Transformation of plant explants

Abstract

Methods for transforming vegetative plant organs and their complex tissues are provided.

Description

[Technical Field]

[0001] The present disclosure relates to the field of plant molecular biology, and more particularly to the transformation of vegetative plant organs and their complex tissues in dicotyledonous plants.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 824,746, filed March 27, 2019, the entire contents of which are incorporated herein by reference.

[0003] Reference to an electronically submitted sequence listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII-formatted Sequence Listing having a size of 1,151,646 bytes with the file name 8045-WO-PCT_ST25.txt, created on March 23, 2020, and is being submitted contemporaneously herewith. The Sequence Listing contained in this ASCII-formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] In recent years, the capabilities of plant genetic engineering have expanded significantly. Current transformation technologies offer opportunities for producing commercially viable transgenic plants and enable the creation of new plant varieties containing desirable traits. One limitation of plant genetic engineering is the availability of plant tissue explants suitable for transformation, as many plant tissue explants are difficult to transform and regenerate. Therefore, there is a need for plant transformation methods that allow for a wider range of transformable and regenerative plant explant tissues. Summary of the Invention

[0005] The present disclosure includes methods and compositions for producing transgenic plants containing heterologous polynucleotides, as well as methods and compositions for producing gene-edited plants. In a further aspect, the present disclosure provides seeds of the plants produced by the methods disclosed herein.

[0006] The present disclosure provides a method for producing a transgenic dicotyledonous plant containing a heterologous polynucleotide, comprising contacting a growing plant organ of a dicotyledonous plant or a composite tissue thereof with a T-DNA containing a heterologous polynucleotide and a morphogenetic gene expression cassette; selecting plant cells containing the heterologous polynucleotide but not the morphogenetic gene expression cassette, wherein the plant cells form regenerable plant structures containing the heterologous polynucleotide but not the morphogenetic gene expression cassette; and regenerating a transgenic plant from the regenerable plant structures containing the heterologous polynucleotide but not the morphogenetic gene expression cassette. In a further aspect, the morphogenetic gene expression cassette comprises (i) a nucleotide sequence encoding a functional WUS / WOX polypeptide; or (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide; or (iii) a combination of (i) and (ii). In a further aspect, the nucleotide sequence encodes a functional WUS / WOX polypeptide. In a further aspect, the nucleotide sequence encoding a functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, and WOX9. In a further aspect, the nucleotide sequence encodes a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In a further aspect, the nucleotide sequence encoding a Babyboom (BBM) polypeptide is selected from BBM2, BMN2, and BMN3, or the nucleotide sequence encoding an ovule development protein 2 (ODP2) polypeptide is ODP2. In a further aspect, the nucleotide sequence encodes a functional WUS / WOX polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide.In a further aspect, the nucleotide sequence encoding a functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, the nucleotide sequence encoding a Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the nucleotide sequence encoding an ovule development protein 2 (ODP2) polypeptide is ODP2. In a further aspect, the heterologous polynucleotide is selected from the group consisting of a heterologous polynucleotide that confers nutritional enhancement, a heterologous polynucleotide that confers modified oil content, a heterologous polynucleotide that confers modified protein content, a heterologous polynucleotide that confers modified metabolite content, a heterologous polynucleotide that confers increased yield, a heterologous polynucleotide that confers abiotic stress tolerance, a heterologous polynucleotide that confers drought tolerance, a heterologous polynucleotide that confers cold tolerance, a heterologous polynucleotide that confers herbicide tolerance, a heterologous polynucleotide that confers pest resistance, a heterologous polynucleotide that confers pathogen resistance, a heterologous polynucleotide that confers insect resistance, a heterologous polynucleotide that confers nitrogen utilization efficiency (NUE), a heterologous polynucleotide that confers disease resistance, a heterologous polynucleotide that confers increased biomass, a heterologous polynucleotide that confers the ability to alter metabolic pathways, and combinations thereof. In a further aspect, the dicotyledonous growing plant organ or composite tissue thereof is selected from the group consisting of leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral meristems), and combinations thereof. In a further aspect, the leaf explant is selected from the group consisting of leaves, basal leaves, cauline leaves, scattered and opposite leaves, cross-opposite leaves, opposite superposed leaves, whorl leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, compound leaves, and combinations thereof. In a further aspect, the stem explant is selected from the group consisting of a stem node region, a stem internode region, a petiole, a hypocotyl, an epicotyl, a stolon, a rhizome, a tuber, a corm, and combinations thereof.In a further aspect, the dicotyledonous plant is selected from the group consisting of soybean, cotton, sunflower, cassava, kidney bean, cowpea, tomato, potato, beet, grapevine, Eucalyptus, citrus, papaya, cacao, cucumber, apple, Capsicum, melon, and Brassica. In a further aspect, the morphogenetic gene expression cassette comprises a polynucleotide encoding a functional WUS / WOX polypeptide, wherein the functional WUS / WOX polypeptide is selected from the group consisting of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 128, 130, 132, 134, 136, 138, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 , 144, 146, or 148, or encoded by the nucleotide sequence of any of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, or 147. In a further aspect, the morphogenetic gene expression cassette further comprises a polynucleotide sequence encoding a site-specific recombinase selected from the group consisting of FLP, FLPe, KD, Cre, SSV1, Lambda Int, PhiC31 Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153, wherein the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally regulated promoter. In a further aspect, the method further comprises excising the morphogenetic gene expression cassette. In a further aspect, a transgenic plant produced by the method is provided. In a further aspect, a seed of the transgenic plant produced by the method is provided, the seed comprising the heterologous polynucleotide.In further aspects, regenerable plant structures are formed at a frequency that is increased by about 0.1% to about 1.0%, about 1.1% to about 10%, about 10.1% to about 20%, about 20.1% to about 30%, about 30.1% to about 40%, about 40.1% to about 50%, about 50.1% to about 60%, about 60.1% to about 70%, about 70.1% to about 80%, about 80.1% to about 90%, and about 90.1% to about 100% compared to the frequency of formation of regenerable plant structures formed when a growing plant organ of a dicotyledonous plant or a composite tissue thereof is not contacted with a morphogenetic gene expression cassette.

[0007] The present disclosure provides a method for producing a genome-edited dicotyledonous plant, comprising contacting a growing plant organ of a dicotyledonous plant or a composite tissue thereof with a T-DNA containing a morphogenetic gene expression cassette to provide a polynucleotide encoding a site-specific polypeptide or the site-specific polypeptide; selecting plant cells containing the genome edits and not containing the morphogenetic gene expression cassette (wherein the plant cells contain the genome edits and form a regenerable plant structure that does not contain the morphogenetic gene expression cassette); and regenerating a genome-edited plant from the regenerable plant structure that contains the genome edits and does not contain the morphogenetic gene expression cassette. In a further aspect, the morphogenetic gene expression cassette comprises (i) a nucleotide sequence encoding a functional WUS / WOX polypeptide; or (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide; or (iii) a combination of (i) and (ii). In a further aspect, the nucleotide sequence encodes a functional WUS / WOX polypeptide. In a further aspect, the nucleotide sequence encoding a functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, and WOX9. In a further aspect, the nucleotide sequence encodes a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In a further aspect, the nucleotide sequence encoding a Babyboom (BBM) polypeptide is selected from BBM2, BMN2, and BMN3, or the nucleotide sequence encoding an ovule development protein 2 (ODP2) polypeptide is ODP2. In a further aspect, the nucleotide sequence encodes a functional WUS / WOX polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide.In a further aspect, the nucleotide sequence encoding the functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, and WOX9; the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2, and BMN3; or the nucleotide sequence encoding the ovule development protein 2 (ODP2) polypeptide is ODP2. In a further aspect, the site-specific polypeptide is selected from the group consisting of a zinc finger nuclease, a meganuclease, a TALEN, and a CRISPR-Cas nuclease. In a further aspect, the CRISPR-Cas nuclease is Cas9 or Cpfl nuclease, and the method further comprises providing a guide RNA. In a further aspect, the site-specific nuclease effects an insertion, deletion, or substitution mutation. In a further aspect, the guide RNA and the CRISPR-Cas nuclease are a ribonucleoprotein complex. In a further aspect, the dicotyledonous growing plant organ or composite tissue thereof is selected from the group consisting of leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral meristems), and combinations thereof. In a further aspect, the leaf explant is selected from the group consisting of leaves, basal leaves, cauline leaves, scattered and opposite leaves, cross-opposite leaves, opposite superposed leaves, whorl leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, compound leaves, and combinations thereof. In a further aspect, the stem explant is selected from the group consisting of a stem node region, a stem internode region, a petiole, a hypocotyl, an epicotyl, a stolon, a rhizome, a tuber, a corm, and combinations thereof. In a further aspect, the dicotyledonous plant is selected from the group consisting of soybean, cotton, sunflower, cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus, citrus, papaya, cacao, cucumber, apple, Capsicum, melon, and Brassica.In a further aspect, the morphogenetic gene expression cassette comprises a polynucleotide encoding a functional WUS / WOX polypeptide, wherein the functional WUS / WOX polypeptide is selected from the group consisting of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 128, 130, 132, 134, 136, 138, 140, 142, and the like. , 144, 146, or 148, or encoded by the nucleotide sequence of any of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, or 147. In a further aspect, the morphogenetic gene expression cassette further comprises a polynucleotide sequence encoding a site-specific recombinase selected from the group consisting of FLP, FLPe, KD, Cre, SSV1, Lambda Int, PhiC31 Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153, wherein the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally regulated promoter. In a further aspect, the method further comprises excising the morphogenetic gene expression cassette. In a further aspect, a genome-edited plant produced by the method is provided. In a further aspect, a seed of the genome-edited plant produced by the method is provided, the seed comprising the genome edits. In further aspects, regenerable plant structures are formed at a frequency that is increased by about 0.1% to about 1.0%, about 1.1% to about 10%, about 10.1% to about 20%, about 20.1% to about 30%, about 30.1% to about 40%, about 40.1% to about 50%, about 50.1% to about 60%, about 60.1% to about 70%, about 70.1% to about 80%, about 80.1% to about 90%, and about 90.1% to about 100% compared to the frequency of formation of genome-edited regenerable plant structures when a dicotyledonous growing plant organ or a composite tissue thereof is not contacted with a morphogenetic gene expression cassette. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a graphical representation of the percentage (%) of tobacco leaf segments with de novo shoots after infection with Agrobacterium carrying different WUS genes, as described in Example 14. DETAILED DESCRIPTION OF THE INVENTION

[0009] The disclosure herein is described more fully below with reference to the accompanying drawings, which illustrate some, but not all, possible embodiments. Indeed, the disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0010] Numerous modifications and other embodiments of the present disclosure will occur to those skilled in the art to which the disclosed methods pertain and having access to the teachings presented in the following description and the accompanying drawings. It is to be understood, therefore, that the disclosure is not to be limited to the particular embodiments disclosed herein, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0011] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and claims, the term "comprising" may include embodiments "consisting of." Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. In the following specification and claims, reference will be made to a number of terms defined herein.

[0012] As used herein, a "regenerable plant structure" is a multicellular structure capable of forming a fully functional, fertile plant. Regenerable plant structures capable of forming a fully functional, fertile plant include, but are not limited to, shoot meristems, shoots, somatic embryos, embryogenic callus, somatic meristems, and / or organogenic callus.

[0013] As used herein, a "somatic embryo" is a multicellular structure that grows through developmental stages similar to those of a zygotic embryo, including the formation of a globular transitional embryo, the formation of an embryonic axis and scutellum, and the accumulation of lipids and starch. The single somatic embryo that arises from a zygotic embryo germinates to produce a non-chimeric plant that may originally arise from a single cell.

