Methods and compositions for monocotyledon transformation

Abstract

Provided herein are polynucleotides encoding recombinant plant transcription factors, recombinant plant transcription factors, and methods of producing recombinant monocotyledonous plants using recombinant plant transcription factors.

Description

[0001] References to sequence lists

[0002] An official copy of the sequence list is submitted electronically along with the instruction manual. The sequence list is an ST26-compliant XML file named "108740-WO-SEC-1_ST-26_Sequence Listing", created on March 21, 2024, and is 610,838 bytes in size. The sequence list contained in this XML file is an integral part of this instruction manual and is incorporated herein by reference in its entirety. Background Technology

[0003] In recent years, the capabilities of plant genetic engineering have expanded dramatically. Current transformation technologies offer opportunities to produce commercially viable transgenic plants, enabling the creation of new plant varieties with desired traits. However, there remains a need for plant transformation methods that allow for a wider range of transformable and regenerable plant explant tissues and to improve the efficiency of these methods. Summary of the Invention

[0004] This disclosure describes methods and compositions for the transformation of monocotyledonous plants. The disclosed methods and compositions significantly increase the frequency of plant transformation (e.g., by increasing the number of recombinant T0 plants regenerated from each starting seedling and / or by providing the T0 plants with (1) a single copy (instead of multiple copies) insertion of T-DNA containing the target gene and (2) no other detectable inserted nucleotide sequence (e.g., from the plasmid backbone).

[0005] This article describes a polynucleotide encoding a recombinant transcription factor. The recombinant transcription factor comprises a nucleic acid-binding domain and a transcription activation domain. The nucleic acid-binding domain comprises a Bbm-truncated polypeptide and is capable of binding to gene regulatory sequences. The transcription activation domain comprises a transcription activator polypeptide and is capable of activating the transcription of target genes. The nucleic acid-binding domain contains at least 50 amino acid residues, and the transcription activation domain contains at least 20 amino acid residues. The nucleic acid-binding domain and the transcription activation domain are not naturally present in the same polypeptide.

[0006] This document also provides a method for generating recombinant monocotyledonous plants. The method includes contacting a monocotyledonous plant cell with a first polynucleotide encoding a target gene, wherein the target gene is heterologous to the monocotyledonous plant cell. The method also includes contacting the monocotyledonous plant cell with a second polynucleotide encoding a recombinant transcription factor disclosed herein. The method further includes selecting monocotyledonous plant cells in which the target gene has been incorporated into their genome and regenerating recombinant monocotyledonous plants from the selected monocotyledonous plant cells. Detailed Implementation

[0007] This disclosure is not limited to specific instances, which can naturally vary. The terminology and exemplary examples used herein are for the purpose of describing aspects of this disclosure only and are not intended to be limiting. As used herein, singular and singular forms of terms such as “a” and “the” include plural referents unless the context explicitly specifies otherwise. Thus, for example, the reference to “plant,” “the plant,” or “a plant” also includes multiple plants; and depending on the context, the use of the term “plant” may also include genetically similar or identical offspring of that plant; the use of the term “nucleic acid” optionally includes multiple copies of the nucleic acid molecule; similarly, the term “probe” optionally (and typically) covers many similar or identical probe molecules.

[0008] As used herein, the term “comprising” includes aspects that are “composed of”.

[0009] Unless otherwise defined, the numerical ranges listed in the specification include the numbers that define the range and include every integer within the defined range. 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 this disclosure pertains.

[0010] As used herein, a “recombinant” plant or plant cell contains a heterologous nucleic acid sequence, a heterologous polypeptide, and / or a heterologous noncoding RNA. A “recombinant” nucleic acid or polypeptide is a nucleic acid or polypeptide that has been altered from its natural form through artificial intervention via insertion, deletion, substitution, or fusion. The “recombinant” transcription factors disclosed herein are not naturally occurring.

[0011] As used herein, "heterologous" means a given nucleic acid sequence that has been artificially located in a genome, locus, or construct. Heterologous sequences can be naturally occurring but now located in a genome, locus, or construct where the sequence was not naturally found. Alternatively, a sequence can be "heterologous" because it is located in a genome, locus, or construct and is not naturally occurring. Heterologous genes can be inserted into the genome via methods such as transformation and / or site-specific nuclease-based approaches.

[0012] As used herein, the term "morphogenetic gene" refers to a gene that, when ectopically expressed, stimulates the formation of somatic cell-derived structures in a plant. More precisely, ectopic expression, mutation, silencing, or reduced expression of a morphogenetic gene stimulates de novo formation of somatic embryos or organogenesis structures (such as bud meristems or axillary meristems) or stimulates plant regeneration. This stimulated de novo formation occurs in the cell expressing, silencing, or repressing the morphogenetic gene or in neighboring cells. A morphogenetic gene can be a transcription factor that regulates the expression of other genes or a gene that affects hormone levels in plant tissues, both of which can stimulate morphological changes. A morphogenetic gene can be stably incorporated into the plant genome or can be transiently expressed. In one aspect, the expression of a morphogenetic gene is controlled. Expression can be controlled at transcription or post-transcriptional. Controlled expression can also be pulsed expression of a morphogenetic gene at a specific time. Alternatively, a morphogenetic gene can be expressed only in some transformed cells and not in others. The control of morphogenetic gene expression can be achieved through a variety of methods disclosed below. The morphogenetic genes that can be used in the methods disclosed herein can be obtained from or derived from any plant species.

[0013] As used in this article, the term "morphogenetic factor" refers to morphogenetic genes and / or proteins expressed by morphogenetic genes.

[0014] Morphogenesis genes are involved in 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 bud or axillary meristem, initiation and / or development of bud, or combinations thereof, such as WUS / WOX genes (WUS, 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 20170121722 and 20070271628; 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 [Genomics Biology] 10:248; Dolzblasz et al., 2016, Mol. Plant [Molecular Plant] 19:1028-39 can be used with respect to the methods disclosed herein. Regulation of WUS / WOX is expected to modulate plant and / or plant tissue phenotypes, including 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 bud meristem, initiation and / or development of bud, or combinations thereof. Expression of WUS in Arabidopsis can induce stem cells in vegetative tissues that can differentiate into somatic embryos (Zuo et al. (2002) Plant J [Plant Journal] 30:349-359).Also significant in this regard are the MYB118 gene (see US Patent 7,148,402), the MYB115 gene (see Wang et al. (2008) Cell Research 224-235), the BABYBOOM gene (BBM; see Bouutilier et al. (2002) Plant Cell 14:1737-1749), the CLAVATA gene (see, for example, US Patent 7,179,963), the bud regeneration enhancer 1 (ESR1) gene (see Banno et al. (2001), The Plant Cell, Vol. 13:2609–2618), the Cornglass1 (Cg1) gene (see Chuck et al. (2007) Nature Genetics, Vol. 39(4):544-549), and the cup cotyledon (CUC) gene (see Hibara et al. (2006) The Plant Cell [Plant Cell], Volume 18: 2946–2957), REVOLUTA (REV) gene (see Otsuga et al. (2001) The Plant Journal of Plants 25(2):223-236), more axillary growth 1 (MAX1) genes (see Stirnberg et al. (2002) Development 129:1131-1141), SUPERSHOOT (SPS) genes (see Tanikanjana et al. (2001) Genes & Development 15:1577–1588), transverse repressor (LAS) genes (see Greb et al. (2003) Genes & Development 17:1175–1187), more axillary growth 4 (MAX4) genes (see Sorefan et al. (2003) Genes & Development 17:1469-1474), stem cell inducing factor 1 (STEMIN1) genes (see Ishikawa et al. (2019) Nature) Plants [Nature Plants] 5:681-690), growth regulator 4 (GRF4) gene and / or GRF interactor 1 (GIF1) gene (see Debernardi et al. bioRxiv 2020.08.23.263905; doi:2020.08.23.263905) and growth regulator 5 (GRF5) gene (see Kong et al. bioRxiv 2020.08.23.263947; doi:2020.08.23.263947).

