Methods and compositions for transformation of monocotyledone plants

Description

1 / 71 “METHODS AND COMPOSITIONS FOR TRANSFORMING MONOCOTYLEDONOUS PLANTS” REFERENCE TO THE LISTING OF SEQUENCES

[0001] The official copy of the sequence listing is sent electronically and simultaneously with the descriptive report. The sequence listing is in an XML-formatted file conforming to the ST26 standard, with the filename “108740-WO-SEC-1_ST-26_Sequence Listing”, created on March 21, 2024, and with a size of 610838 bytes. The sequence listing contained in this XML file forms part of the descriptive report and is incorporated into this document in its entirety by reference. BACKGROUND

[0002] In recent years there has been a tremendous expansion of capabilities for the genetic manipulation of plants. Current transformation technology provides an opportunity to produce commercially viable transgenic plants, allowing the creation of new plant varieties containing desirable traits. However, there is still a need for plant transformation methods that allow for a wider range of transformable and regenerable plant explant tissues, as well as increases in the efficiency of plant transformation methods. SUMMARY

[0003] The present disclosure describes methods and compositions for transforming monocotyledonous plants. The disclosed methods and compositions significantly improve the frequency of plant transformation (e.g., by increasing the number of recombinant T0 plants recovered per initial change and / or providing T0 plants with (1) single-copy (rather than multiple-copy) T-DNA insertions containing a gene of interest and (2) no other sequences of Petition 870250086132, dated 09 / 23 / 2025, pp. 95 / 173 2 / 71 nucleotides inserted detectable (e.g., from a plasmid structure).

[0004] A polynucleotide encoding a recombinant transcription factor is provided in this document. The recombinant transcription factor comprises a nucleic acid-binding domain and a transcriptional activation domain. The nucleic acid-binding domain comprises a truncated Bbm polypeptide and is capable of binding to a gene regulatory sequence. The transcriptional activation domain comprises a transcriptional activator polypeptide and is capable of activating the transcription of a target gene. The nucleic acid-binding domain comprises at least 50 amino acid residues and the transcriptional activation domain comprises at least 20 amino acid residues. The nucleic acid-binding domain and the transcriptional activation domain do not naturally occur in the same polypeptide.

[0005] Also provided in this document is a method for producing a recombinant monocotyledonous plant. The method comprises contacting a monocotyledonous plant cell with a first polynucleotide encoding a gene of interest, wherein the gene of interest is heterologous to the monocotyledonous plant cell. The method comprises contacting the monocotyledonous plant cell with a second polynucleotide encoding the recombinant transcription factor of the present disclosure. The method comprises selecting a monocotyledonous plant cell that has incorporated the gene of interest into its genome and regenerating a recombinant monocotyledonous plant from the selected monocotyledonous plant cell. Petition 870250086132, dated 09 / 23 / 2025, pp. 96 / 173 3 / 71 DETAILED DESCRIPTION

[0006] Disclosure is not limited to specific examples, which may, of course, vary. The terminology and illustrative examples used herein are intended to describe aspects of disclosure only and are not meant to be limiting. As used herein, singular terms and singular forms such as a, an, and the, for example, include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “plant,” “the plant,” or “a plant” also includes a plurality of plants; furthermore, depending on the context, use of the term “plant” may also include the genetically similar or identical progeny of that plant; use of the term “a nucleic acid” optionally includes, for practicality, many copies of that nucleic acid molecule; similarly, the term “probe” optionally (and typically) encompasses several similar or identical probe molecules.

[0007] As used in this document, the term “comprising” includes the aspect of “consisting of”.

[0008] Unless otherwise defined, the numerical ranges cited within the descriptive report include the numbers that define the range and include each integer within the defined range. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the technique to which the disclosure pertains.

[0009] As used in this document, a recombinant plant or plant cell comprises a heterologous nucleic acid sequence, a heterologous polypeptide, and / or a heterologous non-coding RNA. An acid Petition 870250086132, dated 09 / 23 / 2025, page 97 / 173 4 / 71 recombinant nucleic acid or polypeptide is a nucleic acid or polypeptide that has been altered from its natural form by human intervention through insertion, deletion, substitution, or fusion. The recombinant transcription factors of the present disclosure do not occur naturally.

[0010] As used in this document, heterologous means that a particular nucleic acid sequence has been located in a genome, locus, or construct through human intervention. The heterologous sequence may be naturally occurring but is now located in a genome, locus, or construct where the sequence is not naturally found. Alternatively, the sequence may be heterologous as it is located in a genome, locus, or construct and is not naturally occurring. A heterologous gene may be inserted into a genome by means of, for example, transformation methods and / or site-specific nuclease-based methods.

[0011] As used in this document, the term morphogenic gene means a gene that, when ectopically expressed, stimulates the formation of a somatically derived structure that can produce a plant. More precisely, ectopic expression or mutation or silencing or decreased expression of the morphogenic gene stimulates the de novo formation of a somatic embryo or an organogenic structure, such as a bud meristem or an axillary meristem, which can produce a plant or stimulate the regeneration of a plant. This stimulated de novo formation occurs in the cell in which the morphogenic gene is expressed or silenced or repressed or in a neighboring cell. A morphogenic gene can be a transcription factor that regulates the expression of other genes or a gene that influences hormone levels in a plant tissue, both of which can stimulate morphogenic changes. A morphogenic gene can be stably incorporated into the genome of a Petition 870250086132, dated 09 / 23 / 2025, pp. 98 / 173 5 / 71 plant or can be expressed transiently. In one aspect, the expression of the morphogenic gene is controlled. Expression can be controlled transcriptionally or post-transcriptionally. Controlled expression can also be pulsed expression of the morphogenic gene over a particular period of time. Alternatively, the morphogenic gene can be expressed only in some transformed cells and not expressed in others. Control of morphogenic gene expression can be achieved by a variety of methods as disclosed hereafter. The morphogenic genes useful in the methods of the present disclosure can be obtained from or derived from any plant species.

[0012] As used in this document, the term “morphogenic factor” means a morphogenic gene and / or the protein expressed by a morphogenic gene.

[0013] A morphogenic gene is involved in plant metabolism, organ development, stem cell development, stimulation of cell growth, organogenesis, regeneration, initiation of somatic embryogenesis, accelerated maturation of the somatic embryo, initiation and / or development of the apical meristem, initiation and / or development of the bud meristem or axillary meristem, initiation and / or development of buds or a combination thereof, such as the WUS / WOX genes (WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 or WOX9), see US patent documents no. 7,348,468 and 7,256,322 and publications of US Patent Applications no. 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 10:248; Dolzblasz et al., 2016, Mol. Plant 19:1028-39 which are useful in the methods of revelation.It is expected that WUS / WOX modulation will affect the phenotype of plants and / or plant tissues, including plant metabolism and organ development. Petition 870250086132, dated 09 / 23 / 2025, page 99 / 173 6 / 71 stem cell development, stimulation of cell growth, organogenesis, regeneration, initiation of somatic embryogenesis, accelerated maturation of the somatic embryo, initiation and / or development of the apical meristem, initiation and / or development of the bud meristem, initiation and / or development of buds, or a combination thereof. Arabidopsis WUS expression can induce stem cells and vegetative tissues, which can differentiate into somatic embryos (Zuo, et al. (2002) Plant J 30:349-359). It would also be of interest with regard to a MYB118 gene (see US Patent No. 7,148,402), the MYB115 gene (see Wang et al. (2008) Cell Research 224-235), a BABYBOOM gene (BBM; see Boutilier et al. (2002) Plant Cell 14:1737-1749), a CLAVATA gene (see, for example, US Patent No. 7,179,963), a Shoot Regeneration Enhancer 1 (ESR1) gene (see Banno et al. (2001), The Plant Cell, Vol.13:2609–2618), a Corngrass1 (Cg1) gene (see Chuck et al. (2007) Nature Genetics, Vol. 39 (4):544–549), a Cup-Shaped Cotyledon (CUC) gene (see Hibara et al. (2006) The Plant Cell, Vol. 18:2946–2957), a REVOLUTA (REV) gene (see Otsuga et al. (2001) The Plant Journal 25 (2):223–236), a More Axillary Growth1 (MAX1) gene (see Stirnberg et al. (2002) Development 129:1131–1141), a SUPERSHOOT (SPS) gene (see Tanikanjana et al. (2001) Genes & Development 15:1577–1588), a gene of Lateral Suppressor (LAS) (see Greb et al. (2003) Genes & Development 17:1175-1187), a More Axillary Growth4 (MAX4) gene (see Sorefan et al. (2003) Genes & Development 17:1469-1474), a Stem Cell Induction Factor 1 (STEMIN1) gene (see Ishikawa et al. (2019) Nature Plants 5:681-690), a Growth Regulator Factor 4 (GRF4) gene and / or a GRF Interaction Factor 1 (GIF1) gene (see Debernardi et al. Petition 870250086132, dated 09 / 23 / 2025, pages 100 / 173 7 / 71 al. bioRxiv 2020.08.23.263905; doi: 2020.08.23.263905) and a growth regulatory factor 5 (GRF5) gene (see Kong et al. bioRxiv 2020.08.23.263947; doi: 2020.08.23.263947).

[0014] Morphogenic polynucleotide sequences and amino acid sequences of functional WUS / WOX nucleotides / polypeptides can be used in the disclosed methods. As defined herein, a “functional WUS / WOX nucleotide” or “functional WUS / WOX polypeptide” is any polynucleotide encoding a polypeptide or the peptide itself, as the case may be, that contains a DNA homeobox binding domain, a WUS box, and an EAR repressor domain (Ikeda et al., 2009 Plant Cell 21:3493-3505). As demonstrated by Rodriguez et al., (2016 PNAS doi: 1607673113) removal of the dimerization sequence that leaves behind the DNA homeobox binding domain, a WUS box, and an EAR repressor domain results in a functional WUS / WOX polypeptide. The Wuschel protein, hereinafter referred to as WUS, plays a crucial role in the initiation and maintenance of the apical meristem, which contains a set 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 plants mutant for the WUS gene contain stem cells that undergo misspecification and appear to undergo differentiation. WUS encodes a novel homeodomain protein that presumably functions as a transcription regulator (Mayer, et al., (1998) Cell 95:805-815). The Arabidopsis bud meristem stem cell population is believed to be maintained by a regulatory cycle between the CLAVATA (CLV) genes that promote organ initiation and the WUS gene that is required for stem cell identity, with the CLV genes repressing. Petition 870250086132, dated 09 / 23 / 2025, pp. 101 / 173 8 / 71 WUS at the transcript level and WUS expression being sufficient to induce meristem cell identity and CLV3 stem cell marker expression (Brand, et al., (2000) Science 289:617-619; Schoof, et al., (2000) Cell 100:635-644). It has been shown that the constitutive expression of WUS in Arabidopsis leads to adventitious proliferation of buds from leaves (in planta) (Laux, T., Oral Presentation at the XVI International Botanical Congress Meeting, August 1-7, 1999, St. Louis, Mo.).

[0015] In one aspect, the functional WUS / WOX polypeptides useful in the methods of the present disclosure comprise a WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX5A or WOX9 polypeptide (see U.S. Patent Documents 7,348,468 and 7,256,322 and U.S. Patent Application Publication Numbers 2017 / 0121722 and 2007 / 0271628, incorporated herein in their entirety by reference and van der Graaff et al., 2009, Genome Biology 10:248). The polypeptides Useful functional WUS / WOX compounds in the methods of the present disclosure can be obtained from or derived from any plant including, but not limited to, monocots, dicots, angiosperms and gymnosperms.

[0016] As used in this document, T-DNA means a portion of a Ti plasmid that is inserted into the genome of a host plant cell.

[0017] As used in this document, transformation frequency refers to a measure of transformation performance in plants that is calculated based on the number of T0 transgenic plants recovered per initial seedling. Values ​​above 100% indicate that multiple T0 transgenic plants were recovered per initial seedling. Values ​​below 100% indicate that less than 1 T0 transgenic plant was recovered per initial seedling. Petition 870250086132, dated 09 / 23 / 2025, pp. 102 / 173 9 / 71 Recombinant transcription factors and the polynucleotides that encode them.