[0014] As used herein, "embryogenic callus" is a friable or non-friable mixture of undifferentiated or partially undifferentiated cells containing proliferative primary and secondary somatic embryos that can be regenerated into mature fertile plants.

[0015] As used herein, a "somatic meristem" is a multicellular structure similar to the apical meristem that is part of a seed-derived embryo, described as having an undifferentiated apical dome flanked by leaf primordia and surrounded by vascular initial cells, that gives rise to the aboveground growing plant. Such somatic meristems can form a single meristem or a collection of fused meristems.

[0016] As used herein, "organogenic callus" is a compact mixture of differentiated growing plant structures including, but not limited to, the apical meristem, root meristem, leaves, and roots.

[0017] As used herein, "germination" is the development of a regenerable structure to form a plantlet that continues to grow and produce a plant.

[0018] As used herein, a "transgenic plant" is a mature, fertile plant that contains a transgene.

[0019] The methods of the present disclosure can be used to transform vegetative plant organs and their complex tissues. As used herein, "vegetative plant organs and their complex tissues" includes, but is not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral meristems). As used herein, "stem explants" includes, but is not limited to, stem nodal and internodal regions, petioles, hypocotyls, epicotyls, stolons, rhizomes, tubers, and corms. As used herein, "leaf explant" includes, but is not limited to, basal leaves, cauline leaves, scattered leaves, opposite leaves, cross-opposite leaves, opposite superposed leaves, whorl leaves, stalk leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, or compound leaves. Leaf explants include the leaf base or the portion of the leaf immediately proximal to the point of attachment to the petiole or stem. Such growth organs and their composite tissues can be used for transformation with nucleotide sequences encoding agronomically important traits.

[0020] As used herein, a "leaf" is a flattened lateral structure that projects from a plant stem, including the supporting stem between the flattened leaf and the plant stem, but not including the axillary meristem located at the junction of the petiole and the stem, and includes, but is not limited to, basal leaves, cauline leaves, scattered and opposite leaves, cross-opposite leaves, opposite superposed leaves, whorl leaves, stalk leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, or compound leaves.

[0021] As used herein, a "stem internode" is the tissue located between the stem internodes of a plant, and a "stem node" is the region of the stem from which a branch, petiole, leaf, or aerial root grows from the stem.

[0022] As used herein, the term "morphogenetic gene" refers to a gene that, when ectopically expressed, stimulates the formation of structures derived from somatic cells that can give rise to plants. More precisely, ectopic expression of a morphogenetic gene stimulates the de novo formation of organogenic structures, such as somatic embryos or shoot meristems, that can give rise to plants. This stimulated de novo formation occurs in the cell in which the morphogenetic gene is expressed or in neighboring cells. Morphogenic genes can be transcription factors that regulate the expression of other genes or genes that affect hormone concentrations in plant tissues, both of which can promote morphogenetic changes. Morphogenic genes can be stably integrated into the plant genome or expressed transiently. In one aspect, the expression of a morphogenetic gene is controlled. Control of expression can be intermittent expression of the morphogenetic gene for a specific period of time. Alternatively, the morphogenetic gene can be expressed only in some transformed cells and not in other cells. Control of morphogenetic gene expression can be achieved by various methods disclosed herein below. Morphogenic genes useful in the methods of the present disclosure can be obtained or derived from any of the plant species described herein.

[0023] As used herein, the term "morphogen" refers to a morphogenic gene and / or a protein expressed by a morphogenic gene.

[0024] Morphogenic genes, such as WUS / WOX genes (WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, or WOX9), are involved in plant metabolism, organogenesis, stem cell development, cell proliferation stimulation, organogenesis, regeneration, initiation of somatic embryogenesis, promotion of somatic embryo maturation, apical meristem development and / or development, shoot meristem development and / or development, shoot development and / or development, or a combination thereof. See U.S. Patent Nos. 7,348,468 and 7,256,322, U.S. Patent Application Publication Nos. 2017 / 0121722 and 2007 / 0271628; Laux et al. (1996) Development 122:87-96; and Mayer et al. (1998) Cell 95:805-815; van der Graaff et al., 2009, Genome Biology 10:248; Dolzblasz et al., 2016, Mol. Plant 19:1028-39, which are useful in the methods of the present disclosure. Regulation of WUS / WOX is believed to regulate plant and / or plant tissue phenotype, including plant metabolism, organogenesis, stem cell development, stimulation of cell proliferation, organogenesis, regeneration, initiation of somatic embryogenesis, promotion of somatic embryo maturation, apical meristem development and / or development, shoot meristem development and / or development, shoot development and / or development, or combinations thereof. Expression of Arabidopsis WUS can induce stem cells in vegetative tissues, which can differentiate into somatic embryos (Zuo, et al. (2002) Plant J 30:349-359). Also of interest in this regard are the MYB118 gene (see U.S. Pat. No. 7,148,402), the MYB115 gene (see Wang et al. (2008) Cell Research 224-235), the BABYBOOM gene (BBM; see Boutilier et al. (2002) Plant Cell 14:1737-1749), or the CLAVATA gene (see, e.g., U.S. Pat. No. 7,179,963).

[0025] The morphogenic polynucleotide and amino acid sequences of functional WUS / WOX polypeptides are useful in the methods of the present disclosure. As defined herein, a "functional WUS / WOX nucleotide" is a polynucleotide encoding a protein containing a homeobox DNA-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 www.pnas.org / cgi / doi / 10.1073 / pnas.1607673113, removal of the dimerization sequence, leaving the homeobox DNA-binding domain, the WUS box, and the EAR repressor domain, results in a functional WUS / WOX polypeptide. The Wuschel protein (hereafter referred to as WUS) plays an important role in the development and maintenance of the apical meristem, which contains the pluripotent stem cell pool (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 unspecified stem cells that appear to differentiate. WUS encodes a novel homeodomain protein that is predicted to function as a transcriptional regulator (Mayer, et al., (1998) Cell 95:805-815). The stem cell population in the Arabidopsis shoot meristem is thought to be maintained by a regulatory loop between the CLAVATA (CLV) gene, which promotes organogenesis, and the WUS gene, which is required for stem cell identity (Brand, et al., (2000) Science 289:617-619; Schoof, et al., (2000) Cell 100:635-644). The CLV gene transcriptionally represses WUS, allowing WUS to be expressed sufficiently to induce meristem cell identity and the expression of the stem cell marker CLV3.Constitutive expression of WUS in Arabidopsis has been shown to lead to adventitious shoot outgrowth (in planta) from leaves (Laux, T., presented at the XVI International Botanical Congress Meeting, Aug. 1-7, 1999, St. Louis, Mo.).

[0026] In one embodiment, a functional WUS / WOX polypeptide useful in the methods of the present disclosure is a WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX5A, or WOX9 polypeptide (see U.S. Pat. Nos. 7,348,468 and 7,256,322, and U.S. Patent Application Publication Nos. 2017 / 0121722 and 2007 / 0271628, and van der Graaff et al., 2009, Genome Biology 10:248, which are incorporated by reference in their entireties). Functional WUS / WOX polypeptides useful in the methods of the present disclosure can be obtained or derived from any plant, including, but not limited to, monocotyledons, dicotyledons, angiosperms, and gymnosperms. Additional WUS / WOX genes useful in the methods of the present disclosure are listed in Table 3.

[0027] Other morphogenetic genes useful in the present disclosure include LEC1 (U.S. Pat. No. 6,825,397, herein incorporated by reference in its entirety; Lotan et al., 1998, Cell 93:1195-1205), LEC2 (Stone et al., 2008, PNAS 105:3151-3156; Belide et al., 2013, Plant Cell Tiss. Organ Cult 113:543-553), KN1 / STM (Sinha et al., 1993, Genes Dev 7:787-795), the IPT genes from Agrobacterium (Ebinuma and Komamine, 2001, In vitro Cell. Dev Biol-Plant 37:103-113), MONOPTEROS-DELTA (Ckurshumova et al., 2014, New Phytol. 204:556-566), Agrobacterium AV-6b gene (Wabiko and Minemura 1996, Plant Physiol. 112:939-951), a combination of Agrobacterium IAA-h and IAA-m genes (Endo et al., 2002, Plant Cell Rep., 20:923-928), Arabidopsis SERK gene (Hecht et al., 2001, Plant Physiol. 127:803-816), Arabidopsis AGL15 gene (Harding et al., 2003, Plant Physiol. 133:653-663), FUSCA gene (Castle and Meinke, Plant Cell 6:25-41), and the PICKLE gene (Ogas et al., 1999, PNAS 96:13839-13844).

[0028] As used herein, the term "transcription factor" refers to a protein that controls the rate of transcription of a specific gene by binding to a promoter DNA sequence and up-regulating or down-regulating expression. Examples of transcription factors that are also morphogenetic genes include members of the AP2 / EREBP family (e.g., BBM (ODP2)), the plethora and aintegra subfamilies, CAAT box-binding proteins such as LEC1 and HAP3, and members of the MYB, bHLH, NAC, MADS, bZIP, and WRKY families.

[0029] Ovule development protein 2 (ODP2) polypeptides and related polypeptides, such as morphogenetic polynucleotide and amino acid sequences of Babyboom (BBM) protein family proteins, are useful in the methods of the present disclosure. In one embodiment, the polypeptide containing two AP2-DNA-binding domains is an ODP2, BBM2, BMN2, or BMN3 polypeptide; see U.S. Patent Application Publication No. 2017 / 0121722, incorporated herein by reference in its entirety. The ODP2 polypeptide useful in the methods of the present disclosure contains two predicted APETALA2 (AP2) domains and is a member of the AP2 protein family (PFAM Accession PF00847). The AP2 family of putative transcription factors is known to regulate a wide range of developmental processes, and family members are characterized by the presence of an AP2 DNA-binding domain, a conserved core predicted to form an amphipathic alpha-helix that binds to DNA. AP2 domains were first identified in APETALA2, an Arabidopsis protein that regulates meristem determination, floral organ specification, seed coat development, and flower bud homeotic gene expression. AP2 domains have now been found in a variety of proteins.

[0030] ODP2 polypeptides useful in the methods of the present disclosure share homology with several polypeptides of the AP2 family (see, e.g., Figure 1 of U.S. Pat. No. 8,420,893, which is incorporated herein by reference in its entirety, showing an alignment of maize and rice ODP2 polypeptides with eight other proteins having two AP2 domains). The consensus sequence of all proteins shown in the alignment of U.S. Pat. No. 8,420,893 is also shown in Figure 1 thereof. Polypeptides comprising two AP2-DNA binding domains useful in the methods of the present disclosure can be obtained or derived from any of the plants described herein. In one aspect, a polypeptide comprising two AP2-DNA binding domains useful in the methods of the present disclosure is an ODP2 polypeptide. In one aspect, a polypeptide comprising two AP2-DNA binding domains useful in the methods of the present disclosure is a BBM2 polypeptide. The ODP2 and BBM2 polypeptides useful in the methods of the present disclosure can be obtained or derived from any plant, including, but not limited to, monocotyledons, dicotyledons, angiosperms, and gymnosperms.

[0031] As used herein, the term "expression cassette" refers to a discrete component of vector DNA consisting of coding and non-coding sequences, including 5' and 3' regulatory sequences that control expression in transformed / transfected cells.

[0032] As used herein, the term "coding sequence" means the portion of a DNA sequence bounded by a start codon and a stop codon that encodes the amino acids of a protein.