[0015] The morphogenetic polynucleotide and amino acid sequences of functional WUS / WOX nucleotides / peptides can be used with the methods disclosed herein. As defined herein, a “functional WUS / WOX nucleotide” or “functional WUS / WOX peptide” is any polynucleotide or peptide itself (as the case may be) encoding a peptide containing a homeobox DNA-binding domain, a WUS box, and an EAR repression domain (Ikeda et al., 2009 Plant Cell 21:3493-3505). As demonstrated by Rodriguez et al. (2016 PNAS doi:1607673113), removal of the dimerizing sequence preserves the homeobox DNA-binding domain, the WUS box, and the EAR repression domain, resulting in a functional WUS / WOX peptide. The Wuschel protein (hereinafter referred to as WUS) plays a crucial role in the initiation and maintenance of apical meristems containing pluripotent stem cell pools (Endrizzi et al., (1996) Plant Journal 10:967-979; Laaux et al., (1996) Development 122:87-96; and Mayer et al., (1998) Cell 95:805-815). Mutants of the WUS gene in Arabidopsis contain misdesignated stem cells that appear to be undergoing differentiation. WUS encodes a novel homologous domain protein that may act as a transcriptional regulator (Mayer et al., (1998) Cell 95:805-815). The stem cell population in Arabidopsis bud meristems is thought to be maintained through a regulatory loop between the organ-initiating CLAVATA (CLV) gene and the WUS gene, which is required for stem cell characteristics. The CLV gene represses WUS at the transcriptional level, and WUS expression is sufficient to induce the expression of meristem cell characteristics and 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 lead to adventitious bud proliferation in leaves (in vivo) (Laux, T., Talk Presented at the XVI International Botanical Congress Meeting, August 1-7, 1999, St. Louis, Missouri).

[0016] In one aspect, the functional WUS / WOX peptides that can be used in the methods of this disclosure include WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX5A, and / or WOX9 peptides (see U.S. Patents 7,348,468 and 7,256,322 and U.S. Patent Application Publications 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). The functional WUS / WOX peptides that can be used in the methods of this disclosure may be obtained from or derived from any plant, including but not limited to monocots, dicots, angiosperms, and gymnosperms.

[0017] As used in this article, "T-DNA" refers to a portion of the Ti plasmid inserted into the genome of a host plant cell.

[0018] As used in this article, “transformation frequency” is a measure of transformation performance in plants, calculated based on the number of transgenic T0 plants regenerated from each starting seedling. A value above 100% indicates that each starting seedling regenerated multiple transgenic T0 plants. A value below 100% indicates that each starting seedling regenerated fewer than one transgenic T0 plant.

[0019] Recombinant transcription factors and the polynucleotides encoding them

[0020] This article provides polynucleotides encoding recombinant transcription factors. A polynucleotide can contain any polynucleotide suitable for encoding a transcription factor used for translation (e.g., protein production). For example, a polynucleotide can contain a DNA molecule or an RNA molecule. In some instances, the polynucleotide contains a vector. In some instances, the polynucleotide is intracellular.

[0021] As used herein, "vector" refers to a DNA molecule, such as a plasmid, granule, or bacterial phage, used to introduce nucleotide constructs, such as expression cassettes or constructs, into a host cell. Cloning vectors typically contain one or more restriction endonuclease recognition sites, at which foreign DNA sequences can be inserted in a deterministic manner without losing the vector's essential biological functions, as well as marker genes suitable for identifying and selecting cells transformed by the cloning vector.

[0022] As used herein, “contacting,” “contact,” “contacted,” “comes in contact with,” or “in contact with” means “direct contact” or “indirect contact.” For example, placing cells in conditions where they can come into contact with expression cassettes, nucleotides, peptides, RNPs (ribonucleoproteins), or other substances disclosed herein. Allowing such expression cassettes, nucleotides, peptides, or other substances to be present in the cell's viable environment (e.g., culture medium) or expressed in the cell or in neighboring cells, and to function on the cell. For example, polynucleotides can come into direct contact with cells (e.g., the polynucleotide may be located within the cell), or polynucleotides can come into indirect contact with cells (e.g., the polynucleotide may be located within adjacent cells, and the expression of the polynucleotide by the adjacent cells can function on the cell). For example, the WUS gene is known to function on cells via expression originating from adjacent cells. The expression cassettes, polynucleotides, peptides and other substances disclosed herein can be delivered to cells via T-DNA transfer (e.g., bacterial-mediated transformation), particle bombardment, electroporation, PEG transfection or RNP (ribonucleoprotein) delivery.

[0023] As used herein, the term “expression cassette” refers to a unique component of vector DNA consisting of coding and non-coding sequences, including 5' and 3' regulatory sequences that control expression in transformed / transfected cells.

[0024] As used herein, the term "regulatory sequence" refers to a segment of nucleic acid molecule that can increase or decrease gene expression. Regulatory sequences include promoters, terminators, enhancer elements, silencing elements, the 5'UTR, and the 3'UTR (untranslated region).

[0025] Recombinant transcription factors contain a nucleic acid-binding domain and a transcription activation domain. The nucleic acid-binding domain is capable of binding to gene regulatory sequences present on polynucleotides. For example, the nucleic acid-binding domain may contain a portion of a transcription factor that binds to nucleic acid regulatory sequences (e.g., those in promoters operatively coupled to the coding sequence of a gene).

[0026] The nucleic acid binding domain contains a Bbm-truncated polypeptide.

[0027] Transcription activation domains can induce activation and / or recruitment of transcriptional mechanisms (typically by binding to transcriptional mechanisms such as RNA polymerases or associated polypeptides). Transcriptional mechanisms are adapted to transcribe genes whose ORFs are typically located at the 3' of the regulatory sequence bound by the nucleic acid-binding domain. Thus, recombinant transcription factors contain two domains that, when properly positioned via the nucleic acid-binding domain, function to (1) bind to the regulated gene (usually upstream) (via the nucleic acid-binding domain) and (2) activate the transcription of the target gene (e.g., the regulated gene) via the transcription activation domain.

[0028] The transcription activation domain contains transcription activator peptides.

[0029] The nucleic acid binding domain contains at least 50 amino acid residues. In some instances, the nucleic acid binding domain contains at least 55 amino acid residues, at least 60 amino acid residues, at least 65 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, or at least 100 amino acid residues.

[0030] The transcription activation domain and / or transcription activator polypeptide contains at least 20 amino acid residues. In some instances, the transcription activation domain contains at least 25, at least 30, at least 35, at least 40, or at least 50 amino acid residues.

[0031] Nucleic acid binding domains and transcription activation domains do not naturally exist in the same polypeptide.

[0032] Nucleic acid binding domain

[0033] The truncated Bbm polypeptide comprises truncated Baby Boom (Bbm) subfamily polypeptides. The Bbm subfamily of polypeptides is a subset of the AP2 family of plant transcription factors. The Bbm subfamily contains Bbm, Bbm1, and Bbm2 plant genes from a variety of different plant species, which contain some or all of the Bbm, Bbm1, and Bbm2 genes. It should be noted that the Bbm gene from maize (Zeamays) (which has the polypeptide sequence of SEQ ID NO:15 and the cDNA sequence of SEQ ID NO:14, and the subfamily is named after this gene) was initially referred to as “Odp2” when it was discovered. Therefore, when “Bbm” or “Odp2” is used herein, unless the context otherwise requires, both terms refer to the gene exhibiting SEQ ID NO:14 and 15 in maize. Many related Bbm subfamily genes are present in other plants and maize (e.g., maize BBM2). Exemplary polypeptide sequences from such genes are disclosed in SEQ ID NO: 15, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, and 122. All of these polypeptide sequences can be truncated to form Bbm-truncated polypeptides as disclosed herein and as shown in the sequence listing.

[0034] The nucleic acid-binding domain of recombinant transcription factors contains a truncated Bbm polypeptide. As used herein, a “truncated Bbm polypeptide” is a fragment of the Bbm, Bbm1, or Bbm2 polypeptide. The truncated Bbm polypeptide also retains sufficient nucleic acid-binding activity to activate genes controlled by regulatory sequences bound by the truncated Bbm polypeptide (via transcriptional activation domains). Surprisingly, it has been found that preparing such a truncation can improve the performance of Bbm morphogenesis genes during monocotyledonous plant transformation, as illustrated in the examples.

