP fused to an ODP2 protein for use in combination with a chimeric signal peptide-WUSCHEL fusion protein. These genetic constructs are engineered to deliver to microspores and contact microspores with embryogenesis-inducing morphogenetic proteins comprising a CPP fused to an ODP2 protein for use in combination with a chimeric signal peptide-WUSCHEL fusion protein operably linked to an anther-specific promoter, or more specifically, a tapetum-specific promoter.

[0493] As used herein, such gene constructs can also be engineered to deliver embryogenic induction morphological development proteins to embryos and contact embryogenic induction morphological development proteins, and the embryos are more specifically maize haploid embryos. An expression cassette for use in the disclosed method is also provided, the expression cassette comprising a CPP fused to an ODP2 protein for operably combined use with a chimeric signal peptide-WUSCHEL fusion protein, whereby these proteins are engineered using a gene construct designed to have a chimeric endosperm or transfer cell layer signal peptide-WUSCHEL fusion protein operably connected to an endosperm-specific promoter, and a polynucleotide encoding an endosperm or transfer cell layer signal peptide-ODP2-CPP fusion peptide, so that the expressed protein is translocated from the endosperm to the embryo.

[0494] The present disclosure provides methods in which chromosomes can be doubled at the microspore stage, embryo stage, mature seed stage, or at any time between plant pollination and before germination of haploid seeds. Alternatively, spontaneous doubling can also occur.

[0495] The non-original position method of the present disclosure promotes microspore embryogenesis and cell reprogramming by contacting the isolated microspore with embryogenesis induction morphological development protein.The isolated microspore can contact with exogenous embryogenesis induction morphological development protein specificity to improve maize microspore embryogenesis.For example, as disclosed herein, the non-original position embryogenesis induction morphological development protein treatment cell reprogramming method uses a heterologous expression system to produce the recombinant WUSCHEL protein of purification.The method disclosed herein includes delivering the protein to plant cells, for example, using a transfection reagent to further promote the delivery of exogenous WUSCHEL protein to the isolated microspore cells.In some aspects, the protein delivery method with or without a transfection reagent may include electroporation and / or sonication methods carried out in the presence of reagents (such as dimethyl sulfoxide (DMSO), adjuvants, surfactants, etc.) that further promote the delivery of exogenous embryogenesis induction morphological development protein to the microspore cells.

[0496] Also provided are ex situ methods, which include contacting or treating isolated microspores with agents such as small molecules or compounds that achieve cell fate reprogramming and stimulate embryogenic cell proliferation. The present disclosure provides methods including co-culturing isolated microspores in an induction medium supplemented with a small molecule or compound. In some aspects, small molecule inhibitors of protein kinases are used in the methods of the present disclosure to perform cell reprogramming on plant cells.

[0497] The methods disclosed herein also provide for combining protein delivery cell reprogramming methods with or without transfection reagents, with and without electroporation methods and / or sonication methods, which can be performed in the presence of the above reagents (such as dimethyl sulfoxide (DMSO), adjuvants, surfactants, etc.) and cell reprogramming treatment using the above small molecules or compounds to improve cell reprogramming of plant cells.

[0498] The method disclosed herein also provides that the non-original and / or original situ methods may subsequently include the co-cultivation of the isolated microspores contacted with maize suspension "feeder cells" having embryogenesis and cell reprogramming characteristics. In particular, the method includes co-cultivating the isolated microspores in the presence of a transgenic maize suspension cell culture, which is transformed with a gene construct expressing an embryogenesis-inducing morphological development gene protein (such as WUSCHEL protein and / or ODP2).

[0499] On the one hand, feeder cells are engineered to express polynucleotides encoding polypeptides involved in growth stimulation, embryogenesis, cell reprogramming and / or cell cycle stimulation, to increase the frequency of haploid embryos, to increase the starting frequency of microspore-derived embryos and / or to stimulate and increase chromosome doubling efficiency. The polynucleotides that can be used in the method of the present disclosure include but are not limited to embryogenesis induction morphological development genes and cell cycle genes, including cyclin A, cyclin B, cyclin C, cyclin D, cyclin E, cyclin F, cyclin G and cyclin H; Pin1; E2F; Cdc25; RepA gene and similar plant virus polynucleotides encoding replication-related proteins. See U.S. Patent Publication No. 2002 / 0188965, incorporated herein by reference in its entirety.

[0500] In one aspect, the present disclosure provides a method comprising co-cultivating isolated microspores in the presence of a non-transgenic maize suspension cell culture (feeder cells), more specifically using feeder cells derived from a genotype having a responsive androgenic phenotype, such as, for example, ATCC40520 or ATCC40519 (see US5306864 A, incorporated herein by reference in its entirety), or a non-transgenic responsive inbred line such as HF1 (Martin and Widholm, (1996)).

[0501] The in situ methods of the present disclosure promote embryogenesis from plant tissues or organs by ectopically expressing morphogenetic developmental proteins in the tissue or organ or in adjacent tissues or organs.Genetic elements that provide spatiotemporal expression and localization to specific tissues or organs of plants can be used in the methods of the present disclosure.

[0502] In one aspect, the promoter employed in the methods of the present disclosure is the native maize Ms44 promoter (SEQ ID NO: 318), which results in the utilization of the spatiotemporal expression and localization characteristics of Ms44, an anther-specific gene, which was first detected in tapetum cells during meiosis that persists through uninucleate microspore development.

[0503] A signal peptide useful in the methods of the present disclosure is the native maize Ms44 signal peptide (SEQ ID NO: 320).

[0504] In the present disclosure, a heterologous expression cassette encoding the Ms44 promoter (SEQ ID NO: 318) regulating the anther-specific Ms44 signal peptide (SEQ ID NO: 320) is fused to a polynucleotide encoding the WUSCHEL peptide (SEQ ID NO: 322) to ectopically express the embryogenesis-inducing morphogenetic gene during microgametogenesis. The method of the present disclosure allows the synthesis and processing of the embryogenesis-inducing morphogenetic gene protein in the tapetum cells for secretion into the ovary chamber, resulting in contact with the microspores and the activity of the embryogenesis-inducing morphogenetic protein to induce cell reprogramming and activate microspore embryogenesis.

[0505] As used herein, a "chimeric gene expression cassette" is an expression cassette comprising a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence, and the expression cassette may include a transcription initiation region (i.e., a promoter) and a translation initiation region in the 5'-3' direction of transcription, a secretion signal peptide, an embryogenesis-inducing morphological development gene sequence, a fluorescent protein sequence, and a transcription and translation termination region (i.e., a termination region) that functions in plants.

[0506] In one aspect, a gene construct useful in the methods of the present disclosure includes a polynucleotide encoding: an Ms44 promoter and an Ms44 secretion signal peptide fused to a WUSCHEL protein, the WUSCHEL protein also being fused to a C-terminal 36 amino acid VirF translocation peptide sequence (SEQ ID NO: 329) (referred to herein as "virF C36 ”) or optionally fused to a C-terminal 127 amino acid VirF translocation peptide sequence (SEQ ID NO: 331) (referred to herein as “virF C127 ”) or optionally fused to a 27 amino acid translocation signal peptide (SEQ ID NO: 333) from the GALLS protein of Agrobacterium rhizogenes (referred to herein as “GS C27 ”) fusion to promote increased morphological activity and cellular reprogramming.

[0507] In one aspect, the genetic construct having embryogenicity-inducing morphogenetic development gene protein activity (cell reprogramming and embryogenicity-inducing activity) that can be used in the methods of the present disclosure can also include a fusion of the embryogenicity-inducing morphogenetic development gene with a cell-penetrating peptide to increase cell delivery and activity in a cell-non-autonomous manner (increase the embryogenicity-inducing effect on surrounding / neighboring cells).

[0508] In one aspect, the gene construct having embryogenicity-inducing morphogenetic development gene protein activity (cell reprogramming and embryogenicity-inducing activity) that can be used in the methods of the present disclosure can also include a fusion of the embryogenicity-inducing morphogenetic development gene with a glucocorticoid receptor (GR)-based fusion protein system (SEQ ID NO: 363 encoding SEQ ID NO: 364) to conditionally localize the protein activity to the cell nucleus by externally applying animal hormone analogs to in vitro tissue culture.

[0509] Promoters that can be used in the methods of the present disclosure include ZmBETL9 and a 5' untranslated region or a ZmBETL9-like promoter and a 5' untranslated region (SEQ ID NO: 348 and SEQ ID NO: 351, respectively), which are fused to a polynucleotide encoding an embryogenic induced morphological development gene (such as WUSCHEL peptide (SEQ ID NO:) or ovule development protein 2 (ODP2) (SEQ ID NO: 335)), thereby ectopically regulating the expression of the embryogenic induced morphological development gene during embryogenesis.

[0510] An endosperm secretion signal peptide, such as an N-terminal ZmBETL9 secretion signal peptide or a ZmBETL9-like secretion signal peptide (SEQ ID NO: 347 and SEQ ID NO: 350, respectively), can be used in the methods of the present disclosure, wherein the endosperm secretion signal peptide is fused to an embryogenesis-inducing morphological development gene protein so that the protein can be translocated from the endosperm to the embryonic cell during embryogenesis. Optionally, a translational fusion protein comprising a secretion signal peptide and an embryogenesis-inducing morphological development gene protein can be fused to a translocation signal peptide. In one aspect, the translational fusion protein can comprise a cell penetrating peptide. The methods disclosed herein achieve improved embryogenesis and cell reprogramming in plant cells, which also improves cell responses in subsequent plant tissue culture methods.

[0511] The in situ cell reprogramming method of the present disclosure improves the productivity of maternal haploid embryo regeneration and achieves gene editing to provide regenerated gene-edited maize double haploids, wherein the treated cells (but non-transgenic) are contacted with embryogenesis-inducing morphological development gene proteins derived from triploid endosperm cells that contain one paternal allele expressing a trait as a stable transformant.

[0512] In some aspects, a heterologous expression cassette encoding a ZmBETL9 promoter, a 5' untranslated region (SEQ ID NO: 348) and an N-terminal ZmBETL9 secretion signal peptide (SEQ ID NO: 346) or a ZmBETL9-like promoter, a 5' untranslated region (SEQ ID NO: 351) and an N-terminal ZmBETL9-like secretion signal peptide (SEQ ID NO: 349) is fused to a polynucleotide encoding an embryogenic-inducible morphogenetic developmental protein, such as a WUSCHEL peptide (SEQ ID NO: 322) or an ovule development protein 2 (ODP2) peptide (SEQ ID NO: 335), for use in the methods of the present disclosure, thereby ectopically regulating embryogenic-inducible morphogenetic developmental gene expression during embryogenesis.

[0513] On the one hand, haploid cell can be contacted with a certain amount of chromosome doubling agent to promote chromosome doubling, then regenerate homozygous diploid plant from treated haploid cell.Before, during or after microspore embryogenesis or embryo maturation, haploid microspore cell can be contacted with doubling agent.After chromosome doubling, the doubled haploid embryo will contain 2 copies of chromosome derived by father.The efficiency of the method for obtaining double haploid plant from haploid embryo can be greater than 10%, 20%, 30%, 50%, 60%, 70%, 80% or 90%.The duration of contact between haploid cell and chromosome doubling agent can vary.Contact time can be from less than 24 hours (for example 4-12 hours) to about one week.The duration of contact is generally from about 8 hours to 2 days.

[0514] Methods for chromosome doubling are disclosed in Antoine-Michard, S. et al., Plant cell, tissue organ cult., Kluwer Academic Publishers, Koldrecht, The Netherlands, 1997, 48(3): 203-207; Kato, A., Maize Genetics Cooperation Newsletter, 1997, 36-37; and Wan, Y. et al., TAG, 1989, 77: 889-892. Wan, Y. et al., TAG, 1991, 81: 205-211; the disclosures of which are incorporated herein by reference. A typical doubling method involves contacting cells with colchicine, an anti-microtubule agent or anti-microtubule herbicide, natrium, nitrous oxide, or any mitotic inhibitor to produce homozygous doubled haploid cells. The amount of colchicine used in the culture medium is typically 0.01%-0.2%, or about 0.05% methylamine phosphine (APM) (5-225 μM) can be used. The amount of colchicine can be in the range of about 400-600 mg / L or about 500 mg / L. The amount of natrium in the culture medium is about 0.5-20 μM. Examples of mitotic inhibitors are included in Table 2. Other agents can be used together with mitotic inhibitors to improve doubling efficiency. Such agents include dimethyl sulfoxide (DMSO), adjuvants, surfactants, etc.

[0515] Table 2. Chemical chromosome doubling agents

[0516]

[0517]

[0518]

[0519] As an alternative to using chemical chromosome doubling agents, regulating the expression of genes known to affect the plant cell cycle (genetic chromosome doubling proteins) by stimulating the cell cycle (and cell division) or by stimulating endoreduplication can be used to double the chromosome set in the embryo. By regulating the expression of genes that stimulate key control points in cell cycle cells, an increase in ploidy levels in plant cells can be achieved. In the present disclosure, BBM using an egg cell promoter has been demonstrated. 404 Overexpression of 404The simultaneous expression of ZM-DZ470 (maize cyclin-D family member) not only causes haploid embryo formation but also stimulates the doubling of chromosome number. Therefore, in forming haploid embryo, adding the cyclin-D of overexpression seems to provide the cell cycle stimulation of appropriate level, thereby causes 1n haploid chromosome number to double to 2n (diploid). It is expected that other known plant genes of stimulation plant cell cycle (or cell division) can be used to produce similar chromosome number doubling in forming embryo. Examples of plant genes whose overexpression stimulates the cell cycle include cyclin-A in tobacco (Yu et al., 2003), cyclin-D in tobacco (Cockcroft et al., 2000, Nature 405:575-79; Schnittger et al., 2002, PNAS [Proceedings of the National Academy of Sciences of the United States of America] 99:6410-6415; Dewitte et al., 2003, Plant Cell [Plant Cell] 15:79-92), E2FA in Arabidopsis (De Veylder et al., 2002, EMBO J [Journal of the European Molecular Biology Association] 21:1360-1368), E2FB in Arabidopsis (Magyar et al., 2005, Plant Cell [Plant Cell] 17:2527-2541). Similarly, overexpression of viral genes known to regulate plant cell cycle machinery can be used, such as when overexpression of the wheat dwarf virus RepA gene stimulates cell cycle progression (G1 / S transition) and cell division in maize (Gordon-Kamm et al., 2002, PNAS [Proceedings of the National Academy of Sciences of the United States of America] 99: 11975-11980). Conversely, plant genes whose encoded products are known to inhibit the cell cycle have been shown to cause increased cell division when genes such as inhibitor of cyclin-dependent kinase (ICK1 / KRP) in Arabidopsis are downregulated (Cheng et al. 2013, Plant J [Plant Journal] 75: 642-655). Therefore, downregulation of the KRP gene driven by an egg cell-specific promoter is expected to have a similar effect as overexpression of DZ470, resulting in chromosome doubling. Methods for down-regulating genes such as KRP are known in the art and include expressing artificial microRNAs targeting KRP mRNA, or expressing dCas9-repressor fusions targeting the KRP promoter by gRNA targeting the KRP promoter sequence. Finally, plant genes are known to exist that specifically affect the endoreduplication process.When such genes (such as, for example, the ccs52 gene or the Del1 gene) are used in the methods of the present disclosure, it is expected that overexpression of ccs52 will result in increased ploidy levels, as observed in Medicago sativa (Cebolla et al., 1999, EMBO J [Journal of the European Molecular Biology Association] 18: 4476-4484), and downregulation of Del1 will result in increased ploidy levels, as observed in Arabidopsis (Vlieghe et al., 2005, Current Biol [Contemporary Biology] 15: 59-63). It is expected that other genes known to stimulate the cell cycle, G1 / S transition, or endoreduplication can be used in the methods disclosed herein to increase ploidy levels.