[0033] As used herein, the term "non-coding sequence" refers to a portion of a DNA sequence that is transcribed to produce messenger RNA but does not code for the amino acids of a protein, such as the 5' untranslated region, introns, and 3' untranslated region. A non-coding sequence may also refer to an RNA molecule, such as a microRNA, an interfering RNA, or an RNA hairpin, that, when expressed, can down-regulate the expression of an endogenous gene or another introduced gene.

[0034] As used herein, the term "regulatory sequence" refers to a segment of a nucleic acid molecule that can increase or decrease the expression of a gene. Regulatory sequences include promoters, terminators, enhancer elements, silencing elements, 5'UTRs, and 3'UTRs (untranslated regions).

[0035] As used herein, the term "transfer cassette" means a T-DNA comprising one or more expression cassettes flanked by right and left border sequences.

[0036] As used herein, T-DNA refers to the portion of the Ti plasmid that is inserted into the genome of the host plant cell.

[0037] As used herein, the term "selectable marker" refers to a transgene that, when expressed in transformed / transfected cells, confers resistance to selection agents such as antibiotics, herbicides and other compounds that are toxic to non-transformed / transfected cells.

[0038] As used herein, the term "EAR" refers to an "ethylene response element binding factor-associated amphipathic repression motif" that has a common consensus sequence that acts as a transcriptional repression signal in transcription factors. Addition of an EAR-type repressor element to a DNA-binding protein, such as a transcription factor, dCAS9, or LEXA (for example), confers transcriptional repression function to the fusion protein (Kagale, S., and Rozwadowski, K. 2010. Plant Signaling and Behavior 5:691-694).

[0039] In one aspect, the transformation method of the present disclosure uses a recombinant expression cassette or construct comprising a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide.

[0040] In one aspect, a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, can be targeted for excision by a site-specific recombinase. Thus, expression of a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, can be controlled by excision at a desired time after transformation. When a site-specific recombinase is used to control the expression of a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, it is understood that the expression construct will contain appropriate site-specific excision sites adjacent to the polynucleotide sequence to be excised, for example, Cre lox sites if Cre recombinase is used.The site-specific recombinase need not be located in an expression construct comprising a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide. However, in one aspect, the morphogenetic gene expression cassette further comprises a nucleotide sequence encoding a site-specific recombinase.

[0041] The site-specific recombinase used to control the expression of a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, can be selected from a variety of suitable site-specific recombinases. For example, in various embodiments, the site-specific recombinase is FLP, FLPe, KD, Cre, SSV1, Lambda Int, PhiC31 Int, HK022, R, B2 (Nern et al., (2011) PNAS Vol. 108, No. 34 pp. 14198-14203), B3 (Nern et al., (2011) PNAS Vol. 108, No. 34 pp. 14198-14203), Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. The site-specific recombinase can be a destabilized fusion polypeptide. The destabilized fusion polypeptide can be TETR(G17A)~CRE or ESR(G17A)~CRE.

[0042] In one embodiment, the nucleotide sequence encoding the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally regulated promoter. Suitable constitutive, inducible, tissue-specific, and developmentally regulated promoters include UBI, LLDAV, EVCV, DMMV, BSV(AY)PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO(1.2KB), IN2-2, NOS, -135 version of 35S, ZM-ADF PRO(ALT2), AXIG1, DR5, XVE, GLB1, OLE, LTP2 (Kalla et al., 1994, Plant Cell Pathology, 1999, incorporated herein by reference in its entirety). J.6:849-860 and U.S. Pat. No. 5,525,716), HSP17.7, HSP26, HSP18A, AT-HSP811, AT-HSP811L, GM-HSP173B, promoters activated by tetracycline, ethametsulfuron, or chlorsulfuron, PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34 (U.S. Patent Application Publication Nos. 2017 / 0121722 and 2018 / 0371480, which are incorporated by reference in their entireties).

[0043] In one embodiment, the chemically inducible promoter operably linked to the site-specific recombinase is XVE. The chemically inducible promoter can be repressed by the tetracycline repressor (TETR), ethametsulfuron repressor (ESR), or chlorsulfuron repressor (CR), and derepression occurs by the addition of a tetracycline-related ligand or a sulfonylurea ligand. The repressor can be TETR, and the tetracycline-related ligand can be doxycycline or anhydrotetracycline. (Gatz, C., Frohberg, C., and Wendenburg, R. (1992) Stringent repression and homogeneous derepression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants, Plant J. 2, 397-404). Alternatively, the repressor can be ESR and the sulfonylurea ligand is ethametsulfuron, chlorsulfuron, metsulfuron methyl, sulfometuron methyl, chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron, sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron, prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron (U.S. Patent Application Publication No. 2011 / 0287936, incorporated herein by reference in its entirety).

[0044] In one aspect, when the morphogenetic gene expression cassette or construct contains a site-specific recombinase excision site, the nucleotide sequence encoding a functional WUS / WOX polypeptide, or the nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, can be operably linked to an auxin-inducible promoter, a developmentally-regulated promoter, a tissue-specific promoter, or a constitutive promoter.Exemplary auxin-inducible, developmentally-regulated, tissue-specific, and constitutive promoters useful in this regard include UBI, LLDAV, EVCV, DMMV, BSV(AY)PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO(1.2KB), IN2-2, NOS, -135 version of 35S, ZM-ADF PRO(ALT2), AXIG1 (U.S. Pat. No. 6,838,593, which is incorporated herein by reference in its entirety), DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, AT-HSP811 (Takahashi, T, et al., (1992) Plant Physiol. 99(2):383-390), AT-HSP811L (Takahashi, T, et al., (1992) Plant Physiol. 99(2):383-390), GM-HSP173B (Schoeffl, F., et al. (1984) EMBO J. 3(11):2491-2497), promoters activated by tetracycline, ethamethsulfuron, or chlorsulfuron, PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, LEA-D34 (U.S. Patent Application Publication Nos. 2017 / 0121722 and 2018 / 0371480, which are incorporated by reference in their entireties), and any of the promoters disclosed herein.

[0045] When generating transgenic plants using a morphogenetic gene cassette and a trait gene cassette (heterologous polynucleotide), it is desirable to have the ability to separate the morphogenetic gene locus from the trait gene (heterologous polynucleotide) locus in the co-transformed plant to provide transgenic plants containing only the trait gene (heterologous polynucleotide). This can be achieved using the Agrobacterium tumefaciens two-T-DNA binary system, and there are two variations on this general theme (see Miller et al., 2002). For example, in the first two-T-DNA vector, expression cassettes for the morphogenetic genes and herbicide selection (i.e., HRA) are contained within the first T-DNA, and the trait gene cassette (heterologous polynucleotide) is contained within the second T-DNA. Here, both T-DNAs are present on a single binary vector. When plant cells are transformed with Agrobacterium containing a plasmid carrying two T-DNAs, a high percentage of transformed cells contain both T-DNAs integrated at different genomic locations (e.g., on different chromosomes). In the second method, for example, two Agrobacterium strains, each containing one of the two T-DNAs (morphogenetic gene T-DNA or trait gene (heterologous polynucleotide) T-DNA), are mixed in a certain ratio and the mixture is used for transformation. After transformation using this mixed Agrobacterium method, it has been observed that the recovered transgenic events frequently contain both T-DNAs (often at separate genomic locations). In most transgenic events generated, both co-transformation methods have been observed to result in independent segregation of the two T-DNA loci, easy identification of progeny T1 plants with segregating T-DNA loci, and recovery of progeny seeds containing the trait gene (heterologous polynucleotide) but lacking the morphogenetic / herbicide gene. See Miller et al. Transgenic Res 11(4):381-96.

[0046] The methods provided herein rely on the use of bacterial-mediated and / or biolistic gene transfer to generate regenerable plant cells incorporating a nucleotide sequence of interest. Strains useful in the methods of the present disclosure include, but are not limited to, disarmed Agrobacteria, Ochrobactrum, or Rhizobiaceae. Disarmed Agrobacteria useful in the methods include, but are not limited to, AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-. Ochrobactrum strains useful in the methods include, but are not limited to, those disclosed in U.S. Patent Application Publication No. 2018 / 0216123, the entire contents of which are incorporated herein by reference. Strains of the family Rhizobiaceae useful in the present methods include, but are not limited to, those disclosed in U.S. Pat. No. 9,365,859, which is incorporated herein by reference in its entirety.

[0047] Also embodied are plants having an expression cassette described herein stably integrated into the plant's genome, and seeds of such plants, the seeds containing such expression cassettes. Furthermore, embodied are plants in which a gene or gene product of a heterologous polynucleotide or polynucleotide of interest confers nutritional enhancement, increased yield, abiotic stress tolerance, drought tolerance, cold tolerance, herbicide tolerance, pest resistance, pathogen resistance, insect resistance, nitrogen use efficiency (NUE), disease resistance, or the ability to alter a metabolic pathway. Also embodied are plants in which expression of a heterologous polynucleotide or polynucleotide of interest alters the plant's phenotype.

[0048] The present disclosure encompasses isolated or substantially purified nucleic acid compositions. An "isolated" or "purified" nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or is substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" nucleic acid is substantially free of sequences (e.g., protein-coding sequences) that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). For example, in various embodiments, an isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0049] As used herein, the term "fragment" refers to a portion of a nucleic acid sequence. Fragments of sequences useful in the disclosed methods retain the biological activity of the nucleic acid sequence. Alternatively, fragments of nucleotide sequences useful as hybridization probes may not necessarily retain biological activity. Fragments of the nucleotide sequences disclosed herein may be at least about 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1750, 1725, 1750, 1775, 1800, 1825, 1850, 1875, or 1900 nucleotides, up to the full length of the subject sequence. Biologically active portions of a nucleotide sequence can be prepared by isolating a portion of the sequence and assessing the activity of the portion.

[0050] Fragments and variants of nucleotide sequences useful in the disclosed methods, as well as the proteins encoded thereby, are also encompassed. As used herein, the term "fragment" refers to a portion of a nucleotide sequence, and thus to the protein encoded thereby, or to a portion of an amino acid sequence. A fragment of a nucleotide sequence may encode a protein fragment that retains the biological activity of the native protein. Alternatively, fragments of a nucleotide sequence useful as hybridization probes generally do not encode fragment proteins that retain biological activity. Thus, fragments of a nucleotide sequence can range from at least about 20 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, to the full-length nucleotide sequence encoding a protein useful in the disclosed methods.

[0051] As used herein, the term "variant" refers to a sequence having substantial similarity to the promoter sequence disclosed herein. Variants include deletions and / or additions of one or more nucleotides at one or more internal sites of the native polynucleotide and / or substitutions of one or more nucleotides at one or more sites of the native polynucleotide. As used herein, a "native" nucleotide sequence includes a naturally occurring nucleotide sequence. In nucleotide sequences, naturally occurring variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined herein.

[0052] Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated using site-directed mutagenesis. Generally, variants of the nucleotide sequences disclosed herein will have at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%-95%, 96%, 97%, 98%, 99%, or more sequence identity to the nucleotide sequence as determined by sequence alignment programs (using default parameters) described elsewhere herein. Biologically active variants of the nucleotide sequences disclosed herein are also encompassed. Biological activity may be measured using techniques such as Northern blot analysis or reporter activity assays using transcriptional fusions. See, e.g., Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) (hereinafter "Sambrook") (incorporated herein by reference in its entirety). Alternatively, the level of a reporter gene, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), produced under the control of a promoter operably linked to the nucleotide fragment or variant can be measured. See, e.g., Matz et al. (1999) Nature Biotechnology 17:969-973; U.S. Patent No. 6,072,050 (incorporated herein by reference in its entirety); Nagai, et al., (2002) Nature Biotechnology 20(1):87-90. Variant nucleotide sequences also encompass sequences generated by mutagenesis procedures, such as DNA shuffling. Such procedures allow the manipulation of one or more different nucleotide sequences to generate new nucleotide sequences. In this manner, a library of recombinant polynucleotides is generated from a population of polynucleotides of related sequences that have substantial sequence identity and contain sequence regions that are capable of homologously recombining in vitro or in vivo.Strategies for such DNA shuffling are known in the art. See, e.g., Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291, and U.S. Pat. Nos. 5,605,793 and 5,837,458, which are incorporated herein by reference in their entireties.