[0035] For example, the Bbm truncated polypeptide may have at least 90% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0036] For example, the Bbm truncated polypeptide may have at least 91% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0037] For example, the Bbm truncated polypeptide may have at least 92% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0038] For example, the Bbm truncated polypeptide may have at least 93% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0039] For example, the Bbm truncated polypeptide may have at least 94% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0040] For example, the Bbm truncated polypeptide may have at least 95% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0041] For example, the Bbm truncated polypeptide may have at least 96% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0042] For example, the Bbm truncated polypeptide may have at least 97% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0043] For example, the Bbm truncated polypeptide may have at least 98% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, or 125.

[0044] For example, the Bbm truncated polypeptide may have at least 99% sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0045] In some instances, the Bbm truncated polypeptide may contain the sequence of any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, or 125.

[0046] In some instances, the nucleic acid binding domain comprises a truncated Bbm polypeptide and optionally further comprises and is operatively linked to a Bbm A polypeptide, a Bbm B polypeptide, or both Bbm A and Bbm B polypeptides. The truncated Bbm polypeptide may be present in a nucleic acid binding domain without either a Bbm A polypeptide or a Bbm B polypeptide. Alternatively, the truncated Bbm polypeptide may be present in the nucleic acid binding domain along with a Bbm A polypeptide, a Bbm B polypeptide, or both. In instances comprising a Bbm A polypeptide and / or a Bbm B polypeptide, the Bbm A polypeptide and the Bbm B polypeptide are aligned at the N-terminus of the truncated Bbm polypeptide. If both a Bbm B polypeptide and a Bbm A polypeptide are present, the Bbm B polypeptide is immediately aligned at the N-terminus of the Bbm A polypeptide. Therefore, the nucleic acid binding domain may comprise any of the following exemplary configurations:

[0047] The nucleic acid binding domain may contain: a Bbm truncated polypeptide (e.g., SEQ ID NO:21).

[0048] The nucleic acid binding domain may contain: Bbm B polypeptide - Bbm truncated polypeptide (e.g., SEQ ID NO: 17 and 21).

[0049] The nucleic acid binding domain may contain: Bbm A polypeptide-Bbm truncated polypeptide (e.g., SEQ ID NO:19 and 21).

[0050] The nucleic acid binding domain may contain: Bbm B polypeptide-Bbm A polypeptide-Bbm truncated polypeptide (e.g., SEQ ID NO: 17, 19 and 21).

[0051] The nucleic acid binding domain may contain: Bbm B polypeptide-Bbm A polypeptide-Bbm B polypeptide-Bbm A polypeptide-Bbm truncated polypeptides (e.g., SEQ ID NO: 17, 19, 17, 19 and 21).

[0052] In some instances, if not only Bbm-truncated peptides are used, the linker peptide may optionally be located in immediate proximity to any of the three components. (e.g., SEQ ID NO:17-linker-SEQ ID NO:19-linker-SEQ ID NO:21).

[0053] In some instances, the Bbm B polypeptide may contain an amino acid sequence of any one of SEQ ID NO: 17, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, or 123.

[0054] In some instances, the Bbm B polypeptide may comprise an amino acid sequence of any one of SEQ ID NO: 17, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, or 123, or any other identical sequence in which a single amino acid has been substituted, inserted, or deleted.

[0055] In some instances, the Bbm A polypeptide may contain an amino acid sequence of any one of SEQ ID NO: 19, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, or 124.

[0056] In some instances, the Bbm A polypeptide may comprise an amino acid sequence of any one of SEQ ID NO: 19, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, or 124, or any other identical sequence in which a single amino acid has been substituted, inserted, or deleted.

[0057] Transcription activation domain

[0058] In the recombinant transcription factors disclosed herein, the transcription activation domain contains a transcription activator polypeptide.

[0059] Transcription activator peptides can comprise members of the AP2 / ERF superfamily, which includes the following subgroups: the ERF / DREB family, the AP2 family, and the RAV family. The ERF / DREB family is the largest of these families, containing, for example, 122 members from the Arabidopsis genus. These members can be further subdivided into ethylene-responsive proteins (ERF genes), dehydration proteins (DREB genes), and proteins containing C-repetition binding factors (CBF genes). While these subgroups of AP2 / ERF proteins are typically classified based on their binding to classical DNA sequences, certain family members also contain repressive elements (e.g., the EAR repressive motif characterized by Ohta M et al., 2001, Plant Cell 13:1959-1968), while other AP2 / ERF members contain activating motifs, such as the well-characterized EDLL peptide located within the C-terminal domain of the ATERF98 protein (Tiwari Sb et al., 2012, Plant J 70:855-865).

[0060] Examples of transcription activator protein families include, for example, DoF proteins, such as corn DOF1 (Yanagisawa S, 2001, Plant Cell Physiol. 42:813-822), C-repetitive DRE binding factors such as CBF1 (Achard P et al., 2008, Plant Cell 20:2117-2129), proteins containing drought-responsive elements such as DREB1 (Maruyama K et al., 2004, Plant Journal 38:982-993), ethylene-responsive factor proteins such as ERF1 (Fujimoto Sy et al., 2000, Plant Cell 12:393-404) or ERF2 (Nakano T et al., 2006, Plant Cell Physiol. [Plant Cell Physiology] 47:554-558), proteins with octadecanoic acid derivative-responsive Catharanthus AP2 domains such as ORCA (Menke FLH et al., 1999, EMJO J [European Journal of Molecular Biology] 18:4455-4463), and Pseudomonas syringae cv tomato pathogenic species interaction-related proteins such as PIT1 (Gu YQ et al., 2002, Plant Cell [Plant Cell] 14:817-831).

[0061] In some instances, the transcription activator peptide has at least 90% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0062] In some instances, the transcription activator peptide has at least 91% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0063] In some instances, the transcription activator peptide has at least 92% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0064] In some instances, the transcription activator peptide has at least 93% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0065] In some instances, the transcription activator peptide has at least 94% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0066] In some instances, the transcription activator peptide has at least 95% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0067] In some instances, the transcription activator peptide has at least 96% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0068] In some instances, the transcription activator peptide has at least 97% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0069] In some instances, the transcription activator peptide has at least 98% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0070] In some instances, the transcription activator peptide has at least 99% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0071] In some instances, the transcription activator peptide contains the sequence of any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0072] In some instances, the transcription activator peptide comprises the CBF1A peptide. In some instances, the CBF1A peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with SEQ ID NO:127. In some instances, the CBF1A peptide comprises the sequence of SEQ ID NO:127.

[0073] In some instances, the transcription activator peptide comprises the CBF3I peptide. In some instances, the CBF3I peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with SEQ ID NO:129. In some instances, the CBF3I peptide comprises the sequence of SEQ ID NO:129.

[0074] In some instances, the transcription activation domain contains multiple transcription activator peptides. For example, the transcription activation domain may contain two CBF1A peptides, two CBF3I peptides, or one CBF1A peptide and one CBF3I peptide. In some instances, the transcription activation domain contains more than one transcription activator peptide, wherein each transcription activator peptide independently contains a sequence selected from the following: SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0075] In some instances, the recombinant transcription factors of this disclosure may comprise a nucleic acid-binding domain (comprising Bbm B polypeptide, Bbm A polypeptide, and a truncated Bbm polypeptide) and a transcription activator polypeptide (comprising CBF1A polypeptide). In particular instances, the Bbm B polypeptide comprises SEQ ID NO:17, the Bbm A polypeptide comprises SEQ ID NO:19, the truncated Bbm polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:21, and the CBF1A polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:127. More specifically, recombinant transcription factors can be arranged as N-terminal -BA-Bbm404-[transcription activator domain]-C-terminal or N-terminal -BA-Bbm404-CBF1A-C-terminal.