[0520] An exemplary pathway for maintaining stem cell homeostasis in plant cells is the CLAVATA-WUSCHEL feedback signaling pathway, in which shoot meristems are maintained by pluripotent stem cells. This pathway coordinates the maintenance of stem cell proliferation and differentiation. Although first discovered in Arabidopsis, this pathway appears to be conserved in a variety of higher plant species such as maize, rice, and tomato (Somssich et al., 2016. Development [Development 1143: 3238-3248]. The pathway contains the stem cell-promoting transcription factor WUS and the differentiation-promoting peptide CLAVATA3 (CLV3) (Brand, U., et al., 2000. Science [Science] 289: 617-619). Although conserved in multiple higher plant species, multiple receptors have been shown in other plants to play a role in regulating stem cells and development, such as the rice gene FON1 (Suzaki, T, et al. 2006. Plant Cell Physiol. 47: 1591-1602), the maize CLAVATA 2 orthologs THICK TASSEL DWARF1 (TD1) and FASCIATED EAR2 (FEA2) (Bommert, P., et al., 2005. Development 132: 1235-1245), and the maize CLV-type LRR receptor-like gene, FASCIATEDEAR3 (FEA3) (Je, et al., 2016. Nat. Genet. 48: 785-791). Therefore, such species-specific proteins, which can repress induced cellular reprogramming provided by treating cells with morphogenetic developmental proteins, are relevant to the present disclosure.

[0521] In another example, the developmental regulation of the floral meristem of Arabidopsis has shown that the repression of WUS is controlled by the expression of the AGAMOUS (AG) protein. Here, AG directly induces the transcription of KNUCKLES (KNU), encoding a C2H2-type zinc finger protein with a conserved transcriptional repressor motif, which represses WUS transcription to eliminate stem cell activity, thereby controlling the determination of the floral meristem (Sun. B, et al., 2009. Gene Dev [Gene and Development] 23: 1791-804). For this pathway of stem cell regulation in flower development, a key aspect of coordinating stem cell maintenance and differentiation is not only the temporal expression pattern (where WUS activity activates AG activity, and AG activates KNU transcription), but also how the KNU locus is regulated. Notably, KNU transcription by AG requires the removal of repressive histone modifications at the KNU locus, such as the removal of trimethylation at the 27th lysine residue of the histone H3 protein (H3K27me3). Such repressive marks are removed in an AG-dependent manner. For the methods of the present disclosure, where WUS activity has a mechanistic link between transcriptional feedback and epigenetic regulation in plant stem cell proliferation, targeted repression of positively activated WUS repressors (such as KNU or other such repressor proteins) is expected to be of interest as well.

[0522] It is expected that ectopic WUS protein activity in plant cells will positively regulate the signaling pathway response to repress WUS activity.Herein, the genetic loci encoding the proteins for repressing WUS activity are considered useful as genomic target sites for the methods of the present disclosure.

[0523] Several other genetic loci that act as negative switches for embryogenesis have also been identified. When PICKLE (PKL) function is lost, the PKL gene encodes a CHD3 chromatin remodeling factor and an embryonic trait that develops ectopically in the root (Ogas J., et al., 1997. Science. [Science] 277: 91-94; Ogas J, et al., 1999. Proc. Natl Acad. Sci. USA. [Proceedings of the National Academy of Sciences of the United States] 96: 13839-13844). In pkl seedlings, LEC-class genes are derepressed, indicating that such developmental genes are silenced via a repressive chromatin state (Dean Rider, S. et al., 2003. Plant J. [Plant Journal] 35: 33-43). This finding is further supported by results showing robust expression of PKL and establishment of repressive H3K27me3 modifications on PKL-dependent genes during germination (Henderson JT. et al., 2004. Plant Physiol. 134:995-1005; Zhang H. et al., 2008. J Biol Chem. 283:22637-22648).

[0524] Likewise, deficiency in three VP1 / ABI3-LIKE (VAL) genes induces expression of embryogenic traits in seedlings, again characterized by ectopic LEC1 activation (Suzuki M. et al., 2007. Plant Physiol [Plant Physiology] 143: 902-911). The VAL genes encode transcriptional regulators containing PHD-like and CW domains characteristic of chromatin remodeling proteins, an EAR transcriptional repressor domain, and a B3 DNA binding domain (Suzuki M. et al., 2007. Plant Physiol [Plant Physiology] 143: 902-911). In addition, it has been reported that several polycomb group proteins and two histone deacetylases HDA6 and HDA19 are essential for repressing embryonic traits after germination (Makarevich, G. et al. 2006. EMBO Rep [European Molecular Biology Organization Report] 7: 947-952; Tanaka, M. et al. 2008. Plant Physiol [Plant Physiology] 146: 149-161). In another example, PHERES1 and FUSCA3 were demonstrated to be potential PcG target genes, and therefore, it was proposed that different PcG complexes repress such target genes at different stages of plant development to promote differentiation (Makarevich, G. et al., 2006. EMBO Rep. [European Molecular Biology Organization Report] 7: 947-52).

[0525] Repressor motifs are known in the art, see for example Kagale and Rozwadowski (Epigenetics. 2011. 6: 141-146). Transcriptional repression mediated by the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is known in plants, and the motif includes an EAR motif defined by the consensus sequence patterns of LxLxL and DLNxxP (see Hiratsu et al., 2003. Plant J. 35: 177-192). Of interest in the present disclosure are peptides that include an amphiphilic repressor motif (disclosed in WO 2013 / 109754 A1 and all references cited herein) and the Dr1 / DRAP1 global repressor complex (see US 7,288,695 B2 and all references cited herein), and the Dr1 / DRAP1 global repressor complex includes a Dr1 motif (similar to the motif found in Arabidopsis MYBL2) (see Matsui K, Umemura Y, Ohme-Takagi M. 2008. Plant J. 55: 954-967).

[0526] The in situ method of the present disclosure provides stable transgenic "microspore-activated" parental inbred lines for genetic crosses with a second wild-type parental inbred line to produce first-generation F1 hybrids.

[0527] In one aspect, the method of the present disclosure uses this hemizygous transgenic F1 hybrid to generate immature tassels, which can generate florets together with anthers containing developed microspores. Microspores are the products of meiosis, and thus, due to chromosomal crossover events during meiosis, each male gamete has a unique combination of genes inherited from the parents along the recombinant chromosomes. During meiosis, a single copy of the transgene at a single locus in the hemizygous state can segregate at a ratio of 1:1, resulting in half of the gametes being wild-type and the other half having the inherited transgenic locus. After meiosis, wild-type and transgenic gametes continue to develop in situ, where all developed microspores are exposed to the embryogenesis-inducing morphogenic gene protein, which is secreted from the tapetal cells of the sporophyte and is the protein translation from the single copy of the transgene in the hemizygous F1 genome. After isolating microspores from the tassel tissue, the method of the present disclosure induces cell reprogramming activity during microgametogenesis to improve microspore embryogenesis responsiveness and in vitro cell reprogramming. Non-transgenic microspore-derived embryoids are selected using standard methods.

[0528] On the one hand, two different inbred lines are cross-fertilized to produce the first generation F1 zygotic embryos developed in the fertilized female spike of the maternal parent. Each F1 zygotic embryo has two groups of (genome) chromosomes, each from each parent. After fertilization, for example 8 to 16 days after fertilization, for the purpose of transformation, the immature F1 zygotic embryo can be separated from the female spike of the maternal parent subsequently, so that the polynucleotides encoding the cell reprogramming factors of induced embryogenesis are stably integrated into the F1 plant genome. In this way, the F1 plant with the cell reprogramming gene construct of the induced embryogenesis of a single copy in the hemizygous state can be selected to sample the male spike tissue producing microspores in the anther. About the cell reprogramming transgenic of the induced embryogenesis of insertion, the microspore will be separated at a ratio of 1: 1 during gametogenesis, causing half of the gametes to be wild type, and the other half of the gametes have the cell reprogramming locus of the induced transgenic embryogenesis of inheritance. Thus, the methods of the present disclosure allow selection of F2 generation wild-type microspores with improved embryogenic responsiveness from hemizygous F1 hybrids to produce doubled haploid populations.

[0529] On the one hand, the method of the present disclosure also provides in situ protein delivery. The method includes transforming maize haploid inducer system to stably integrate and express one or more heterologous expression cassettes, and the heterologous expression cassette encodes two main functional activities: one activity includes proteins for inducing somatic embryogenesis and cell reprogramming, and another activity includes proteins for gene editing purposes. Both components are operably connected to one or more promoters expressed in endosperm (particularly embryo periregion (ESR) and / or basal endosperm transfer layer (BETL)). The method of the present disclosure uses the transformed haploid inducer system for fertilizing the female spike of the target plant to produce haploid embryos with improved double haploid plantlet regeneration and / or improved double haploid offspring regeneration through gene editing. In these methods, the expression of a heterologous expression cassette of an embryogenesis induction morphological development gene protein comprising a paternal allele from a triploid endosperm cell results in the protein being translocated to the haploid embryo by a transfer cell using a secretion signal peptide unique to an endosperm transfer cell. Once the rescued haploid embryos are cultured in vitro, the method of the present invention provides maternal haploid embryos with improved embryogenesis levels and plantlet regeneration capabilities.

[0530] sgRNA hybridizes with intragenic DNA, can have regulatory effects and affect gene regulation at the target site. This finding is consistent with the regulatory effects shown by bidirectionally expressed long noncoding RNA (lncRNA) transcripts, which can silence or activate genes. For example, transcription of RNA molecules from the polycomb / trithorax complex response element (PRE / TRE) downstream of the degenerate (vestigial, vg) gene of Drosophila was shown to affect gene regulation, where the forward strand transcript was associated with vg repression and the reverse strand transcript was associated with activation of vg expression (Herzog, et al. 2014. Nat. Genet. [Nature-Genetics] 46: 973-981). More specifically, reverse transcribed long noncoding RNA (lncRNA) bound to a subunit of the polycomb group complex (PcG) inhibited PcG histone methyltransferase activity and isolated the inhibitory PcG complex from the vg locus, thereby activating vg gene expression. It is also relevant that promiscuous in vitro binding of PcG proteins and RNA may occur and that nonspecific RNA binding to mammalian PcG complexes inhibits PcG activity (Davidovich, et al. 2013. Nat. Struct. Mol. Biol. [Natural Structural Molecular Biology] 20: 1250-1257; Cifuentes-Rojas, et al. 2014. Mol. Cell [Molecular Cell] 55: 171-185). Such binding may explain how the sgRNA used in the cell reprogramming method of the present disclosure acts as a functional switch to control gene regulation at the genomic target site.

[0531] In plant cells, the conservation of PcG activity is known and is associated with the regulation of developmental regulatory genes, including morphogenic genes used in the cell reprogramming methods disclosed herein. In Arabidopsis, the WUS locus is silenced after the recruitment of PcG proteins leads to the establishment of H3K27me3 post-translational modification, resulting in WUS gene repression (Liu et al. 2011. Plant Cell [Plant Cell] 23: 3654-3670). In maize, the PcG proteins that deposit H3K27me3 are conserved, and it is reported that the level of H3K27me3 deposited on the BBM locus in the tassel is increased compared to leaf tissue (Makarevitch et al. 2013. The Plant Cell [Plant Cell], Vol. 25: 780-793), indicating that BBM repression mediated by PcG activity is possible in isolated and cultured maize microspores.

[0532] Collectively, these results suggest that providing RNA molecules with sequence homology to loci encoding morphological developmental genes can improve cellular reprogramming, for example by activating expression of targeted genes with or without the activity of heterologous dCas9 fusion proteins. Promiscuous binding of PcG and RNA as previously shown can explain how sgRNAs used in the methods of the present disclosure can improve cellular reprogramming in treated cells, for example, by inhibiting PcG activity, thereby increasing WUS and / or BBM expression.

[0533] Methods for generating haploid inducer lines are known in the art, for example by ectopic expression of transcription factors containing an AP2 domain. For example, the method of Gordon-Kamm et al. (see U.S. Pat. No. 7,579,529; the contents of which are hereby incorporated by reference) is preferably used.

[0534] In addition, it was shown that the AP2 transcription factor apospory-specific-genomic-region BabyBoomLike (PsASGR-BBML) transgene of African pennisetum (Pennisetum squamulatum) induced parthenogenesis and embryo formation without fertilization. In maize, individuals with the PsASGR-BBML transgene fertilized with pollen marked with R1-navajo anthocyanin color exhibited haploid embryo production (Steffen JG, et al. 2007. Plant J [Plant Journal] 51: 281-292, US 2016 / 0304901 A1, which is incorporated herein by reference in its entirety). Recently, the method of Khanday and Sundaresan demonstrated similar findings, for example in rice (see WO 2018 / 098420 A1; the contents of which are hereby incorporated by reference).

[0535] The descriptions of SEQ ID NOs: 316-364 are shown in Table 3.

[0536] Table 3.

[0537]

[0538]

[0539]

[0540] Methods for inserting polynucleotides at the specific position of plant genome targeting are known in the art. The insertion of polynucleotides at the desired genome position is achieved using a site-specific recombination system. See, for example, WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855 and WO 99 / 25853, which are incorporated herein by reference in their entirety. In short, the target polynucleotide flanked by two different recombination sites may be included in a T-DNA transfer box. The T-DNA transfer box is introduced into a plant, which has stably incorporated a target site into its genome, and the target site flanks two different recombination sites corresponding to the site of the transfer box. Suitable recombinase is provided, and the transfer box is integrated into the target site. Thus, the target polynucleotide is integrated into the specific chromosome position in the plant genome.

[0541] The disclosed method can be used to introduce polynucleotides into explants, and the polynucleotides are used to target specific sites in the genome of plants derived from plant cells for modification. Site-specific modifications that can be introduced with the disclosed method include modifications produced using any method for introducing site-specific modifications, including but not limited to using gene repair oligonucleotides (e.g., U.S. Publication 2013 / 0019349), or by using double-strand break technology, such as TALEN, large-range nucleases, zinc finger nucleases, CRISPR-Cas, etc. For example, the disclosed method can be used to introduce the CRISPR-Cas system into plant cells or plants for the following purposes: genome modification of target sequences in the genome of plants or plant cells, selection of plants, deletion of bases or sequences, gene editing, and insertion of target polynucleotides into the genome of plants or plant cells. Therefore, the disclosed method can be used together with the CRISPR-Cas system to provide an effective system for modifying or changing target sites and target nucleotides in the genome of plants, plant cells or seeds. The Cas endonuclease gene is a plant-optimized Cas9 endonuclease, wherein the plant-optimized Cas9 endonuclease is capable of binding to a genomic target sequence of a plant genome and generating a double-strand break therein.

[0542] The Cas endonuclease is guided by a guide nucleotide to identify double-strand breaks at a specific target site and optionally introduce the double-strand breaks into the genome of the cell. The CRISPR-Cas system provides an effective system for modifying the target site in the genome of a plant, a plant cell or a seed. Further provided is a method for using a guide polynucleotide / Cas endonuclease system to provide an effective system for modifying the target site in the cell genome and editing the nucleotide sequence in the cell genome. Once the genomic target site is identified, a variety of methods can be used to further modify the target site so that they include a variety of purpose polynucleotides. The disclosed method can be used to introduce a CRISPR-Cas system, and the system is used to edit the nucleotide sequence in the cell genome. The nucleotide sequence to be edited (purpose nucleotide sequence) can be located inside or outside the target site identified by the Cas endonuclease.

[0543] CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) (also known as SPIDR-interspersed direct repeats) constitute a family of recently described DNA loci. CRISPR loci consist of short, highly conserved DNA repeats (usually 24 to 40 bp, repeated from 1 to 140 times, also known as CRISPR repeats) that are partially palindromic. The repeats (usually species-specific) are spaced by variable sequences of constant length (usually 20 to 58, depending on the CRISPR locus (WO2007 / 025097, published on March 1, 2007)).

[0544] Cas genes include genes that are typically coupled to, associated with, or near or adjacent to a flanking CRISPR locus. The terms "Cas gene" and "CRISPR-associated (Cas) gene" are used interchangeably herein.

[0545] In another aspect, the Cas endonuclease gene is operably linked to an SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal downstream of the Cas codon region (Tinland et al., (1992) Proc. Natl. Acad. Sci. USA 89:7442-6).

[0546] With respect to Cas endonucleases, the terms "functional fragment", "functionally equivalent fragment" and "functionally equivalent fragment" are used interchangeably herein. These terms refer to a portion or subsequence of a Cas endonuclease sequence in which the ability to generate double-strand breaks is retained.

[0547] With respect to Cas endonucleases, the terms "functional variant", "functionally equivalent variant" and "functionally equivalent variant" are used interchangeably herein. These terms refer to variants of Cas endonucleases in which the ability to generate double-strand breaks is retained. These fragments and variants can be obtained via methods such as site-directed mutagenesis and synthetic construction.

[0548] In one aspect, the Cas endonuclease gene is a plant codon-optimized Streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the type N(12-30)NGG and that can in principle be targeted.