[0053] Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel, et al., (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and the references cited therein, which are incorporated herein by reference in their entireties. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) (incorporated herein by reference). Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be optimal.

[0054] The nucleotide sequences of the present disclosure can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocotyledonous or dicotyledonous plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on sequence homology to the sequences described herein. Sequences isolated based on sequence identity to the entire sequences described herein, or fragments thereof, are encompassed by the present disclosure.

[0055] In PCR, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any target plant. Methods for designing PCR primers and PCR cloning methods are generally known in the art and are disclosed in Sambrook (supra). See also Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York), all of which are incorporated herein by reference in their entireties. Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, etc.

[0056] In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a collection of cloned genomic DNA or cDNA fragments from a selected organism (i.e., a genomic or cDNA library). Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group, such as 32P or any other detectable marker. Thus, for example, hybridization probes can be made by labeling synthetic oligonucleotides based on the disclosed sequences. Methods for preparing hybridization probes and constructing genomic libraries are generally known in the art and are disclosed in Sambrook, supra.

[0057] Generally, sequences that are active and hybridize to the sequences disclosed herein will be at least 40%-50% homologous, and about 60%, 70%, 80%, 85%, 90%, 95%-98% or more homologous to the disclosed sequences. In other words, the sequence similarity of the sequences can range from at least about 40%-50%, about 60%-70%, and about 80%, 85%, 90%, 95%-98% sequence similarity.

[0058] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, the algorithm of Smith, et al., (1981) Adv. Appl. Math. 2:482, the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453, the algorithm of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 872:264 (modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), which are incorporated herein by reference in their entireties. Computerized implementations of these mathematical algorithms are well known in the art and can be used for comparing sequences to determine sequence identity.

[0059] "Sequence identity" or "identity," as used herein in the context of two nucleic acid or polypeptide sequences, refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and therefore the functional properties of the molecule are not altered. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Methods for making such adjustments are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, where identical amino acids are assigned a score of 1 and non-conservative substitutions are assigned a score of 0, conservative substitutions are assigned a score between 0 and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.).

[0060] As used herein, "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) relative to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0061] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity compared to a reference sequence using an alignment program with standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes typically means at least 60%, 70%, 80%, 90%, and at least 95% sequence identity.

[0062] Another indication that nucleotide sequences are substantially identical is whether two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5°C lower than the Tm of the specific sequence at a defined ionic strength and pH. However, stringent conditions also include temperatures ranging from about 1°C to about 20°C lower than the Tm, and will vary depending on the desired degree of stringency, as otherwise defined herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when copies of the nucleic acids are generated using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is if the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.

[0063] The methods, sequences, and genes disclosed herein are useful in the genetic engineering of plants, e.g., in the creation of transformed or transgenic plants, to express a desired phenotype. As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant that contains a heterologous polynucleotide in its genome. Generally, the heterologous polynucleotide is stably integrated into the genome of the transgenic or transformed plant so that the polynucleotide is passed on through generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. As used herein, the term "transgenic" should be understood to include any cell, cell line, callus, tissue, plant part, or plant body whose genotype has been altered by the presence of a heterologous nucleic acid (e.g., the initial transgenic body so altered, and any plant body produced from the initial transgenic body by sexual or asexual propagation).

[0064] Transgenic "events" are produced by transforming plant cells with a heterologous DNA construct, such as a nucleic acid expression cassette, containing a gene of interest, regenerating a population of plants by inserting the transgene into the plant's genome, and selecting plants characterized by the insertion at a specific genomic location. Events are characterized phenotypically by expression of the inserted gene. At the genetic level, an event is part of the plant's genetic makeup. The term "event" also refers to progeny produced by sexual mating between a transformant and another plant, which progeny contain heterologous DNA.

[0065] The term "plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos, and their progeny. Plant cells can be differentiated or undifferentiated (e.g., callus, undifferentiated callus, immature and mature embryos, immature zygotic embryos, immature cotyledons, hypocotyls, suspension culture cells, protoplasts, leaves, leaf cells, root cells, phloem cells, and pollen). Plant cells include, but are not limited to, seeds, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic callus, meristems, somatic meristems, meristem regions, organogenic callus, callus tissue, protoplasts, embryos from mature ear-producing seeds, leaves, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, clusters, immature ears, silks, cotyledons, immature cotyledons, hypocotyls, leaf-derived cells, stem-derived cells, root-derived cells, shoot-derived cells, roots, shoots, gametophytes, sporophytes, pollen, microspores, multicellular structures (MCS), regenerable plant structures (RPS), and cells from embryo-like structures.

[0066] Plant parts include differentiated and undifferentiated tissues, including, but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, and callus tissue). Plant tissues may be whole plants or plant organs, tissues, or cell cultures.

[0067] Grain is intended to mean mature seed produced by a grower for purposes other than cultivating or propagating the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of this disclosure, provided that these progeny, variants, and mutants comprise the introduced polynucleotide.

[0068] The present disclosure also includes plants obtained by any of the methods disclosed herein. The present disclosure also includes seeds from plants obtained by any of the methods disclosed herein.

[0069] The methods of the present disclosure may be used to transform plant species including, but not limited to, alfalfa, soybean, cotton, sunflower, cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus, poplar, pine, Douglas-fir, citrus, papaya, cacao, cucumber, apple, Capsicum, melon, and Brassica. In one aspect, dicotyledonous plants used in the methods of the present disclosure include, but are not limited to, kale, cauliflower, broccoli, mustard, cabbage, pea, clover, alfalfa, faba bean, tomato, peanut, cassava, soybean, canola, sunflower, safflower, tobacco, Arabidopsis, or cotton.

[0070] Higher plants, such as Angiospermae and Gymnospermae, may be used in the present disclosure. Suitable plant species useful in the methods of the present disclosure include those from the Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, and the Brassicaceae. Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae e), Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nysaceae The plant may be from the family Abelmoschus, Abies, Acer, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Abelmoschus spp. ...Artemisia, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythrochy Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Jatropha, Lactuca, Linum, Lupin, Lycopersicon esculentum on), Lycopodium, Manihot, Medicago, Mentha, Musa, Nicotiana, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus,Plants from members of the genera Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Solanum, Spinacea, Tanacetum, Taxus, Theobroma, Uniola, Veratrum, Vinca, and Vitis.

[0071] Plants important or interesting for agriculture, horticulture, biomass production (for the production of liquid fuel molecules and other chemicals), and / or forestry can be used in the methods of the present disclosure. Non-limiting examples include, for example, Populus balsamifera (poplar), cotton (Gossypium barbadense, Gossypium hirsutum), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (beta vulgaris), and others. vulgaris (sugar beet), Erianthus spp., Salix spp. (willows), Eucalyptus spp. (eucalyptus, e.g., E. grandis (and its hybrids known as "urograndis"), E. globulus, E. camaldulensis, E. tereticornis, E. viminalis, E. nitens, E. saligna, and E. urophylla), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis communis (castor bean), Manihot esculenta (cassava), Solanum lycopersicum (tomato), Lactuca sativa (lettuce), Phaseolus vulgaris (green bean), Phaseolus limensis (lima bean), Lathyrus species (pea), Solanum tuberosumtuberosum (potato), Brassica species (B. napus (canola), B. rapa, B. juncea), Brassica oleracea (broccoli, cauliflower, Brussels sprouts), Camellia sinensis (tea plant), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee tree), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annuum annum (chili and bell peppers), Arachis hypogaea (peanut), Ipomoea batatus (sweet potato), Cocos nucifera (coconut), Citrus species (citrus trees), Persea americana (avocado), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), Carica papaya (papaya), Anacardium occidentalis occidentale (cashew), Macadamia integrifolia (macadamia tree), Prunus amygdalus (almond), Allium cepa (onion), Cucumis melo (muskmelon), Cucumis sativus (cucumber), Cucumis cantalupensis (cantaloupe), Cucurbita maxima (cucumber),maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Cyamopsis tetragonoloba (guar bean), Ceratonia siliqua (carob), Trigonella foenum-graecum (fenugreek), Vigna radiata radiata (mungbean), Vigna unguiculata (cowpea), Vicia faba (broad bean), Cicer arietinum (chickpea), Lens culinaris (lentil), Papaver somniferum (poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis sativa, Camptotheca acuminate, Catharanthus roseus roseus, Vinca rosea, Cinchona officinalis, Colchicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea species, Andrographis paniculatapaniculata, Atropa belladonna, Datura stomonium, Berberis species, Cephalotaxus species, Ephedra sinica species, Ephedra species, Ephedra species, Erythroxylum coca, Galanthus wornorii, Scopolia species, Lycopodium serratum (Huperzia serrata), Lycopodium species, Rauwolfia serpentina serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber tree), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana orellana (Bixa), Alstroemeria spp., Rosa spp. (Roses), Rhododendron spp. (Azaleas), Macrophylla hydrangea (Azaleas), Hibiscus rosasanensis (Hibiscus), Tulipa spp. (Tulips), Narcissus spp. (Daffodils), Petunia hybridshybrida (petunia), Dianthus caryophyllus (carnation), Euphorbia pulcherrima (poinsettia), chrysanthemum, Nicotiana tabacum (tobacco), Lupinus albus (lupine), Populus tremuloides (aspen), Pinus species (pine), Abies species (fir), Acer species (maple), and conifers.

[0072] Coniferous plants may be used in the methods of the present disclosure, including, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Canadian hemlock (Tsuga canadensis); American hemlock (Tsuga heterophylla); mountain hemlock (Tsuga mertensiana); tamarack or larch (Larix ocdentalis); occidentalis); Sitka spruce (Picea glauca); sequoia (Sequoia sempervirens); true firs such as European fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis).

[0073] In certain aspects, plants useful in the methods of the present disclosure are crop plants (e.g., alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, tobacco, etc.) Other plants useful in the methods of the present disclosure include cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus, poplar, pine, Douglas-fir, citrus, papaya, cacao, cucumber, apple, Capsicum, and melon.

[0074] Additional heterologous coding sequences, heterologous polynucleotides, and polynucleotides of interest can be used in the methods of the present disclosure to alter the phenotype of a plant. Various phenotypic changes of interest include altering gene expression in a plant, altering a plant's defense mechanisms against pathogens or insects, increasing a plant's tolerance to herbicides, altering plant development in response to environmental stress, and modulating a plant's response to salt, temperature (hot and cold), drought, etc. These results can be achieved by expressing a heterologous nucleotide sequence of interest comprising an appropriate gene product. In certain embodiments, the heterologous nucleotide sequence of interest is an endogenous plant sequence whose expression level is increased in the plant or plant parts. Results can be obtained by altering the expression of one or more endogenous gene products (especially hormones, receptors, signaling molecules, enzymes, transporters, or cofactors) or by affecting the plant's nutrient uptake. These changes result in a phenotypic change in the transformed plant.