[0076] In some instances, the recombinant transcription factor of this disclosure may comprise a nucleic acid-binding domain containing a Bbm-truncated polypeptide and a transcriptional activation domain containing a CBF1A polypeptide. In particular instances, the Bbm-truncated polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:21, and the CBF1A polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:127.

[0077] Methods for producing recombinant plants

[0078] This article provides a method for generating recombinant monocotyledonous plants. The method includes contacting a monocotyledonous plant cell with a first polynucleotide encoding a target gene (the target gene is heterologous to the monocotyledonous plant cell), contacting the monocotyledonous plant cell with a second polynucleotide encoding a recombinant transcription factor disclosed herein, selecting a monocotyledonous plant cell in which the target gene has been incorporated into its genome, and regenerating a recombinant monocotyledonous plant from the selected monocotyledonous plant cell.

[0079] A transformation method may be constituted by contacting one or more monocotyledonous plant cells with one or both of the first and second polynucleotides. In addition to electroporation, PEG transfection, or RNP (ribonucleoprotein) delivery, transformation methods may also include bacterial-mediated and / or gene ballistic-mediated gene transfer to produce regenerative plant cells with the incorporated target nucleotide sequence. Bacterial strains that can be used in the methods of this disclosure include, but are not limited to, disarmed Agrobacterium, Ochrobactrum, or Rhizobiaceae bacteria. Agrobacterium species that can be used in this method include, but are not limited to, AGL-1, EHA105, GV3101, LBA4404, LBA4404 THY- (see US 8,334,429, which is incorporated herein by reference in its entirety) and LBA4404 TD THY- (in which two copies of the Tn904 transposon have been removed from LBA4404 THY-) (see PCT / US20 / 24993, filed March 26, 2020, which claims the benefit of U.S. Provisional Patent Application No. 62 / 825054, filed March 28, 2019, all of which are hereby incorporated herein by reference in their entirety). Agrobacterium tumefaciens strain LBA4404 TD THY- is an Agrobacterium tumefaciens strain LBA4404 THY- deposited at ATCC, designated accession number PTA-10531, wherein the functional Tn904 transposon is absent or both copies of the Tn904 transposon have been deleted. Anthrobacterium strains that can be used in this method include, but are not limited to, those disclosed in U.S. Patent Publication No. US20180216123, which is incorporated herein by reference in its entirety. Rhizobium strains that can be used in the method of this invention include, but are not limited to, those disclosed in U.S. Patent No. US 9,365,859, which is incorporated herein by reference in its entirety.

[0080] Various methods exist for regenerating plants from plant tissues / cells. Specific regeneration methods will depend on the starting plant tissue and the specific plant species to be regenerated. Plant regeneration, development, and cultivation from a single plant protoplast transformant or from various transformed explants are well-known in the art (Weissbach and Weissbach, (1988), in Methods for Plant Molecular Biology, (ed.), Academic Press, Inc., San Diego, Calif., which is incorporated herein by reference in its entirety). This regeneration and growth process typically involves the following steps: selecting transformed cells, culturing those individualized cells through the usual stages of embryogenic development, and through a rooting seedling stage. Similarly, regenerating transgenic embryos and seeds. The resulting transgenic rooted seedlings are then planted in a suitable plant growth medium (such as soil). Preferably, the regenerated plant is self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated plant is hybridized with a seed-producing plant of an agronomically important line. Instead, pollen from these important plant strains is used to pollinate the regenerated plants.

[0081] In some instances, methods for producing recombinant monocotyledonous plants may include contacting monocotyledonous plant cells with a third polynucleotide encoding a functional Wuschel or Wuschel homeobox (WUS / WOX) polypeptide.

[0082] In other instances, the method does not involve contacting monocotyledonous plant cells with a third polynucleotide encoding a functional WUS / WOX polypeptide.

[0083] In some instances, monocotyledonous plant cells contain cells from Poaceae plants. In some instances, monocotyledonous plant cells contain plant cells from any of the following species: maize, rice (Oryza sativa), wheat (Triticum aestivum), millet (Setaria italica), barley (Hordeum vulgare), pearl millet (Cenchrus americanus), sugarcane (Saccharum officinarum), or sorghum (Sorghum bicolor).

[0084] In some instances, exposing cells to any combination of the three polynucleotides includes bacterial-mediated transformation or particle bombardment.

[0085] In some instances, the first polynucleotide is present on a first vector, and the second polynucleotide is present on a second vector. In some instances, the second vector further comprises a third polynucleotide. In other instances, each polynucleotide is provided on a vector that does not contain either of the other two polynucleotides.

[0086] In some instances, monocotyledonous plant cells are immature embryonic cells or leaf cells.

[0087] In some instances, the target gene comprises a trait gene, a site-specific endonuclease, a guide polynucleotide, or a combination thereof. Many trait genes are known in the art and can be used in the methods disclosed herein. By way of illustration and not limitation, trait genes conferring resistance to insects or diseases, trait genes conferring resistance to herbicides, and trait genes conferring or promoting altered cereal characteristics (e.g., altered fatty acids, altered phosphorus content, altered carbohydrate or carbohydrate composition, altered antioxidant content or composition, or altered essential seed amino acid content or composition) are examples of trait gene types that can be operatively linked to a promoter for expression in plants transformed by the methods disclosed herein. Other genes known in the art may be included in expression cassettes that can be used in the methods disclosed herein. Non-limiting examples include genes that produce sites for site-specific DNA integration, genes that affect resistance to abiotic stresses (including but not limited to flowering, spike and seed development, improved nitrogen use efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, and salt resistance or tolerance) and genes that increase yield under stress, or other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth and / or plant structure.

[0088] Site-specific endonucleases are polypeptides that can cleave DNA (e.g., genomic DNA) at specific sites based on their affinity for a particular DNA sequence and / or the base pairing of a guide polynucleotide that complexes with the site-specific endonuclease. Examples include, but are not limited to, Cas9 (complexed with guide RNA) and zinc finger nucleases.

[0089] In some instances, the method involves removing one or both of the second and third polynucleotides from the genome of the selected cells. In other instances, the third polynucleotide is not used, and therefore is not removed.

[0090] This disclosure will be more fully understood by referring to the following terms.

[0091] 1. A polynucleotide encoding a recombinant transcription factor comprising a nucleic acid binding domain and a transcription activation domain,

[0092] The nucleic acid binding domain contains a truncated Bbm polypeptide and is capable of binding to gene regulatory sequences.

[0093] The transcription activation domain contains a transcription activator polypeptide, which can activate the transcription of the target gene.

[0094] The nucleic acid binding domain contains at least 50 amino acid residues, and the transcription activation domain contains at least 20 amino acid residues; and

[0095] The nucleic acid binding domain and the transcription activation domain do not naturally exist in the same polypeptide.

[0096] 2. The polynucleotide as described in Clause 1, wherein the nucleic acid binding domain comprises a Bbm truncated polypeptide operatively linked to: (i) a Bbm A polypeptide, (ii) a Bbm B polypeptide, or (iii) both a Bbm A polypeptide and a Bbm B polypeptide.

[0097] 3. The polynucleotide as described in Clause 1, wherein the nucleic acid binding domain does not contain Bbm A polypeptide or Bbm B polypeptide.

[0098] 4. The polynucleotide as described in any one of clauses 1-3, wherein the Bbm truncated polypeptide has at least 90% amino acid sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

[0099] 5. The polynucleotide as described in any one of Clauses 2 or 4, wherein the Bbm B polypeptide comprises any one of SEQ ID NO: 17, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, or 123, or any other identical sequence wherein a single amino acid substitution, insertion, or deletion has been performed.

[0100] 6. The polynucleotide as described in any one of Clauses 2, 4 or 5, wherein the Bbm A polypeptide comprises any one of SEQ ID NO: 19, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120 or 124, or any other identical sequence wherein a single amino acid substitution, insertion or deletion has been performed.

[0101] 7. The polynucleotide as described in any one of Clauses 1-6, wherein the transcription activator polypeptide has at least 90% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

[0102] 8. The polynucleotide as described in any one of Clauses 1-7, wherein the transcription activator polypeptide comprises the CBF1A polypeptide.