[0549] Endonucleases are enzymes that cut the phosphodiester bonds in polynucleotide chains, and are included in restriction endonucleases that cut DNA at specific sites without damaging bases. Restriction endonucleases include type I, type II, type III, and type IV endonucleases, which further include subtypes. In type I and type III systems, both methylase and restriction enzyme activity are contained in a single complex. Endonucleases also include meganucleases, also referred to as homing endonucleases (HE enzymes), which are similar to restriction endonucleases, bind and cut at specific recognition sites, but for meganucleases, these recognition sites are usually longer, about 18bp or longer (patent application PCT / US 12 / 30061 filed on March 22, 2012). Based on conservative sequence motifs, meganucleases have been divided into four families. These motifs participate in the coordination of metal ions and the hydrolysis of phosphodiester bonds. The notable feature of meganucleases is their long recognition sites, and also in tolerance to some sequence polymorphisms in their DNA substrates. The naming convention for meganucleases is similar to that for other restriction endonucleases. Meganucleases are also characterized by the prefixes F-, I-, or PI- for enzymes encoded by separate ORFs, introns, and inteins, respectively. One step in the recombination process involves cleavage of a polynucleotide at or near a recognition site. This cleavage activity can be used to produce double-strand breaks. For a review of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol [New Opinions in Biotechnology] 5:521-7; and Sadowski (1993) FASEB [Journal of the Federation of American Societies for Experimental Biology] 7:760-7. In some instances, the recombinase is from the Integrase or Resolvase family. TAL effector nucleases are a new class of sequence-specific nucleases that can be used to create double-strand breaks at specific target sequences in the genome of plants or other organisms. (Miller, et al. (2011) Nature Biotechnology 29: 143-148). Zinc finger nucleases (ZFNs) are engineered double-strand break-inducing agents consisting of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprises two, three, or four zinc fingers, e.g., having a C2H2 structure, although other zinc finger structures are known and have been engineered. The zinc finger domain is suitable for designing polypeptides that specifically bind to a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain (e.g., a nuclease domain from an Ms-type endonuclease, e.g., Fok1).Additional functionalities can be fused to the zinc finger binding domain, including transcriptional activator domains, transcriptional repressor domains, and methylases. In some instances, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3-finger domain recognizes a sequence of 9 consecutive nucleotides, and since dimerization of the nuclease requires, two sets of zinc finger triplets are used to bind to a recognition sequence of 18 nucleotides.

[0550] "Dead-CAS9" (dCAS9) used herein is used to provide a transcriptional repressor domain. dCAS9 has mutated and can no longer cut DNA. dCAS0 can still bind when guided to the sequence by gRNA, or it can be fused to a repressor element. As described herein, dCAS9 fused to a repressor element is abbreviated as dCAS9-REP, where the repressor element (REP) can be any known repressor motif that has been characterized in plants. The expressed guide RNA (gRNA) binds to the dCAS9-REP protein and targets the binding of the dCAS9-REP fusion protein to a specific predetermined nucleotide sequence within the promoter (promoter within T-DNA). For example, if it is expressed outside the borders using a ZM-UBI PRO::dCAS9~REP::PINII TERM cassette and a U6-POL PRO::gRNA::U6 TERM cassette, and the gRNA is designed to direct the dCAS9-REP protein to bind to the SB-UBI promoter in the expression cassette SB-UBIPRO::moPAT::PINII TERM within the T-DNA, any event that integrates the sequence outside the borders will be bialaphos sensitive. Only transgenic events that integrate the T-DNA will express moPAT and be resistant to bialaphos. The advantage of using a dCAS9 protein fused to a repressor (as opposed to TETR or ESR) is the ability to target these repressors to any promoter within the T-DNA. TETR and ESR are limited to cognate operator binding sequences. Alternatively, a synthetic zinc finger nuclease fused to a repressor domain can be used instead of gRNA and dCAS9-REP (Urritia et al., 2003, Genome Biol. 4:231), as described above.

[0551] The type II CRISPR / Cas system from bacteria uses crRNA and tracrRNA to guide the Cas endonuclease to its DNA target. The crRNA (CRISPR RNA) comprises a strand complementary to a double-stranded DNA target, and base pairs with tracrRNA (trans-activating CRISPR RNA) to form a region of RNA duplexes, which guide the Cas endonuclease to cut the DNA target. As used herein, the term "guide nucleotide" refers to a synthetic fusion of two RNA molecules - crRNA (CRISPR RNA) and tracrRNA comprising a variable targeting domain. On the one hand, the guide nucleotide comprises a variable targeting domain of 12 to 30 nucleotide sequences and an RNA fragment that can interact with the Cas endonuclease.

[0552] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease, and enables the Cas endonuclease to recognize and optionally cut a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide can include at least one nucleotide, a phosphodiester bond, or a connection modification, such as, but not limited to, a locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, a thiophosphate bond, a connection to a cholesterol molecule, a connection to a polyethylene glycol molecule, a connection to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent connection resulting in cyclization. The guide polynucleotide comprising only ribonucleic acid is also referred to as a "guide nucleotide".

[0553] The nucleotide sequence modifications of the guide polynucleotide, VT domain and / or CER domain can be selected from, but not limited to, the group consisting of: a 5′ cap, a 3′ poly(A) tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a protein binding site, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2′-fluoro A nucleotides, 2′-fluoro U nucleotides, 2′-O-methyl RNA nucleotides, a phosphorothioate bond, a linkage to a cholesterol molecule, a linkage to a polyethylene glycol molecule, a linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage, or any combination thereof. These modifications can produce at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group consisting of modified or regulated stability, subcellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity to complementary target sequences, modified resistance to cellular degradation and increased cellular permeability.

[0554] In one aspect, the guide nucleotide and the Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double-strand break at a DNA target site.

[0555] In one aspect of the disclosure, the variable target domain is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

[0556] In one aspect of the present disclosure, the guide nucleotide comprises a cRNA (or cRNA fragment) and a tracrRNA (or tracrRNA fragment) that can form a complex with a type II Cas endonuclease in a type II CRISPR / Cas system, wherein the guide nucleotide / Cas endonuclease complex can guide the Cas endonuclease to a plant genome target site, such that the Cas endonuclease can introduce a double-strand break into the genome target site. The guide nucleotide can be directly introduced into a plant or plant cell using any method known in the art (e.g., but not limited to, particle bombardment or topical application).

[0557] In one aspect, the guide nucleotide can also be introduced indirectly by introducing a recombinant DNA molecule comprising a corresponding guide DNA sequence operably linked to a plant-specific promoter capable of transcribing the guide nucleotide in a plant cell. The term "corresponding guide DNA" includes a DNA molecule that is identical to an RNA molecule, but in which a "T" replaces each "U" of the RNA molecule.

[0558] In one aspect, the guide nucleotides are introduced via particle bombardment or using the disclosed methods for Agrobacterium transformation of a recombinant DNA construct comprising the corresponding guide DNA operably linked to a plant U6 polymerase III promoter.

[0559] On the one hand, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. An advantage of using a guide nucleotide versus a duplex crRNA-tracrRNA is that only one expression cassette needs to be made in order to express the fused guide nucleotide.

[0560] The terms "target site", "target sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", and "genomic target locus" are used interchangeably herein and refer to a polynucleotide sequence in the genome of a plant cell (including chloroplast DNA and mitochondrial DNA) at which a double-strand break is induced in the genome of the plant cell by a Cas endonuclease. The target site may be an endogenous site in the plant genome, or alternatively, the target site may be heterologous to the plant, so that it does not naturally occur in the genome, or the target site may be found in a heterologous genomic location compared to where it occurs in nature.

[0561] As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein to mean a target sequence that is endogenous or native to the genome of a plant and is at the endogenous or native location of the target sequence in the genome of a plant.

[0562] "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a plant. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the plant, but located at a different location in the genome of the plant (i.e., a non-endogenous or non-native location).

[0563] "Altered target site", "altered target sequence", "modified target site" and "modified target sequence" are used interchangeably herein and refer to a target sequence as disclosed herein: the target sequence comprises at least one change when compared to the unaltered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0564] In one aspect, the disclosed method can be used to introduce into a plant a polynucleotide for gene suppression of a target gene in a plant. For several aspects of plant genetic engineering, it is desirable to reduce the activity of a specific gene (also referred to as gene silencing or gene suppression). Many gene silencing techniques are well known to those skilled in the art, and include but are not limited to antisense technology.

[0565] On the one hand, the disclosed method can be used for introducing the polynucleotides used for the targeted integration of nucleotide sequences into plants. For example, the disclosed method can be used for introducing a T-DNA expression cassette to transform a plant comprising a target site, wherein the transfer cassette comprises the target nucleotide sequence flanking different recombination sites. On the one hand, the target site comprises at least one group of different recombination sites corresponding to those on the T-DNA expression cassette. The exchange of the nucleotide sequence flanking the recombination site is carried out by a recombinase. Therefore, the disclosed method can be used for introducing a T-DNA expression cassette to target and integrate a nucleotide sequence, wherein the flank of the T-DNA expression cassette is a different recombination site identified by a recombinase, and the recombinase identifies and realizes the reorganization at different recombination sites. Therefore, the disclosed method and composition can be used for improving the development efficiency and the development speed of the plant containing different recombination sites.

[0566] Thus, the disclosed methods can further include methods for directed, targeted integration of exogenous nucleotides into transformed plants. In one aspect, the disclosed methods use novel recombination sites in a gene targeting system that facilitates directed targeting of desired genes and nucleotide sequences to corresponding recombination sites previously introduced into the target plant genome.

[0567] On the one hand, the nucleotide sequence of two non-identical recombination sites is introduced into one or more cells of the explant derived from the target organism genome, thereby setting up the target site for inserting the target nucleotide sequence. Once a stable plant or cultured tissue has been established, a second construct or the target nucleotide sequence with recombination sites corresponding to those recombination sites of the flank target site is introduced into the plant or tissue of stable transformation in the presence of the recombinase protein. This process results in the exchange of the nucleotide sequence between the non-identical recombination sites of the target site and the T-DNA expression cassette.

[0568] It should be appreciated that the transformed plants prepared in this manner may contain multiple target sites; i.e., multiple sets of non-identical recombination sites. In this manner, multiple manipulations of target sites in the transformed plants are available. The target site in the transformed plant refers to a DNA sequence that has been inserted into the transformed plant genome and contains non-identical recombination sites.

[0569] Examples of recombination sites used in the disclosed methods are known. The two-micron plasmid found in most naturally occurring strains of Saccharomyces cerevisiae encodes a site-specific recombinase that promotes the inversion of DNA between two inverted repeats. This inversion plays a central role in plasmid copy number amplification.

[0570] A protein named FLP protein catalyzes site-specific recombination events. The minimal recombination site (FRT) has been defined and contains two reverse 13 base pair (bp) repeats around an asymmetric 8-bp spacer. The FLP protein cuts the site at the junction of the repeat and the spacer and covalently connects to the DNA via the 3' phosphate. Site-specific recombinases (such as FLP) cut and reconnect DNA at specific target sequences, which leads to precisely defined recombination between two identical sites. In order to work, the system requires recombination sites and recombinases. No auxiliary factors are required. Therefore, the entire system can be inserted into plant cells and work therein. The yeast FLP\FRT site-specific recombination system has been shown to work in plants. So far, the system has been used to remove unwanted DNA. See Lyznik et al. (1993) Nucleic Acids Res. [Nucleic Acids Research] 21: 969-975. In contrast, the present disclosure utilizes non-identical FRTs for the control of exchange, targeting, arrangement, insertion and expression of nucleotide sequences in plant genomes.

[0571] On the one hand, it is necessary to transform a target organism, such as an explant from a plant, containing a target site that is integrated into its genome. The target site is characterized in that the flanks have non-identical recombination sites. In addition, a targeting box is needed, which contains a nucleotide sequence that is flanked by non-identical recombination sites corresponding to those sites included in the target site of the transformed organism. It is necessary to recognize a recombinase that recognizes non-identical recombination sites and catalyzes site-specific recombination.

[0572] It should be appreciated that the recombinase can be provided by any means known in the art. That is, by transient expression or by providing a messenger RNA (mRNA) of the recombinase or recombinase protein, it can be provided in an organism or plant cell by transforming an organism with an expression cassette capable of expressing the recombinase in the organism.

[0573] "Non-identical recombination sites" means that the flanking recombination sites are not identical in sequence and will not recombine, or the recombination between the sites will be minimized. That is, one flanking recombination site can be a FRT site, and the second recombination site can be a mutated FRT site. The non-identical recombination sites used in the method of the present disclosure prevent or greatly inhibit the reorganization between the two flanking recombination sites and the excision of the nucleotide sequence contained therein. Therefore, it should be recognized that any suitable non-identical recombination sites can be used in the present disclosure, including FRT and mutated FRT sites, FRT and lox sites, lox and mutated lox sites, and other recombination sites known in the art.

[0574] By suitable non-identical recombination sites is meant that, in the presence of an active recombinase, excision of the sequence between the two non-identical recombination sites (if any) occurs at a significantly lower efficiency than the targeted arrangement of the nucleotide sequence recombination-mediated exchange into the plant genome. Thus, suitable non-identical sites for use in the present disclosure include those sites where the recombination efficiency between the sites is low; for example, where the efficiency is less than about 30% to about 50%, preferably less than about 10% to about 30%, more preferably less than about 5% to about 10%.

[0575] As noted above, the recombination sites in the targeting cassette correspond to the recombination sites in the target site of the transformed plant. That is, if the target site of the transformed plant contains non-identical recombination sites flanked by FRT and mutant FRT, the targeting cassette will contain non-identical recombination sites for the same FRT and mutant FRT.

[0576] It should also be recognized that the recombinase used in the disclosed method will depend on the recombination sites in the target site of the transformed plant and in the targeting box. That is, if the FRT site is used, the FLP recombinase is required. In the same way, in the case of using lox sites, the Cre recombinase is required. If the different recombination sites contain both FRT and lox sites, both FLP and Cre recombinases will be required in the plant cell.

[0577] The FLP recombinase is a protein that catalyzes a site-specific reaction that is involved in amplifying the copy number of the two-micron plasmid of Saccharomyces cerevisiae during DNA replication. The FLP protein has been cloned and expressed. See, e.g., Cox (1993) Proc. Natl. Acad. Sci. USA 80:4223-4227. The FLP recombinase used in the present disclosure may be an enzyme derived from Saccharomyces. It may be preferred to synthesize the recombinase using plant-preferred codons for optimal expression in the plant of interest. See, e.g., U.S. Application Serial No. 08 / 972,258, filed on November 18, 1997, entitled "Novel Nucleic Acid Sequence Encoding FLP Recombinase," which is incorporated herein by reference.

[0578] The bacteriophage recombinase Cre catalyzes site-specific recombination between two lox sites. The Cre recombinase is known in the art. See, for example, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J. Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet. 22:477-488; and Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702. All of which are incorporated herein by reference. Such Cre sequences can also be synthesized using plant-preferred codons.

[0579] Where appropriate, the nucleotide sequence to be inserted into the plant genome can be optimized to increase expression in the transformed plant. In the case of mammalian, yeast or bacterial genes used in the present disclosure, they can be synthesized using plant preferred codons to improve expression. It should be recognized that for expression in monocots, dicot genes can also be synthesized using monocot preferred codons. Methods for synthesizing plant preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 477-498, which are incorporated herein by reference. Plant preferred codons can be determined from the codons used more frequently in the proteins expressed in the target plant. It should be recognized that monocot or dicot preferred sequences as well as plant preferred sequences of specific plant species can be constructed. See, e.g., EPA 0359472; EPA 0385962; WO 91 / 16432; Perlak et al., (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; etc., which are incorporated herein by reference. It is further recognized that all or any portion of the gene sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences may also be used.

[0580] Additional sequence modifications are known to enhance gene expression in cellular hosts and can be used in the present disclosure. These include: elimination of sequences encoding spurious polyadenylation signals and exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to an average level for a given cellular host calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid the presence of foreseeable hairpin secondary RNA structures.