[0075] General types of heterologous polynucleotides or nucleotide sequences of interest for use in the methods of the present disclosure include, for example, genes involved in signaling such as zinc fingers, genes involved in transduction such as kinases, and genes involved in housekeeping such as heat shock proteins. More specific types of transgenes (heterologous polynucleotides or nucleotide sequences of interest) include, for example, genes encoding agronomically important traits, insect resistance, disease resistance, herbicide resistance, environmental stress tolerance (varied tolerance to cold, salt, drought, etc.), and grain characteristics. Still other types of transgenes include genes that induce expression of exogenous products such as enzymes, cofactors, and hormones from plants and other eukaryotes, as well as prokaryotes. It is recognized that any gene or polynucleotide of interest can be operably linked to a promoter and expressed in plants by the methods disclosed herein.

[0076] Many agronomic traits can affect "yield," including, but not limited to, plant height, number of pods, pod position on the plant, number of internodes, incidence of pod breakage, grain size, nodulation and nitrogen fixation efficiency, nutrient digestion efficiency, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, lodging resistance, seed germination rate, seedling vigor, and seedling traits. Other traits that can affect yield include germination efficiency (e.g., germination efficiency under stress conditions), growth rate (e.g., growth rate under stress conditions), number of panicles, number of seeds per panicle, seed size, seed composition (starch, oil, protein), and grain filling characteristics. Development of transgenic plants that exhibit desired phenotypic characteristics, which may or may not increase the overall yield of the plant, is also of interest. Such characteristics include enhanced plant morphology, enhanced plant physiology, or improved composition of mature seeds obtained from the transgenic plants.

[0077] The "increased yield" of the transgenic plants of the present disclosure may be assessed and measured in many ways, such as test weight, seeds per plant, seed weight, seeds per unit area (i.e., seeds or seed weight per acre), bushels per acre, tons per acre, and kilos per hectare. For example, corn yield may be measured as the production of hulled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, and is often reported on a moisture-adjusted basis (e.g., 15.5% moisture). Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorus, and carbohydrates, or increased tolerance to environmental stresses, such as cold, heat, drought, salinity, and pest or pathogen attack. Enhanced recombinant DNA can also be used to provide transgenic plants with improved growth and development, and therefore increased yield, as a result of altering the expression of plant growth regulators or altering the cell cycle or photosynthetic pathway.

[0078] "Enhanced traits," as used herein in describing aspects of the present disclosure, include improved or enhanced water use efficiency or drought tolerance, enhanced cold tolerance such as osmotic stress tolerance, high salinity stress tolerance, heat stress tolerance, low temperature germination tolerance, increased yield, improved seed quality, enhanced nitrogen use efficiency, faster plant growth and development, slower plant growth and development, enhanced seed protein, and enhanced seed oil productivity.

[0079] Many genes of interest (heterologous polynucleotides or nucleotide sequences of interest), e.g., insect resistance traits, herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stem strength, etc.) or output traits (e.g., increased yield, modified starch, improved oil profile, balanced amino acids, high lysine or high methionine, increased digestibility, improved fiber quality, drought tolerance, nutritional enhancement, etc.) can be used in the methods of the present disclosure and expressed in plants.

[0080] In one aspect, the methods of the present disclosure can be used to transform vegetative plant organs and complex tissues thereof, such as, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including but not limited to, apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem) with an insect resistance gene (heterologous polynucleotide or nucleotide sequence of interest) encoding resistance to severely yield-reducing pests such as cutworms, armyworms, and European corn borers. Such genes (heterologous genes or nucleotide sequences of interest) include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892, 5,747,450, 5,736,514, 5,723,756, 5,593,881, and Geiser, et al., (1986) Gene 48:109, the disclosures of which are incorporated herein by reference in their entireties). Genes (heterologous polynucleotides or nucleotide sequences of interest) encoding disease resistance traits can also be used in the methods of the present disclosure, e.g., detoxification genes, such as genes that detoxify fumonisins (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones, et al., (1994) Science 266:789; Martin, et al., (1993) Science 262:1432; and Mindrinos, et al., (1994) Cell 78:1089), which are incorporated by reference in their entireties.

[0081] Herbicide-tolerance traits, such as genes encoding tolerance to herbicides that act by inhibiting the action of acetolactate synthase (ALS), particularly sulfonylurea herbicides (e.g., an acetolactate synthase (ALS) gene containing a mutation that leads to such tolerance, particularly an S4 and / or Hra mutation), genes encoding tolerance to herbicides that act by inhibiting the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), genes encoding tolerance to glyphosate (e.g., the EPSPS gene and the GAT gene; see, e.g., U.S. Patent Application Publication No. 2004 / 0082770 and WO 03 / 092360, which are incorporated by reference in their entireties), or other such genes known in the art, can be used in the methods of the present disclosure. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutant encodes resistance to the herbicide chlorsulfuron, any of which can be operably linked to a promoter of the present disclosure and used in the methods of the present disclosure.

[0082] Glyphosate tolerance is conferred by a mutant 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) and an aroA gene operably linked to a promoter of the present disclosure and used in the methods of the present disclosure. See, for example, U.S. Patent No. 4,940,835 to Shah et al., which discloses nucleotide sequences of forms of EPSPS that can confer glyphosate tolerance. U.S. Patent No. 5,627,061 to Barry et al. also describes a gene encoding an EPSPS enzyme operably linked to a promoter of the present disclosure and used in the methods of the present disclosure. U.S. Patent Nos. 6,248,876B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061 See also U.S. Patent Nos. 5,633,448, 5,510,471, U.S. Patent Nos. Re. 36,449, 37,287E, and U.S. Patent No. 5,491,288, and International Publication Nos. WO 97 / 04103, WO 97 / 04114, WO 00 / 66746, WO 01 / 66704, WO 00 / 66747, and WO 00 / 66748 (which are incorporated herein by reference in their entireties). Glyphosate tolerance can also be conferred to plants by expressing a gene encoding a glyphosate oxidoreductase enzyme, which is described in more detail in U.S. Patent Nos. 5,776,760 and 5,463,175 (which are incorporated herein by reference in their entireties). Glyphosate tolerance can also be conferred to plants by overexpression of the gene encoding glyphosate N-acetyltransferase.See, for example, US Patent Application Nos. 11 / 405,845 and 10 / 427,692, which are incorporated herein by reference in their entireties.

[0083] The methods of the present disclosure can use sterility genes (heterologous polynucleotides or nucleotide sequences of interest) to provide an alternative to physical detasseling. Examples of genes for use in such methods include male tissue-preferential genes and genes with a male sterility phenotype, such as QM, as described in U.S. Patent No. 5,583,210, which is incorporated herein by reference in its entirety. Other genes that can be operably linked to promoters of the present disclosure and used in the methods of the present disclosure include those encoding kinases and compounds that are toxic to male or female gametophyte formation.

[0084] Commercial traits that can increase starch production, for example for ethanol production, or provide protein expression can also be produced using the methods of the present invention. Another important commercial use of transformed plants is the production of polymers and bioplastics, as described in U.S. Patent No. 5,602,321 (incorporated herein by reference in its entirety). Genes such as β-ketothiolase, PHBase (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase (see Schubert, et al., (1988) J. Bacteriol. 170:5837-5847 (incorporated herein by reference in its entirety)), which can be operably linked to the promoters of the present disclosure and used in the methods of the present disclosure, promote the expression of polyhydroxyalkanoates (PHAs).

[0085] Many trait genes (heterologous polynucleotides or nucleotide sequences of interest) are known in the art and can be used in the methods disclosed herein. By way of illustration, and not limitation, trait genes (heterologous polynucleotides) that confer insect or disease resistance, trait genes (heterologous polynucleotides) that confer herbicide resistance, trait genes (heterologous polynucleotides) that confer or contribute to modified grain characteristics such as modified fatty acids, modified phosphorus content, modified carbohydrates or carbohydrate composition, modified antioxidant content or composition, or modified essential seed amino acid content or composition are examples of types of trait genes (heterologous polynucleotides) that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein. Additional genes known in the art can be included in expression cassettes useful in the methods disclosed herein. Non-limiting examples include genes that create sites for site-specific DNA integration, genes that affect abiotic stress tolerance (e.g., but are not limited to, flowering, ear and seed development, enhanced nitrogen use efficiency, modified nitrogen responsiveness, drought tolerance or resistance, cold tolerance or resistance, and salinity tolerance or resistance), and increased yield under stress, or other genes and transcription factors that affect plant growth and agronomic traits (e.g., yield, flowering, plant growth and / or plant architecture).

[0086] The disclosed methods can be used to transform plants with a heterologous nucleotide sequence that is an antisense sequence of a target gene. As used herein, "antisense orientation" includes reference to a polynucleotide sequence operably linked to a promoter in the direction in which the antisense strand is transcribed. The antisense strand is sufficiently complementary to the endogenous transcript so that translation of the endogenous transcript is often suppressed. "Operably linked" refers to two or more nucleic acid fragments on a single nucleic acid fragment being in a relationship such that the function of one fragment is affected by the other fragment. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either sense or antisense orientation.

[0087] The term "antisense DNA nucleotide sequence" is intended to mean a sequence that is reversed relative to the normal 5' to 3' orientation of the nucleotide sequence. When delivered to a plant cell, expression of the antisense DNA sequence disrupts the normal expression of the target gene DNA nucleotide sequence. The antisense nucleotide sequence encodes an RNA transcript that is complementary to and hybridizable with the endogenous messenger RNA (mRNA) produced by transcription of the target gene DNA nucleotide sequence. In this case, production of the native protein encoded by the target gene is inhibited, resulting in the desired phenotypic response. The antisense sequence can be modified so long as it hybridizes to the corresponding mRNA and disrupts its expression. Thus, antisense constructs having 70%, 80%, or 85% sequence identity to the corresponding antisense sequence can be used. Furthermore, portions of the antisense nucleotide can be used to disrupt the expression of the target gene. Generally, sequences of at least 50, 100, 200, or more nucleotides can be used. Thus, the promoter sequences disclosed herein can be operably linked to antisense DNA sequences to suppress or inhibit expression of native proteins in plants.

[0088] "RNAi" refers to a series of related techniques for reducing gene expression (see, e.g., U.S. Pat. No. 6,506,559, incorporated herein by reference in its entirety). Older techniques, referred to by other names, are believed to rely today on the same mechanism but are given different names in the literature. These techniques include "antisense inhibition," i.e., the production of antisense RNA transcripts that can suppress the expression of a target protein, and "cosuppression" or "sense suppression," which refers to the production of sense RNA transcripts that can suppress the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference in its entirety). These techniques rely on the use of constructs that result in the accumulation of double-stranded RNA, one strand of which is complementary to the target gene to be silenced. The methods of the present disclosure can be used to express RNA interference constructs, including microRNAs and siRNAs.

[0089] As used herein, the term "promoter" or "transcription initiation region" refers to a regulatory region of DNA that typically contains a TATA box or other DNA sequence capable of inducing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter may further contain other recognition sequences (called upstream promoter elements, which affect the rate of transcription initiation) that are generally located upstream or 5' of the TATA box or DNA sequence capable of inducing RNA polymerase II to initiate RNA synthesis. It is recognized that the nucleotide sequences of the promoter regions disclosed herein have been identified, and the isolation and identification of additional promoters in the 5' untranslated region upstream of the specific promoter regions identified herein is within the state of the art. Furthermore, chimeric promoters may be provided. Such chimeras contain portions of a promoter sequence fused to fragments and / or variants of heterologous transcriptional regulatory regions. Thus, the promoter regions disclosed herein may include upstream promoters, such as enhancers, involved in the organization and temporal expression of the coding sequence.