[0103] 9. The polynucleotide of any one of Clauses 1-8, wherein the transcription activator polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:127.

[0104] 10. The polynucleotide as described in any one of clauses 1-9, wherein the transcription activator polypeptide comprises the CBF3I polypeptide.

[0105] 11. The polynucleotide of any one of clauses 1-10, wherein the transcription activator polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:129.

[0106] 12. The polynucleotide as described in any one of clauses 1-11, wherein the transcription activation domain comprises a plurality of transcription activator polypeptides.

[0107] 13. The polynucleotide as described in Clause 12, wherein the transcriptional activation domain comprises two CBF1A polypeptides, two CBF3I polypeptides, or one CBF1A polypeptide and one CBF3I polypeptide.

[0108] 14. The polynucleotide as described in any one of clauses 1, 2, or 4-13, wherein the nucleic acid binding domain comprises the BbmB polypeptide, the BbmA polypeptide, and a truncated Bbm polypeptide, and

[0109] The transcription activator polypeptide includes the CBF1A polypeptide.

[0110] 15. The polynucleotide as described in Clause 14, wherein the Bbm B polypeptide comprises SEQ ID NO:17, the Bbm A polypeptide comprises SEQ ID NO:19, and the truncated Bbm polypeptide has at least 90% amino acid sequence identity with SEQ ID NO:21, and

[0111] The CBF1A polypeptide has at least 90% amino acid sequence identity with SEQ ID NO:127.

[0112] 16. The polynucleotide as described in any one of clauses 1, 3, 4, or 7-13, wherein the nucleic acid binding domain comprises a Bbm-truncated polypeptide, and

[0113] The transcription activator polypeptide includes the CBF1A polypeptide.

[0114] 17. The polynucleotide as described in Clause 16, wherein the truncated polypeptide of Bbm has at least 90% amino acid sequence identity with SEQ ID NO:21, and

[0115] The CBF1A polypeptide has at least 90% amino acid sequence identity with SEQ ID NO:127.

[0116] 18. The polynucleotide of any one of clauses 1-17, wherein the Bbm truncated polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:21.

[0117] 19. The polynucleotide of any one of clauses 1-18, wherein the Bbm truncated polypeptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with SEQ ID NO:20.

[0118] 20. The polynucleotide as described in any one of clauses 1, 2 or 4-19, wherein the Bbm B polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:17.

[0119] 21. The polynucleotide as described in any one of clauses 1, 2 or 4-20, wherein the Bbm B polypeptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity with SEQ ID NO:16.

[0120] 22. The polynucleotide as described in any one of clauses 1, 2 or 4-21, wherein the Bbm A polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:19.

[0121] 23. The polynucleotide as described in any one of clauses 1, 2 or 4-22, wherein the Bbm A polypeptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity with SEQ ID NO:18.

[0122] 24. The polynucleotide of any one of clauses 8-23, wherein the CBF1A polypeptide is encoded by a polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:126.

[0123] 25. The polynucleotide of any one of clauses 10-24, wherein the CBF3I polypeptide is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO:128.

[0124] 26. A recombinant transcription factor encoded by a polynucleotide as described in any one of clauses 1-25.

[0125] 27. A method for producing recombinant monocotyledonous plants, the method comprising:

[0126] Contact a monocotyledonous plant cell with a first polynucleotide encoding a target gene, wherein the target gene is heterologous to the monocotyledonous plant cell.

[0127] This contacts the monocotyledonous plant cell with a second polynucleotide encoding a recombinant transcription factor as described in Clause 26;

[0128] Select monocotyledonous plant cells that have already incorporated the target gene into their genome; and

[0129] Regenerate recombinant monocotyledonous plants from selected monocotyledonous plant cells.

[0130] 28. The method of Clause 27, further comprising contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional Wuschel or Wuschel homeobox (WUS / WOX) polypeptide.

[0131] 29. The method of Clause 27, wherein the method does not involve contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional WUS / WOX polypeptide.

[0132] 30. The method of any one of Clauses 27-29, wherein the monocotyledonous plant cell comprises a grass plant cell.

[0133] 31. The method of any one of clauses 27-30, wherein the monocotyledonous plant cell comprises a plant cell of any of the following species: corn, rice (Oryza sativa), wheat (Triticum aestivum), millet (Setaria italica), barley (Hordeum vulgare), pearl millet (Cenchrus americanus), sugarcane (Saccharum officinarum), or sorghum (Sorghum bicolor).

[0134] 32. The method of any one of Clauses 27-31, wherein the contact steps include bacterial-mediated transformation or particle bombardment.

[0135] 33. The method of any one of clauses 27-32, wherein the first polynucleotide is present on the first carrier and the second polynucleotide is present on the second carrier.

[0136] 34. The method of claim 33, wherein the second vector further comprises the third polynucleotide.

[0137] 35. The method of any one of Clauses 27-34, wherein the monocotyledonous plant cell is an immature embryonic cell or a leaf cell.

[0138] 36. The method of any one of Clauses 27-35, wherein the target gene comprises a trait gene, a site-specific endonuclease, a guide polynucleotide, or a combination thereof.

[0139] 37. The method of any one of clauses 27-36, further comprising removing one or both of the second and third polynucleotides from the genome of the selected cell.

[0140] Examples - This disclosure will be more fully understood by referring to the following non-limiting examples.

[0141] Example 1: Sequence

[0142] The useful sequences in the methods of this disclosure are presented in Table 1 and provided in the sequence list.

[0143] Table 1.

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] Example 2: Particle bombardment

[0159] Standard schemes of particle bombardment (Finer and McMullen, 1991, In Vitro Cell Dev.Biol.–Plant [In Vitro Cell Development Biology - Plant] 27:175-182 and those found in WO 2022072335A2) can be used in conjunction with the methods of this disclosure.

[0160] Example 3: Agrobacterium-mediated maize transformation

[0161] A. Preparation of Agrobacterium-containing motherboard.

[0162] Agrobacterium tumefaciens with a binary donor vector was frozen at -80°C, aliquoted, and streaked onto solid 12R medium. The samples were then incubated in the dark at 28°C for 2-3 days to prepare a master plate.

[0163] B. Grow Agrobacterium on solid culture medium.

[0164] Pick out one or more Agrobacterium colonies from the mother plate and streak them onto a second plate containing 810K medium, then incubate overnight in the dark at 28°C.

[0165] Add 5 ml of Agrobacterium infection medium and 100 mM 3'-5'-dimethoxy-4'-hydroxyacetophenone (acetylsuccinone; 5 μL) to a 14 mL conical tube in a fume hood. Suspend approximately three confluent loops of Agrobacterium from the second plate in the tube and then vortex the tube to form a homogeneous suspension. Transfer the suspension (1 ml) to a spectrophotometer tube and adjust the optical density (550 nm) of the suspension to a reading of approximately 0.35–1.0. The Agrobacterium concentration is approximately 0.5 to 2.0 × 10⁻⁶. 9 cfu / mL. Aliquot the final Agrobacterium suspension into 2mL microcentrifuge tubes, each containing approximately 1mL of suspension. Use the suspension as soon as possible.

[0166] C. Agrobacterium growth in liquid culture medium.

[0167] Alternatively, Agrobacterium can be prepared for transformation by growth in liquid medium. One day prior to infection, prepare a 125 ml flask with 30 ml of 557A medium and 30 μL of spectinomycin (50 mg / mL) and 30 μL of acetylsuccinone (20 mg / mL). Suspend the *Agrobacterium hemicyclicae* from the second plate in the flask and incubate overnight at 28°C on an orbital shaker set to 200 rpm. Centrifuge the *Agrobacterium* culture at 5000 rpm for 10 min. Remove the supernatant and add *Agrobacterium* infection medium containing acetylsuccinone solution. Resuspend the bacteria by vortexing and adjust the optical density (550 nm) of the *Agrobacterium* suspension to a reading of approximately 0.35 to 2.0.

[0168] D. Corn transformation.