[0581] The present disclosure also includes a novel FLP recombination target site (FRT). The FRT has been identified as a minimal sequence comprising two 13 base pair repeats separated by an eight (8) base spacer. The nucleotides in the spacer can be replaced by a combination of nucleotides as long as the two 13 base repeats are separated by eight nucleotides. It appears that the actual nucleotide sequence of the spacer is not critical; however, for the practice of the present disclosure, certain substitutions of nucleotides in the spacer region may work better than other substitutions. During strand exchange, the eight base pair spacer participates in DNA-DNA pairing. The asymmetry of this region determines the direction of site alignment in a recombination event, which will subsequently lead to inversion or excision. As shown above, most spacers can be mutated without loss of function. See, for example, Schlake and Bode (1994) Biochemistry 33: 12746-12751, incorporated herein by reference.

[0582] The new FRT mutation site can be used to practice the disclosed method. Such mutation site can be constructed by mutagenesis based on PCR. Although the mutation FRT site is known (see SEQ ID No 2,3,4 and 5 of WO 1999 / 025821), it is recognized that other mutation FRT sites can be used to practice the disclosure. The disclosure is not limited to the use of specific FRT or recombination sites, but can use different recombination sites or FRT sites with the nucleotide sequence targeted for insertion and expression in the plant genome. Therefore, other mutation FRT sites can be constructed and used based on the disclosure.

[0583] As discussed above, in the presence of a recombinase, a genomic DNA containing a target site with non-identical recombination sites causes recombination together with a carrier containing a T-DNA expression cassette with corresponding non-identical recombination sites. The nucleotide sequence of the T-DNA expression cassette between the flanking recombination sites is exchanged with the nucleotide sequence of the target site between the flanking recombination sites. In this way, the target nucleotide sequence can be accurately incorporated into the host's genome.

[0584] It is recognized that many variations of the present disclosure can be implemented. For example, a target site with a plurality of different recombination sites can be constructed. Therefore, a plurality of genes or nucleotide sequences can be stacked or sorted at the precise position in the plant genome. Similarly, once a target site has been established in the genome, other recombination sites can be introduced by incorporating such site into the nucleotide sequence of the T-DNA expression cassette and transferring the site to the target sequence. Therefore, once a target site has been established, it is possible to add a site or change the site subsequently by restructuring.

[0585] Another variation includes providing a promoter or transcription initiation region operably linked to a target site in an organism. Preferably, the promoter will be located 5' of the first recombination site. By transforming an organism with a T-DNA expression cassette comprising a coding region, expression of the coding region will occur when the T-DNA expression cassette is integrated into the target site. This aspect provides a method for selecting transformed cells (particularly plant cells) by providing a selectable marker sequence as a coding sequence.

[0586] Other advantages of this system include the ability to reduce the complexity of the integration of the transgenic or transferred DNA in an organism by utilizing the T-DNA expression cassette as discussed above and selecting the organism with a simple integration pattern. In the same manner, the preferred sites in the genome can be identified by comparing several transformation events. The preferred sites in the genome include sites that do not destroy the expression of the essential sequence and provide sufficient expression of the transgenic sequence.

[0587] Epigenetics studies changes in an organism that result from modifications in gene expression rather than changes in the genetic code itself and can include analysis of DNA modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), post-translational histone tail modifications, energy-dependent nucleosome remodeling, and long noncoding RNA regulation of local chromatin structure and chromosome organization.

[0588] Modifications of DNA and / or chromatin are of interest given the impact these modifications may have on gene expression, development, and ultimately genome stability. Patterns of histone-mediated chromatin remodeling, the so-called "histone code," coordinate this control, which can interact and sometimes be coordinated by DNA methylation patterns, suggesting a complex interplay of different epigenetic mechanisms that work together to orchestrate genome stability.

[0589] Epigenetic modifications of DNA, or histone modifications, can be established or removed by enzymes that catalyze specific modifications (“writers”), proteins that recognize and bind to the modifications (“recognizers”), and enzymes that remove certain modifications (“erasers”).

[0590] Makarevitch et al. previously demonstrated evidence of epigenetic regulation in a tissue-specific manner, specifically including tassel tissue containing anthers and microspores, that is allelically specific with BABY BOOM (Makarevitch et al., 2013. Plant Cell. March; 25: 780-793). These authors characterized trimethylation of histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing that plays a key role in regulating gene expression during plant and animal development (see also, Schuettengruber et al., 2007; Berr et al., 2011; Zheng and Chen, 2011).

[0591] H3K27me3 is catalyzed by Polycomb group (PcG) proteins and is thought to regulate such developmental processes. PcG genes are characterized by the inability of mutants to properly maintain the repressed state of gene expression of homologous genes in Drosophila melanogaster (Simon, 1995), and a subset of PcG proteins can form Polycomb repressive complex 2 (PRC2) involved in catalyzing lysine methylation of histone H3. Many plant species have orthologs of PRC2 genes (reviewed in Hennig and Derkacheva, 2009; Kohler and Hennig, 2010; Zheng and Chen, 2011) and may encode the same function in plants (Schuberr et al., 2006).

[0592] The maize (Zea mays) genome encodes three E(z) homologs: Mez1, Mez2, and Mez3 (Springer et al., 2002). Mez1 is the imprinted gene most closely related to the CURLY LEAF (CLF) gene from Arabidopsis thaliana (Haun et al., 2007). Mez2 and Mez3 are highly similar to each other (92% nucleotide identity), are located in collinear regions of the maize genome, and are likely paralogs generated by an ancient allopolyploid event in maize (Springer et al., 2002).

[0593] Yu et al. 2008. identified two BRI1-EMS-SUPPRESSOR 1 (BES1) interacting proteins (EARLY FLOWERING 6 (ELF6) and its homolog ELATIVE OF EARLYFLOWERING 6 (REF6), Jumonji N / C (JmjN / C) domain-containing proteins previously reported to regulate flowering time (see Plant Cell 16: 2601-13). After confirming that BES1 and ELF6 / REF6 proteins interact using GST pull-down and BiFC (bimolecular fluorescence complementation) experiments, these authors further demonstrated that mutations in the ELF6 or REF6 genes lead to abnormal BR-related phenotypes, including impaired cell elongation and reduced expression of BR target genes, thus demonstrating that BES1 recruits ELF6 and REF6 transcriptional regulators to regulate target gene expression.

[0594] The functional activity of REF6 was further characterized in Lu et al. (Nat Genet. 43:715-9) in 2011. These authors demonstrated that overexpression of REF6 specifically demethylated dimethylated and trimethylated post-translational modifications at the lysine 27 residue of histone 3 (referred to here as H3K27me2 and H3K27me3, respectively), while other histone modifications were not reported using standard methods. To further characterize how demethylation of H3K27me2 and H3K27me3 modifications affects gene regulation, several H3K27me3 target genes that are normally silent in Arabidopsis seedlings, such as the MADS-box floral organ identity genes AP1 (APETALA1), AP3 (APETALA3), PI (PISTILLATA), AG (AGAMOUS), and SEP3 (SEPALLATA3), were shown to be ectopically activated in Arabidopsis seedlings stably transformed with a REF6 overexpression transgenic construct.

[0595] The results show that the catalytic activity of H3K27me3 demethylation is coupled to the deposition of activating histone modifications, which can be used for cellular reprogramming, resulting in active transcription from a silent or repressed regulatory state. One class of proteins that can be used to deposit activating histone modifications is histone acetyltransferases (HATs), enzymes that transfer acetyl groups to substrates using acetyl coenzyme A (CoA). Therefore, it is expected that combining such H3K27me3 demethylation activities and establishing histone acetylation modifications in a targeted, programmable manner can be used for cellular reprogramming of plant cells.

[0596] Histone acetyltransferases (HATs) acetylate lysine residues in the amino-terminal histone tails, leading to transcriptional activation, and are divided into at least four distinct families based on sequence conservation within the HAT domain.

[0597] The General Control Nonrepressed (Gcn5)-associated N-acetyltransferase (GNAT) domain catalyzes the transfer of an acetyl group from a CoA donor to a primary amine of an acceptor. GNAT proteins share a domain consisting of four conserved sequence motifs AD [PMID: 9175471, PMID: 10940244]. The acetyltransferase / GNAT domain forms a structurally conserved fold of six to seven β strands (B) and four helices (H) in the topology B1-H1-H2-B2-B3-B4-H3-B5-H4-B6, followed by a C-terminal strand that may be derived from the same monomer or contributed by another [PMID: 10940244, PMID: 15581578]. Motifs D (B2-B3), A (B4-H3) and B (B5-H4) are collectively referred to as the HAT core [PMID: 10940244, PMID: 12527305, PMID: 15581578], whereas the N-terminal motif C (B1-H1) is less conserved.

[0598] The MYST family (named after MOZ, Ybf2 / Sas3, Sas2, and Tip60 proteins) mediate many biological functions, including gene regulation, DNA repair, cell cycle regulation, and development [PMID: 21132344], and can acetylate non-histone substrates [PMID: 19303850]. The MYST-type HAT domain contains three regions: in addition to the flanking N-terminal and C-terminal regions with C2HC-type zinc fingers and helix-turn-helix DNA binding motifs, respectively, there is also a central region associated with acetyl-CoA cofactor binding and catalysis. The N-terminal and C-terminal fragments directly flanking the catalytic core may play an important role in histone substrate binding [PMID: 11106757, PMID: 17925393].

[0599] The following examples are offered by way of illustration and not by way of limitation.

[0600] Examples

[0601] Aspects of the present disclosure are further defined in the following examples, wherein parts and percentages are by weight, and degrees are in degrees Celsius, unless otherwise stated. Although these examples illustrate aspects of the present disclosure, they are provided by way of illustration only. From the above discussion and these examples, those skilled in the art will be able to determine the essential characteristics of aspects of the present disclosure, and, without departing from the spirit and scope of the present disclosure, various changes and modifications thereof may be made to adapt them to various uses and conditions. Therefore, from the above description, various modifications will be clear to those skilled in the art, except those shown and described herein. Such modifications are also considered to fall within the scope of the appended claims.

[0602] Example 1: Plasmid

[0603] See Table 4 for a description of plasmids useful in the present disclosure.

[0604] Table 4.

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615] Example 2: Culture medium

[0616] See Table 5 for a description of the formation of media for transformation, selection and regeneration that can be used in the methods of the present disclosure.

[0617] Table 5.

[0618]

[0619]

[0620] Table 5. Continued

[0621]

[0622]

[0623]

[0624] Example 3: Agrobacterium-mediated transformation of maize

[0625] A. Preparation of Agrobacterium master plates.

[0626] Master plates were prepared by streaking A. tumefaciens carrying the binary donor vector from -80 °C frozen aliquots onto solid 12R medium and growing at 28 °C in the dark for 2-3 days.

[0627] B. Growing Agrobacterium on solid medium.

[0628] Single colonies or multiple colonies of Agrobacterium were picked from the mother plate and streaked onto a second plate containing 810K medium and incubated overnight at 28°C in the dark. Agrobacterium infection medium (700A; 5 ml) and 100 mM 3′-5′-dimethoxy-4′-hydroxyacetophenone (acetosyringone; 5 μL) were added to a 14 mL conical tube in a fume hood. Approximately 3 full loops of Agrobacterium from the second plate were suspended in a test tube, which was then vortexed to form a uniform suspension. The suspension (1 ml) was transferred to a spectrophotometer tube, and the optical density (550 nm) of the suspension was adjusted to a reading of about 0.35-1.0. The Agrobacterium concentration was about 0.5 to 2.0×10 9 cfu / mL. The final Agrobacterium suspension was aliquoted into 2 mL microcentrifuge tubes, each containing approximately 1 mL of the suspension. The suspension was then used as quickly as possible.

[0629] C. Growing Agrobacterium in liquid culture.

[0630] Alternatively, Agrobacterium can be prepared for transformation by growing in liquid medium. Infect the day before, prepare 125ml flask with following: 30ml 557A culture medium (10.5g / l dipotassium hydrogen phosphate, 4.5g / l potassium dihydrogen phosphate anhydrous, 1g / l ammonium sulfate, 0.5g / l sodium citrate dehydration, 10g / l sucrose, 1mM magnesium sulfate) and 30 μ L spectinomycin (50mg / mL) and 30 μ L acetosyringone (20mg / mL). The semi-ring Agrobacterium from the second flat plate is suspended in the flask, and is placed on the orbital shaker set at 200rpm, and incubated overnight at 28 ℃. Agrobacterium culture is centrifuged 10 minutes at 5000rpm. Remove supernatant and add the Agrobacterium infection culture medium (700A) with acetosyringone solution to precipitation. The Agrobacterium was resuspended by vortexing, and the optical density (550 nm) of the Agrobacterium suspension was adjusted to a reading of approximately 0.35 to 1.0.

[0631] D. Maize transformation.

[0632] The ear of maize (Zea mays L.) cultivar is surface sterilized in 20% (v / v) bleach (5.25% sodium hypochlorite) plus 1 drop of Tween 20 for 15-20 minutes, then washed 3 times in sterile water. Immature embryos (IE) are separated from the ear and placed in 2ml Agrobacterium 700A infection medium with acetosyringone solution. The optimal size of embryo varies with inbred lines, but for transformation with embryogenic factors listed in Table 6 and / or WUS2 and / or ODP2 developmental genes, the immature embryo size of a wide range of sizes can be used. Take out Agrobacterium infection medium (700A), 1ml Agrobacterium suspension is added to the embryo, and the test tube is vortexed for 5-10 seconds. Microcentrifuge tubes are incubated in a fume hood for 5 minutes. Agrobacterium suspension and embryo are poured on 710I (or 562V) co-culture medium (see Table 5). Any embryo remaining in the tube is transferred to a flat plate using a sterile scraper. The Agrobacterium suspension was drawn and the embryos were placed on the axial side of the medium. The plates were incubated in the dark at 21°C for 1-3 days for co-cultivation, and then the embryos were transferred to resting medium (605B medium) without selection.

[0633] Example 4: Methods for improved corn transformation

[0634] The following experiments demonstrate that delivery of T-DNA containing embryogenic factors operably linked to a strong, tissue-preferred promoter and morphogenic genes operably linked to a weak, constitutive promoter improves somatic embryogenesis compared to T-DNA containing only morphogenic genes operably linked to a weak, constitutive promoter.

[0635] Two series of experiments were performed. Each series compared two plasmids used to assess the cellular reprogramming effects on treated immature embryos as measured by callus induction and somatic embryogenic responses over a period of 7 to 10 days.

[0636] An expression cassette containing each embryogenic factor DNA polynucleotide (SEQ ID NO: 1-16) (see Table 6) encoding each embryogenic factor polypeptide (SEQ ID NO: 17-32) operably linked to a strong, tissue-preferred promoter was synthesized and used in experimental treatments. The embryogenic expression cassette was used in experimental treatments as described below. An expression cassette containing a morphogenic gene operably linked to a weak, constitutive promoter was synthesized and used in experimental treatments as described below with the embryogenic expression cassette. As described below, an experimental control treatment containing a sequence of the same morphogenic gene operably linked to the same weak, constitutive promoter was also used.

[0637] Table 6.

[0638]

[0639] In the first series of experiments, the first plasmid was used as an experimental treatment and contained three expression cassettes, the expression cassettes comprising: i.) ZM-PLTP PRO:: ZM-PLT 5UTR:: (cloning site; for insertion of each embryogenic factor):: OS-T28TERM, ii.) NOS PRO:: ZM-WUS2:: PINII TERM, and iii.) UBIZM PRO:: UBIZM 5UTR:: UBIZMINTRON 1:: DS-RED2:: PINII TERM. For each embryogenic factor shown in Table 6, each synthetic gene fragment included flanking sequences encoding compatible restriction sites, typically BamHI and KpnI recognition motifs. These restriction sites allowed directional cloning of each embryogenic factor (SEQ ID NOS: 1-16) into the cloning site of an expression cassette operably linked to the PLTP regulatory elements described in i.) above. Exemplary sequences are provided, including polynucleotides encoding: ZM-PLTP PRO::ZM-PLT 5UTR (SEQ ID NO:33), OS-T28 TERM (SEQ ID NO:34), expression cassette NOS PRO::ZM-WUS2::PINII TERM (SEQ ID NO:35), and expression cassette UBIZM PRO::UBIZM 5UTR::UBIZMINTRON 1::DS-RED2::PINII TERM (SEQ ID NO:36).

[0640] Plasmid RV010564 (SEQ ID NO: 37) used as an experimental control treatment contains a T-DNA comprising the following components; RB+NOS PRO::WUS2::PINII TERM+UBIZM PRO::UBIZM 5UTR::UBIZM INTRON1::ZS-YELLOWN1::PINII TERM+UBIZM PRO::UBIZM 5UTR::UBIZM INTRON1::PMI::PINII TERM+OS-ACTINPRO::OS-ACTIN INTRON1::MO-PAT::CAMV35TERM+LB.