[0090] As used herein, the term "regulatory element" also refers to a DNA sequence upstream (5') of the coding sequence of a structural gene, usually (but not always) containing sequences that control expression of the coding region by recognizing RNA polymerase and / or other factors necessary for initiating transcription at a specific site. One example of a regulatory element that recognizes RNA polymerase or other transcription factors to ensure initiation at a specific site is a promoter element. Promoter elements include core promoter elements, which are involved in initiating transcription, and other regulatory elements that alter gene expression. It should be understood that nucleotide sequences located within introns or 3' to the coding region sequence can also contribute to regulating expression of a coding region of interest. Examples of suitable introns include, but are not limited to, the maize IVS6 intron or the maize actin intron. Regulatory elements can also include elements located downstream (3') of the transcription start site, within the transcribed region, or both. In the context of the present disclosure, post-transcriptional regulatory elements include elements active after transcription initiation, such as translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability determinants.

[0091] A "heterologous nucleotide sequence," "heterologous polynucleotide of interest," or "heterologous polynucleotide," as used throughout this disclosure, is a sequence that is not naturally present with or operably linked to a promoter sequence. The nucleotide sequence is heterologous to the promoter sequence, but may be homologous, native, heterologous, or foreign to the host plant. Similarly, the promoter sequence may be homologous, native, heterologous, or foreign to the host plant and / or the polynucleotide of interest.

[0092] It is recognized that enhancers may be present to increase transcription levels. Enhancers are nucleotide sequences that act to increase the expression of a promoter region. Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, and the like. Some enhancers are also known to alter the normal promoter expression pattern, for example, by constitutively expressing the promoter (in the absence of an enhancer, the same promoter is expressed only in one or a few specific tissues).

[0093] Modification of promoter sequences can provide a range of expression for heterologous nucleotide sequences. Therefore, they can be modified into weak or strong promoters. Generally, a "weak promoter" refers to a promoter that drives expression of a coding sequence at a low level. "Low-level" expression is intended to mean expression at a level of about 1 / 10,000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts. Conversely, a strong promoter drives expression of a coding sequence at a high level, i.e., about 1 / 10 transcripts to about 1 / 100 transcripts to about 1 / 1,000 transcripts.

[0094] The transformation methods disclosed herein are useful for the genetic manipulation of any plant, thereby resulting in phenotypic changes in the transformed plant.

[0095] The term "operably linked" means that the transcription or translation of a heterologous nucleotide sequence is under the influence of a promoter sequence. Thus, a promoter nucleotide sequence can be provided in an expression cassette together with a heterologous nucleotide sequence of interest for expression in a plant of interest, more particularly in the reproductive tissues of the plant.

[0096] In one aspect of the present disclosure, the expression cassette comprises a transcription initiation region comprising a promoter nucleotide sequence or a variant or fragment thereof operably linked to a morphogenetic gene and / or a heterologous nucleotide sequence. Such an expression cassette may have multiple restriction sites for inserting a nucleotide sequence under the transcriptional control of the regulatory region. The expression cassette may further comprise a selectable marker gene and a 3' termination region.

[0097] An expression cassette may include, in the 5'-3' direction of transcription, a transcription initiation region (i.e., a promoter or a variant or fragment thereof), a translation initiation region, a heterologous nucleotide sequence of interest, a translation termination region, and an optional transcription termination region that are functional in the host organism. The regulatory regions (i.e., promoter, transcriptional regulatory region, and translation termination region) and / or the polynucleotides of this embodiment may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or the polynucleotides of this embodiment may be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is a sequence that has been substantially altered from its native form by deliberate human intervention in composition and / or genomic locus. For example, a promoter operably linked to a heterologous polynucleotide may be from a species different from that from which the polynucleotide was derived, or, if from the same / similar species, one or both may be substantially altered from their original form and / or genomic locus, or the promoter may not be the native promoter of the operably linked polynucleotide.

[0098] The termination region may be of the same origin as the transcription initiation region, of the same origin as the operably linked DNA sequence of interest, of the same origin as the host plant, or of another source (i.e., foreign or heterologous to the promoter, the DNA sequence to be expressed, the host plant, or any combination thereof). Suitable termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthase termination region and the nopaline synthase termination region. See also Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903; and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639, all of which are incorporated by reference herein in their entireties.

[0099] Expression cassettes useful in the methods of the present disclosure can also include a gene, heterologous nucleotide sequence, heterologous polynucleotide of interest, or at least one additional nucleotide sequence for a heterologous polynucleotide to be co-transformed into the organism, or this additional nucleotide sequence can be provided in a separate expression cassette.

[0100] Where appropriate, nucleotide sequences can be optimized to increase expression in transformed plants. That is, to improve expression, these nucleotide sequences can be synthesized using plant-preferred codons. For example, for a discussion of host-preferred codon usage, see Campbell and Gowri (1990) Plant Physiol. 92:1-11, which is incorporated herein by reference in its entirety. Various methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference in their entireties.

[0101] It is known that additional sequence modifications can be made to enhance gene expression in cellular hosts. Such modifications include the removal of sequences encoding pseudo polyadenylation signals, sequences encoding exon-intron splice site signals, sequences encoding transposon-like repeats, and other such well-characterized sequences that may be harmful to gene expression. The GC content of heterologous nucleotide sequences, calculated with reference to known genes expressed in host cells, can be adjusted to the average level for a given cellular host. If possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0102] The expression cassette may further comprise a 5' leader sequence, which may act to facilitate translation. Translation leaders are known in the art and include, but are not limited to, picornavirus leaders, such as the EMCV leader (encephalomyocarditis 5' non-coding region) (Elroy-Stein, et al., (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus leaders, such as the TEV leader (Tobacco Etch Virus) (Allison, et al., (1986) Virology 154:9-20); MDMV leader (Maize Dwarf Mosaic Virus); human immunoglobulin heavy chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); and the non-translated leader derived from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1991) Nature 353:90-94). al., (1987) Nature 325:622-625; tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) Molecular Biology of RNA, pages 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385), all of which are incorporated herein by reference in their entireties. See also Della-Cioppa, et al., (1987) Plant Physiology 84:965-968, all of which are incorporated herein by reference in their entireties.Methods known to increase mRNA stability may also be used, such as, for example, introns (e.g., the maize ubiquitin intron (Christensen and Quail, (1996) Transgenic Res. 5:213-218; Christensen, et al., (1992) Plant Molecular Biology 18:675-689) or the maize AdhI intron (Kyozuka, et al., (1991) Mol. Gen. Genet. 228:40-48; Kyozuka, et al., (1990) Maydica 35:353-357), which are incorporated herein by reference in their entireties).

[0103] A DNA expression cassette or construct useful in the disclosed method may also optionally contain an enhancer (translation enhancer or transcription enhancer). Enhancer regions are well known to those skilled in the art and may include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. Translation control signals and initiation codons may be derived from a variety of sources, both natural and synthetic. The translation initiation region may be provided from the source of the transcription initiation region or from a structural gene. This sequence may also be derived from the regulatory elements selected for gene expression and may be modified to specifically enhance mRNA translation. It has been found that enhancers can be used in combination with the promoter region of this embodiment to increase transcription levels. Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, and the like.

[0104] To prepare an expression cassette, various DNA fragments can be manipulated to provide DNA sequences in the appropriate orientation and, if necessary, in the appropriate reading frame. Toward this end, adapters or linkers can be used to join the DNA fragments, or other manipulations can be performed to provide appropriate restriction enzyme recognition sites, remove unwanted DNA, remove restriction enzyme recognition sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, such as transitions and transversions, can be performed.

[0105] A reporter gene or a selectable marker gene can also be included in the expression cassette useful in the methods of the present disclosure. Examples of suitable reporter genes known in the art can be found, for example, in Jefferson, et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell. Biol. 7: 725-737; Goff, et al., (1990) EMBO J. 9: 2517-2522; Kain, et al., (1995) BioTechniques 19: 650-655; and Chiu, et al., (1996) Current Biology 6: 325-330 (these are incorporated herein by reference in their entirety).

[0106] Selectable marker genes for selecting transformed cells or tissues may include genes that confer antibiotic resistance or herbicide resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding chloramphenicol resistance (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); genes encoding methotrexate resistance (Herrera Estrella, et al., (1983) Nature 303:209-213; Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); genes encoding hygromycin resistance (Waldron, et al., (1985) Plant Mol. Biol. 5:103-108; and Zhijian, et al., (1995) Plant Science 108:219-227); genes encoding streptomycin resistance (Jones, et al., (1995) Plant Science 108:219-227); al., (1987) Mol. Gen. Genet. 210:86-91; genes encoding spectinomycin resistance (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131-137); genes encoding bleomycin resistance (Hille, et al., (1990) Plant Mol. Biol. 7:171-176); genes encoding sulfonamide resistance (Guerineau, et al., (1990) Plant Mol. Biol. 15:127-36); genes encoding bromoxynil resistance (Stalker, et al., (1988) Science 242:419-423); genes encoding glyphosate resistance (Shaw, et al., (1986) Science 233:478-481; and U.S. Patent Application Nos. 10 / 004,357 and 10 / 427,692; a gene encoding phosphinothricin resistance (DeBlock, et al., (1987) EMBO J. 6:2513-2518), which are incorporated herein by reference in their entireties.

[0107] Other genes that may provide utility in the recovery of transgenic events include, but are not limited to, GUS (beta-glucuronidase; Jefferson, (1987) Plant Mol. Biol. Rep. 5:387), GFP (green fluorescent protein; Chalfie, et al., (1994) Science 263:802), luciferase (Riggs, et al., (1987) Nucleic Acids Res. 15(19):8115; and Luehrsen, et al., (1992) Methods Enzymol. 216:397-414), and maize genes encoding anthocyanin production (Ludwig, et al., (1990) Science 247:449), all of which are incorporated herein by reference in their entireties.

[0108] As used herein, "vector" refers to a DNA molecule, such as a plasmid, cosmid, or bacteriophage, for introducing a nucleotide construct, e.g., an expression cassette or construct, into a host cell. Cloning vectors generally contain one or a few restriction endonuclease recognition sites into which foreign DNA sequences can be inserted in a specific manner without losing the essential biological functions of the vector, and a marker gene suitable for use in identifying and selecting cells transformed with the cloning vector. Marker genes include: Commonly, genes that confer tetracycline resistance, hygromycin resistance, or ampicillin resistance are included.

[0109] The disclosed methods include introducing a polypeptide or polynucleotide into a plant. As used herein, "introducing" means presenting a polynucleotide or polypeptide to a plant in a manner that allows the sequence to enter the interior of a cell of the plant. The disclosed methods do not rely on a particular method of introducing a sequence into a plant, but simply allow the polynucleotide or polypeptide to enter at least one cell of the plant. Methods for introducing polynucleotides or polypeptides into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0110] "Stable transformation" refers to transformation in which a nucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" refers to the introduction of a polynucleotide into a plant but not into the genome of the plant, or the introduction of a polypeptide into a plant.

[0111] Transformation protocols and protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell targeted for transformation, i.e., whether it is a monocotyledonous or dicotyledonous plant. Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No. 5,563,055, and Zhao, et al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO). J. 3:2717-2722), ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050, 5,879,918, 5,886,244, 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology 6:923-926), and Lec1 transformation (WO 00 / 28058). Also, Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477, Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion), Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean), McCabe, et al., (1988) Bio / Technology 6:923-926 (soybean), Finer and McMullen, (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean), Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean), Datta, et al., (1990) Biotechnology 8:736-740 (rice), Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (corn), Klein, et al., (1988) Biotechnology 6:559-563 (corn), U.S. Patent Nos. 5,240,855, 5,322,783, and 5,324,646, Klein, et al., (1988) Plant Physiol. 91:440-444 (corn), Fromm, et al. al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311:763-764, U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418, and Kaeppler, et al. al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation), D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation), Li, et al., (1993) Plant Cell Reports 12:250-255, and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice), Osjoda, et al.See also, (1996) Nature Biotechnology 14:745-750 (maize with Agrobacterium tumefaciens), all of which are incorporated herein by reference in their entireties. Methods and compositions for rapid plant transformation are also found in U.S. Patent Application Publication No. 2017 / 0121722, which is incorporated herein by reference in its entirety. Vectors useful for plant transformation are found in U.S. Patent Application No. 15 / 765,521, which is incorporated herein by reference in its entirety.