[0018] A polynucleotide encoding a recombinant transcription factor is provided in this document. The polynucleotide may comprise any polynucleotide suitable for encoding the transcription factor for translation (e.g., protein production). For example, the polynucleotide may comprise a DNA molecule or an RNA molecule. In some examples, the polynucleotide comprises a vector. In some examples, the polynucleotide is inside a cell.

[0019] As used in this document, vector refers to a DNA molecule such as a plasmid, cosmid, or bacterial phage for introducing a nucleotide construct, for example, a cassette or expression construct, into a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites into which foreign DNA sequences can be inserted in a determinable manner without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in identifying and selecting cells transformed with the cloning vector.

[0020] As used in this document, the terms contact, contact, contacted, comes into contact with, or come into contact with mean direct contact or indirect contact. For example, cells are placed in a condition where the cells may come into contact with an expression cassette, a nucleotide, a peptide, an RNP (ribonucleoprotein), or other substance disclosed in this document. Such an expression cassette, nucleotide, peptide, or other substance is permitted to be present in Petition 870250086132, dated 09 / 23 / 2025, pp. 103 / 173 10 / 71 an environment where cells survive (e.g., medium) or expressed in the cell or expressed in an adjacent cell) and can act on cells. For example, a polynucleotide may have direct contact with a cell (e.g., the polynucleotide may be located inside a cell) or a polynucleotide may have indirect contact with a cell (e.g., the polynucleotide may be located inside an adjacent cell and the expression of the polynucleotide by the adjacent cell may act on the cell). The WUS gene, for example, is known to act on cells through expression originating in adjacent cells. The expression cassettes, polynucleotides, polypeptides, and other substances disclosed herein may come into contact with a cell through T-DNA transfer (e.g., bacteria-mediated transformation), particle bombardment, electroporation, PEG transfection, or RNP (ribonucleoprotein) administration.

[0021] As used in this document, the term “expression cassette” means a distinct component of the vector DNA consisting of coding and non-coding sequences including 5' and 3' regulatory sequences that control expression in a transformed / transfected cell.

[0022] As used in this document, the term “regulatory sequence” means a segment of a nucleic acid molecule that is capable of increasing or decreasing the expression of a gene. Regulatory sequences include promoters, terminators, enhancer elements, silencer elements, UTR 5' and UTR 3' (untranslated regions).

[0023] The recombinant transcription factor comprises a nucleic acid binding domain and a transcriptional activation domain. The nucleic acid binding domain Petition 870250086132, dated 09 / 23 / 2025, pp. 104 / 173 11 / 71 is capable of binding to a gene regulatory sequence present in a polynucleotide. For example, the nucleic acid binding domain may comprise a portion of a transcription factor that binds to nucleic acid regulatory sequences (e.g., those in a promoter operatively coupled to the coding sequence of a gene).

[0024] The nucleic acid binding domain comprises a truncated Bbm polypeptide.

[0025] The transcriptional activation domain is capable of causing the activation and / or recruitment of the transcriptional machinery (frequently by the binding of the transcriptional machinery (e.g., an RNA polymerase) or an associated polypeptide). The transcriptional machinery is suited to transcribe a gene whose ORF is typically 3' from the regulatory sequence bound to the nucleic acid binding domain. Thus, the recombinant transcription factor comprises two domains that function to (1) bind (usually upstream) to a regulated gene (via the nucleic acid binding domain) and (2) activate the transcription of a target gene (e.g., the regulated gene) via the transcriptional activation domain when positioned appropriately by the nucleic acid binding domain.

[0026] The transcriptional activation domain comprises a transcriptional activator polypeptide.

[0027] The nucleic acid binding domain comprises at least 50 amino acid residues. In some examples, the nucleic acid binding domain comprises 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. Petition 870250086132, dated 09 / 23 / 2025, pp. 105 / 173 12 / 71

[0028] The transcriptional activation domain and / or the transcriptional activator polypeptide comprises at least 20 amino acid residues. In some examples, the transcriptional activation domain comprises at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, or at least 50 amino acid residues.

[0029] The nucleic acid binding domain and the transcriptional activation domain do not naturally occur in the same polypeptide. Nucleic acid binding domain

[0030] The truncated Bbm polypeptide comprises a truncated polypeptide of the Baby Boom (Bbm) subfamily. The Bbm subfamily of polypeptides is a subset of the AP2 family of plant transcription factors. The Bbm subfamily comprises the plant genes Bbm, Bbm1, and Bbm2 from several different plant species comprising some or all of the Bbm, Bbm1, and Bbm2 genes. Note that the Bbm gene of Zea mays (which has the polypeptide sequence SEQ ID NO: 15 and the cDNA sequence SEQ ID NO: 14 and for which the subfamily is named) was initially designated Odp2 when the gene was discovered. Consequently, when “Bbm” or “Odp2” are used in this document, both terms refer to the gene exhibiting SEQ ID NOs: 14 and 15 in Zea mays, unless the context indicates otherwise. Many related genes from the Bbm subfamily exist in other plants and in Zea mays (for example, Zea mays BBM2).Exemplary polypeptide sequences of these genes are revealed in the SEQ ID NOs: 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 these polypeptide sequences can be truncated to Petition 870250086132, dated 09 / 23 / 2025, pp. 106 / 173 13 / 71 form a truncated Bbm polypeptide, as disclosed in this document and shown in the sequence listing.

[0031] The nucleic acid-binding domain of the recombinant transcription factor comprises a truncated Bbm polypeptide. A “truncated Bbm polypeptide,” as used herein, is a fragment of a Bbm polypeptide, Bbm1 or Bbm2. The truncated Bbm polypeptide also retains sufficient nucleic acid-binding activity to activate genes (via the transcriptional activation domain) controlled by the regulatory sequence bound to the polypeptide. Bbm truncated. It was surprisingly discovered that making such truncations can improve the performance of the morphogenic gene. Bbm during the transformation of monocotyledons, as described in the examples.

[0032] For example, the truncated Bbm polypeptide may comprise at least 90% sequence identity with any of the following SEQ IDs: 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.

[0033] For example, the truncated Bbm polypeptide may comprise at least 91% sequence identity with any of the following SEQ IDs: 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.

[0034] For example, the truncated Bbm polypeptide may comprise at least 92% sequence identity with any of the following SEQ IDs: 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.

[0035] For example, the truncated Bbm polypeptide may comprise at least 93% sequence identity with any of the following SEQ IDs: 21, 25, 29, 33, 37, 41, 45, 49, Petition 870250086132, dated 09 / 23 / 2025, pp. 107 / 173 14 / 71 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, or 125.

[0036] For example, the truncated Bbm polypeptide may comprise at least 94% sequence identity with any of the following SEQ IDs: 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 truncated Bbm polypeptide may comprise at least 95% sequence identity with any of the following SEQ IDs: 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 truncated Bbm polypeptide may comprise at least 96% sequence identity with any of the following SEQ IDs: 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 truncated Bbm polypeptide may comprise at least 97% sequence identity with any of the following SEQ IDs: 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 truncated Bbm polypeptide may comprise at least 98% sequence identity with any of the following SEQ IDs: 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 truncated Bbm polypeptide may comprise at least 99% sequence identity with any of the following SEQ IDs: 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. Petition 870250086132, dated 09 / 23 / 2025, pp. 108 / 173 15 / 71

[0042] In some examples, the truncated Bbm polypeptide may comprise the sequence of any of the following SEQ ID NOs: 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] In some examples, 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 a Bbm A polypeptide and a Bbm B polypeptide. The truncated Bbm polypeptide may be present in the nucleic acid binding domain without a Bbm A polypeptide and without a Bbm B polypeptide. Alternatively, the truncated Bbm polypeptide may be present in the nucleic acid binding domain together with a Bbm A polypeptide, a Bbm B polypeptide, or both. In examples comprising a Bbm A polypeptide and / or a Bbm B polypeptide, the Bbm A and Bbm B polypeptides are arranged at the N-terminal end of the truncated Bbm polypeptide. If a Bbm B polypeptide and a Bbm A polypeptide are present, the Bbm B polypeptide will be immediately attached to the N-terminal end of the Bbm A polypeptide.Consequently, the nucleic acid binding domain can comprise any of the following example configurations:

[0044] The nucleic acid binding domain may comprise: truncated Bbm polypeptide (e.g., SEQ ID NO: 21).

[0045] The nucleic acid binding domain may comprise: Bbm polypeptide B- truncated Bbm polypeptide (e.g., SEQ ID NO: 17 and 21).

[0046] The nucleic acid binding domain may comprise: Bbm polypeptide A- truncated Bbm polypeptide (e.g., SEQ ID NO: 19 and 21). Petition 870250086132, dated 09 / 23 / 2025, pp. 109 / 173 16 / 71

[0047] The nucleic acid binding domain may comprise: Bbm polypeptide B- Bbm polypeptide A truncated Bbm polypeptide (e.g., SEQ ID NO: 17, 19 and 21).

[0048] The nucleic acid binding domain may comprise: Bbm polypeptide Bbm B-polypeptide Bbm A-polypeptide Bbm B-polypeptide Bbm A-polypeptide Bbm truncated (e.g., SEQ ID NO: 17, 19, 17, 19 and 21).

[0049] In some examples, a linker peptide may optionally be located immediately between any of the three components if more than the truncated Bbm polypeptide is used. (For example, SEQ ID NO: 17-linker-SEQ ID NO: 19-linker-SEQ ID NO: 21).

[0050] In some examples, the polypeptide Bbm B may comprise the amino acid sequence of any of the following 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.

[0051] In some examples, the polypeptide Bbm B may comprise the amino acid sequence of any of the following 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 where a single amino acid substitution, insertion, or deletion has been made.

[0052] In some examples, the polypeptide Bbm A may comprise the amino acid sequence of any of the following 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.

[0053] In some examples, the polypeptide Bbm A may comprise the amino acid sequence of any of the following 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 Petition 870250086132, dated 09 / 23 / 2025, pp. 110 / 173 17 / 71 or any other identical sequence where a single amino acid substitution, insertion, or deletion has been made. Transcriptional activation domain

[0054] In the recombinant transcription factors of the present disclosure, the transcriptional activation domain comprises a transcriptional activator polypeptide.

[0055] The transcriptional activator polypeptide may comprise a member of the AP2 / ERF superfamily which includes the 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 in Arabidopsis, which can be divided into subgroups of proteins that respond to ethylene (ERF genes), dehydration (DREB genes), and proteins containing C-repeat binding factors (CBF genes). Although these AP2 / ERF protein subgroups are typically categorized based on their binding to canonical DNA sequences, specific family members also contain repressor elements (such as the EAR repressor motif characterized by Ohta M et al., 2001, Plant Cell 13:1959-1968), while other AP2 / ERF members contain activation 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).

[0056] Examples of families of transcriptional activator proteins include examples such as Dof proteins, such as maize DOF1 (Yanagisawa S, 2001, Plant Cell Physiol. 42:813-822), C-repeat DRE-binding factors, such as CBF1 (Achard P et al., 2008, Plant Cell 20:2117-2129), drought response element-containing proteins, such as DREB1 (Maruyama K et al., 2004, Plant Journal 38:982-993), and ethylene response 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. 47:554-558), proteins with the AP2 domain of Catharanthus Petition 870250086132, dated 09 / 23 / 2025, pp. 111 / 173 18 / 71 responsive to octadecanoid derivatives, such as ORCA (Menke FLH et al., 1999, EMJO J 18:4455-4463) and proteins associated with the interaction of Pseudomonas syringae cv Tomato, such as PIT1 (Gu YQ et al., 2002, Plant Cell 14:817-831).