[0641] Experimental control treatment using RV010564 (SEQ ID NO: 37) is insufficient to produce high levels of somatic embryogenesis in treated plant cells. The experimental control treatment expression cassette is designed to allow treated cells to be only in a state of readiness or reprogramming close to a balance sufficient for embryogenesis, but not sufficient to induce embryogenesis independently. Therefore, using tissue-preferred PLTP promoters to regulate the expression of each embryogenic factor is therefore expected to be useful for evaluating the cell reprogramming response relative to the baseline observed using the experimental control treatment.

[0642] Using a similar strategy, a second series of experiments was performed using a second plasmid containing three expression cassettes as experimental treatments, comprising: i.) ZM-PLTP PRO::ZM-PLT 5UTR::(cloning site; for transfer of polynucleotides encoding embryogenic factors; see SEQ ID NOs: 1-16)::OS-T28 TERM, ii.) UBIZM PRO::UBIZM 5UTR::UBIZM INTRON 1::ZM-ODP2::PINII TERM, and iii.) UBIZM PRO::UBIZM 5UTR::UBIZMINTRON 1::DS-RED2::PINII TERM. Exemplary sequences include polynucleotides encoding ZM-PLTP PRO::ZM-PLT 5UTR (SEQ ID NO:33), OS-T28 TERM (SEQ ID NO:34), UBIZM PRO::UBIZM 5UTR::UBIZM INTRON 1::ZM-ODP2::PINII TERM (SEQ ID NO:38), and UBIZM PRO::UBIZM 5UTR::DS-RED2::PINII TERM (SEQ ID NO:36).

[0643] Each embryogenic factor shown in Table 6 was evaluated by transforming embryos (average length 1.8 mm) isolated from the same donor ear using the two plasmid entities, accounting for variation in donor ear quality. The isolated embryos were divided into two groups, each of which was treated with either the experimental treatment or the experimental control treatment for each series, with comparisons made relative to the WUSCHEL polypeptide in one series and relative to the ovule development protein 2 (ODP2) polypeptide in the second series.

[0644] Briefly, after incubation with 700A medium for 5 minutes at room temperature, infected embryos were cultured with co-cultivation (710I or 562V) medium for 24 hours in the dark at 21° C. Treated embryos were transferred to resting (605B) medium at 28° C. in the dark for 7 to 11 days.

[0645] The relative efficacy of somatic embryogenesis is calculated by scoring the percentage of the response embryos that show callus induction phenotype 7 days after infection and the further somatic embryogenesis development in response to the experimental treatment and experimental control treatment 14 days after infection. The ratio of the percentage of embryos scored in response to the reprogrammed cell fate of the experimental treatment is divided by the percentage of embryos scored in response to the reprogrammed cell fate of the experimental control treatment to calculate the relative efficacy of the somatic embryogenesis induction for each embryogenic factor (shown as independent EF+WUS / WUS or independent EF+BBM / BBM; see Figure 1 ). Co-expression of DsRED protein expression within proliferating cells was also assessed using epifluorescence microscopy to confirm the presence of T-DNA containing each embryogenic factor.

[0646] The cellular reprogramming response was measured by observing callus induction, epifluorescence reporter gene expression levels, and somatic embryogenesis responses in embryos treated with experimental treatments relative to embryos treated with experimental control treatments at day 14, as shown in Figure 2. Figure 1 shown.

[0647] These results indicate that several embryogenic factors, specifically dpzm02g056660.1.1 (embryogenic factor 1; SEQ ID NO: 1 encoding SEQ ID NO: 17), dpzm03g050950.1.1 (embryogenic factor 4; SEQ ID NO: 4 encoding SEQ ID NO: 20), dpzm07g031470.1.1 (embryogenic factor 5; SEQ ID NO: 5 encoding SEQ ID NO: 21), and dpzm09g039270.1.1 (embryogenic factor 11; SEQ ID NO: 11 encoding SEQ ID NO: 27), each interact with the morphogenetic genes WUS2 and ODP2 / BBM. For the remaining embryogenic factors, each interacted with either the morphogenetic gene WUS2 or ODP2 / BBM. However, it is expected that changes in the regulatory elements for any of the expression cassettes used in these experiments will result in changes in the response. Specifically, regulatory elements that promote higher gene activity would be expected to result in an increased somatic embryogenic response, and conversely, weaker regulatory elements would be expected to result in a decreased somatic embryogenic response.

[0648] The embryogenic factor listed in the table 6 can be used for activating the embryogenic response in the plant cell, and is used as the cell reprogramming factor, as proved by the high embryogenic callus produced in response to experimental treatment. The result shown herein shows, the coexpression of these embryogenic factors and morphogenetic genes can be used for cell reprogramming and obtain embryogenic cell fate. Therefore expect that these embryogenic factors can be used for improving the transformation efficiency of maize.

[0649] For example, it is expected that a plasmid (which comprises: a first expression cassette i) (which contains a polynucleotide encoding a CRE / lox recombination system, which is flanked by a morphogenetic gene / embryogenic factor (EF) / CRE co-expression cassette, wherein CRE is operably linked to a desiccation-inducible RAB17 promoter, allowing excision of the morphogenetic gene / EF / CRE co-expression cassette), and a second expression cassette ii) (which contains a polynucleotide encoding a gene of interest, such as UBI1ZM PRO+UBI1ZM 5UTR+UBI1ZM INTRON1+DS-RED2+PINII TERM (SEQ ID NO: 99))) can be used to improve the transformation efficiency and regeneration of transgenic plants transformed with the second expression cassette (e.g., transgenic plants expressing DS-RED2). It should be understood that one or more morphogenetic genes can be used in the morphogenetic gene / EF / CRE co-expression cassette.

[0650] Example 5: Methods for regulating cell cycle activity to further improve somatic embryogenesis

[0651] Further improvements in cell reprogramming and embryogenic cell fate acquisition were obtained using delivery of experimental treatments comprising T-DNA containing (a) morphological development genes operably linked to weak, constitutive promoters, (b) embryogenic factors operably linked to strong, tissue-preferred promoters, and (c) cyclin-dependent kinases operably linked to strong, tissue-preferred promoters. This experimental treatment showed improved somatic embryogenesis compared to an experimental control treatment comprising T-DNA containing (a) the same morphological development genes operably linked to weak, constitutive promoters and (b) the same embryogenic factors operably linked to strong, tissue-preferred promoters.

[0652] A series of plasmids were constructed and used to deliver T-DNA containing: (a) the WUS2 or ODP2 morphological developmental gene operably linked to a weak, constitutive promoter, (b) the embryogenic factor dpzm07g031470.1.1, also referred to herein as "DZ470", annotated as cyclin delta-2 protein (embryogenic factor 5, SEQ ID NO: 5 encoding SEQ ID NO: 21), operably linked to a strong, tissue-preferred promoter, and (c) a cyclin-dependent kinase encoded by the gene dpzm01g001860.1.1 or dpzm04g074910.1.2, referred to herein as CDKA1 or CDKA3, respectively, operably linked to a strong, tissue-preferred promoter (see Table 7).

[0653] Table 7: Plasmids used to regulate cell cycle activity

[0654]

[0655]

[0656]

[0657] Compared with experimental control treatment, the experiment of the activation of cell reprogramming and somatic embryogenesis in response to experimental treatment was performed as described above. The efficacy of each carrier in providing cell reprogramming and obtaining embryogenic cell fate was compared with the following: experimental control treatment using one of the two morphological development genes (e.g., SEQ ID NO: 37 (only WUS) or SEQ ID NO: 41 (only BBM)), and positive control treatment using two morphological development genes (e.g., SEQ ID NO: 45 (WUS+BBM)).

[0658] These results suggest that when combined with the developmental gene (WUS) ( Figure 2A ) or developmental genes (BBM) ( Figure 2B) and cyclin-dependent kinase (CDKA1 or CDKA3) (SEQ ID NO: 114 and SEQ ID NO: 115, respectively), the level of somatic embryogenesis conferred by the embryogenic factor "DZ470" (dpzm07g031470.1.1; polynucleotide SEQ ID NO: 5, encoding polypeptide SEQ ID NO: 21) is further improved.

[0659] like Figure 2A As shown, CDKA1 combined with the WUS morphogenetic gene more efficiently converts cells to be more embryogenic, whereas Figure 2B The CDKA3 shown in Figure 1 is more efficient in combination with the ODP2 (BBM) morphogenetic gene to convert cells to be more embryogenic.

[0660] Example 6: Paternally doubled haploid method using exogenous proteins as cell reprogramming agents

[0661] The following experiments demonstrate that microspore embryogenesis is improved by contacting wild-type microspores with an exogenous polypeptide used as a cell reprogramming agent.

[0662] For example, wild-type microspores are contacted with exogenous polypeptides used as cell reprogramming agents before, during, or after in vitro tissue culture. Specifically, embryogenic factors comprising SEQ ID NO: 1 to SEQ ID NO: 16 are used to produce polypeptides comprising SEQ ID NO: 17 to SEQ ID NO: 32, respectively. The polypeptides of SEQ ID NO: 17 to SEQ ID NO: 32 are then contacted with microspores before, during, or after in vitro tissue culture. It is expected that contacting the polypeptides of SEQ ID NO: 17 to SEQ ID NO: 32 with microspores will promote cell reprogramming of the treated microspores, thereby generating embryogenic microspores.

[0663] It is expected that modifications to the polypeptides of SEQ ID NO: 17 to SEQ ID NO: 32 will further enhance microspore cell reprogramming, resulting in enhanced microspore embryogenesis. It is expected that translational fusion peptides of SEQ ID NO: 17 to SEQ ID NO: 32, wherein the peptide is fused to a polyhistidine motif (his tag) at the N-terminus or C-terminus of the peptide, such as those of SEQ ID NO: 46 encoding SEQ ID NO: 47, will further increase microspore cell reprogramming. In addition, it is expected that microspore cell reprogramming will be further enhanced by treating isolated microspores with a his-tag translational fusion peptide of SEQ ID NO: 17 to SEQ ID NO: 32, wherein the his-tag translational fusion peptide has been further modified by adding a cell penetrating peptide (CPP) to the N-terminus or C-terminus of the polypeptide, thereby generating an embryogenic factor-his-tag-cell penetrating translational fusion peptide having the following properties, including but not limited to (a) cell reprogramming activity, (b) a His-tag that acts as a metal binding site for the recombinant protein, and / or (c) a cell penetrating peptide that promotes improved cell delivery, thereby improving the translocation of the translational fusion peptide into isolated maize microspores, resulting in improved cell reprogramming and / or microspore embryogenesis.

[0664] Cell penetrating peptides (CCPs) that can be used in the methods of the present disclosure include, but are not limited to, Zea mays knotted1 CPP (ZM-KNT1 CPP; SEQ ID NO:48 encoding SEQ ID NO:49), Saccharomyces pombe TP10 CPP (SP-TP10 CPP; SEQ ID NO:50 encoding SEQ ID NO:51), Candida albicans Zebra CPP (CA-Zebra CPP; SEQ ID NO:52 encoding SEQ ID NO:53), PEP1 CPP (PEP1 CPP; SEQ ID NO:54 encoding SEQ ID NO:55), HIV-1 TATCPP (HIV-1 TAT CPP; SEQ ID NO:56 encoding SEQ ID NO:57), and gamma-zein cell penetrating peptide (GZ CPP; SEQ ID NO:58 encoding SEQ ID NO:59) (see US 8581036, incorporated herein by reference in its entirety). It is expected that delivery of such his-tag-CPP peptides into plant cells (eg, isolated microspores) will improve cellular reprogramming and thereby improve regeneration of non-transgenic plants.

[0665] like Figure 3As shown in , mononucleate microspores extracted from maize anthers will be converted into pollen mitosis I with the characteristics of microgametogenesis without further treatment, and develop into mature pollen grains. The method disclosed herein induces cell reprogramming by treating isolated microspores with a translational fusion peptide comprising an embryogenic factor-his tag-CPP.

[0666] It is expected that by treating the microspores with a translational fusion peptide comprising an embryogenic factor-his tag-CPP combined with a morphogenic polypeptide, the cell reprogramming of the microspores can be further enhanced. In particular, the translational fusion peptide comprising an embryogenic factor-his tag-CPP can be combined with a morphogenic polypeptide selected from the group consisting of: (i) a functional WUS / WOX polypeptide (e.g., polypeptide SEQ ID NO: 322); (ii) a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide (e.g., polypeptide SEQ ID NO: 335); (iii) a LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii).

[0667] It is expected that further enhancement of microspore embryogenesis can be achieved by preparing a translational fusion peptide comprising a morphogenic polypeptide-his tag-CPP for an embryogenic factor as described above, and then treating the microspores with a combination of a translational fusion peptide comprising an embryogenic factor-his tag-CPP and a translational fusion peptide comprising a morphogenic polypeptide-his tag-CPP, thereby resulting in improved reprogramming of microspore cells.

[0668] The translational fusion peptides of morphological development genes that can be used in the methods of the present disclosure include, but are not limited to, BBM 404 -FLAG-KNOTTED-1 CPP (comprising a 6XHis tag fused to a truncated ODP2 / BBM peptide fused to a FLAG epitope fused to a KNOTTED-1 cell penetrating peptide (polynucleotide sequence SEQ ID NO: 122 encoding a polypeptide sequence SEQ ID NO: 123)) and WUS-HISTAG-GZCPP (comprising a 6XHis tag fused to a WUSCHEL peptide fused to a γ-zein cell penetrating peptide (CPP) (polynucleotide sequence SEQ ID NO: 361 encoding a polypeptide sequence SEQ ID NO: 362)).

[0669] It is expected that further enhancement of microspore embryogenesis can be achieved by treating microspores with a translational fusion peptide comprising an embryogenic factor-his tag-CPP and / or a combination comprising a morphogenic polypeptide-his tag-CPP and / or any of the translational fusion peptides disclosed above, resulting in improved reprogramming of microspore cells.

[0670] In order to obtain the above-mentioned translational fusion peptides for embryogenic factors (embryogenic factors-his tag-CPP) and / or translational fusion peptides for morphogenic polypeptides (morphogenic polypeptide-his tag-CPP), a DNA plasmid having an expression cassette encoding each such translational fusion polypeptide was constructed. The DNA plasmid can be a protein expression vector, which is constructed by transferring a DNA polynucleotide encoding each of such translational fusion polypeptides to a self-replicating gene element such as an expression vector (e.g., pDAB135006 vector (SEQ ID NO: 60)). The pDAB135006 vector (SEQ ID NO: 60) has the following features (a) T7 promoter and tobacco mosaic virus 5' leader sequence (ω), (b) NcoI and KpnI cloning sites for transferring polynucleotides encoding translational fusion polypeptides, (c) 3'UTR, (d) Fl ​​replication origin (Fl ori), and (d) blasticidin S resistance gene (bla).

[0671] Such protein expression vectors are used to express translational fusion polypeptides, and the expressed recombinant proteins are used for exogenous applications in cell reprogramming processes, such as Figure 3 As shown in. The method disclosed herein includes using an in vitro translation system derived from Nicotiana tabacum L.cv. Bright Yellow 2 (BY-2) tobacco cells. In brief, a lysate from vacuolated BY-2 protoplasts (including removal of nuclei and undamaged cells) is prepared and the lysate is frozen at -80°C in 1 mL aliquots, thereby coupling transcription and translation from a protein expression vector.

[0672] The method of harvesting tassels (including sterilization methods) and tassel pretreatment (such as temperature pretreatment) will vary according to the intended tassel use. Before selecting tassels for microspore culture, microspores must be brought to the appropriate stage, usually between the uninucleate stage and the binucleate stage. Typically, for tassels with microspores at the appropriate stage, these tassels are dissociated and each tassel is individually wrapped in aluminum foil. In a reduced temperature environment, the separation of microspores typically occurs after tassel pretreatment to improve the response of male-producing characteristics. A commonly used technique is to place the foil-wrapped tassels at 10°C for 1 to 21 days. In addition, anthers can be pre-cultured in a mannitol solution (such as 0.3M liquid mannitol plus 50mg / L ascorbic acid).