[0112] In certain embodiments, the DNA expression cassette or construct can be delivered to plants using various transient transformation methods. Such transient transformation methods include, but are not limited to, viral vector systems and precipitation of polynucleotides in a manner that prevents subsequent detachment of the DNA. Thus, although transcription from particle-bound DNA can occur, the frequency of detachment and integration into the genome is very low. Such methods include the use of particles coated with polyethyleneimine (PEI; Sigma #P3143).

[0113] In other embodiments, polynucleotides can be introduced into plants by contacting the plant with a virus or viral nucleic acid. Generally, such methods involve incorporating a nucleotide construct into a viral DNA or RNA molecule. Methods for introducing polynucleotides, including viral DNA or RNA molecules, into plants and expressing the proteins encoded thereby are known in the art. See, e.g., U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta, et al., (1996) Molecular Biotechnology 5:209-221, which are incorporated herein by reference in their entireties.

[0114] The transformed cells can be grown into plants using conventional methods. See, e.g., McCormick, et al., (1986) Plant Cell Reports 5:81-84, incorporated herein by reference in its entirety. These plants can then be grown and pollinated with the same transformed strain or a different strain, and the resulting progeny expressing the desired phenotypic characteristics can be identified. To ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, the plants can be grown for two or more generations, after which the seeds can be harvested to ensure that expression of the desired phenotypic traits has been achieved. Thus, the present disclosure provides transformed seeds (also referred to as "transgenic seeds") having a nucleotide construct, e.g., an expression cassette, stably integrated into the genome.

[0115] There are various methods for regenerating plants from plant tissue. The specific method of regeneration will depend on the starting plant tissue and the particular plant species being regenerated. The regeneration, development, and culture of plants from single plant protoplast transformants or various transformed explants is well known in the art (Weissbach and Weissbach, (1988) In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., San Diego, Calif., which is incorporated herein by reference in its entirety). This regeneration and cultivation process typically involves selecting transformed cells and culturing those individual cells from the normal developmental stage of an embryo to the stage where plantlets can be rooted. Transgenic embryos and seeds are similarly regenerated. The resulting rooted transgenic shoots are then planted in an appropriate plant growth medium, such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Alternatively, pollen from the regenerated plants is crossed with seed-grown plants of an agronomically important line. Conversely, pollen from plants of these important lines is used to pollinate the regenerated plants. Transgenic plants of this embodiment containing the desired polynucleotide are cultivated by methods well known to those skilled in the art.

[0116] Methods for targeting the insertion of a polynucleotide at a specific location in a plant genome are known in the art. The insertion of a polynucleotide at a desired location in the genome is carried out using a site-specific recombination system. See, for example, U.S. Patent Nos. 9,222,098 B2, 7,223,601 B2, 7,179,599 B2, and 6,911,575 B1. All of these documents are incorporated herein by reference in their entirety. Briefly, a polynucleotide of interest flanked by two non-identical recombination sites can be contained in a T-DNA transfer cassette. The T-DNA transfer cassette is introduced into a plant whose genome has stably integrated a target site flanked by two non-identical recombination sites corresponding to the sites of the transfer cassette. An appropriate recombinase is provided, and the transfer cassette is integrated into the target site. This allows the polynucleotide of interest to be integrated into a specific chromosomal location in the plant genome.

[0117] In one aspect, the methods of the present disclosure can be used to efficiently and rapidly introduce polynucleotides useful for targeting specific sites for modification in the genome of plants into growing plant organs and their complex tissues, such as, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral meristems). Site-specific modifications that can be introduced by the methods of the present disclosure can be introduced by any method of introducing site-specific modifications, including, but not limited to, the use of gene repair oligonucleotides (e.g., U.S. Patent Application Publication No. 2013 / 0019349) or double-strand break technologies (such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, etc.). For example, the disclosed methods can be used to introduce a CRISPR-Cas system into a plant cell or plant for genome modification of a target sequence in the genome of a plant or plant cell, for plant selection, for deleting bases or sequences, for gene editing, and for inserting a polynucleotide of interest into the genome of a plant or plant cell. Therefore, the disclosed methods can be used in conjunction with a CRISPR-Cas system to provide an effective system for altering or modifying target sites and target nucleotides in the genome of a plant, plant cell, or seed. The Cas endonuclease gene is a plant-optimized Cas9 endonuclease, which can bind to the plant genome and cause a double-strand break in the genomic target sequence.

[0118] Guided by a guide nucleotide, the Cas endonuclease recognizes a specific target site in a cell's genome and optionally introduces a double-strand break. The CRISPR-Cas system provides an effective system for modifying target sites in the genome of a plant, plant cell, or seed. Additionally, methods and compositions using the guide polynucleotide / Cas endonuclease system are provided to provide an effective system for modifying target sites in a cell's genome and editing nucleotides in a cell's genome. Once a genomic target site has been identified, various methods can be used to further modify the target site to contain various polynucleotides of interest. The compositions and methods disclosed herein can be used to introduce a CRISPR-Cas system that edits nucleotide sequences in a cell's genome. The nucleotide sequence to be edited (the target nucleotide sequence) can be located within or outside the target site recognized by the Cas endonuclease.

[0119] CRISPR loci (clustered regularly interspaced short palindromic repeats) (also known as SPIDR - spacer-interspersed direct repeats) constitute a recently described family of DNA loci. CRISPR loci are composed of short, highly conserved DNA repeats (usually 24-40 bp, 1-140 repeats - also called CRISPR repeats) that form partial palindromes. The repeats (usually species-specific) are separated by a variable sequence of fixed length (usually 20-58 bp, depending on the CRISPR locus) as a spacer (WO 2007 / 025097, published March 1, 2007).

[0120] Cas genes include genes that are typically linked to, associated with, or located adjacent to or near a flanking CRISPR locus. The terms "Cas gene" and "CRISPR-associated (Cas) gene" are used interchangeably herein.

[0121] In another embodiment, the Cas endonuclease gene is operably linked to an SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region.

[0122] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" are used interchangeably herein in relation to Cas endonucleases and refer to a portion or subsequence of a Cas endonuclease sequence that retains the ability to make a double-strand break.

[0123] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" are used interchangeably herein with respect to Cas endonucleases. These terms refer to variants of Cas endonucleases that retain the ability to generate double-strand breaks. Fragments and variants can be obtained by site-directed mutagenesis, synthetic construction, and the like.

[0124] In one embodiment, the Cas endonuclease gene is the plant codon-optimized Streptococcus pyogenes Cas9 gene, which can recognize, in principle, any genomic sequence of the N(12-30)NGG type that can be targeted.

[0125] Endonucleases are enzymes that cleave phosphodiester bonds in polynucleotide chains, including restriction endonucleases, which cleave DNA at specific sites without damaging bases. Restriction endonucleases include type I, type II, type III, and type IV endonucleases, each of which contains further subtypes. In type I and type III systems, both methylase and restriction activities are contained within a single complex. Endonucleases also include meganucleases, also known as homing nucleases (HEases), which, like restriction endonucleases, bind to and cleave specific recognition sites; however, the recognition sites of meganucleases are typically long, approximately 18 bp or longer (Patent Application No. PCT / US12 / 30061, filed March 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs. These motifs are involved in metal ion coordination and phosphodiester bond hydrolysis. Meganucleases are known for their long recognition sites and tolerance of sequence polymorphism in their DNA substrates. The naming convention for meganucleases is similar to that of other restriction endonucleases. Meganucleases are also characterized by the prefixes F-, I-, or PI-, which are used for enzymes encoded by independent ORFs, introns, and inteins, respectively. One step in the genetic recombination process involves cleaving a polynucleotide at or near its recognition site. This cleavage activity can be used to create double-strand breaks. For reviews of site-specific recombinases and their recognition sites, see Sauer (1994) Curr Op Biotechnol 5:521-7 and Sadowski (1993) FASEB 7:760-7. In some instances, the recombinase is from the integrase or resolvase family. TAL effector nucleases are a new class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genomes of plants or other organisms (Miller, et al. (2011) Nature Biotechnology 29:143-148).Zinc finger nucleases (ZFNs) are artificial double-strand break inducers consisting of a zinc finger DNA-binding domain and a double-strand break inducer domain. Recognition site specificity is typically conferred by the zinc finger domain, which typically contains two, three, or four zinc fingers, e.g., with a C2H2 structure, although other zinc finger structures are known and have been engineered. Zinc finger domains can be applied to design polypeptides that specifically bind to selected polynucleotide recognition sequences. ZFNs include artificial DNA-binding zinc finger domains linked to a nonspecific endonuclease domain, e.g., a nuclease domain derived from an Ms-type endonuclease such as Fokl. Additional functionality can be fused to the zinc finger binding domain, e.g., a transcriptional activator domain, a transcriptional repressor domain, and a methylase. In some instances, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a three-finger domain recognizes a sequence of nine consecutive nucleotides, and due to the dimerization requirements of the nuclease, two sets of three zinc fingers are used to bind to a recognition sequence of 18 nucleotides.

[0126] As used herein, "dead CAS9" (dCAS9) is used to provide a transcriptional repressor domain. dCAS9 has been mutated so that it can no longer cleave DNA. dCAS9 can still bind when guided to a sequence by a gRNA, and can also be fused to a repressor element. dCAS9 fused to a repressor element is abbreviated as dCAS9~REP, as described herein, where the repressor element (REP) can be any of the known repressor motifs characterized in plants. An expression guide RNA (gRNA) binds to the dCAS9~REP protein and targets the dCAS9-REP fusion protein to a specific, predetermined nucleotide sequence within the promoter (promoter within the T-DNA). For example, if the ZM-UBI PRO::dCAS9~REP::PINII TERM cassette is used with the U6-POL PRO::gRNA::U6 TERM cassette to achieve transboundary expression, with the gRNA guiding the dCAS9-REP protein to bind to the SB-UBI expression cassette SB-UBI PRO::moPAT::PINII TERM within the T-DNA, events integrating sequences across the border will be bialaphos sensitive. Transgenic events integrating only the T-DNA will express moPAT and be bialaphos resistant. The advantage of using a dCAS9 protein fused to a repressor (as opposed to TETR or ESR) is the ability to target these repressors to any promoter within the T-DNA. TETR and ESR are limited to their cognate operator binding sequences. Alternatively, a synthetic zinc finger nuclease fused to a repressor domain can be used in place of the gRNA and dCAS9~REP, as described above (Urritia et al., 2003, Genome Biol. 4:231).

[0127] Bacterial type II CRISPR / Cas systems use crRNA and tracrRNA to guide the Cas endonuclease to its DNA target. The crRNA (CRISPR RNA) contains a region complementary to one strand of double-stranded DNA and base pairs with the tracrRNA (trans-activating CRISPR RNA) to form an RNA duplex that directs the Cas endonuclease to cleave the DNA target. As used herein, the term "guide nucleotide" refers to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) and a tracrRNA, each containing a variable targeting domain. In one embodiment, the guide nucleotide comprises a variable targeting domain consisting of a 12-30 nucleotide sequence and an RNA fragment capable of interacting with the Cas endonuclease.