[0057] In some examples, the transcriptional activator polypeptide comprises at least 90% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0058] In some examples, the transcriptional activator polypeptide comprises at least 91% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0059] In some examples, the transcriptional activator polypeptide comprises at least 92% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0060] In some examples, the transcriptional activator polypeptide comprises at least 93% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0061] In some examples, the transcriptional activator polypeptide comprises at least 94% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0062] In some examples, the transcriptional activator polypeptide comprises at least 95% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0063] In some examples, the transcriptional activator polypeptide comprises at least 96% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166. Petition 870250086132, dated 09 / 23 / 2025, pp. 112 / 173 19 / 71

[0064] In some examples, the transcriptional activator polypeptide comprises at least 97% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0065] In some examples, the transcriptional activator polypeptide comprises at least 98% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0066] In some examples, the transcriptional activator polypeptide comprises at least 99% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0067] In some examples, the transcriptional activator polypeptide comprises the sequence of any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

[0068] In some instances, the transcriptional activator polypeptide comprises a CBF1A polypeptide. In some instances, the CBF1A polypeptide comprises 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 polypeptide comprises the sequence of SEQ ID NO: 127.

[0069] In some instances, the transcriptional activator polypeptide comprises a CBF3I polypeptide. In some instances, the CBF3I polypeptide comprises 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 polypeptide comprises the sequence of SEQ ID NO: 129.

[0070] In some examples, the transcriptional activation domain comprises multiple transcriptional activator polypeptides. For example, the activation domain Petition 870250086132, dated 09 / 23 / 2025, pp. 113 / 173 The 20 / 71 transcriptional domain may comprise two CBF1A polypeptides, two CBF3I polypeptides, or one CBF1A polypeptide and one CBF3I polypeptide. In some examples, the transcriptional activation domain comprises more than one transcriptional activator polypeptide, each independently comprising a sequence selected from: SEQ ID NO: 127, 129, 131, 133, 135, 137, 158, 160, or 164-166.

[0071] In some examples, a recombinant transcription factor of the present disclosure may comprise a nucleic acid binding domain comprising a Bbm B polypeptide, a Bbm A polypeptide and a truncated Bbm polypeptide and a transcriptional activator polypeptide comprising a CBF1A polypeptide. In specific examples, the Bbm B polypeptide comprises SEQ ID NO: 17, the Bbm A polypeptide comprises SEQ ID NO: 19, the truncated Bbm polypeptide comprises 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 comprises 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, the recombinant transcription factor can be organized as Nterm-BA-Bbm404-[transcriptional activator domain]-Cterm or as Nterm-BA-Bbm404-CBF1A-Cterm.

[0072] In some examples, a recombinant transcription factor of the present disclosure may comprise a nucleic acid binding domain comprising a truncated Bbm polypeptide and a transcriptional activation domain comprising a CBF1A polypeptide. In specific examples, the truncated Bbm polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 21 and Petition 870250086132, dated 09 / 23 / 2025, pp. 114 / 173 21 / 71 the CBF1A polypeptide comprises at least 90%, 91%, 92%, %, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 127. Recombinant plant production methods

[0073] A method for producing a recombinant monocotyledonous plant is provided in this document. The method comprises contacting a monocotyledonous plant cell with a first polynucleotide encoding a gene of interest (the gene of interest is heterologous to the monocotyledonous plant cell), contacting the monocotyledonous plant cell with a second polynucleotide encoding a recombinant transcription factor of the present disclosure, selecting a monocotyledonous plant cell that has incorporated the gene of interest into its genome, and regenerating a recombinant monocotyledonous plant from the selected monocotyledonous plant cell.

[0074] Contact of monocotyledonous plant cell(s) with one or both of the first and second polynucleotides may involve transformation methods. Transformation methods may include bacterial-mediated gene transfer and / or biolistics, as well as electroporation, PEG transfection, or administration of RNP (ribonucleoprotein) to produce regenerable plant cells with an incorporated nucleotide sequence of interest. Useful bacterial strains in the development methods include, but are not limited to, an unarmed Agrobacterium, an Ochrobactrum bacterium, or a Rhizobiaceae bacterium. Useful unarmed Agrobacteria in the present methods include, but are not limited to, AGL-1, EHA105, GV3101, LBA4404, LBA4404 THY- (see US document 8,334,429 incorporated herein in its entirety by reference) and LBA4404 TD THY- wherein both copies of Petition 870250086132, dated 09 / 23 / 2025, pp. 115 / 173 22 / 71 transposon Tn904 removed were removed from LBA4404 THY (see document PCT / US20 / 24993 filed March 26, 2020, which claims the benefit of Provisional Patent Application No. US 62 / 825054 filed March 28, 2019, all of which are incorporated herein in their entirety by reference). The Agrobacterium LBA4404 TD THY- strain is the A. tumefaciens LBA4404 THY- strain filed with the ATCC, Accession Number PTA-10531, in which a functional Tn904 transposon is not present or both copies of the Tn904 transposon have been deleted. Bacterial strains of Ochrobactrum useful in the present methods include, but are not limited to, those disclosed in U.S. Patent Publication No. US20180216123, which is incorporated herein in its entirety by reference. Bacterial strains of Rhizobiaceae useful in the present methods include, but are not limited to, those disclosed in U.S. Patent No.9365859 incorporated into this document in its entirety by way of reference.

[0075] There are a variety of methods for regenerating plants from plant tissues / cells. The specific regeneration method will depend on the starting plant tissue and the specific plant species to be regenerated. Regeneration, growth, and culture of plants from individual plant protoplast transformants or from multiple transformed explants are well known in the art (Weissbach and Weissbach, (1988) In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., San Diego, Calif., incorporated herein in its entirety by reference). This regeneration and growth process typically includes the steps of selecting transformed cells, culturing these individual cells through the normal stages of embryonic development through the Petition 870250086132, dated 09 / 23 / 2025, pages 116 / 173 23 / 71 rooted seedling stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are subsequently planted in a suitable plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from regenerated plants is crossed with plants grown from seeds of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.

[0076] In some examples, the method of producing a recombinant monocotyledonous plant may comprise contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional Wuschel polypeptide or Wuschel homeobox (WUS / WOX).

[0077] In other examples, the method does not involve contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional WUS / WOX polypeptide.

[0078] In some examples, the monocotyledonous plant cell comprises a Poaceae plant cell. In certain examples, the monocotyledonous plant cell comprises a plant cell of any of the following species: Zea mays, Oryza sativa, Triticum aestivum, Setaria italica, Hordeum vulgare, Cenchrus americanus, Saccharum officinarum, or Sorghum bicolor.

[0079] In some examples, contact of cells with any combination of the three polynucleotides involves transformation mediated by bacteria or particle bombardment.

[0080] In some examples, the first polynucleotide is present in a first vector and the second polynucleotide Petition 870250086132, dated 09 / 23 / 2025, pages 117 / 173 24 / 71 is present in a second vector. In some examples, the second vector additionally comprises the third polynucleotide. In other examples, each polynucleotide is provided in a vector that does not comprise either of the other two polynucleotides.

[0081] In some examples, the monocotyledonous plant cell is an immature embryonic cell or a leaf cell.

[0082] In some examples, the gene of interest comprises a trait gene, a site-specific endonuclease, a guide polynucleotide, or a combination thereof. Numerous trait genes are known in the art and can be used in the methods disclosed herein. By way of illustration, without being intended to be limiting, trait genes conferring resistance to insects or diseases, trait genes conferring resistance to a herbicide, trait genes conferring or contributing to an altered grain characteristic such as altered fatty acids, altered phosphorus content, altered carbohydrates or carbohydrate composition, altered antioxidant content or composition, or altered essential seed amino acid content or composition are examples of the types of trait genes that can be operationally linked to a promoter for expression in transformed plants by the methods disclosed herein.Additional genes known in the art may be included in the expression cassettes useful in the methods disclosed herein. Non-limiting examples include genes that create a site for site-specific DNA integration, genes that affect resistance to abiotic stress (including, but not limited to, flowering, ear and seed development, enhanced nitrogen utilization efficiency, altered nitrogen responsiveness, etc.). Petition 870250086132, dated 09 / 23 / 2025, pages 118 / 173 25 / 71 resistance or tolerance to drought, resistance or tolerance to cold, and resistance or tolerance to salt) and increased 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.

[0083] A site-specific endonuclease refers to a polypeptide that is capable of cutting DNA (e.g., genomic DNA) at a specific location based on its affinity for a specific DNA sequence and / or base pairing by a guide polynucleotide that has complexed with the site-specific endonuclease. Examples include, but are not limited to, Cas9 (complexed with a guide RNA) and zinc finger nucleases.

[0084] In some examples, the method involves excising one or both of the second and third polynucleotides from the genome of the selected cell. In some examples, the third polynucleotide is not used and is therefore not excised.