[0673] Before use, the tassels are surface sterilized in a 30% Clorox (diluted 8.25% sodium hypochlorite v / v) solution plus two drops of Tween 80 for about fifteen minutes while gently agitating on a reciprocating shaker. The tassels are then rinsed three or more times in sterile water at room temperature and placed in a large petri dish and typically left uncovered for 1-1.5 hours under sterile conditions to evaporate excess moisture before microspores are isolated. Alternatively, the florets are separated from the tassels and placed in a permeable basket, which is then immersed in a 30% Clorox (diluted 8.25% sodium hypochlorite v / v) solution plus two drops of Tween 80 for fifteen minutes and then rinsed as described above. The spikelets are placed in a large petri dish and typically left uncovered for 1-1.5 hours to evaporate excess moisture before microspores are isolated.

[0674] Microspores can be isolated from maize anthers and florets by a variety of methods including, but not limited to, a glass rod immersion method, a blending method, a blade tissue cutting method, a tissue homogenizer method, and a tissue grinding method.

[0675] After the microspores are separated from the surrounding somatic tissue, the microspores are typically purified immediately after being separated from any anther fragments and placed in a fresh separation medium. A common method for separating microspores from anther fragments is to pass a blended microspore anther fragment slurry from a separation procedure through a sieve. Alternatively, the microspore anther fragment slurry is passed through several layers of cheesecloth or a mesh filter. Further separation can be performed using a discontinuous density centrifugation method or an additional filtration method, including but not limited to methods using sucrose or Percoll gradients. In methods using a Percoll gradient, cells captured at the 20%-30% interface after centrifugation at 225g for 3 minutes (20%-50% cell range) can be further separated using a final high sucrose (0.44M) centrifugation method. Any of the above separation methods can be optimized as needed.

[0676] The culture medium used during microspore isolation is typically composed of 6% sucrose, 50 mg / L ascorbic acid, 400 mg / L proline, 0.05 mg / L biotin, and 10 mg / L nicotinic acid. Other culture media and solutions used to culture maize microspores are similar to those used in other cereal tissue culture procedures, and various culture media modifications may be used. Common features of maize culture media typically include the use of Chu N6 basal medium with vitamins (N6), NLN basal medium (NLN), or YP basal medium (YP) formulations with relatively high sugar concentrations (6%-12%), and may have ingredients including triiodobenzoic acid, various plant hormones, and / or proline.

[0677] It is expected that treating microspores with the cell reprogramming treatments disclosed in this Example 6 (alone and / or in combination with the cell reprogramming treatments disclosed in this Example 6 and / or in combination with the cell reprogramming treatments disclosed in Example 5) will improve cell reprogramming. It is expected that these treatments and treatment combinations will induce a parthenogenetic response in the microspores, such as Figure 3 As shown, the cell fate of the microspore is changed from microgametogenesis to microspore embryogenesis response with male characteristics. It is also expected that the use of multiple embryogenic factors selected from the polypeptides of SEQ ID NO: 17 to SEQ ID NO: 32 in combination with the cell reprogramming treatment disclosed in this Example 6 will further improve microspore cell reprogramming.

[0678] After any of the cell reprogramming treatments disclosed above, such as Figure 3 As shown in, the haploid plantlets derived from microspores can be transferred to a resting medium with a chromosome doubling (or mitotic inhibitor) agent to produce double haploid plants. For example, the plantlets are treated with colchicine at a concentration of 0.1-1.0 g / ml for 24 hours to arrest the mitosis of dividing cells in metaphase by interfering with microtubule organization, and then transferred to a resting medium without a chromosome doubling agent, followed by incubation at 28° C. in the dark, and then transferred to a maturation medium without selection. Alternatively, based on the morphological development of microspore-derived embryos or embryoids produced by treated microspores, the embryoids derived from microspores are treated with a chromosome doubling agent for 24 hours, then transferred to a resting medium without chromosome doubling, then incubated at 28° C. in the dark, and then transferred to a maturation medium without selection.

[0679] Example 7: dCas9 technology as a paternal double haploid approach for cell reprogramming

[0680] The cell reprogramming method of the present disclosure comprises treating a cell having a target locus with a heterologous protein (including a heterologous protein complexed with a guide RNA) that hybridizes to the target locus and alters gene regulation at the target locus, resulting in cell reprogramming of the cell. Target loci that can be used for the cell reprogramming method of the present disclosure include those loci encoding embryogenic factors listed in Table 6 and / or loci encoding morphogenic developmental genes (including but not limited to ODP2 / BBM, WUS or LEC1 morphogenic genes and combinations thereof).

[0681] It is expected that cell fate reprogramming can be achieved using a dCas9 translational fusion protein / gRNA complex recruited to a target locus region. For example, it is expected that cells from plant tissues, such as leaf cells, in which a gene at a target locus is not expressed, can be transcriptionally activated after treatment with a dCas9 translational fusion protein / gRNA complex specific for a gene at the target locus.

[0682] Using the cell reprogramming method disclosed in the present invention, it is expected that changing the gene regulation state of the endogenous locus in the gamete cell (such as a mononuclear microspore or an egg cell) will lead to gene activation and reprogramming of the cell, thereby giving the treated cell an improved embryogenic phenotype. The cell reprogramming method disclosed in the present invention uses the CRISPR-Cas9 system guided by RNA to hybridize to the DNA encoding the target locus or sequence, thereby changing the expression of at least one gene product. The target locus or the sequence of interest includes but is not limited to the embryogenic factors and morphological development genes listed in Table 6. The CRISPR-Cas9 system guided by RNA that can be used in the cell reprogramming method disclosed in the present invention includes the design of a heterologous protein, the heterologous protein comprising a degenerate, also referred to as an inactivated Cas9 protein (dCas9), which is used as a recognition domain fused to a regulatory domain (such as a transcriptional activator domain, a transcriptional repressor domain and / or a chromatin modification domain for changing the expression of the target locus or sequence).

[0683] Guide RNA (gRNA) is used to recruit dCas9 fusion protein to each of the embryogenic factors listed in encoding Table 6 and / or the endogenous locus of morphological development genes. Once ribonucleotide complex is recruited to the target locus, cell reprogramming is achieved by changing the expression of at least one gene product (including but not limited to the post-translational modification of histone modification), the post-translational modification includes but not limited to (a) removing the histone modification associated with repressor gene expression, (b) establishing the histone modification associated with promoting gene expression, and / or (c) recruiting the transcription machinery associated with expressed genes, and (a), (b) and / or (c) combination. The expression of this change of at least one gene product reprograms the treated microspore from the microspore cell fate to the embryogenic cell fate, thereby improving the production of double haploids of maize male parents.

[0684] The heterologous protein of the dCas9 translation fusion protein containing chromatin modification domain (including histone demethylase domain), particularly the Jumanji family of lysine demethylase that gives histone demethylase catalytic activity, referred to herein as dCAS9-jmj fusion protein, can be used for the cell reprogramming method disclosed herein. The heterologous protein (referred to herein as dCas9-HAT fusion protein) of the inactivation Cas9 (dCas9) protein fused with histone acetyltransferase (HAT) domain (including but not limited to HAT domain (characterized by general control non-repression (Gcn5) related N-acetyltransferase (GNAT) domain, MYST domain and / or B-type catalytic subunit domain, wherein each is known to give histone acetyltransferase catalytic activity)) can also be used for the cell reprogramming method disclosed herein. These dCas9 translation fusion proteins are used as cell reprogramming agents in the method disclosed herein. The chromatin modification domain is shown in Table 8. The dCas9 translation fusion expression cassette containing the chromatin modification domain of Table 8 is shown in Table 9.

[0685] Table 8. Regulatory domains that can be used for dCas translational fusion proteins

[0686]

[0687] Table 9. dCas9 translational fusion expression cassettes that can be used as cell reprogramming factors

[0688]

[0689]

[0690] Inactivated Cas9 (dCas9) proteins fused to transcriptional activator domains can also be used in the cell reprogramming methods disclosed herein. One such domain that can be used in the cell reprogramming methods disclosed herein is from the Arabidopsis CBF1 protein. In particular, the D10A and H840A mutations in Spy Cas9 (polynucleotide SEQ ID NO: 83 encoding polypeptide SEQ ID NO: 84) are fused to the transcriptional activator component of the Arabidopsis CBF1 protein in an expression cassette (which is operably linked to the maize UBIQUITIN promoter and uses a PIN II terminator sequence) (referred to as dCas9-CBF1A, polynucleotide SEQ ID NO: 85 encoding polypeptide SEQ ID NO: 86). Alternatively, the transcriptional activator domain of the VP64 domain (polynucleotide SEQ ID NO: 244 encoding polypeptide SEQ ID NO: 245) is another option for the regulatory domain, which can be fused to dCas9 for the cell reprogramming methods disclosed herein.

[0691] For the methods described in this Example 7, a non-natural fusion RNA molecule of two RNA molecules (crRNA (CRISPR RNA) fused to tracrRNA (trans-activating CRISPR RNA) comprising a variable targeting domain (linked to a tracr pairing sequence that hybridizes with tracrRNA)) can be used for guide RNA molecule delivery. The guide RNA (gRNA) may include a crRNA or crRNA fragment of a type II CRISPR / Cas system that can form a complex with a type II inactivated Cas endonuclease and a tracrRNA or tracrRNA fragment, wherein the gRNA / dCas9 endonuclease complex can be guided to a DNA target site, allowing the dCas9 endonuclease to recognize and bind to the DNA target site, thereby allowing the functional activity conferred by the one or more heterologous dCas9 endonuclease fusion proteins described above to alter the gene regulation of the target locus.

[0692] Methods of providing gRNA to cells include, but are not limited to, introducing single-stranded or double-stranded polynucleotide molecules (e.g., gRNA molecules produced as gRNA expressed using DNA plasmids, or as gRNA transcribed in vitro, or as synthetic (sgRNA) produced using solid phase RNA synthesis chemistry) into cells.

[0693] For the guide RNA delivered into the cell by introducing a recombinant DNA molecule, the method includes operably linking a heterologous nucleic acid fragment encoding a guide RNA (or crRNA+tracrRNA) to a specific promoter capable of transcribing the guide RNA (crRNA+tracrRNA molecule). The guide RNA molecules C5, C4, C3, C2, and C1 (SEQ ID NOs: 87-91, respectively) targeting the WUS2 promoter region can be operably linked to a maize RNA polymerase III promoter (SEQ ID NO: 92), which allows transcription of RNA with precisely defined, unmodified 5' and 3' ends. Alternatively, any promoter capable of transcribing a guide RNA in a cell can be used.

[0694] It is expected that the use of guide RNA molecules designed to simultaneously target multiple loci can further improve the cell reprogramming of cell reprogramming agents. For example, the method of using gRNA molecules capable of binding to target sites encoding embryogenic factors (polynucleotide SEQ ID NO: 1 to 16) and morphological development genes encoded in the plant genome can improve the cell reprogramming of maize microspores, thereby generating an improved male double haploid production method.

[0695] It should be further understood that the gRNA molecules capable of binding to the loci encoding embryogenic factors and / or the loci encoding morphogenic genes should include regions flanking the loci encoding embryogenic factors and / or the loci encoding morphogenic factors, including proximal regulatory regions up to 10,000 nucleotides upstream or downstream of the loci. In addition, gRNA molecules capable of binding to the cis-regulatory regions of the target loci can also be used in the cell reprogramming methods disclosed herein.

[0696] In view of the genetic diversity of different maize varieties or inbred lines, it is expected that different inbred lines will have different alleles at the target locus, wherein different alleles have DNA sequence variation at the locus. Therefore, it should be understood that the allele-specific gRNA sequence composition will supplement the corresponding DNA allele sequence, thereby giving the best hybridization at the target locus.

[0697] Multiple gene targeting agents can be used for the cell reprogramming method disclosed herein.One such multiple gene targeting agent is Cas6, previously known as Csy4.Cas6 can be cut in a precursor RNA molecule called pre-crRNA (which contains a repetitive sequence that generates a mature guide RNA).Mature single guide RNA (gRNA) comprising crRNA and tracrRNA can be associated with dCas9 protein to form a programmable source of gRNA-Cas9 ribonucleotide protein complex (RNP), allowing RNP to be recruited to multiple targets in cells.As disclosed herein, multiple targets in the target cell for reprogramming the microspore treated from the microspore cell fate cell to the embryogenic cell fate include but are not limited to the target locus encoding the embryogenic factor locus and / or the target locus encoding the morphological development locus.

[0698] The cell reprogramming method of this example 7 includes treating cells with a heterologous protein cell reprogramming agent, the heterologous protein cell reprogramming agent includes a heterologous protein compounded with a guide RNA. The heterologous protein cell reprogramming agent changes the gene regulation at the endogenous locus encoding the target site in the cell. More specifically, the heterologous protein cell reprogramming agent is a dCas9 translational fusion protein compounded with a gRNA introduced into a cell (e.g., isolated, wild-type microspore), wherein the treated microspore is reprogrammed from the microspore cell fate to the embryogenic cell fate to obtain a microspore-derived haploid plant.

[0699] A. Cell reprogramming method using DNA plasmid particle bombardment.

[0700] In this Example 7, particle bombardment is used to deliver degenerate Cas9-mediated cell reprogramming agents to isolated maize microspores. It should be understood that different methods can be used to deliver degenerate Cas9-mediated cell reprogramming agents to isolated maize microspores. It can also be understood that similar schemes can be used to treat microspores of other plants, including but not limited to: soybean, cotton, canola, wheat, rice, sorghum or sunflower. A single plasmid or multiple plasmids can be used for each particle bombardment. For example, multiple plasmids including the following can be co-bombarded: 1) a plasmid containing an expression cassette of a degenerate Cas9-chromatin remodeling fusion protein, and 2) a plasmid containing an expression cassette of one or more gRNAs, and optionally 3) a plasmid containing a reporter gene cassette.

[0701] To test the functional activity of dCAS9-chromatin modifier sex-translational fusion proteins, different combinations of guide RNAs were delivered to isolated microspores and the microspore embryogenesis response resulting from improved cellular reprogramming to produce male-like characteristics was monitored.

[0702] A DNA plasmid containing a single gRNA expression cassette (SEQ ID NOs: 201-206, see Table 4) corresponding to gRNA molecules C5, C4, C3, C2, and C1 (SEQ ID NOs: 87-91, respectively) operably linked to a pol III promoter (SEQ ID NO: 92) targeting endogenous WUS2 was co-bombarded with a DNA plasmid containing the aforementioned dCas9 translational fusion protein, e.g., a dCas9-CBF1A expression cassette.

[0703] Optionally, a DNA plasmid containing a multiplex gene targeting agent can be used, for example a DNA plasmid containing an expression cassette (SEQ ID NO: 206), wherein the expression cassette comprises a polynucleotide encoding 1) a maize-optimized Cas6 / Csy4 peptide, 2) a precursor RNA molecule capable of targeting the maize endogenous ODP2 / BBM locus, and optionally 3) a ZsYELLOW color marker protein.

[0704] The DNA used in this Example 7 was attached to gold particles using standard methods. Particle bombardment was performed using a PDS-1000 / He system (Bio-Rad, #166-2257) under vacuum at -28 inches of mercury (in. Hg), with a helium pressure of 650 psi and a target distance of 6 cm.

[0705] Optionally, methods for improving transient expression can be performed to provide increased transcription and expression while preventing subsequent release of DNA. One such method uses polyethyleneimine (PEI; Sigma #P3143) to concentrate the target DNA plasmid. PEI condenses DNA into positively charged polymer DNA complexes, known as polymers. After entering the cell, the ability of the polymer DNA complex to avoid acidic lysosomes causes the polymer transcriptional activity to increase. Specifically, the buffering capacity of PEI can cause lysosomal osmotic expansion and rupture, thereby improving DNA transcription in the cytoplasm. Before adding DNA, gold particles are coated with PEI.

[0706] Prior to bombardment, isolated microspores were placed on tissue culture plates or, optionally, cells were placed on sterile polycarbonate membranes (Millipore Sigma, #ATTP02500) to allow transfer after bombardment. The bombarded cells were incubated at 28°C in the dark. After 14-28 days, multicellular structures (MCS) were transferred to solidified 289Q medium and incubated at 28°C in the dark until plantlets germinated.

[0707] Particle bombardment was performed as described above using a transient expression DNA plasmid using a DNA plasmid containing an expression cassette encoding the following: dCas9-CBF1A fusion protein (SEQ ID NO: 85) and gRNA molecules targeting the WUS2 and BBM / ODP2 genomic loci PHP89615 (SEQ ID NO: 201), PHP89613 (SEQ ID NO: 203), PHP89611 (SEQ ID NO: 205), and RV038531 (SEQ ID NO: 206).