[0169] Corn seeds were surface-sterilized in 20% (v / v) bleach (5.25% sodium hypochlorite) with 1 drop of Tween 20 for 15-20 minutes, then washed three times in sterile water. Germination was allowed, and seedlings were permitted to grow for approximately 14 days before preparation to produce leaf segments. The leaf segments were then placed in a solution containing 200 μM acetylsyleugenone + 0.02% sodium hypochlorite. The surfactant (Plant Health Technologies, PO Box 70013, Boise, ID83707-0113) was added to Agrobacterium infection medium (700A). The Agrobacterium infection medium was removed, and 1 ml of Agrobacterium suspension was added to the leaf segments, allowing it to stand for 20 min. The Agrobacterium and leaf segment suspension was poured into a sterile metal sieve, and the liquid was discarded. Using a spatula, the leaf segments collected on the metal sieve were transferred to a stack of three sterile filter papers to absorb excess liquid containing Agrobacterium, and then the leaf segments were transferred to the filter paper placed on the co-culture medium using a spatula. The plates were co-cultured in the dark at 21°C for 1–3 days.

[0170] The filter paper supporting the leaf segments was then transferred to a static culture medium without selection. Seven days later, the filter paper supporting the leaf segments was transferred to a selection medium and maintained for three weeks. After selection, healthy somatic embryos were transferred to a maturation medium using tweezers and kept in darkness for two weeks. At this point, the entire maturation plate (still containing mature somatic embryos) was transferred to light for another week. After one week in light, the regenerated plantlets were transferred to a rooting medium. Once rooted, the plantlets were ready for transplanting into the greenhouse.

[0171] Example 4: BBM (ODP2) truncation

[0172] The full-length ZM-ODP2(BBM) gene (2,133 bp) (SEQ ID No: 14 and 15) encoding the full-length wild-type ODP2 protein (710 aa) was truncated to produce three fragments:

[0173] ZM-ODP2(TR11) = motif "B" encoding amino acids 60-69 of the ZM-ODP2 protein (SEQ ID NO: 16 and 17)

[0174] ZM-ODP2(TR12) = motif "A" encoding amino acids 156-171 of the ZM-ODP2 protein (SEQ ID NO: 18 and 19).

[0175] ZM-ODP2(TR5) = BBM404 (SEQ ID NO: 20 and 21) encoding amino acids 266-669 of the ZM-ODP2 protein.

[0176] Example 5: The fusion of the CBF1A activation domain with the truncated Bbm enhances the transformation.

[0177] (A) Maize hard-stem self-pollinated line ED85E.

[0178] Seeds of the maize inbred line ED85E were surface sterilized and germinated on a medium containing MS salt, sucrose, and 2 mg / L pyrimidinol. Thirteen-day-old seedlings were harvested, and the first 3 cm of leaf tissue above the mesocoaxial layer was longitudinally bisected, mechanically chopped in a mixer, and simultaneously suspended in 100 ml of Agrobacterium, with an OD of 0.5 (see Example 5 for details).

[0179] Ten seedlings were used for each treatment to test five T-DNA conformations. Leaf tissue samples from each T0 plant were taken after T0 plantlet regeneration and root formation (i.e., the stage when plants are typically sent to the greenhouse) for multiplex PCR to confirm the copy number of integrated T-DNA. All five plasmids contained the same Nos::Wus2 expression cassette and the same 3xENH:UBI promoter driving Bbm gene expression; experimental results are summarized in Table 2. For all five treatments (five different Bbm coding sequences), T-DNA delivery was good (score = 3) to excellent (score = 4), as measured by transient ZS-Green1 expression 3–4 days post-infection. In the first control treatment with the full-length Bbm gene (PHP97334), T0 plants were regenerated at a frequency of 600% (e.g., 10 starting seedlings yielded 60 T0 plants). In the second treatment, the Bbm gene was truncated to include 404 amino acids, which include two AP2 DNA-binding domains and most of the C-terminus of the protein (PHP102072), and for this treatment, the transformation frequency was reduced by 438% relative to the control. Adding one copy of the highly conserved amino peptides “B” and “A” from PHP101977 (as described in WO 2020 / 214986) or two copies of these motifs (PHP103858) resulted in a further reduction in transformation frequency, yielding values ​​of 113% and 125%, respectively. Surprisingly, when the maize codon-optimized transcriptional activation domain from the Arabidopsis thaliana CBF1A protein (SEQ ID NO:127) was fused to the C-terminus of the truncated BA-Bbm404 protein (PHP101978), a transformation frequency of 2031% was produced, approximately 3.4 times higher than the control full-length Bbm gene.

[0180] Table 2. Leaf transformation frequencies of corn inbred lines ED85E using various modified Bbm genes.

[0181]

[0182] (B) Maize non-hard-stemmed self-pollinated line GR013D.

[0183] Starting with 10 seedlings per replicate, 14-day-old seedlings were mechanically minced in Agrobacterium containing either the helper plasmid PHP71539 plus PHP97334 (full-length wild-type Bbm gene) or PHP101978 (BA-Bbm404:CBF1A). The experimental treatments were replicated three times (two plasmid treatments side-by-side), and transformation frequencies were scored after selection and plant regeneration to produce T0 plantlets. PHP97334 produced transformation frequencies of 50%, 40%, and 10%, with a mean and standard deviation of 33% ± 17%. PHP101978 produced transformation frequencies of 210%, 220%, and 180%, with a mean and standard deviation of 203% ± 17%. For this inbred line, using a truncated Bbm404 fused with the CBF1A activation domain resulted in a transformation frequency increase of more than 6-fold relative to the control plasmid.

[0184] (C) The full-length Bbm gene was compared with the truncated BA-Bbm404, each of which was fused with the activation domain CBF1A.

[0185] Agrobacterium-mediated transformation was performed on leaf tissue of the maize inbred line ED85E. Three plasmids were compared, starting with seven seedlings per replicate for each treatment. Results were consistent across both replicates, with the full-length Bbm gene and the full-length Bbm fused with CBF1A producing similar transformation frequencies. However, when the truncated BA-Bbm404 gene was fused with CBF1A, the transformation frequency was significantly higher in both replicates.

[0186] Table 3. Compared with the full-length Bbm gene or the full-length gene fused with the same activation domain, fusing the CBF1A activation domain with the truncated BA-Bbm404 protein substantially increased the conversion frequency.

[0187]

[0188] Similar enhanced transformation frequencies have been observed in maize inbred lines ED85E, GR013D, and GR0112 using BA-Bbm404:CBF1A. Furthermore, improved transformation has been observed in tropical maize inbred lines JFDYY, EEP7E, and EENW5.

[0189] (D) Compare the truncated Bbm404 with the truncated BA-Bbm404, where each is fused with the active structural domain CBF1A.

[0190] Leaf tissues of the maize inbred line GR0112 were processed using the helper plasmids PHP71539, plus PHP97334 (Nos::Wus2, full-length Bbm with no activation domain), PHP104222 (Act::Wus2, full-length Bbm with no activation domain), PHP105696 (Ubi::Wus2, full-length Bbm with no activation domain), and PHP101978 (Nos::Wus2, BA-B). Agrobacterium tumefaciens transformations were performed using the following constructs: PHP10404:CBF1A, PHP104187(Act::Wus2, BA-Bbm404:CBF1A), PHP107604(Nos::Wus2, Bbm404:CBF1A), PHP106860(Act::Wus2, Bbm404:CBF1A), or PHP106868(Ubi::Wus2, Bbm404:CBF1A). The results of these transformations are shown in Table 4. GR0112 with PHP97334 showed a lower transformation frequency, but transformations with PHP105696 (Ubi::Wus2) showed an increased transformation frequency. Compared to the control PHP97334, the construct with BA-Bbm404:CBF1A transformed GR0112 at twice the transformation frequency. In the absence of BA struct fields with Nos::Wus2 or Act::Wus2, the conversion frequency is reduced by 3-5 times compared to PHP97334. However, PHP106868, with Ubi::Wus2 and Bbm404:CBF1A, gives the highest conversion frequency of 160%.

[0191] Table 4. Comparison of conversion frequencies between the full-length Bbm gene and the truncated Bbm404 or BA-Bbm404 protein fused with the CBF1A activation domain in the maize inbred line GR0112.