[0085] The present disclosure will be more fully understood by reference to the following clauses. 1. A polynucleotide encoding a recombinant transcription factor, wherein the recombinant transcription factor comprises a nucleic acid-binding domain and a transcriptional activation domain, in which the nucleic acid-binding domain comprises a truncated Bbm polypeptide and the nucleic acid-binding domain is capable of binding to a gene regulatory sequence; wherein the transcriptional activation domain comprises a transcriptional activator polypeptide, in which the transcriptional activation domain is capable of activating the transcription of a target gene; Petition 870250086132, dated 09 / 23 / 2025, pp. 119 / 173 26 / 71 wherein the nucleic acid-binding domain comprises at least 50 amino acid residues and the transcriptional activation domain comprises at least 20 amino acid residues and wherein the nucleic acid-binding domain and the transcriptional activation domain do not naturally occur in the same polypeptide. 2. The polynucleotide of clause 1, wherein the nucleic acid-binding domain comprises a truncated Bbm 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. 3. The polynucleotide of clause 1, wherein the nucleic acid-binding domain does not comprise a Bbm A polypeptide or a Bbm B polypeptide. 4. The polynucleotide of any of the clauses 13, wherein the truncated Bbm polypeptide comprises at least 90% amino acid sequence identity with any of the 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 either clause 2 or 4, wherein the polypeptide Bbm B comprises either of the following SEQs: 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 where a single amino acid substitution, insertion, or deletion has been made. 6. The polynucleotide of any of clauses 2, 4 or 5, wherein the Bbm A polypeptide comprises any of the following 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 Petition 870250086132, dated 09 / 23 / 2025, pp. 120 / 173 27 / 71 or 124 or any other identical sequence where a single amino acid substitution, insertion, or deletion has been made. 7. The polynucleotide of any of clauses 16, wherein the transcriptional activator polypeptide comprises at least 90% amino acid sequence identity with any of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166. 8. The polynucleotide of any of clauses 17, wherein the transcriptional activator polypeptide comprises a CBF1A polypeptide. 9. The polynucleotide of any of clauses 18, wherein the transcriptional activator polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 127. 10. The polynucleotide of any of clauses 19, wherein the transcriptional activator polypeptide comprises a CBF3I polypeptide. 11. The polynucleotide of any of the clauses 110, wherein the transcriptional activator polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 129. 12. The polynucleotide of any of the clauses 111, wherein the transcriptional activation domain comprises multiple transcriptional activator polypeptides. 13. The polynucleotide of clause 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 of clauses 1, 2, or 4-13, in which the nucleic acid-binding domain Petition 870250086132, dated 09 / 23 / 2025, pp. 121 / 173 28 / 71 comprises a Bbm B polypeptide, a Bbm A polypeptide and a truncated Bbm polypeptide, wherein the transcriptional activator polypeptide comprises a CBF1A polypeptide. 15. The polynucleotide of clause 14, wherein the polypeptide Bbm B comprises SEQ ID NO: 17, the polypeptide Bbm A comprises SEQ ID NO: 19 and the truncated polypeptide Bbm comprises at least 90% amino acid sequence identity with SEQ ID NO: 21 and wherein the polypeptide CBF1A comprises at least 90% amino acid sequence identity with SEQ ID NO: 127. 16. The polynucleotide of any of clauses 1, 3, 4 or 7-13, wherein the nucleic acid binding domain comprises a truncated Bbm polypeptide and wherein the transcriptional activator polypeptide comprises a CBF1A polypeptide. 17. The polynucleotide of clause 16, wherein the truncated Bbm polypeptide comprises at least 90% amino acid sequence identity with SEQ ID NO: 21 and wherein the CBF1A polypeptide comprises at least 90% amino acid sequence identity with SEQ ID NO: 127. 18. The polynucleotide of any of the clauses 117, wherein the truncated Bbm polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 21. 19. The polynucleotide of any of the clauses 118, wherein the truncated Bbm polypeptide is encoded by a polynucleotide comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity with SEQ ID NO: 20. Petition 870250086132, dated 09 / 23 / 2025, pages 122 / 173 29 / 71 20. The polynucleotide of any of clauses 1, 2 or 4-19, wherein the Bbm B polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the SEQ ID NO: 17. 21. The polynucleotide of any of clauses 1, 2 or 4-20, wherein the polypeptide Bbm B is encoded by a polynucleotide comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity with SEQ ID NO: 16. 22. The polynucleotide of any of clauses 1, 2 or 4-21, wherein the Bbm A polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the SEQ ID NO: 19. 23. The polynucleotide of any of clauses 1, 2 or 4-22, wherein the polypeptide Bbm A is encoded by a polynucleotide comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity with SEQ ID NO: 18. 24. The polynucleotide of any of the 823 clauses, wherein the CBF1A polypeptide is encoded by a polynucleotide comprising a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 126. 25. The polynucleotide of any of the 1024 clauses, wherein the CBF3I polypeptide is encoded by a polynucleotide comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 128. Petition 870250086132, dated 09 / 23 / 2025, pages 123 / 173 30 / 71 26. The recombinant transcription factor encoded by the polynucleotide in any of clauses 1-25. 27. A method for producing a recombinant monocotyledonous plant, wherein the method comprises: to contact a monocotyledonous plant cell with a first polynucleotide encoding a gene of interest, wherein the gene of interest is heterologous to the monocotyledonous plant cell; to contact the monocotyledonous plant cell with a second polynucleotide that encodes the recombinant transcription factor of clause 26; Select a monocotyledonous plant cell that has incorporated the gene of interest into its genome and regenerate a recombinant monocotyledonous plant from the selected monocotyledonous plant cell. 28. The method in clause 27, which further comprises contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional Wuschel polypeptide or Wuschel homeobox (WUS / WOX). 29. The method in clause 27, wherein the method does not involve contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional WUS / WOX polypeptide. 30. The method of any of clauses 27-29, wherein the monocotyledonous plant cell comprises a Poaceae plant cell. 31. The method of any of clauses 27-30, wherein the monocotyledonous plant cell comprises a plant cell of any of the following species: Zea mays, Oryza sativa, Triticum aestivum, Setaria italica, Hordeum vulgare, Cenchrus americanus, Saccharum officinarum, or Sorghum bicolor. Petition 870250086132, dated 09 / 23 / 2025, pp. 124 / 173 31 / 71 32. The method described in any of clauses 27-31, wherein the contact steps comprise transformation mediated by bacteria or bombardment with particles. 33. The method of either of clauses 27-32, wherein the first polynucleotide is present in a first vector and the second polynucleotide is present in a second vector. 34. The method of clause 33, in which the second vector additionally comprises the third polynucleotide. 35. The method of any of clauses 27-34, wherein the monocotyledonous plant cell is an immature embryonic cell or a leaf cell. 36. The method of any of clauses 27-35, wherein the gene of interest comprises a characteristic gene, a site-specific endonuclease, a guide polynucleotide, or a combination thereof. 37. The method described in any of clauses 27-36, which additionally comprises the excision of one or both of the second and third polynucleotides from the genome of the selected cell.

[0086] Examples - The present disclosure will be appreciated more fully with reference to the following non-limiting examples. EXAMPLE 1: SEQUENCES