[0708] Bombarding freshly separated corn microspores with these cell reprogramming agents improves the initiation of embryogenesis response, characterized in that the sporopollenin rupture of the exine in the cell proliferation and responsive microspore subsets that increase. The feature of other embryogenesis response progress is that multicellular structure (MCS) further develops into the transformation of embryo-like structure (ELS). No transformation to the organized structure that can sprout in vitro to produce plantlets is observed.

[0709] The corn microspores separated were cultured for about 14 days to produce multicellular structures (MCS). Bombarding these MCS with these cell reprogramming agents also showed the initiation of improved embryogenesis response, characterized in that the sporopollenin rupture of the exine in the cell proliferation and responsive microspore subsets increased. Other embryogenesis response progress is characterized in that the treated MCS further develops into the transformation of embryo-like structure (ELS), which is then converted into embryoids, which are organized structures ( Figure 4B-Figure 4E ).

[0710] After these experiments, it was proved that Figure 4A The embryogenic response shown in . When the cells were bombarded with an expression vector containing polynucleotides encoding WUS2 and ODP2 / BBM proteins (labeled as DG, SEQ ID NO: 210) or an expression vector containing polynucleotides encoding dCas9-CBF1A polynucleotides (labeled as dCas9, SEQ ID NO: 85), and when the cells were bombarded with unlabeled gold particles (labeled gold), the ratio of embryo-like structure (ELS) productivity of the experimental treatments (DG, dCas9, gRNA, RNP and gold) showed slight improvements in embryogenic response in some replicates compared to the untreated control (NTC) treatment. Overall, these results of DG, dCas9 and gold indicate that the cellular reprogramming response is relatively unchanged. As Figure 4A As shown in , embryogenic responses were demonstrated after gRNA and RNP experimental treatment.

[0711] like Figure 4A As shown in (labeled gRNA), improved cell reprogramming was observed in response to bombardment (i.e., bombardment with PHP89615 (SEQ ID NO: 201), PHP89613 (SEQ ID NO: 203), PHP89611 (SEQ ID NO: 205), and RV038531 (SEQ ID NO: 206)) using DNA vectors containing gRNA molecules targeting the WUS2 genomic locus. The embryogenic response was observed after bombarding freshly isolated maize microspores with DNA plasmids containing gRNA molecules PHP89615 (SEQ ID NO: 201), PHP89613 (SEQ ID NO: 203), and PHP89611 (SEQ ID NO: 205) targeting the WUS2 genomic locus. However, the developed embryo-like structure (ELS), which is more than 0.9 mm ( Figure 4C ), unable to maintain sustained embryonic development over time.

[0712] Improved microspore embryogenesis response was also observed after cells were co-bombarded with an expression vector containing a polynucleotide encoding a dCas9-CBF1A polynucleotide (SEQ ID NO: 85) and gRNA molecules targeting the WUS2 and BBM genomic loci (i.e., co-bombarded with PHP89615 (SEQ ID NO: 201), PHP89613 (SEQ ID NO: 203), PHP89611 (SEQ ID NO: 205), and RV038531 (SEQ ID NO: 206). Figure 4AShown in (labeled RNP) is a transient expression treatment containing a fully functional dCas9 ribonucleoprotein complex treatment designed to target the WUS2 and BBM / ODP2 genomic loci. The embryoid development rate in response to the RNP treatment was observed within 14 days of bombardment, as shown in Figure 1. Figure 4B , indicating an improved cellular reprogramming response.

[0713] When multicellular structures derived from maize microspores cultured for approximately 14 days were bombarded, an approximately 2-fold increase in ELS productivity was observed compared to the response observed using freshly isolated maize microspores.

[0714] It is expected that delivery of PEI-DNA complexes to maize microspores cultured for approximately 14 days will result in an improved microspore embryogenic response.

[0715] Additional experiments were performed to assess cellular reprogramming responses and further characterize microspore embryogenesis responses to bombardment with synthetic gRNA molecules with or without dCas9-CBF1A polynucleotide (SEQ ID NO: 85).

[0716] B. Cell reprogramming method using co-bombardment of DNA plasmid and sgRNA particles.

[0717] sgRNA molecules were designed and synthesized for the target loci of WUS2, ODP2 / BBM, and LEC1 (SEQ ID NOs: 207-209, respectively). Each of these sgRNA molecules was attached to gold particles and bombarded into cells as described above. Improved cellular reprogramming responses were observed in microspores bombarded with WUS2 and ODP2 / BBM sgRNA molecules relative to untreated microspore cells ( Figure 4D ). In the absence of a transient expression vector containing a dCas9-CBF1A polynucleotide, sgRNA was bombarded into microspores alone. These results show that contacting cells with a single sgRNA improves cell reprogramming. These results show that RNA (including but not limited to RNA produced as a strand-specific transcript from a genetic response element) acts as a key functional switch to promote cell reprogramming in plant cells by causing the DNA response element to act as an enhancer or a repressor when using an opposite strand RNA molecule. C. Cell reprogramming method using ribonucleotide protein complexes.

[0718] Particle bombardment is used to deliver RNP-complexed cellular reprogramming agents to plant cells.

[0719] A protein expression system can be used to express a degenerate Cas9 translational fusion protein (e.g., as described above by transferring a polynucleotide sequence encoding a dCas9 protein fusion as described in Table 9 into a protein expression vector, such as the pDAB135006 vector (SEQ ID NO: 60)). Standard protein expression methods can be performed to express and then purify recombinant proteins, such as the dCas9 fusion proteins in Table 9 (SEQ ID NOs: 78-82), optionally in combination with a dCas9 transcriptional activator (e.g., a polypeptide expressed from a polynucleotide encoding SEQ ID NO: 84 fused to SEQ ID NO: 86). Other protein expression vectors and protein expression systems can be used.

[0720] The dCas9-CBF1a polypeptide was expressed from a protein expression cassette encoding SEQ ID NO: 84 and SEQ ID NO: 86 fused to a His-MBP-tag and purified using an E. coli expression system. First, the expression construct was transformed into E. coli BL21 (DE3) or ArcticExpress (DE3) strains and the culture was grown in LB broth supplemented with a selection agent such as ampicillin (100 μg / ml). The culture was grown to an OD of 1. 600 After the concentration of 1% arabinose was 0.5, the temperature was lowered to 16°C, and expression was induced with IPTG (0.5 mM) or arabinose (0.2% (w / v)). After 16 hours, the cells were pelleted and resuspended in loading buffer (20 Tris-HCl, pH 8.0, 25°C, 1.5 M NaCl, 5 mM 2-mercaptoethanol, 10 mM imidazole, 2 mM PMSF, 5% (v / v) glycerol) and disrupted by ultrasound. Cell debris was removed by centrifugation. The supernatant was loaded onto a Ni-bearing 2+The dCas9-CBF1a polypeptide was charged onto a HiTrap chelating HP column (GE Healthcare) and eluted with a linear gradient of increasing imidazole concentration (from 10 to 500 mM) in 20 mM Tris-HCl (pH 8.0, 25°C), 0.5 M NaCl, 5 mM 2-mercaptoethanol buffer. The fractions containing the dCas9-CBF1a polypeptide were pooled and subsequently loaded onto a HiTrap heparin HP column (GE Healthcare) using a linear increasing concentration gradient of NaCl (from 0.1 M to 1.5 M) for elution. The next fraction containing the dCas9-CBF1a protein was pooled and the tag was cleaved by incubation with TEV protease overnight at 4°C. To remove the cleaved His-MBP tag and TEV protease, the reaction mixture was loaded onto a HiTrap heparin HP 5 column (GE Healthcare) and eluted using a linear gradient of increasing NaCl concentration (from 0.1 to 1.5 M). Next, the eluate from the HiTrap column was loaded onto an MBPTrap column (GE Healthcare) and the dCas9-CBF1a polypeptide that flowed through was collected. The collected fractions were then dialyzed in 20 mM Tris-HCl (25°C, pH 8.0), 500 mM NaCl, 2 mM DTT, and 50% (v / v) glycerol and stored at -20°C until use.

[0721] Briefly, RNP assembly of the RNP complexed cell reprogramming agent was performed by combining Riboguard RNase inhibitor (1 μL; Lucigen #RG90925), 10X reaction buffer 3.1 (2.0 μL; New England Biolabs #B7200S), 1 μL of dCas9-CBF1a fusion protein (5 μg protein / μL), gRNA molecules for targeting genomic WUS2, ODP2 / BBM and LEC1 loci (1.5 μg / gRNA) (SEQ ID NOs: 207-209, respectively), and nuclease-free water (Thermo Fisher scientific #AM9932) in a 20 μL final volume, RNase-free microcentrifuge tube. The RNP assembly mixture was incubated at room temperature for about 15 minutes, and then the tube was placed on ice. The premixed RNP assembly was combined with sonicated gold particles (30 μL; 6 μg / μl) and mixed gently, then incubated on ice for approximately 10 minutes.

[0722] The carrier discs were loaded by centrifuging the RNP-complexed cell reprogramming agent / gold suspension at 10,000 rpm for 60 seconds, removing the supernatant, and resuspending the pellet in 50 μl of RNase-free H2O by brief sonication (approximately 10 seconds) followed by pipetting to mix thoroughly, dispensing a volume of 10 μL per disc. Particle bombardment was then performed as described previously.

[0723] In addition, the cell reprogramming agent of RNP compound is prepared, filtered and sterilized, and incubated at room temperature for about 15 minutes, and then directly added to maize microspore cells (microspores of fresh separation or microspores of culture 14 days filtered and washed with 70 μm cell strainer (Thermo Fisher Scientific, #22-363-548). After the cell is contacted and incubated with the cell reprogramming agent of RNP compound, the treated cells are transferred to a tissue culture plate and bombarded with unlabeled ultrasonically treated gold particles as described above.

[0724] Improved microspore embryogenic response of microspore cells to cell reprogramming using the DNA plasmids described herein is shown in Figure 4A-4E In view of those results, it is expected that the cell reprogramming agent that provides RNP complex to the cell derived from microspore will improve cell reprogramming by changing the initial cell fate of the cell to become the embryogenic cell fate to provide the embryoid body derived from microspore. When the embryoid body derived from microspore is treated with a chromosome doubling agent, it is expected that a double haploid plant of the male parent of maize will be produced.

[0725] D. Cell Reprogramming Methods Using Combination dCas9 Technology as Cell Reprogramming Agents

[0726] The cell reprogramming effect of the cell reprogramming agent combination was evaluated. The cells were co-bombarded with a plasmid combination comprising a dCas9 transcriptional activator and a dCas9 histone acetyltransferase fusion protein, and the cell reprogramming effect was evaluated. The co-bombardment of these cell reprogramming agents changed the post-translational modification of chromatin at the genomic target site, while promoting transcriptional upregulation at the genomic target site. This type of combined activity that conferred cell reprogramming in microspore-derived cells is expected to further improve the microspore embryogenesis response of producing male characteristics, thereby improving the production of male parent double haploids in recalcitrant maize genotypes.

[0727] According to the above method, the following plasmids are used: 1) a first DNA plasmid, a dCas9-CBF1A plasmid, which has a polynucleotide encoding a dCas9-CBF1A translational fusion protein (a polynucleotide SEQ ID NO: 85 encoding a polypeptide SEQ ID NO: 86) (a first transcription up-regulation treatment); 2) a second DNA plasmid, a dCas9-VP64 plasmid, which has a polynucleotide encoding a dCas9-VP64 translational fusion protein (dCas9 sequence (a polynucleotide SEQ ID NO: 83 encoding a polypeptide SEQ ID NO: 84)) fused to a transcriptional activator component of the VP64 domain (a polynucleotide SEQ ID NO: 244 encoding a polypeptide SEQ ID NO: 245) (a second transcription up-regulation treatment); and 3) a third DNA plasmid, a dCas9-HAT2 plasmid, which has a polynucleotide encoding a dCas9 histone acetyltransferase 2 translational fusion protein (SEQ ID NO: 76) (chromatin modification treatment (enrichment of target loci by increasing the level of histone acetylation modification associated with active gene expression)).

[0728] The following treatments were used on the first bombardment carrier disk: 1) a first treatment of bombarding the dCas9-CBF1A plasmid; 2) a second treatment of bombarding the dCas9-VP64 plasmid; 3) a third treatment of co-bombarding the dCas9-CBF1A plasmid and the dCas9-HAT2 plasmid; 4) a fourth treatment of co-bombarding the dCas9-VP64 plasmid and the dCas9-HAT2 plasmid; and 5) a fifth treatment used as a control, which did not receive a bombardment treatment. Each of treatments 1-4 further included on the second bombardment carrier disk, providing equal amounts of DNA plasmids PHP89613 (SEQ ID NO: 203), PHP89611 (SEQ ID NO: 205), and RV038531 (SEQ ID NO: 206) to target WUS2 and ODP2 / BBM genomic target sites for the gold particles used for bombardment. Microspore-derived cells cultured for 14 days at 28°C in the dark were collected using a 70 micron cell strainer, washed, and transferred to tissue culture medium plates as described above. Each plate of 14 day old cells was bombarded with two carrier discs as described above. The plates were then incubated at 28°C in the dark for 8 days and then scored for the presence of responding cells to each treatment relative to the untreated control.

[0729] Treated cells from the first treatment using only the dCas9-CBF1A plasmid showed a 71% improvement in responding cells compared to the untreated control. Treated cells from the second treatment using only dCas9-VP64 showed an 8% improvement in responding cells compared to the untreated control. Treated cells from the third treatment using the combined dCas9-CBF1A plasmid and dCas9-HAT2 plasmid produced responding cells equal to those in the no-treatment control. Treated cells from the fourth treatment using the combined dCas9-VP64 plasmid and dCas9-HAT2 plasmid showed a 50% improvement in responding cells relative to the no-treatment control.

[0730] These results show that the combination treatment of the maize microspore culture from the recalcitrant genotype may need to optimize the combination of so-called "synthetic transcription factor" and the dCas9 translation fusion protein used herein, to provide microspore-derived embryoids. It is expected that the plasmid combination comprising dCas9 histone demethylase, dCas9 histone acetyltransferase and dCas9 transcription activator or any combination thereof will further improve the cell reprogramming towards embryogenic cell fate in the plant cell with initial, non-embryogenic regulatory state. It is also expected that some genotypes and / or cells showing different degrees of cell reprogramming (such as microspore embryogenesis response caused during response tassel pretreatment or in vitro cell culture) may need different cell reprogramming agent activities in a manner depending on the time or duration of cell fate reprogramming.

[0731] It is also contemplated that improved cell reprogramming can be achieved in response to treating cells with a combination of gRNA molecules. For example, where different gRNA molecules are capable of recruiting dCas9 translational fusion protein activity to multiple target loci, such as loci encoding embryogenic factors and / or morphogenic genes described in Table 6.

[0732] It is expected that bombarding and / or contacting microspores with a DNA plasmid having multiple expression cassettes (e.g., a polynucleotide comprising a first expression cassette encoding a dCas9-transcriptional activator translational fusion protein, a second expression cassette encoding a dCas9 histone acetyltransferase translational fusion protein, and a third expression cassette capable of providing one or more gRNA molecules) will provide further improvements in cell reprogramming. This improvement is expected to result in an increase in the frequency with which each treated cell is contacted with a plasmid having three activities, compared to the likelihood that each treated cell can be successfully contacted with multiple DNA plasmids, each of which has one activity.

[0733] E. Chromosome Doubling Methods for Obtaining Microspore-Derived Doubled Haploids

[0734] For any of the methods of this Example 7, embryogenic microspore-derived cells (which germinate into plants) can be transferred to a medium containing a chromosome doubling (or mitotic inhibitor) agent to produce doubled haploid plants. For example, colchicine at a concentration of 0.1-1.0 g / ml can be used for treatment, which arrests the mitosis of dividing cells in metaphase by interfering with microtubule organization.

[0735] Example 8: Paternally Doubled Haploid Method Using Improved Cellular Uptake of Cellular Reprogramming Agents

[0736] The methods disclosed herein improve the efficacy of maize male haploid plant production by contacting plant cells with a heterologous protein comprising a dCAS9 translational fusion protein fused to a cell penetrating peptide (CPP) motif as described above. The fusion protein is purified using the above protein expression method and recruited to the target locus as described above.