[0192]

[0193] Example 6: The use of BA-Bbm404:CBF1A resulted in an increased conversion frequency in other cereal crops.

[0194] Transformation frequencies of Agrobacterium-mediated transformation in leaf tissues of foxtail millet were compared with those of control plasmids PHP97334 (WT Bbm gene) or PHP101978 (BA-Bbm404:CBF1A), resulting in a 40% transformation frequency of PHP97334 (8 T0 plants from 20 initial seedlings) or a 190% increase in transformation frequency of PHP97334 (19 T0 plants from 10 initial seedlings). These relative transformation frequencies in foxtail millet are reflected in the + (single+) score for plasmid RV035126 (also known as PHP973343) and the +++ (triple+) score for plasmid RV045474 (also known as PHP101978) in Table 5 below. For pearl millet, higher transformation scores were also observed than those of comparable plasmids containing only the full-length Bbm gene. Testing is underway in barley and wheat, and the BA-Bbm404:CBF1A treatment is expected to result in a higher conversion frequency than the unmodified Bbm treatment.

[0195] Table 5. Transformation scores of various gramineous crops using full-length Bbm or truncated BA-Bbm404 fused with the CBF1A activation domain. NT = Not yet tested.

[0196]

[0197] Example 7. Screening different BBM orthologous sequences and different activation domains demonstrated a wide range of components that improve transformation.

[0198] To evaluate its potential for first generating three optimal BBM component fragments (i.e., A and B motifs and a larger BBM404-like fragment) and then combining them with heterologous activation domains to improve the range of orthologous BBM proteins in grass explant transformation, PHP101978 was used for Agrobacterium-mediated leaf transformation assays.

[0199] (A) Different activation domains (VP64, ERF2, CBF3I, PTI4, PTI4m, DREB1A, DREB1Am, VP16, DOF1, HSFA6B, DREB2A, corresponding to SEQ ID NO:160, SEQ ID NO:153, SEQ ID NO:128, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:144, SEQ ID NO:166, SEQ ID NO:164, SEQ ID NO:165) were tested using expression cassette configurations found in PHP101978, maintaining all molecular components except for the heterologous activation domain fused to the carboxyl terminus of ZM-Bbm404. T-DNA is composed of RB+Nos::Wus2+3xENH:UBIPRO::BA-BBM404:[ACT]+Sb-UBI PRO::ZsGreen1+Si-UBIPRO::NPTII, where [ACT] represents the different activation domains that replace At-CBF1A in the construct PHP101978.

[0200] To facilitate a convenient transformation schedule, the activation domain constructs were divided into three groups. Each group included constructs PHP101978 and PHP97334, used to compare transformation frequencies within the experiment. Transformation was performed in leaf tissue of the maize inbred line ED85E, starting with 5 seedlings per replicate per treatment. Each experiment had three replicates. Results from all experiments with combined three replicates are given in Table 6.

[0201] Table 6. Compared with the full-length Bbm protein without heterologous activation domains, fusing different activation domains into the truncated BA-Bbm404 protein significantly increased the conversion frequency.

[0202]

[0203] This method is applicable to activation domains in both monocotyledonous and dicotyledonous plant species. A series of different transcription factors were tested on candidate heterologous activation domains from orthologs of proteins from various exemplary plant species (Arabidopsis thaliana, Brassica, soybean, maize, rice, sorghum, etc.). These transcription factors included CBF1A, CBF3I, CBF1E, ERF98, ERF1, DOF1, ORCA, PTI4, C1, OP2, ARF, LFY, LEC1, LEC2, MADS, bHLH, bZIP, HBP-1a, WRKY, NAM, and CUC genes. For example, when activation domains from At-CBF1A and At-CBF3I were fused with BA-BBM404, CBF1A demonstrated a strong stimulus to somatic embryogenesis in leaf tissues, and CBF3I produced a similarly strong response (Table 6). The corn C1 activation domain is expected to produce a slightly reduced response relative to CBF3I or CBFA1. In contrast, using the At-CBF1E or Zm-O2 activation domains will produce progressively weaker somatic embryonic stimulation. Therefore, it is envisioned that this assay could be used to identify activation domains used in this method that will produce a series of somatic embryonic stimulations, from a weak response (Zm-O2) to a gradually increasing response (CBF1E, then C1), and finally strong somatic embryonic stimulation as with CBF1A and CBF3I. Activation domains share similar properties in eukaryotic cells, and therefore activation domains such as herpes simplex virus VP16 (and the synthetic multimeric form VP64) are effective in this method when fused with BA-BBM404 (Table 6).

[0204] (B) Modified activation domains that increase or decrease activation intensity can also be used in this method. Using wild-type activation domain sequences and modified amino acid sequences fused with BA-BBM404 as described by Li et al., 2013 (Plant Biotechnology Journal, 11, 671–680) will help stimulate somatic embryogenesis and transformation frequency. Therefore, the use of the CBF1, DOF1, DREB1, ERF1, ERF2, ORCA, and PTI4 trans-activation domains, as well as these same domains that have been modified to have a stronger similarity to the VP16 core trans-activation domain, is expected to be used to stimulate somatic embryogenesis and increase transformation frequency. Among these domains, DREB1A and DREB1Am (where “m” represents the modified sequence described by Li et al.) showed strong stimulation of somatic embryogenesis and transformation (Table 6).

[0205] (C) As demonstrated by VP64, which exhibits a significantly enhanced activation level after the core activation domain of VP16 is polymerized (Table 6), attaching multimers (2x, 3x, 4x, etc.) to a single BA-BBM404 protein, whether using homopolymers (i.e., CBF1A:CBF1A: etc.) or heteropolymers (i.e., CBF1A:ERF2m:PTI4), enhances the transactivation intensity of endogenous Bbm target genes, resulting in a proportionally stronger stimulus to somatic embryonic growth and an increased conversion frequency.

[0206] (D) Testing using the same plasmid design (PHP101978) but replacing Bbm paralogs or orthologs resulted in similar positive results. Therefore, Bbm, or Bbm1 or Bbm2 proteins from different plant species can be truncated in a similar manner, assembled in the same pattern (BA-BBM404), fused with exemplary activation domains (such as CBF1A), and when this fusion product is cloned downstream of 3xENH:UBIPRO, a similar increase in leaf transformation frequency is expected (see SEQ ID No:xx-yy).

[0207] (E) The present invention also uses different viral enhancer elements with different strong constitutive promoters instead of corn UBIPRO.

[0208] Example 8. The use of BBM404:CBF1A and / or BA-BBM404:CBF1A improved the transformation of immature embryos in difficult-to-transform inbred lines.

[0209] The following experiments demonstrate that immediate expression of BBM404:CBF1A (or BA-BBM404:CBF1A) and WUS2 after Agrobacterium infection leads to direct somatic embryogenesis and regeneration in the difficult-to-transform common maize inbred line W22.

[0210] The PLTP promoter driving BBM404:CBF1A and the AXIG1 promoter driving WUS2 expression lead to rapid and direct somatic embryogenesis after transformation of immature maize embryos.

[0211] Approximately 11 days after pollination, immature embryos (2-2.5 mm in length) were harvested from the public corn inbred line W22 and infected with Agrobacterium strain LBA4404 THY-TD containing the helper plasmid PHP71539 and T-DNA with the following composition: RB+AXIG1PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTP PRO::ZM-BBM404:CBF1A::OS-T28 TERM+GZ-W64A TERM+SB-ALS PRO::HRA::SB-PEPC1 TERM+LTP2 PRO::ZS-YELLOW::PINII TERM-LB(PHP000001). Agrobacterium was grown overnight on solid medium, then suspended to an optical density of 0.5 (at 520 nm), and immature embryos were incubated in the Agrobacterium suspension for 5 minutes, then removed from the liquid and placed on solid 710I medium at 21 °C overnight.

[0212] Twenty-four hours later, the embryos were transferred to 605T medium to begin screening for Agrobacterium. Six days later, numerous small somatic embryos were observed on the surface of each of the 124 treated immature embryos. Each immature embryo contained many different individual somatic embryos; many embryos were supported by well-defined stalks.