[0087] Useful sequences in the methods of revelation are presented in Table 1 and provided in the sequence listing. Table 1. SEQ ID NO: Polynucleotide (DNA) or Polypeptide (PRT) NAME DESCRIPTION 1 DNA PHP71539 VIRB1 + VIRB2 + VIRB3 + VIRB4 + VIRB5 + VIRB6 + Petition 870250086132, dated 09 / 23 / 2025, pages 125 / 173 32 / 71 VIRB7 + VIRB8 + VIRB9 + VIRB10 + VIRB11 + VIRG + VIRC2 + VIRC1 + VIRD1 + VIRD2 + VIRD3 + VIRD4 + VIRD5 + VIRE1 + VIRE2 + VIRE3 + GENT + COLE1 ORI + PVS1 ORI 2 DNA PHP96037 Complete plasmid sequence 3 DNA PHP96037 RB + LOXP + NOS PRO::ZMWUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM-ODP2::OS-T28 TERM + A-5-IV2 INS + ZM-HSP17,7 PRO::MO-CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + A-5-IV2 INS + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SBALS PRO::ZM-ALS (HRA)::SB- PEPC1 TERM + LB 4 DNA PHP97334 RB + LOXP + NOS PRO::ZM- WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- ODP2::OS-T28 TERM + A-5-IV2 INS + ZM-HSP17,7 PRO::MOCRE-EXON1::ST-LS1 Petition 870250086132, de 23 / 09 / 2025, pág. 126 / 173 33 / 71 INTRON1::MO-CRE EXON2::PINII TERM + A-5-IV- 2 INS + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 5 DNA RV045471 = PHP102072 RB + LOXP + NOS PRO::ZMWUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- BBM404::OS-T28 TERM + A-5- IV-2 INS + ZM-HSP17,7 PRO::MO-CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 6 DNA RV045472 = PHP101977 RB + LOXP + NOS PRO::ZM- WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- B:A:BBM404::OS-T28 TERM + A-5-IV-2 INS + ZM-HSP17,7 PRO::MO-CRE-EXON1::ST-LS1 Petition 870250086132, de 23 / 09 / 2025, pág. 127 / 173 34 / 71 INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 7 DNA RV045473 = PHP103858 RB + LOXP + NOS PRO::ZM- WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- B:A:B:A:BBM404::OS-T28 TERM + A-5-IV-2 INS + ZM- HSP17,7 PRO::MO-CRE- EXON1::ST-LS1 INTRON1::MO- CRE EXON2::PINII TERM + OSUBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SIUBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 8 DNA RV045474 = PHP101978 RB + LOXP + NOS PRO::ZM- WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- B:A:BBM404:PROTEIN LINKER 1:A-CBF1A (MO):OS-T28 TERM + A-5-IV-2 INS + ZM-HSP17,7 Petition 870250086132, de 23 / 09 / 2025, pág. 128 / 173 35 / 71 PRO::MO-CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 9 DNA RV048557 = PHP104187 RB + LOXP + OS-ACTIN PRO::ZM-WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- B:A:BBM404:PROTEIN LINKER 1:A-CBF1A (MO):OS-T28 TERM + A-5-IV-2 INS + ZM-HSP17,7 PRO::MO-CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 10 DNA RV048363 = PHP103910 RB + LOXP + NOS PRO::ZM- WUS2::IN2-1 TERM + UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM-BBM:PROTEIN LINKER 1:A-CBF1A (MO):OS- T28 TERM + A-5-IV-2 INS + Petition 870250086132, de 23 / 09 / 2025, pág. 129 / 173 36 / 71 ZM-HSP17,7 PRO::MO-CRE- EXON1::ST-LS1 INTRON1::MO- CRE EXON2::PINII TERM + OS- UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 11 DNA RV047654 = PHP104112 RB + LOXP + NOS PRO::ZM- WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- BBM:PROTEIN LINKER 1:A- CBF1A (MO):OS-T28 TERM + A5-IV-2 INS + ZM-HSP17,7 PRO::MO-CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS-GREEN1::OS-UBI TERM + SI-UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 12 DNA Os-Actin Pro OS-ACTIN PRO 13 DNA UBI1ZM PRO / 5' UTR Corn ubiquitin promoter and 5' untranslated region Petition 870250086132, dated 09 / 23 / 2025, pp. 130 / 173 37 / 71 14 DNA Zm-Bbm Corn ovule development protein 2 (corn ortholog of At-BabyBoom), also known as the Bbm gene 15 PRT ZM-BBM Corn ODP2-encoded protein 16 DNA Zm-Bbm Motif B ZM-ODP2 (TR11) or Motif B 17 PRT ZM-BBM Motif B ZM-ODP2 (TR11) or Motif B 18 DNA Zm-Bbm Motif A ZM-ODP2 (TR12) or Motif A 19 PRT ZM-BBM Motif A ZM-ODP2 (TR11) or Motif A 20 DNA Zm-Bbm404 ZM-ODP2 (TR5) or BBM404 21 PRT ZM-BBM404 ZM-ODP2 (TR5) or BBM404 22 PRT Zemay-BBM2 Protein encoded by Zea mays BBM2 23 PRT Zemay-BBM2 Motif B Peptide encoded from Motif B of Zea mays BBM2 24 PRT Zemay-BBM2 Motif A Peptide encoded from Motif A of Zea mays BBM2 25 PRT Zemay-BBM2 BBM404 Truncated protein encoded by Zea mays BBM2 BBM404 Petition 870250086132, dated 09 / 23 / 2025, pp. 131 / 173 38 / 71 26 PRT Sobic-BBM Protein encoded by Sorghum bicolor BBM 27 PRT Sobic-BBM Motif B Peptide encoded from the B Motif of Sorghum bicolor BBM 28 PRT Sobic-BBM Motif A Peptide encoded from the A Motif of Sorghum bicolor BBM 28 PRT Sobic-BBM BBM404 Truncated protein encoded by Sorghum bicolor BBM BBM404 30 PRT Sobic-BBM2 Protein encoded by Sorghum bicolor BBM2 31 PRT Sobic-BBM2 Motif B Peptide encoded from the B Motif of Sorghum bicolor BBM2 32 PRT Sobic-BBM2 Motif A Peptide encoded from the A Motif of Sorghum bicolor BBM2 33 PRT Sobic-BBM2 BBM404 Truncated protein encoded by Sorghum bicolor BBM2 BBM404 34 PRT Misin-BBM Protein encoded by Miscanthus sinensis BBM 35 PRT Misin-BBM Motif B Peptide encoded from the Motif B of Miscanthus sinensis BBM Petition 870250086132, dated 09 / 23 / 2025, pages 132 / 173 39 / 71 36 PRT Misin-BBM Motif A Peptide encoded from the A Motif of Miscanthus sinensis BBM 37 PRT Misin-BBM BBM404 Truncated protein encoded by Miscanthus sinensis BBM BBM404 38 PRT Misin-BBM2 Protein encoded by Miscanthus sinensis BBM2 39 PRT Misin-BBM2 Motif B Peptide encoded from the B Motif of Miscanthus sinensis BBM2 40 PRT Misin-BBM2 Motif A Peptide encoded from the A Motif of Miscanthus sinensis BBM2 41 PRT Misin-BBM2 BBM404 Truncated protein encoded by Miscanthus sinensis BBM2 BBM404 42 PRT Orsat-BBM Protein encoded by Oryza sativa BBM 43 PRT Orsat-BBM Motif B Peptide encoded from the B motif of Oryza sativa BBM 44 PRT Orsat-BBM Motif A Peptide encoded from the A motif of Oryza sativa BBM 45 PRT Orsat-BBM BBM404 Truncated protein encoded by Oryza sativa BBM BBM404 46 PRT Orsat-BBM2 Protein encoded by Oryza sativa BBM2 Petition 870250086132, dated 09 / 23 / 2025, pages 133 / 173 40 / 71 47 PRT Orsat-BBM2 Motif B Peptídeo codificado a partir do Motivo B de Oryza sativa BBM2 48 PRT Orsat-BBM2 Motif A Peptídeo codificado a partir do Motivo A de Oryza sativa BBM2 49 PRT Orsat-BBM2 BBM404 Proteína truncada codificada por Oryza sativa BBM2 BBM404 50 PRT Orsat-BBM1 Proteína codificada por Oryza sativa BBM1 51 PRT Orsat-BBM1 Motif B Peptídeo codificado a partir do Motivo B de Oryza sativa BBM1 52 PRT Orsat-BBM1 Motif A Peptídeo codificado a partir do Motivo A de Oryza sativa BBM1 53 PRT Orsat-BBM1 BBM404 Truncated protein encoded by Oryza sativa BBM1 BBM404 54 PRT Brdis-BBM Protein encoded by distant Brachypodium BBM 55 PRT Brdis-BBM Motif B Peptídeo encoded starting from Motif B of Brachypodium distachyon BBM 56 PRT Brdis-BBM Motif A Peptídeo encoded starting from Motif A of Brachypodium distachyon BBM Petition 870250086132, 09 / 23 / 2025, pág. 134 / 173 41 / 71 57 PRT Brdis-BBM BBM404 Trunk protein encoded by Brachypodium distachyon BBM BBM404 58 PRT Brdis-BBM2 Protein encoded by Brachypodium distachyon BBM2 59 PRT Brdis-BBM2 Motif B Peptide encoded starting from Motif B of Brachypodium distachyon BBM2 60 PRT Brdis-BBM2 Peptide motif encoded starting from Motif A of Brachypodium distachyon BBM2 61 PRT Brdis-BBM2 BBM404 Truncated protein encoded by Brachypodium distachyon BBM2 BBM404 62 PRT Muaca-Chr2-BBM Protein encoded by Muca acuminata chromosome 2 BBM 63 PRT Muaca- Chr2-BBM Motif B Peptide encoded from Motif B of Muca acuminata chromosome 2 BBM 64 PRT Muaca- Chr2-BBM Motif A Peptide encoded from Motif A of Muca acuminata chromosome 2 BBM 65 PRT Muaca- Chr2-BBM BBM404 Truncated protein encoded by Muca acuminata chromosome 2 BBM BBM404 Petition 870250086132, 09 / 23 / 2025, pág. 135 / 173 42 / 71 66 PRT Muaca-Chr9-BBM2 Protein encoded by Muca acuminata chromosome 9 BBM2 67 PRT Muaca-Chr9-BBM2 Motif B Peptide encoded from Motif B of Muca acuminata chromosome 9 BBM2 68 PRT Muaca-Chr9-BBM2 Motif A Peptide encoded from Motif A of Muca acuminata chromosome 9 BBM2 69 PRT Muaca-Chr9-BBM2 BBM404 Truncated protein encoded by Muca acuminata chromosome 9 BBM2 BBM404 70 PRT Sevir-BBM Protein encoded by Setaria viridis BBM 71 PRT Sevir-BBM Motif B Peptide encoded from Motif B of Setaria viridis BBM 72 PRT Sevir-BBM Motif A Peptide encoded from Motif A of Setaria viridis BBM 73 PRT Sevir-BBM BBM404 Truncated protein encoded by Setaria viridis BBM BBM404 74 PRT Sevir-BBM2 Protein encoded by Setaria viridis BBM2 75 PRT Sevir-BBM2 Motif B Peptide encoded from the Motif B of Setaria viridis BBM2 Petition 870250086132, dated 09 / 23 / 2025, pages 136 / 173 43 / 71 76 PRT Sevir-BBM2 Motif A Peptide encoded from the A motif of Setaria viridis BBM2 77 PRT Sevir-BBM2 BBM404 Truncated protein encoded by Setaria viridis BBM2 BBM404 78 PRT Seita-BBM Protein encoded by Setaria italica BBM 79 PRT Seita-BBM Motif B Peptide encoded from the B motif of Setaria italica BBM 80 PRT Seita-BBM Motif A Peptide encoded from the A motif of Setaria italica BBM 81 PRT Seita-BBM BBM404 Truncated protein encoded by Setaria italica BBM BBM404 82 PRT Seita-BBM2 Protein encoded by Setaria italica BBM2 83 PRT Seita-BBM2 Motif B Peptide encoded from the B motif of Setaria italica BBM2 84 PRT Seita-BBM2 Motif A Peptide encoded from the A Motif of Setaria italica BBM2 85 PRT Seita-BBM2 BBM404 Truncated protein encoded by Setaria italica BBM2 BBM404 86 PRT Traes-BBM Protein encoded by Triticum aestivum BBM Petition 870250086132, dated 09 / 23 / 2025, pages 137 / 173 44 / 71 87 PRT Traes-BBM Motif B Peptide encoded from the B Motif of Triticum aestivum BBM 88 PRT Traes-BBM Motif A Peptide encoded from the A Motif of Triticum aestivum BBM 89 PRT Traes-BBM BBM404 Truncated protein encoded by Triticum aestivum BBM BBM404 90 PRT Traes-BBM2 Protein encoded by Triticum aestivum BBM2 91 PRT Traes-BBM2 Motif B Peptide encoded by the B Motif of Triticum aestivum BBM2 92 PRT Traes-BBM2 Motif A Peptide encoded from the A Motif of Triticum aestivum BBM2 93 PRT Traes-BBM2 BBM404 Truncated protein encoded by Triticum aestivum BBM2 BBM404 94 PRT Pahal-BBM Protein encoded by Panicum hallii BBM 95 PRT Pahal-BBM Motif B Peptide encoded from Motif B of Panicum hallii BBM 96 PRT Pahal-BBM Motif A Peptide encoded from Motif A of Panicum hallii BBM Petition 870250086132, dated 09 / 23 / 2025, pages 138 / 173 45 / 71 97 PRT Pahal-BBM BBM404 Truncated protein encoded by Panicum hallii BBM BBM404 98 PRT Pahal-BBM2 Protein encoded by Panicum hallii BBM2 99 PRT Pahal-BBM2 Motif B Peptide encoded from Motif B of Panicum hallii BBM2 100 PRT Pahal-BBM2 Motif A Peptide encoded from Motif A of Panicum hallii BBM2 101 PRT Pahal-BBM2 BBM404 Truncated protein encoded by Panicum hallii BBM2 BBM404 102 PRT Metru-BBM Protein encoded by Medicago trunculata BBM 103 PRT Metru-BBM Motif B Peptide encoded from Motif B of Medicago trunculata BBM 104 PRT Metru-BBM Motif A Peptide encoded from Motif A of Medicago trunculata BBM 105 PRT Metru-BBM BBM404 Truncated protein encoded by Medicago trunculata BBM BBM404 106 PRT Vivin-BBM Protein encoded by Vitus vinifera BBM Petition 870250086132, dated 09 / 23 / 2025, pp. 139 / 173 46 / 71 107 PRT Vivin-BBM Motif B Peptide encoded from the B Motif of Vitus vinifera BBM 108 PRT Vivin-BBM Motif A Peptide encoded from the A Motif of Vitus vinifera BBM 109 PRT Vivin-BBM BBM404 Truncated protein encoded by Vitus vinifera BBM BBM404 110 PRT Brnap-BBM Protein encoded by Brassica napus BBM 111 PRT Brnap-BBM Motif B Peptide encoded from the B Motif of Brassica napus BBM 112 PRT Brnap-BBM Motif A Peptide encoded from the A Motif of Brassica napus BBM 113 PRT Brnap-BBM BBM404 Truncated protein encoded by Brassica napus BBM BBM404 114 PRT Cusat-BBM Protein Cucumis sativa BBM 115 PRT Cusat-BBM Motif B Peptide encoded from the B Motif of Cucumis sativa BBM 116 PRT Cusat-BBM Motif A Peptide encoded from the A Motif of Cucumis sativa BBM Petition 870250086132, dated 09 / 23 / 2025, pp. 140 / 173 47 / 71 117 PRT Cusat-BBM BBM404 Truncated protein encoded by Cucumis sativa BBM BBM404 118 PRT Gohir-BBM Protein encoded by Gossypium hirsutum BBM 119 PRT Gohir-BBM Motif B Peptide encoded from the B Motif of Gossypium hirsutum BBM 120 PRT Gohir-BBM Motif A Peptide encoded from the A Motif of Gossypium hirsutum BBM 121 PRT Gohir-BBM BBM404 Truncated protein encoded by Gossypium hirsutum BBM BBM404 122 PRT Glmax-BBM Protein encoded by Glycine max BBM 123 PRT Glmax-BBM Motif B Peptide encoded from the B Motif of Glycine max BBM 124 PRT Glmax-BBM Motif A Peptide encoded from the A motif of Glycine max BBM 125 PRT Glmax-BBM BBM404 Truncated protein encoded by Glycine max BBM BBM404 126 DNA AT-CBF1A CBF1A activation domain gene from Arabidopsis thaliana, encoding aa 116-214 Petition 870250086132, dated 09 / 23 / 2025, pages 141 / 173 48 / 71 127 PRT AT-CBF1A CBF1A activation domain of Arabidopsis thaliana aa 116-214 128 DNA AT-CBF3I CBF3I activation domain gene of Arabidopsis thaliana 129 PRT AT-CBF3I CBF3I activation domain of Arabidopsis thaliana 130 DNA SB-CBF1A CBF1A activation domain gene of Sorghum bicolor 131 PRT SB-CBF1A CBF1A activation domain encoded by Sorghum bicolor 132 DNA ZM-CBF1A CBF1A activation domain gene of Zea mays 133 PRT ZM-CBF1A CBF1A activation domain encoded by Zea mays 134 DNA ZM-C1 C1 transcription factor activation domain gene of Zea mays 135 PRT ZM-C1 C1 transcription factor activation domain C1-encoded transcription - Zea mays 136 DNA ZM-O2 Gene of the activation domain of the O2 transcription factor of Zea mays 137 PRT ZM-O2 Activation domain of the O2 transcription factor - Zea mays 138 PRT ZM-DOF1 Zea mays DNA ligation with activation domain One Finger 1 Petition 870250086132, dated 09 / 23 / 2025, pages 142 / 173 49 / 71 139 PRT AT-ERF97 Ethylene Responsive Factor 97 activation domain of Arabidopsis thaliana 140 PRT AT-ERF98 Ethylene Responsive Factor 98 activation domain of Arabidopsis thaliana 141 PRT GM-EDLLlike ERF activation domain of Glycine max with EDLL motif 142 PRT MT-EDLLlike ERF activation domain of Medicago trunculata with EDLL motif 143 PRT OS-EDLL-like ERF activation domain of Oryza sativa with EDLL motif 144 PRT VP16 core Herpes Simplex Virus VP16 activation core peptide 145 PRT AT-CBF1 core CBF1A activation domain of Arabidopsis thaliana with “VP16-like” region 146 PRT AT-CBF1m Activation domain CBF1A of Arabidopsis thaliana with a modified “VP16-like” region 147 PRT ZM-DOF1 core DOF1 activation domain of Zea mays with a “VP16-like” region 148 PRT ZM-DOF1m DFO1 activation domain of Zea mays with a modified “VP16-like” region Petition 870250086132, dated 09 / 23 / 2025, pages 143 / 173 50 / 71 149 PRT AT-DREB1 core DREB1 activation domain of Arabidopsis thaliana with a “VP16-like” region 150 PRT AT-DREB1m DREB1 activation domain of Arabidopsis thaliana with a modified “VP16-like” region 151 PRT NT-ERF1 core ERF1 activation domain of Nicotiana tabacum with a “VP16-like” region 152 PRT NT-ERF1m ERF1 activation domain of Nicotiana tabacum with a modified “VP16-like” region 153 PRT NT-ERF2 core ERF2 activation domain of Nicotiana tabacum with a “VP16-like” region 154 PRT NT-ERF2m ERF2 activation domain of Nicotiana tabacum with a modified “VP16-like” region 155 PRT CR-ORCA core ORCA activation domain of Catharanthus roseus with a “VP16-like” region 156 PRT CR-ORCAm ORCA activation domain of Catharanthus roseus with a modified “VP16-like” region 157 PRT LS-PTI4 core PTI4 activation domain of Lycopersicum solanum with a “VP16-like” region Petition 870250086132, dated 09 / 23 / 2025, pages 144 / 173 51 / 71 158 PRT LS-PTI4m PTI4 activation domain of Lycopersicum solanum with a modified “VP16-like” region 159 DNA VP64 Synthetic Herpes Simplex Virus VP64 160 PRT VP64 Synthetic multimeric activation domains 161 DNA PHP000001 T-DNA RB + AXIG1 PRO::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 162 DNA PHP000002 T-DNA RB + UBI1ZM PRO::BBM404:CBF1A + UBI1ZM PRO::MO-PAT::PINII + LB 163 DNA PHP000003 T-DNA RB + LOXP + HSP17 PRO::CRE + NOS PRO::ZM-WUS2::IN2-1 TERM + ZM-UBI1 PRO::BBM404:CBF1A::ZM-UBI TERM + LOXP + SB-UBI PRO::ZsGREEN1::OS-UBI TERM + SI-ALS PRO::ZM-HRA::SB UBI TERM + LB 164 PRT AT-HSFA6B Arabidopsis thaliana Heat Shock A6B transcription factor activation domain 165 PRT AT-DREB2A Arabidopsis thaliana DREB2A activation domain Petition 870250086132, dated 09 / 23 / 2025, pages 145 / 173 52 / 71 166 PRT ZM-DOF1 Zea mays DOF1 DNA binding activation domain com um gene finger1 167 DNA RV048912 = PHP104222 RB + LOXP + OS-ACTIN PRO::ZM-WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- ODP2::OS-T28 TERM + A-5-IV- 2 INS + ZM-HSP17,7 PRO::MO- CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + A-5-IV- 2 INS + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI-UBI PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 168 DNA RV050602 = PHP105696 RB + LOXP + UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM-WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM-ODP2::OS-T28 TERM + A-5-IV-2 INS + ZM- HSP17,7 PRO::MO-CRE- EXON1::ST-LS1 INTRON1::MO- CRE EXON2::PINII TERM + A5-IV-2 INS + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- Petition 870250086132, de 23 / 09 / 2025, pag. 146 / 173 53 / 71 GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 169 DNA RV054309 = PHP107604 RB + LOXP +:NOS PRO::ZM- WUS2::HM +2-1 ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM- BBM404:PROTEIN LINKER 1:ACBF1A (MO):OS-T28 TERM + A5-IV-2 INS + ZM-HSP17,7.: PRO-::UBI1ZM INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 DNA INTRON1:M:0 +:NPTI RV052550 = PHP106860 RB + LOXP + OS-ACTIN PRO::ZM-WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR:0PRON1ZM:0 LINKER 1:ACBF1A (MO):OS-T28 TERM + A5-IV-2 INS + ZM-HSP17,7 PRO::MO-CRE-EXON1::ST-LS1 INTRON1::MO-CRE EXON2::PINII TERM + OS-UBI Petition 870250086132, dated 9 / 23 / 2025, p. 147 / 173 54 / 71 TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SI- UBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB 171 DNA RV052548 = PHP106868 RB + LOXP + UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM-WUS2::IN2-1 TERM + FMV ENH::PCSV ENH::MMV ENH::UBI1ZM PRO::UBI1ZM 5UTR::UBI1ZM INTRON1::ZM-BBM404:PROTEIN LINKER 1:A-CBF1A (MO):OS- T28 TERM + A-5-IV-2 INS + ZM-HSP17.7 PRO::MO-CRE- EXON1::ST-LS1 INTRON1::MO- CRE EXON2::PINII TERM + OSUBI TERM + LOXP + SB-UBI PRO::SB-UBI INTRON1::ZS- GREEN1::OS-UBI TERM + SIUBI1 PRO::SI-UBI1 INTRON1::NPTII::SB-UBI TERM + LB EXAMPLE 2: PARTICLE BOMBARDMENT