[0737] To improve cellular delivery and / or cellular uptake of the cell reprogramming peptides disclosed herein, the methods of the present disclosure further comprise the use of a translational fusion peptide containing a cell penetrating peptide (CPP), typically at the N-terminus or C-terminus of the protein. Cell penetrating peptides that can be used in the cell reprogramming methods of the present disclosure for improving the delivery and / or uptake of cell reprogramming polypeptides into plant cells (e.g., isolated microspores) include, but are not limited to, CPPs (e.g., Zea mays knotted1 CPP (ZM-KNT1 CPP; SEQ ID NO: 48 and SEQ ID NO: 49)), Saccharomyces cerevisiae TP10 CPP (SP-TP10 CPP; SEQ ID NO: 50 and SEQ ID NO: 51), Candida albicans Zebra CPP (CA-Zebra CPP; SEQ ID NO: 52 and SEQ ID NO: 53), PEP1 CPP (PEP1 CPP; SEQ ID NO: 54 and SEQ ID NO: 55), HIV-1 TAT CPP (HIV-1 TAT CPP; SEQ ID NO: 56 and SEQ ID NO: 57), and γ-zein cell penetrating peptide (GZ CPP; SEQ ID NO: 58 and SEQ ID NO: 59).

[0738] The delivery of any of the above-mentioned cell reprogramming components is improved by using CPP-mediated protein delivery, and is therefore an alternative method that potentially mitigates any risk associated with the above-mentioned particle bombardment method. It is expected that the use of CCP in the cell reprogramming method disclosed herein will improve cell reprogramming by changing the initial cell fate of non-embryogenic wild-type microspores. For example, by providing CPP-dCas9 histone demethylase, CPP-dCas9 histone acetyltransferase, CPP-dCas9 transcriptional activator or any combination thereof to microspores with an initial non-embryogenic regulatory state, it is expected that the cell reprogramming of the cell will be improved, thereby obtaining a plant derived from the microspores of non-genetic engineering. The morphological changes of such treated microspore cells can be analyzed, and such embryogenic cells can be cultured in vitro using standard methods.

[0739] It is also expected that improved cell reprogramming can be achieved using a combination of gRNA molecules, wherein each gRNA causes the CPP-dCas9 translational fusion protein to bind to a unique, independent genomic target site (e.g., an endogenous locus with sequence homology to the DNA sequence and / or morphogenic gene sequence described in Table 6). It is expected that this simultaneous binding to multiple loci results in changes in the gene regulatory state of multiple target loci, thereby improving the acquisition of microspore-derived haploid plants (particularly for superior maize inbreds), which are otherwise typically non-responsive and non-embryogenic. When such cells are treated with a chromosome doubling agent, it is expected to result in the generation of non-genetically engineered maize double haploid plants.

[0740] Example 9: Method for improving the productivity of double haploid maize male parent using embryogenic factors

[0741] The following experiments demonstrate that delivery of T-DNA containing a chemically inducible expression cassette containing the embryogenic factors and / or morphogenic developmental genes described in Table 6 improves microspore embryogenesis.

[0742] Two plasmids RV028329 (embryogenic control plasmid) and RV028330 (experimental plasmid) were compared. Both plasmids contain expression cassettes for the expression of WUS and ODP2 peptides operably linked to chemically inducible regulatory elements. These plasmids all contain the same expression cassette with the following main characteristic elements; (a) WUSCHEL expression cassette; TET OP1+NOS PRO(ALT1)+ZM-WUS2+PINII TERM (SEQ ID NO: 93), (b) ODP2 expression cassette; UBI1ZM PRO(TR2)+(3X)TETOP1+UBI1ZM 5UTR+SB-UBI INTRON1+ZM-ODP2+PINII TERM (SEQ ID NO: 94), (c) sulfonylurea responsive repressor expression cassette; UBI1ZM PRO+UBI1ZM 5UTR+UBI1ZM INTRON1+ESR(L15-20)+PINII TERM (SEQ ID NO: 95), (d) selectable marker expression cassette; SB-ALS PRO+ZM-ALS(HRA)(TR1)+PINII TERM (SEQ ID NO: NO:96), and (e) an expression cassette containing a CRE / lox recombination system flanking a WUS2 / ODP2 co-expression cassette (SEQ ID NO:97), wherein CRE is operably linked to a promoter, allowing excision of the WUS2 / ODP2 / CRE cassette after transformation, thereby allowing regeneration of transgenic plants containing only the expression cassettes (a) to (d).

[0743] Plants transformed with RV028330 (experimental plasmid) will additionally contain an expression cassette encoding the coding sequence of the gene dpzm07g031470.1.1, referred to herein as (ZM-CYCD2), i.e. CAMV35S PRO+(3X)TET OP1+OMEGA 5UTR+ADH1 INTRON1+ZM-CYCD2+PINII TERM (SEQ ID NO: 98).

[0744] Both plasmids include a polypeptide that is a sulfonylurea-responsive repressor protein (which binds to an operator sequence controlled by a ligand containing a sulfonylurea compound). In the absence of a sulfonylurea ligand, the repressor protein specifically binds to the operator gene. After providing a sulfonylurea compound to a transgenic cell expressing a sulfonylurea-responsive repressor protein, the sulfonylurea binds to the repressor protein to form a complex that modifies the binding properties of the repressor protein to the operator gene and leads to the derepression of the operator gene sequence. In this way, the "chemical switch-mediated expression" system is used to test the effect of each embryogenic factor in Table 6 (e.g., a polynucleotide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16). Each polynucleotide can be transferred into an expression cassette as shown in SEQ ID NO:98, wherein the specific embryogenic factor used corresponds to the embryogenic factor dpzm07g031470.1.1 (ZM-CYCD2) (polynucleotide SEQ ID NO:5; polypeptide SEQ ID NO:21).

[0745] Transgenic plants containing plasmid RV028329 or RV028330 were generated by Agrobacterium-mediated transformation as described above. After Agrobacterium-mediated transformation, somatic embryo maturation and regeneration, T0 plants were analyzed by qPCR to identify single-copy, hemizygous individuals containing either plasmid. Such plants were grown and fertilized with wild-type pollen to produce seeds, of which approximately half were hemizygous T1 seeds and half were wild-type T1 seeds.

[0746] As mentioned above, the initial event characterization of TO tassel is carried out using isolated microspores. In order to induce morphological development gene expression, the microspore cells of culture are processed with ethametsulfuron (e.g., 0.1 to 1ppm). Preliminary results show that the embryogenic factors disclosed in Table 6 are used to double the haploid productivity of the maize male parent, and after the gene activation in response to ethametsulfuron processing, the morphogenic genes are improved.

[0747] In view of the sample size of a tassel of each unique T0 event, it is impossible to further characterize the embryogenesis effect of this generation. Future generations will be used to further assess the improvement of the microspore embryogenesis in response to the coexpression of embryogenic factor and morphogenic gene.Expectation will further improve the frequency of microspore embryogenesis response in the processed microspore with any embryogenic factor and morphogenic gene coexpression disclosed in the table 6.Also expect that the response of this type of improvement may comprise the quantity or number of improving response cells, and improve the quality of the embryo derived from microspore, include but not limited to the cell organization, morphological embryogenesis development of improvement and / or can be used to obtain the plantlet regeneration of fertile double haploid plant.

[0748] Example 10: Paternal doubled haploid method using improved in situ plant cell reprogramming method

[0749] The following experiments demonstrate that the acquisition of non-transgenic, microspore-derived haploid plants can be improved by contact with a plasmid containing: i) an expression cassette encoding an embryogenic factor disclosed in Table 6, ii) a morphological development gene and iii) and combinations thereof.

[0750] In the cell reprogramming method of the present disclosure, a plasmid is delivered and stably integrated into the DNA of a plant cell. The integrated DNA used contains an expression cassette designed to improve microspore embryogenesis in situ in planta prior to microspore isolation.

[0751] For these experiments, two plasmid designs were tested. The first plasmid contained an expression cassette containing a polynucleotide (SEQ ID NO: 319) encoding the Ms44 signal peptide sequence (SEQ ID NO: 320) fused to a polynucleotide (SEQ ID NO: 363) encoding a WUSCHEL polypeptide fused to a glucocorticoid receptor (GR) (SEQ ID NO 364) and an Ms44 terminator sequence (SEQ ID NO: 327), operably linked to a maize Ms44 promoter (SEQ ID NO: 318).

[0752] The second plasmid contains an expression cassette comprising a polynucleotide (SEQ ID NO:319) encoding an Ms44 signal peptide sequence (SEQ ID NO:320) fused to a polynucleotide (SEQ ID NO:363) encoding a WUSCHEL polypeptide fused to a glucocorticoid receptor (GR) (SEQ ID NO:364); a polycistronic linker ("T2A"; SEQ ID NO:101 encoding SEQ ID NO:102); a translational fusion protein comprising a DNA sequence encoding an embryogenic factor selected from any one of the DNA sequences shown in Table 6; and consisting of SEQ ID NOs:1-16 with a C-terminal fusion using any one of the following cell penetrating peptides, including but not limited to: Zea mays knotted1 CPP (ZM-KNT1 CPP; SEQ ID NO:48 encoding SEQ ID NO:49), Saccharomyces pombe TP10 CPP (SP-TP10 CPP; SEQ ID NO:50 encoding SEQ ID NO:51), Candida albicans Zebra CPP (CA-Zebra CPP; SEQ ID NO: 52 encoding SEQ ID NO: 53), PEP1 CPP (PEP1 CPP; SEQ ID NO: 54 encoding SEQ ID NO: 55), HIV-1 TAT CPP (HIV-1 TAT CPP; SEQ ID NO: 56 encoding SEQ ID NO: 57), and gamma-zein cell penetrating peptide (GZ CPP; SEQ ID NO: 58 encoding SEQ ID NO: 59) (see US 8581036, incorporated herein by reference in its entirety); and an Ms44 terminator sequence (SEQ ID NO: 327), operably linked to a maize Ms44 promoter ("ZM-Ms44 PRO"; SEQ ID NO: 318).

[0753] Each construct promotes protein expression and transport of expressed proteins from tapetum cells to the ovary chambers of anthers, thereby allowing delivery and uptake into microspore cells. In the cell reprogramming method disclosed herein, after the WUSCHEL-GR fusion protein is taken up into the microspore, the protein is conditionally localized to the nucleus by external application of animal hormone analogs during in vitro tissue culture, resulting in controlled induction of cell reprogramming. For methods using a second plasmid containing the WUSCHEL-GR fusion protein and the embryogenic factor CPP fusion protein, the WUSCHEL-GR fusion protein is conditionally localized to the nucleus by external application of animal hormone analogs to in vitro tissue culture, and the embryogenic factor CPP fusion protein can be constitutively active.

[0754] F1 hybrid embryos are produced by hybridizing two inbred parental systems, wherein one of the parents is the maternal parent (ear donor) and the second parent is the male parent (pollen donor). Using standard methods, two plant varieties can be cross-fertilized in a manner to prevent any outcrossing to produce F1 hybrid embryos. When both parents are homozygous at all full genome loci, it is expected that the resulting embryo will be heterozygous at all full genome loci. The disclosed method collects immature F1 hybrid embryos for processing as described below.

[0755] In the current example, one of the two plasmids is used to transform immature F1 hybrid embryos, each of which is incorporated into an Agrobacterium transformation vector. Agrobacterium-mediated transformation is performed using standard protocols. Alternatively, the transformation vector can be introduced into plant cells using a biolistic transformation method or any other standard method.

[0756] After transformation of each construct and selection of hemizygous transformed plants, the plants are grown, staged, and the tassels are harvested when the anthers contain uninucleate microspores. Preferably, the tassels can be pretreated with a low temperature stress treatment at about 8° C. to 10° C. for 14 days. Microspores are isolated from the T0 hemizygous tassels, and the cells are cultured in 9% sucrose induction medium.

[0757] Alternatively, wild-type embryos can be transformed, regenerated and single-copy hemizygous plants can be propagated to allow for the presence of homozygous transgenic constructs in offspring. Such homozygous lines can be used as parents in a genetic cross with a second line to produce hemizygous F1 hybrids.

[0758] Whether the immature F1 hybrid embryos are transformed directly as described above, or homozygous transgenic lines are preferentially created and used in genetic crosses, the methods of the present disclosure provide transgene copies from the transformed parents that can be used to express cellular reprogramming agents.

[0759] One such agent is a WUS-GR translational fusion protein whose activity depends on the presence of a glucocorticoid (e.g., the synthetic steroid hormone dexamethasone). In the absence of dexamethasone, the WUS-GR translational fusion protein is inactive and located in the cytoplasm. In the presence of dexamethasone, hormone binding allows the WUS-GR translational fusion protein to enter the nucleus, thereby allowing the fusion protein to activate microspore embryogenesis.

[0760] In the current example, by combining the protein activity conferred by the expression cassette containing the embryogenic factor that can be used for cell reprogramming as described in Table 6, activated embryogenesis is further improved by expressing the embryogenic factor-CPP translational fusion protein. Therefore, the method makes it possible to simultaneously activate one or more cell reprogramming agents in the treated cell nucleus. The activity in the cell nucleus allows for cell reprogramming in response to the simultaneous activity of at least two cell reprogramming agents.

[0761] The haploid microspores produced by any of the methods disclosed herein are contacted with a certain amount of a chromosome doubling agent to promote chromosome doubling and regenerate homozygous diploid plants from the treated haploid microspores. The haploid microspore cells may be contacted with a doubling agent before, during or after microspore embryogenesis, embryo maturation or plant regeneration. Various chemicals, such as those listed in Table 2, have chromosome doubling properties, such as colchicine at ...

Claims

1. A method for obtaining doubled haploid plant offspring with modified genome, the method comprising: (a) obtaining a first plant transformed with one or more expression cassettes comprising: i. a polynucleotide sequence encoding a morphological development polypeptide; ii. a polynucleotide sequence encoding an embryogenic factor, wherein the polynucleotide sequence encoding the embryogenic factor is a polynucleotide sequence encoding a cyclin delta-2 polypeptide, and the amino acid sequence of the cyclin delta-2 polypeptide is shown in SEQ ID NO: 21; and iii. a polynucleotide sequence encoding a Cas polypeptide, wherein the Cas polypeptide is capable of genomic modification of a DNA target site in a first plant; wherein the cyclin delta-2 polypeptide promotes embryogenic cell fate and genetic chromosome doubling; (b) obtaining a second plant; (c) pollinating the first plant with pollen from the second plant; (d) obtaining a haploid offspring produced by the pollination step (c), wherein the haploid offspring comprises the genome of the first plant but does not comprise the genome of the second plant, and wherein the genome of the first plant has been modified by the Cas polypeptide; (e) obtaining doubled haploid offspring from the haploid offspring, wherein the cyclin delta-2 polypeptide stimulates diploidization of the haploid offspring to produce doubled haploid offspring; and (f) Selecting genome-modified doubled haploid plant progeny.

2. The method of claim 1, wherein the Cas polypeptide is a Cas9 nuclease, a Casα nuclease, a Cpf1 nuclease, a dCas9-FokI, a dCpf1-FokI, a chimeric Cas9-cytidine deaminase, a chimeric Cas9 adenine deaminase, a nickase Cas9 (nCas9), a chimeric dCas9 non-FokI nuclease, or a dCpf1-non-FokI nuclease.

3. The method of claim 1, wherein the second plant comprises a marker gene selected from the group consisting of GUS, PMI, PAT, GFP, RFP, CFP, C1, CRC, R-nj, R1-scm and anthocyanin pigments.

4. The method of claim 1, further comprising regenerating plants from progeny of the doubled haploid plants.

5. The method of claim 2, wherein the Cas polypeptide is provided with at least one guide RNA molecule.

6. The method of claim 1, wherein the polynucleotide sequence encoding the cyclin delta-2 polypeptide is operably linked to an egg cell promoter.

7. The method of claim 6, wherein the polynucleotide sequence encoding a cyclin delta-2 polypeptide is stably incorporated into the genome of the first plant.

8. The method of claim 7, wherein the cyclin delta-2 polypeptide of the first plant stimulates reprogramming in egg cells that promotes an embryogenic cell fate.

9. The method of claim 8, wherein the cyclin delta-2 polypeptide of the first plant stimulates maternal haploid induction and causes the egg cell to become a parthenogenetic egg cell having only the maternal genome.

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