[0213] Seven days after Agrobacterium infection, transgenic embryos were screened using imidazolinone herbicides and transferred to maturation medium (289Q medium + 0.1 mg / L methomyl). After 14 days on maturation medium, mature embryos were transferred to rooting medium (13158H medium; 13158 medium plus 25 mg / L cefotaxime), and leaf samples were taken for PCR analysis. Herbicide-resistant plants were PCR-treated and sent to the greenhouse between 32 and 34 days after the start of the experiment (when Agrobacterium transformation began). For PCR sampling of plants, two samples were collected from each plant, one from each of two opposite spikes (taken from opposite sides of the plant), to detect the possibility of partial transformation (chimerism) in any plant. PCR results from each pair of samples from all plants were consistent, indicating that no chimeric plants were produced and that T0 plants were homogeneous transgenes. It is expected that after the transformation of immature embryos in W22, the expression of BBM404:CBF1A plus Wus2 will lead to the efficient production of somatic embryos, thereby rapidly producing a large number of transgenic T0 plants.

[0214] B. Using BBM404:CBF1A alone improved the transformation of immature embryos in the Pioneer hard-stem inbred line PHP38.

[0215] In previous studies, Agrobacterium-mediated transformation of immature embryos in the Pioneer hard-stemmed inbred line PHP38 using Nos::Wus2 plus Ubi::Bbm resulted in a transformation frequency exceeding 50%, while using Ubi::Bbm alone produced a frequency of 10% (Lowe et al., 2016, Plant Cell 28:1998-2015). Using immature embryos from the same inbred line PHP38, Agrobacterium transformation to deliver T-DNA containing Ubi::BBM404:CBF1A (PHP000002) is expected to result in a high transformation frequency exceeding 50%.

[0216] Furthermore, Lowe et al. (2016, Plant Cell 28:1958-1998-2015) demonstrated that using Nos::Wus2 plus Ubi::Bbm can be used to regenerate transgenic embryogenic callus from the hypocotyl of mature seeds after Agrobacterium transformation. When BBM404:CBF1A is used instead of Bbm (PHP000003) in such a construct, a higher frequency of transgenic embryogenic callus and T0 plants is expected from embryonic tissue derived from mature seeds.

Claims

1. A polynucleotide encoding a recombinant transcription factor, said recombinant transcription factor comprising a nucleic acid binding domain and a transcription activation domain, The nucleic acid binding domain contains a Bbm truncated polypeptide and is capable of binding gene regulatory sequences. The transcription activation domain contains a transcription activator polypeptide, and the transcription activation domain is capable of activating the transcription of the target gene. The nucleic acid binding domain comprises at least 50 amino acid residues, and the transcription activation domain comprises at least 20 amino acid residues; and The nucleic acid binding domain and the transcription activation domain do not naturally exist in the same polypeptide.

2. The polynucleotide of claim 1, wherein the nucleic acid binding domain comprises a Bbm truncated polypeptide operatively linked to (i) Bbm A polypeptide, (ii) Bbm B polypeptide, or (iii) both Bbm A polypeptide and Bbm B polypeptide.

3. The polynucleotide of claim 1, wherein the nucleic acid binding domain does not contain Bbm A polypeptide or Bbm B polypeptide.

4. The polynucleotide of any one of claims 1-3, wherein the Bbm truncated polypeptide has at least 90% amino acid sequence identity with any one of SEQ ID NO: 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121 or 125.

5. The polynucleotide of any one of claims 2 or 4, wherein the Bbm B polypeptide comprises any one of SEQ ID NO: 17, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, or 123, or any other identical sequence wherein a single amino acid substitution, insertion, or deletion has been performed.

6. The polynucleotide of any one of claims 2, 4 or 5, wherein the Bbm A polypeptide comprises any one of SEQ ID NO: 19, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120 or 124, or any other identical sequence wherein a single amino acid substitution, insertion or deletion has been performed.

7. The polynucleotide of any one of claims 1-6, wherein the transcription activator polypeptide has at least 90% amino acid sequence identity with any one of SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160 or 164-166.

8. The polynucleotide of any one of claims 1-7, wherein the transcription activator polypeptide comprises the CBF1A polypeptide.

9. The polynucleotide of any one of claims 1-8, wherein the transcription activator polypeptide has at least 90% amino acid sequence identity with SEQ ID NO:

127.

10. The polynucleotide of any one of claims 1-9, wherein the transcription activator polypeptide comprises the CBF3I polypeptide.

11. The polynucleotide of any one of claims 1-10, wherein the transcription activator polypeptide has at least 90% amino acid sequence identity with SEQ ID NO:

129.

12. The polynucleotide of any one of claims 1-11, wherein the transcription activation domain comprises a plurality of transcription activator peptides.

13. The polynucleotide of claim 12, wherein the transcriptional activation domain comprises two CBF1A polypeptides, two CBF3I polypeptides, or one CBF1A polypeptide and one CBF3I polypeptide.

14. The polynucleotide of any one of claims 1, 2, or 4-13, wherein the nucleic acid binding domain comprises a Bbm B polypeptide, a Bbm A polypeptide, and a truncated Bbm polypeptide, and The transcription activator polypeptide mentioned therein includes the CBF1A polypeptide.

15. The polynucleotide of claim 14, wherein the Bbm B polypeptide comprises SEQ ID NO:17, the Bbm A polypeptide comprises SEQ ID NO:19, and the truncated Bbm polypeptide comprises having at least 90% amino acid sequence identity with SEQ ID NO:21, and The CBF1A polypeptide described therein has at least 90% amino acid sequence identity with SEQ ID NO:

127.

16. The polynucleotide of any one of claims 1, 3, 4 or 7-13, wherein the nucleic acid binding domain comprises a Bbm-truncated polypeptide, and The transcription activator polypeptide mentioned therein includes the CBF1A polypeptide.

17. The polynucleotide of claim 16, wherein the Bbm-truncated polypeptide has at least 90% amino acid sequence identity with SEQ ID NO:21, and The CBF1A polypeptide described therein has at least 90% amino acid sequence identity with SEQ ID NO:

127.

18. A recombinant transcription factor encoded by a polynucleotide as described in any one of claims 1-17.

19. A method for producing recombinant monocotyledonous plants, the method comprising: Contact a monocotyledonous plant cell with a first polynucleotide encoding a target gene, wherein the target gene is heterologous to the monocotyledonous plant cell; The monocotyledonous plant cells are brought into contact with a second polynucleotide encoding the recombinant transcription factor as described in claim 18; Select monocotyledonous plant cells in which the target gene has been incorporated into their genome; as well as Regenerate recombinant monocotyledonous plants from selected monocotyledonous plant cells.

20. The method of claim 19, further comprising contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional Wuschel or Wuschel homeobox (WUS / WOX) polypeptide.

21. The method of claim 19, wherein the method does not include contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional WUS / WOX polypeptide.

22. The method of any one of claims 19-21, wherein the monocotyledonous plant cells comprise Poaceae plant cells.

23. The method of any one of claims 19-22, wherein the monocotyledonous plant cells comprise plant cells of any one of the following species: maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), millet (Setaria italica), barley (Hordeum vulgare), pearl millet (Cenchrus americanus), sugarcane (Saccharum officinarum), or sorghum (Sorghum bicolor).

24. The method of any one of claims 19-23, wherein the contact step comprises bacterial-mediated transformation or particle bombardment.

25. The method of any one of claims 19-24, wherein the first polynucleotide is present on the first carrier and the second polynucleotide is present on the second carrier.

26. The method of claim 25, wherein the second carrier further comprises the third polynucleotide.

27. The method of any one of claims 19-26, wherein the monocotyledonous plant cell is an immature embryonic cell or a leaf cell.

28. The method of any one of claims 19-27, wherein the target gene comprises a trait gene, a site-specific endonuclease, a guide polynucleotide, or a combination thereof.

29. The method of any one of claims 19-28, further comprising removing one or both of the second polynucleotide and the third polynucleotide from the genome of the selected monocotyledonous plant cell.

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