[0088] Standard protocols for particle bombardment (Finer and McMullen, 1991, In Vitro Cell Dev. Biol. - Plant 27:175-182 and those found in document no. WO2022072335A2) can be used with the revelation methods. EXAMPLE 3: CORN TRANSFORMATION MEDIATED BY AGROBACTERIUM Petition 870250086132, dated 09 / 23 / 2025, pages 148 / 173 55 / 71

[0089] A. Preparation of Agrobacterium Master Plate.

[0090] Agrobacterium tumefaciens hosting a binary donor vector is taken from a frozen aliquot at -80 °C onto solid 12R medium and cultured at 28 °C in the dark for 2 to 3 days to produce a master plate.

[0091] B. Cultivate Agrobacterium in solid medium.

[0092] A single colony or multiple colonies of Agrobacterium are collected from the main plate and seeded onto a second plate containing 810K medium and incubated at 28 °C in the dark overnight.

[0093] Agrobacterium infection medium (5 ml) and 100 mM 3'-5'-Dimethoxy-4'-hydroxyacetophenone (acetosyringone; 5 pl) are added to a 14 ml conical tube in a fume hood. Approximately 3 complete loops of Agrobacterium from the second plate are suspended in the tube, and the tube is then vortexed to produce a uniform suspension. The suspension (1 ml) is transferred to a spectrophotometer tube, and the optical density (550 nm) of the suspension is adjusted to a reading of approximately 0.35 to 1.0. The concentration of Agrobacterium was approximately 0.5 to 2.0 χ¹⁰⁹ cfu / ml. The final Agrobacterium suspension is aliquoted into 2 ml microcentrifuge tubes, each containing approximately 1 ml of the suspension. The suspensions are then used as soon as possible.

[0094] C. Cultivate Agrobacterium in liquid medium.

[0095] Alternatively, Agrobacterium can be prepared for transformation by growth in liquid medium. One day before infection, a 125 ml flask is prepared with 30 ml of 557A medium and 30 µl of spectinomycin (50 mg / ml) and 30 µl of acetosyringone (20 mg / ml). A half-cycle of Agrobacterium from a second plate is suspended in the flasks and placed on an orbital shaker set at 200 rpm and incubated at 28 °C. Petition 870250086132, dated 09 / 23 / 2025, pp. 149 / 173 56 / 71 overnight. The Agrobacterium culture is centrifuged at 5,000 rpm for 10 min. The supernatant is removed and Agrobacterium infection medium with acetosyringone solution is added. The bacteria are resuspended by vortexing and the optical density (550 nm) of the Agrobacterium suspension was adjusted to a reading of approximately 0.35 to 2.0.

[0096] D. Corn Transformation.

[0097] Corn seed is surface-sterilized for 15-20 min in 20% (v / v) bleach (5.25% sodium hypochlorite) plus 1 drop of Tween 20 followed by 3 washes in sterile water, germinated and allowed to grow to seedlings for approximately 14 days, and then prepared to produce leaf fragments. Leaf segments are placed in Agrobacterium infection medium (700A) with 200 μM acetosyringone solution + 0.02% Break-Thru® surfactant (Plant Health Technologies, PO Box 70013, Boise, ID 837070113). The Agrobacterium infection medium is removed and 1 ml of the Agrobacterium suspension is added to the leaf segments and left to stand for 20 min. The suspension of Agrobacterium and leaf segments are passed through a sterile metal sieve, and the liquid is discarded. The leaf segments collected in the metal sieve are transferred using a spatula to a stack of 3 sterile filter papers, used to remove excess Agrobacterium-containing liquid, and then a spatula is used to transfer the leaf segments to a filter paper resting on the co-culture medium. The plate is incubated in the dark at 21 °C for 1 to 3 co-culture days.

[0098] The filter papers supporting the leaf segments are then transferred to a resting medium without selection. Seven days later, the filter papers supporting the leaf segments were transferred to a selection medium by Petition 870250086132, dated 09 / 23 / 2025, pages 150 / 173 57 / 71 three weeks. After selection, healthy growing somatic embryos are transferred using forceps to maturation medium for two weeks in the dark, at which point the maturation plates are transferred in toto (still containing the maturing somatic embryos) to the light for an additional week. After one week in the light, the regenerating plantlets are transferred to rooting medium. After rooting, the plantlets are ready for transplanting to the greenhouse. Example 4: BBM TRUNCATIONS (ODP2)

[0099] The full-length (2133 bp) ZM-ODP2 (BBM) gene (SEQ ID Nos: 14 and 15) encoding the full-length (710 aa) wild-type ODP2 protein was truncated to produce three fragments: ZM-ODP2 (TR11) = B motif that encodes amino acids 60-69 of the ZM-ODP2 protein (SEQ ID Nos: 16 and 17) ZM-ODP2 (TR12) = Motif A encoding amino acids 156-171 of the ZM-ODP2 protein (SEQ ID Nos: 18 and 19) ZM-ODP2 (TR5) = BBM404 which encodes amino acids 266669 of the ZM-ODP2 protein (SEQ ID Nos: 20 and 21) EXAMPLE 5: Fusion of the CBF1A activation domain with enhanced transformation of truncated Bbm. (A) Inbred stiff-stem maize line ED85E

[0100] Seeds of the inbred maize line ED85E were surface sterilized and germinated in a medium containing MS salts, sucrose, and 2 mg / l of ancymidol. Thirteen-day-old seedlings were harvested, and the first 3 cm of leaf tissue above the mesocotyl were longitudinally bisectioned and then mechanically chopped in a blender while suspended in 100 ml of Agrobacterium at an OD of 0.5 (see Example 5 for details). Petition 870250086132, dated 09 / 23 / 2025, pages 151 / 173 58 / 71

[0101] Five T-DNA configurations were tested using 10 seedlings per treatment. After regeneration of the T0 seedlings and root formation (i.e., the stage at which plants are normally sent to the greenhouse), leaf tissue from each T0 plant was sampled for multiplex PCR to confirm the number of copies of the integrated T-DNA. All five plasmids contained the same Nos::Wus2 expression cassette and the same 3xENH:UBI promoter that controlled Bbm gene expression, and the experimental results are summarized in Table 2. For all five treatments (5 different Bbm coding sequences), T-DNA administration, measured by transient Zs-Green1 expression 3-4 days post-infection, was good (score = 3) to excellent (score = 4). In the first control treatment with the full-length Bbm gene (PHP97334), T0 plants were recovered at a frequency of 600% (i.e., 10 initial seedlings produced T0 plants).In the second treatment, the Bbm gene was truncated to include 404 amino acids, including the two AP2 DNA-binding domains and most of the carboxyl terminus of the protein (PHP102072), and for this treatment, the transformation frequency was reduced compared to the control by 438%. Adding one copy of the highly conserved aminopeptides B and A to PHP101977 (as described in document WO 2020 / 214986) or two copies of these motifs (PHP103858) resulted in a further reduction in transformation frequencies, yielding values ​​of 113% and 125%, respectively. Surprisingly, when a corn codon-optimized transcriptional activation domain from the Arabidopsis protein CBF1A (SEQ ID NO: 127) was fused to the carboxyl end of the truncated BA-Bbm404 protein (PHP101978), a transformation frequency of 2031% was achieved. Petition 870250086132, dated 09 / 23 / 2025, pp. 152 / 173 59 / 71 produced, an increase of approximately 3.4 times above the total length control Bbm gene.

[0102] Table 2. Leaf transformation frequencies for the inbred maize line ED85E using several modified Bbm genes. RV# Plasmid Description Delivery of T-DNA Transformation Frequency RV03512 6 PHP97334 Nos::Wus2 + 3xEnh:Ubi::Bbm (control) 4 600% RV04547 1 PHP10207 2 Nos::Wus2 + 3xEnh:Ubi::Bbm40 4 3 438% RV04547 2 PHP10197 7 Nos::Wus2 + 3xEnh:Ubi::BA-Bbm404 3.5 113% RV04547 3 PHP10385 8 Nos::Wus2 + 3xEnh:Ubi::BA-BA-Bbm404 3.5 125% RV04547 4 PHP10197 8 Nos::Wus2 + 3xEnh:Ubi::BA- Bbm404:CBF1A 4 2031% (B) Non-rigid stem inbred maize line GR013D

[0103] Starting with 10 seedlings per replicate, fourteen-day-old seedlings were mechanically injected with Agrobacterium containing the helper plasmid PHP71539 plus PHP97334 (wild-type full-length Bbm gene) or PHP101978 (BA-Bbm404:CBF1A). The experimental treatments were replicated three times (the two plasmid treatments side-by-side) and after plant selection and regeneration for Petition 870250086132, dated 09 / 23 / 2025, pages 153 / 173 60 / 71 producing T0 plantlets, transformation frequencies were scored. PHP97334 produced transformation frequencies of 50%, 40%, and 10% for a mean and standard deviation of 33% ± 17%. PHP101978 produced transformation frequencies of 210%, 220%, and 180% for a mean and standard deviation of 203% ± 17%. For this inbred lineage, the use of the truncated Bbm404 fused to the CBF1A activation domain produced a more than 6-fold increase in transformation frequency compared to the control plasmid.

[0104] (C) Comparison of the full-length Bbm gene with the BA-Bbm404 truncation, with each being fused to the CBF1A activation domain.

[0105] Three plasmids were compared using Agrobacterium-mediated transformation in ED85E leaf tissue from the inbred maize line, starting with seven seedlings per treatment per replicate. The results for both replicates were consistent, with the full-length Bbm gene and the full-length Bbm fused to CBF1A producing similar transformation frequencies. However, when the truncated BA-Bbm404 gene was fused to CBF1A, the transformation frequency in both replicates was substantially higher in both replicates of the experiment.

[0106] Table 3. Fusion of the CBF1A activation domain to the truncated BA-Bbm404 protein substantially increased transformation frequencies compared to the full-length Bbm gene or the full-length gene fused to the same activation domain. Petition 870250086132, dated 09 / 23 / 2025, pages 154 / 173 61 / 71 RV# Plasmid Description Replica 1 Transformation Frequency Replica 2 Transformation Frequency RV0351 26 PHP9733 4 Nos::Wus2 + 3xEnh:Ubi::Bbm (control) 471% 329% RV0476 54 PHP1041 12 Nos::Wus2 + 3xEnh:Ubi::Bbm:C BF1A 471% 282% RV0454 74 PHP1019 78 Nos::Wus2 + 3xEnh:Ubi::BA- Bbm404:CBF1A 1389% 871%

[0107] Similar enhanced transformation frequencies using BA-Bbm404:CBF1A were observed in inbred maize lines ED85E, GR013D, and GR0112. In addition, an enhanced transformation was also observed in the tropical maize inbred lines JFDYY, EEP7E, and EENW5.

[0108] (D) Comparison of the Bbm404 truncation with the BA-Bbm404 truncation, with each being fused to the CBF1A activation domain.

[0109] Leaf tissue from the inbred maize line GR0112 was transformed with Agrobacterium containing the helper plasmid PHP71539 plus PHP97334 (Nos::Wus2, full-length Bbm, no activation domain), PHP104222 (Act::Wus2, full-length Bbm, no activation domain), PHP105696 (Ubi::Wus2, full-length Bbm, no activation domain), PHP101978 (Nos::Wus2, B-ABbm404:CBF1A), PHP104187 (Act::Wus2, BA-Bbm404:CBF1A), PHP107604 (Nos::Wus2, Bbm404:CBF1A), PHP106860 (Act::Wus2, Bbm404:CBF1A) or PHP106868 (Ubi::Wus2, Bbm404:CBF1A). You Petition 870250086132, dated 09 / 23 / 2025, pages 155 / 173 62 / 71 results of these transformations are shown in Table 4. GR0112 showed the lowest transformation frequency with PHP97334, but the highest transformation frequency with PHP105696 (Ubi::Wus2). Constructs with BA-Bbm404:CBF1A transformed GR0112 with twice the transformation frequency compared to the control PHP97334. In the absence of BA domains, with Nos::Wus2 or Act::Wus2, the transformation frequency decreased 3 to 5 times compared to PHP97334. However, PHP106868 with Ubi::Wus2 and Bbm404:CBF1A showed the highest transformation frequency of 160%.

[0110] Table 4. Comparison of transformation frequencies in the GR0112 maize inbred line between the complete Bbm gene and truncated Bbm404 proteins or BA-Bbm404 with a fused CBF1A activation domain. RV# Plasmid Description Transformation Frequency (%) RV035126 PHP97334 Nos::Wus2 3XEnh:Ubi::Bbm (control) + 30% RV048912 PHP104222 Act::Wus2 3XEnh:Ubi::Bbm + 56% RV050602 PHP105696 Ubi::Wus2 3XEnh:Ubi::Bbm + 117% RV045474 PHP101978 Nos::Wus2 3XEnh:Ubi::BA- Bbm404:CBF1A + 62% RV048557 PHP104187 Act::Wus2 3XEnh:Ubi::BA- Bbm404:CBF1A + 60% RV054309 PHP107604 Nos::Wus2 3XEnh:Ubi::Bbm404 + :CBF1A 6% Petition 870250086132, dated 09 / 23 / 2025, pp. 156 / 173 63 / 71 RV052550 PHP106860 Act::Wus2 3XEnh:Ubi: + :Bbm404:CBF1A 10% RV052548 PHP106868 Ubi::Wus2 3XEnh:Ubi: + :Bbm404:CBF1A 160% Example 6: The use of BA-Bbm404:CBF1A results in increased transformation frequencies in other cereal crops.

[0111] Comparison of transformation frequencies after transformation of Agrobacterium with control plasmid PHP97334 (WT Bbm gene) or PHP101978 (B-ABbm404:CBF1A) in Setaria italica (foxtail millet) leaf tissue resulted in transformation frequencies of 40% for PHP97334 (8 T0 plants out of 20 initial seedlings) or an increase in transformation frequency of 190% (19 T0 plants out of 10 initial seedlings) for PHP97334. These relative transformation frequencies for Setaria italica are reflected in the + (single +) scores for plasmid RV035126, also known as PHP973343, and a +++ (triple plus) score for plasmid RV045474, also known as PHP101978, in Table 5 below. For pearl millet, a higher transformation score was also observed than for the comparable plasmid containing only the complete Bbm gene.Tests on barley and wheat are ongoing, and the BA-Bbm404:CBF1A treatment is also expected to result in higher transformation frequencies than the unmodified Bbm treatment.

[0112] Table 5. Transformation scores for various Poaceae crops using full-length Bbm or truncated BA-Bbm404 fused to the CBF1A activation domain. NT = not yet tested. Petition 870250086132, dated 09 / 23 / 2025, pp. 157 / 173 64 / 71 Nos:wus+3x Enh ubi:Bbm Act:wus+3x Enh ubi:Bbm Nos:wus+3x Enh ubi:B- A-Bbm404- CBF1A Act:wus+3x Enh ubi:B- A-Bbm404- CBF1A Monocotyledonous plant RV035126 RV048848 RV045474 RV048557 Barley ++ +++ NT NT Setaria italica + + + + + NT Pearl millet + + + + + + Wheat 25R61 - + - + Example 7. Screening of different orthologous BBM sequences and different activation domains demonstrated a wide range of components that improved the transformation.

[0113] To evaluate the range of orthologous BBM proteins that can be used to first create the three ideal fragments of the BBM component (i.e., motifs A and B and larger fragments similar to BBM404) and then combined with a heterologous activation domain for better transformation of Poaceae explants, PHP101978 was used in an Agrobacterium-mediated leaf transformation assay.

[0114] (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, respectively) were tested using the expression cassette configuration found in PHP101978, maintaining all molecular components except the heterologous activation domain fused to the carboxylic end of ZmPetition 870250086132, from 23 / 09 / 2025, p. 158 / 173 65 / 71 Bbm404. The T-DNA is composed of RB + Nos::Wus2 + 3xENH:UBI PRO::BA-BBM404:[ACT] + Sb-UBI PRO::ZsGreen1 + Si-UBI PRO::NPTII, where [ACT] represents the different activation domains that were replaced by At-CBF1A in the PHP101978 construct.

[0115] To facilitate convenient transformation schedules, the activation domain constructs were divided into three groups. The PHP101978 and PHP97334 constructs were included in each group for comparison of transformation frequencies within that experiment. Transformations were performed on leaf tissue of the inbred maize line ED85E, starting with 5 seedlings per treatment per replicate. Each experiment had three replicates. The results of all experiments with three replicates combined are presented in Table 6.

[0116] Table 6. Fusion of different activation domains to the truncated BA-Bbm404 protein substantially increased transformation frequencies compared to full-length Bbm without a heterologous activation domain. Vector Frequency of Transformation (%) Group 1 PHP97334 (Full-length BBM, no heterologous activation domain, SEQ ID NO: 4) 160% PHP101978 (CBF1A, SEQ ID NO: 8) 321% VP64 (SEQ ID NO: 160) 455% ERF2 (SEQ ID NO: 153) 301% Petition 870250086132, dated 09 / 23 / 2025, pp. 159 / 173 66 / 71 CBF3I (SEQ ID NO: 129) 231% Group 2 PHP97334 (Full-length BBM, no heterologous activation domain, SEQ ID NO: 4) 287% PHP101978 (CBF1A, SEQ ID NO: 8) 460% PTI4 (SEQ ID NO: 157) 517% PTI4m (SEQ ID NO: 158) 299% DREB1A (SEQ ID NO: 149) 287% DREB1Am (SEQ ID NO: 150) 448% Group 3 PHP97334 (Full-length BBM, no heterologous activation domain, SEQ ID NO: 4) 254% PHP101978 (CBF1A, SEQ ID NO: 8) 522% VP16 (SEQ ID NO: 144) 558% DOF1 (SEQ ID NO: 166) 283% HSFA6B (SEQ ID NO: 164) 232% DREB2A (SEQ ID NO: 165) 304%

[0117] Activation domains in monocotyledonous and dicotyledonous plant species will work in the method. Candidate heterologous activation domains of orthologs of Petition 870250086132, dated 09 / 23 / 2025, pages 160 / 173 67 / 71 proteins from various exemplary plant species (Arabidopsis, Brassica, soybean, maize, rice, sorghum, etc.) are tested for a variety of different transcription factors, including 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 the activation domains of At-CBF1A and At-CBF3I were fused to BA-BBM404, it was demonstrated that CBF1A produced a strong stimulation of somatic embryo formation from leaf tissue and that CBF3I produced a similarly strong response (Table 6). The maize C1 activation domain is also expected to produce a slightly reduced response compared to CBF3I or CBFA1. In contrast, the use of the At-CBF1E or Zm-O2 activation domains will produce progressively weaker embryonic somatic stimulation. Thus, it is anticipated that this assay can be used to identify activation domains for use in the method that will produce a range of embryonic somatic stimulation, from weak responses (Zm-O2) to gradually increasing responses (CBF1E, then C1) and finally strong embryonic somatic stimulation, as with CBF1A and CBF3I. Activation domains share similar properties among eukaryotic cells, and therefore activation domains such as the Herpes Simplex virus VP16 (and the synthetic multimeric form VP64) worked well in the method when fused to BA-BBM404 (Table 6).

[0118] (B) Modified activation domains that increase or decrease the activation strength can also be used in the method. The use of wild-type activation domain sequences and modified amino acid sequences described by Li et al., 2013 (Plant Biotechnology Journal, 11, 671—680) fused to BA-BBM404 will function to stimulate the formation of somatic embryos and the frequencies of Petition 870250086132, dated 09 / 23 / 2025, pages 161 / 173 68 / 71 transformation. Therefore, the use of the transactivation domains CBF1, DOF1, DREB1, ERF1, ERF2, ORCA, and PTI4, as well as those same domains that have been modified to have a stronger resemblance to the VP16 core transactivation domain, is expected to be useful in stimulating the formation of leaf somatic embryos and increasing transformation frequencies. Among these domains, DREB1A and DREB1Am (where m represents the modified sequence as described by Li et al.) showed strong stimulation of leaf somatic embryo formation and transformation (Table 6).

[0119] (C) As demonstrated with the central activation domain of VP16 which was multimerized to produce VP64 with greatly increased activation levels (Table 6), the use of homomultimers (i.e., CBF1A:CBF1A:etc) or heteromultimers (i.e., CBF1A:ERF2m:PTI4) where the multimeric sequence (2x, 3x, 4x, etc.) is attached to a single BA-BBM404 protein will increase the strength of transactivation of endogenous Bbm target genes, resulting in a proportionally greater stimulation of somatic embryo growth and increased transformation frequency.

[0120] (D) Using the same plasmid design for testing (PHP101978), but replacing paralogs or orthologs of Bbm will result in similar positive results. Thus, Bbm proteins, or Bbm1 or Bbm2 from different plant species, can be similarly truncated and assembled in the same way (BA-BBM404) and fused to an exemplary activation domain, such as CBF1A, and when cloned behind 3xENH:UBI PRO, are expected to produce similar enhanced leaf transformation frequencies (see SEQ ID Nos: xx-yy). Petition 870250086132, dated 09 / 23 / 2025, pp. 162 / 173 69 / 71

[0121] (E) Different viral potentiating elements with a different strong constitutive promoter in place of corn UBI PRO also work in the invention. Example 8. The use of BBM404:CBF1A and / or BA-BBM404:CBF1A improves the transformation of immature embryos into recalcitrant inbred lines.

[0122] The following experiment demonstrates that the expression of BBM404:CBF1A (or BA-BBM404:CBF1A) and WUS2 immediately after infection with Agrobacterium results in direct somatic embryogenesis and regeneration in the recalcitrant public maize inbred line W22.

[0123] The PLTP promoter that directs BBM404:CBF1A and the AXIG1 promoter that directs WUS2 expression result in the rapid and direct formation of somatic embryos after the transformation of immature maize embryos.

[0124] Immature embryos (2-2.5 mm in length) are harvested from the W22 public maize inbred line approximately 11 days after pollination and are infected with the Agrobacterium strain LBA4404 THY-TD- containing the helper plasmid PHP71539 and a T-DNA with the following composition; RB + AXIG1 PRO::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 is cultured on solid medium overnight and then suspended at an optical density of 0.5 (at 520 nm), and the immature embryos are incubated in the Agrobacterium suspension for 5 minutes before the liquid is removed and they are placed on solid medium 710I at 21 °C overnight.

[0125] After 24 hours, the embryos are removed to 605T medium to begin selection against Agrobacterium. After 6 days, several small somatic embryos are Petition 870250086132, dated 09 / 23 / 2025, pages 163 / 173 70 / 71 observed on the surface of each of the 124 immature embryos treated. Each immature embryo contained diverse, distinct, and individual somatic embryos, several of which were supported by clearly defined suspensors.

[0126] Seven days after agroinfection, the embryos are transferred to the maturation medium (medium 289Q + 0.1 mg / l imazapyr), using the herbicide imidazolinone to select transgenic embryos. After 14 days in the maturation medium, the mature embryos are moved to rooting medium (medium 13158H; medium 13158 plus 25 mg / l cefotaxime) and leaf pieces are sampled for PCR analysis. Herbicide-resistant plants are subjected to PCR and sent to the incubator between 32 and 34 days after the start of the experiment (when Agrobacterium transformation begins). Plants are sampled by PCR by collecting two samples from each plant, one from each of two opposite ears (from opposite sides of the plant) to check the possibility of any of the plants being only partially transformed (chimeric).PCR results for each pair of samples from all plants are consistent with the others, indicating that no chimeric plants are produced and that the T0 plants are homogeneously transgenic. Expression of... is expected. BBM404:CBF1A plus Wus2 results in the efficient production of somatic embryos after the transformation of immature embryos into W22, resulting in the rapid production of a large number of T0 transgenic plants.

[0127] B. The use of BBM404:CBF1A alone improves the transformation of immature Pioneer Stiff-Stalk Inbred PHP38 embryos.

[0128] In previous studies, Agrobacterium-mediated transformation in immature embryos using Nos::Wus2 plus Ubi::Bbm in PHP38 of Pioneer Stiff-Stalk inbred lineage Petition 870250086132, dated 09 / 23 / 2025, pages 164 / 173 71 / 71 resulted in a transformation frequency of over 50%, while the use of Ubi::Bbm alone produced a frequency of % (Lowe et al., 2016, Plant Cell 28:1998-2015). Using immature embryos of this same PHP38 inbred line for transformation using Agrobacterium to provide a T-DNA containing Ubi::BBM404:CBF1A (PHP000002), high transformation frequencies above 50% are expected to be observed.

[0129] Also in Lowe et al (2016, Plant Cell 28:19581998-2015) it was demonstrated that the use of Nos::Wus2 plus Ubi::Bbm could be used to recover transgenic embryogenic calluses from the embryonic axis of mature seeds after Agrobacterium transformation. When BBM404:CBF1A is replaced by Bbm in such a construct (PHP000003), it is expected that the embryonic tissue derived from mature seeds will produce much higher frequencies of transgenic embryogenic calluses and T0 plants. Petition 870250086132, dated 09 / 23 / 2025, pages 165 / 173

Claims

1 / 6 CLAIMS 1. A polynucleotide characterized in that it encodes a recombinant transcription factor, wherein the recombinant transcription factor comprises a nucleic acid binding domain and a transcriptional activation domain, wherein the nucleic acid binding domain comprises a truncated Bbm polypeptide and the nucleic acid binding domain is capable of binding to a gene regulatory sequence; wherein the transcriptional activation domain comprises a transcriptional activator polypeptide, wherein the transcriptional activation domain is capable of activating the transcription of a target gene; wherein the nucleic acid binding domain comprises at least 50 amino acid residues and the transcriptional activation domain comprises at least 20 amino acid residues and wherein the nucleic acid binding domain and the transcriptional activation domain do not naturally occur in the same polypeptide.

2. Polynucleotide, according to claim 1, characterized in that the nucleic acid binding domain comprises a truncated Bbm 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.

3. Polynucleotide, according to claim 1, characterized in that the nucleic acid binding domain does not comprise a Bbm A polypeptide or a Bbm B polypeptide. Petition 870250086132, dated 09 / 23 / 2025, pp. 166 / 173 2 / 6 4. Polynucleotide, according to any one of claims 1 to 3, characterized in that the truncated Bbm polypeptide comprises at least 90% amino acid sequence identity with any one of the 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. Polynucleotide, according to any one of claims 2 and 4, characterized in that the Bbm B polypeptide comprises any of the 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 where a single amino acid substitution, insertion or deletion has been made.

6. Polynucleotide, according to any one of claims 2, 4 and 5, characterized in that the Bbm A polypeptide comprises any one of the 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 where a single amino acid substitution, insertion or deletion has been made.

7. Polynucleotide, according to any one of claims 1 to 6, characterized in that the transcriptional activator polypeptide comprises at least 90% amino acid sequence identity with any one of the SEQ ID NO: 127, 129, 131, 133, 135, 137-158, 160, or 164-166.

8. Polynucleotide, according to any one of claims 1 to 7, characterized in that the transcriptional activator polypeptide comprises a CBF1A polypeptide. Petition 870250086132, dated 09 / 23 / 2025, pp. 167 / 173 3 / 6 9. Polynucleotide, according to any one of claims 1 to 8, characterized in that the transcriptional activator polypeptide comprises at least 90% amino acid sequence identity with SEQ ID NO:

127.

10. Polynucleotide, according to any one of claims 1 to 9, characterized in that the transcriptional activator polypeptide comprises a CBF3I polypeptide.

11. Polynucleotide, according to any one of claims 1 to 10, characterized in that the transcriptional activator polypeptide comprises at least 90% amino acid sequence identity with SEQ ID NO:

129.

12. Polynucleotide, according to any one of claims 1 to 11, characterized in that the transcriptional activation domain comprises multiple transcriptional activator polypeptides.

13. Polynucleotide, according to claim 12, characterized in that the transcriptional activation domain comprises two CBF1A polypeptides, two CBF3I polypeptides, or one CBF1A polypeptide and one CBF3I polypeptide.

14. Polynucleotide, according to any one of claims 1, 2 and 4 to 13, characterized in that the nucleic acid binding domain comprises a Bbm B polypeptide, a Bbm A polypeptide and a truncated Bbm polypeptide and in that the transcriptional activator polypeptide comprises a CBF1A polypeptide. Petition 870250086132, dated 09 / 23 / 2025, pp. 168 / 173 4 / 6 15. Polynucleotide, according to claim 14, characterized in that the polypeptide Bbm B comprises SEQ ID NO: 17, the polypeptide Bbm A comprises SEQ ID NO: 19 and the truncated polypeptide Bbm comprises at least 90% amino acid sequence identity with SEQ ID NO: 21 and in that the polypeptide CBF1A comprises at least 90% amino acid sequence identity with SEQ ID NO:

127.

16. Polynucleotide, according to any one of claims 1, 3, 4 and 7 to 13, characterized in that the nucleic acid binding domain comprises a truncated Bbm polypeptide and in that the transcriptional activator polypeptide comprises a CBF1A polypeptide.

17. Polynucleotide, according to claim 16, characterized in that the truncated Bbm polypeptide comprises at least 90% amino acid sequence identity with SEQ ID NO: 21 and in that the CBF1A polypeptide comprises at least 90% amino acid sequence identity with SEQ ID NO:

127.

18. Recombinant transcription factor characterized in that it is encoded by the polynucleotide as defined in any one of claims 1 to 17.

19. Method for producing a recombinant monocotyledonous plant, wherein the method is characterized in that it comprises: contacting a monocotyledonous plant cell with a first polynucleotide encoding a gene of interest, wherein the gene of interest is heterologous to the monocotyledonous plant cell; contacting the monocotyledonous plant cell with a second polynucleotide encoding the recombinant transcription factor, as defined in claim 18; selecting a monocotyledonous plant cell that has incorporated the gene of interest into its genome and regenerating a recombinant monocotyledonous plant from the selected monocotyledonous plant cell.

20. Method according to claim 19, characterized in that it further comprises contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional Wuschel polypeptide or Wuschel homeobox (WUS / WOX).

21. Method according to claim 19, wherein the method is characterized in that it does not comprise contacting the monocotyledonous plant cell with a third polynucleotide encoding a functional WUS / WOX polypeptide.

22. Method, according to any one of claims 19 to 21, characterized in that the monocotyledonous plant cell comprises a Poaceae plant cell.

23. Method, according to any one of claims 19 to 22, characterized in that the monocotyledonous plant cell comprises a plant cell of any one of the following species: Zea mays, Oryza sativa, Triticum aestivum, Setaria italica, Hordeum vulgare, Cenchrus americanus, Saccharum officinarum, or Sorghum bicolor. Petition 870250086132, dated 09 / 23 / 2025, pp. 170 / 173 6 / 6 24. A method according to any one of claims 19 to 23, characterized in that the contact steps comprise bacterial-mediated transformation or particle bombardment.

25. A method according to any one of claims 19 to 24, characterized in that the first polynucleotide is present in a first vector and the second polynucleotide is present in a second vector.

26. Method according to claim 25, characterized in that the second vector additionally comprises the third polynucleotide.

27. A method according to any one of claims 19 to 26, characterized in that the monocotyledonous plant cell is either an immature embryonic cell or a leaf cell.

28. A method according to any one of claims 19 to 27, characterized in that the gene of interest comprises a characteristic gene, a site-specific endonuclease, a guide polynucleotide, or a combination thereof.

29. Method, according to any one of claims 19 to 28, characterized in that it further comprises the excision of one or both of the second and third polynucleotides from the genome of the selected monocotyledonous plant cell. Petition 870250086132, dated 23 / 09 / 2025, pp. 171 / 173