[0128] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease, enabling the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide polynucleotide may be a single-stranded or double-stranded molecule. The guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide may contain at least one nucleotide, phosphodiester bond, or linkage modification, including, but not limited to, locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent linkage resulting in cyclization. A guide polynucleotide containing only ribonucleic acid is also referred to as a "guide nucleotide."

[0129] Nucleotide sequence modifications of the guide polynucleotide, VT domain, and / or CER domain may be selected from the group consisting of, but are not limited to, a 5' cap, a 3' polyadenylation tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a binding site for a protein, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2'-fluoro A nucleotides, 2'-fluoro U nucleotides, 2'-O-methyl RNA nucleotides, phosphorothioate linkages, linkages to cholesterol molecules, linkages to polyethylene glycol molecules, linkages to spacer 18 molecules, 5' to 3' covalent linkages, or any combination thereof. These modifications may result in at least one additional advantageous characteristic selected from the group of altered or modulated stability, intracellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, altered binding affinity for complementary target sequences, altered resistance to cellular degradation, and increased cell permeability.

[0130] In one embodiment, the guide nucleotide and the Cas endonuclease may form a complex that allows the Cas endonuclease to introduce a double-stranded break at the DNA target site.

[0131] In one aspect of the disclosed methods, the variable target domain is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0132] In one embodiment of the disclosed method, the guide nucleotide comprises a cRNA (or a cRNA fragment) and a tracrRNA (or a tracrRNA fragment) of a type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease, and this guide nucleotide-Cas endonuclease complex can guide the Cas endonuclease to a plant genome target site, allowing the Cas endonuclease to introduce a double-strand break at the genome target site. The guide nucleotide can be directly introduced into a plant or plant cell by any method known in the art (for example, but not limited to, particle bombardment or topical application).

[0133] In one embodiment, a guide nucleotide can be introduced indirectly by introducing a recombinant DNA molecule comprising a corresponding guide DNA sequence operably linked to a plant-specific promoter capable of transcribing the guide nucleotide in a plant cell. The term "corresponding guide DNA" includes a DNA molecule that is identical to an RNA molecule except that each "U" in the RNA molecule is replaced with a "T."

[0134] In one aspect, the guide nucleotide is introduced by particle bombardment or by the disclosed methods and compositions for Agrobacterium-mediated transformation of a recombinant DNA construct containing the corresponding guide DNA operably linked to a plant U6 polymerase III promoter.

[0135] In one embodiment, the RNA that guides the RNA Cas9 endonuclease complex is a double-stranded RNA comprising a double-stranded crRNA-tracrRNA. One advantage of using guide nucleotides over a double-stranded crRNA-tracrRNA is that only one expression cassette needs to be created to express the fusion guide nucleotides.

[0136] The terms "target site," "target sequence," "target DNA," "target locus," "genomic target site," "genomic target sequence," and "genomic target locus" are used interchangeably herein and refer to a polynucleotide sequence in a plant cell genome (including chloroplast DNA and mitochondrial DNA) at which a double-stranded break is induced in the plant cell genome by a Cas endonuclease. The target site can be an endogenous site in the plant genome, or the target site can be heterologous to the plant and therefore not naturally occurring in the genome, or the target site can be found at a heterologous genomic location compared to where it occurs in nature.

[0137] As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein and refer to a target sequence that is endogenous or native to a plant genome and that is present in the plant genome at the endogenous or natural location of the target sequence. In one embodiment, the target site can be a DNA recognition site or a site similar to a target site that is specifically recognized and / or bound by a double-strand break inducer, such as LIG3-4 endonuclease (U.S. Patent Application Publication No. 2009 / 0133152A1, published May 21, 2009) or MS26++ meganuclease (U.S. Patent Application No. 13 / 526912, filed June 19, 2012).

[0138] "Artificial target site" or "artificial target sequence" are used interchangeably herein to refer to a target sequence that has been introduced into the genome of a plant. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the plant genome, but may be located at a different location in the plant genome (i.e., a non-endogenous or non-native location).

[0139] The terms "modified target site," "modified target sequence," "altered target site," and "altered target sequence" are used interchangeably herein and refer to a target sequence disclosed herein that contains at least one modification when compared to an unmodified target sequence. Such a modification may include, for example, (i) a substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0140] In one aspect, the method of the present disclosure can be used to introduce into plants the polynucleotide that is useful for the gene suppression of target genes in plants.In some aspects of genetic engineering in plants, it is desired to reduce the activity of specific genes (also known as gene silencing or gene suppression).Many gene silencing techniques are well known to those skilled in the art, for example, but not limited to, antisense technology.

[0141] In one embodiment, the disclosed method can be used to introduce into a plant a polynucleotide useful for the targeted integration of a nucleotide sequence into the plant. For example, the disclosed method can be used to introduce a T-DNA expression cassette containing a nucleotide sequence of interest flanked by non-identical recombination sites to be used to transform a plant containing a target site. In one embodiment, the target site contains at least one set of corresponding non-identical recombination sites on the T-DNA expression cassette. Replacement of the nucleotide sequences flanked by the recombination sites is affected by a recombinase. Thus, the disclosed method can be used to introduce a T-DNA expression cassette for targeted integration of a nucleotide sequence, and the T-DNA expression cassette flanked by non-identical recombination sites is recognized by a recombinase that recognizes and recombines at the non-identical recombination sites. Therefore, the disclosed method and composition can be used to improve the efficiency and speed of growth of plants containing non-identical recombination sites.

[0142] As such, the disclosed methods can further include methods for directional, targeted integration of exogenous nucleotides into transformed plants. In one aspect, the disclosed methods use novel recombination sites in a gene targeting system that facilitates directional targeting of desired genes and nucleotide sequences to corresponding recombination sites already introduced into the target plant genome.

[0143] In one embodiment, a nucleotide sequence flanked by two non-identical recombination sites is introduced into one or more cells of an explant from the genome of a target organism, where a target site has been engineered for insertion of a nucleotide sequence of interest. Once a stable plant or culture is established, a second construct (i.e., the nucleotide sequence of interest) flanked by sites corresponding to the recombination sites flanking the target site is introduced into the stably transformed plant or tissue in the presence of a recombinase protein. This process results in the replacement of nucleotide sequences between the non-identical recombination sites of the target site and a T-DNA expression cassette.

[0144] It is recognized that transformed plants prepared by this method may contain multiple target sites (i.e., sets of non-identical recombination sites). In this method, multiple manipulations may be performed on target sites in a transformed plant. A target site in a transformed plant refers to a DNA sequence that is inserted into the genome of the transformed plant and contains non-identical recombination sites.

[0145] Examples of recombination sites for use in the disclosed methods are known. The 2 micron plasmid, found in most naturally occurring strains of Saccharomyces cerevisiae, encodes a site-specific recombinase that promotes DNA inversion between two inverted repeats. This inversion plays a central role in plasmid copy number amplification.

[0146] A protein, designated FLP protein, catalyzes site-specific recombination events. A minimal recombination site (FRT) has been defined, containing two inverted 13-base pair (bp) repeats separated by an asymmetric 8-bp spacer. The FLP protein cleaves the site at the junction between the repeat and the spacer and covalently binds to DNA via its 3'-terminal phosphate. Site-specific recombinases such as FLP cleave DNA at specific target sequences and religate it, resulting in precisely defined recombination between two identical sites. To function, the system requires a recombination site and a recombinase; no accessory factors are required. In this way, the entire system can be inserted into plant cells and function. The yeast FLP / FRT site-specific recombination system has been shown to function in plants. Today, this system is used to excise unwanted DNA. See Lyznik et al. (1993) Nucleic Acids Res. 21:969-975. In contrast, the present disclosure uses non-identical FRTs to replace, target, position, insert, and control expression of nucleotide sequences in plant genomes.

[0147] In one embodiment, a transformed organism of interest (e.g., an explant from a plant) containing a target site to be integrated into its genome is required. The target site is characterized by being flanked by non-identical recombination sites. A targeting cassette containing a nucleotide sequence flanked by non-identical recombination sites corresponding to sites contained in the target site of the transformed organism is also required. A recombinase that recognizes the non-identical recombination sites and catalyzes site-specific recombination is required.

[0148] It is recognized that the recombinase can be provided by any means known in the art, i.e., it can be provided in the cells of an organism or plant by transforming the organism with an expression cassette capable of expressing the recombinase in the organism, by transient expression, or by providing messenger RNA (mRNA) of the recombinase or recombinase protein.

[0149] "Non-identical recombination sites" means that the sequences of the adjacent recombination sites are not identical, and recombination will not occur or will be minimal between the sites. That is, one adjacent recombination site can be an FRT site and the second recombination site can be a mutated FRT site. Non-identical recombination sites used in the methods of the present disclosure prevent or greatly reduce recombination between the two adjacent recombination sites and excision of the nucleotide sequence contained therein. Thus, it is recognized that any suitable non-identical recombination site can be used in the present disclosure, including FRT and mutated FRT sites, FRT and lox sites, lox and mutated lox sites, and other recombination sites known in the art.

[0150] A suitable non-identical recombination site means that, in the presence of an active recombinase, deletion of the sequence between two non-identical recombination sites occurs at a significantly lower efficiency than replacement of the target sequence by recombination of nucleotide sequences into the plant genome. Thus, suitable non-identical sites as used in the present disclosure include sites with low recombination efficiency between the sites, for example, efficiencies of less than about 30 to about 50%, preferably less than about 10 to about 30%, and more preferably less than about 5 to about 10%.

[0151] As mentioned above, the recombination site of the targeting cassette corresponds to that of the target site in the transformed plant, i.e., if the target site in the transformed plant contains adjacent non-identical recombination sites of FRT and mutant FRT, then the targeting cassette will contain the same non-identical recombination sites of FRT and mutant FRT.

[0152] Furthermore, it is recognized that the recombinase used in the disclosed methods will vary depending on the recombination site in the transformed plant and the target site of the targeting cassette. That is, if FRT sites are used, FLP recombinase will be required. Similarly, if lox sites are used, Cre recombinase will be required. If the non-identical recombination sites contain both FRT and lox sites, both FLP and Cre recombinases will be required in the plant cell.

[0153] FLP recombinase is a protein that catalyzes a site-specific reaction involved in amplifying the copy number of the 2-micron plasmid in S. cerevisiae during DNA replication. FLP proteins have been cloned and expressed. See, e.g., Cox (1993) Proc. Natl. Acad. Sci. USA 80:4223-4227. The FLP recombinase used in this disclosure may be derived from Saccharomyces. For optimal expression in the target plant, it may be preferable to synthesize the recombinase using codons preferred in the plant. See, e.g., U.S. Patent Application Serial No. 08 / 972,258, filed November 18, 1997, entitled "Novel Nucleic Acid Sequence Encoding FLP Recombinase," which is incorporated herein by reference.

[0154] The bacteriophage recombinase Cre catalyzes site-specific recombination between two lox sites. Cre recombinase is known in the art. See, for example, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J. Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet. 22:477-488, and Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702, all of which are incorporated herein by reference. Such Cre sequences can also be synthesized using desired codons in plants.

[0155] Where appropriate, nucleotide sequences inserted into the plant genome can be optimized to increase expression in transformed plants. When mammalian, yeast, or bacterial genes are used in the present disclosure, they can be synthesized using plant-preferred codons for improved expression. For expression in monocotyledonous plants, it is recognized that dicotyledonous genes can also be synthesized using monocotyledonous codons. Various methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498 (incorporated herein by reference). Plant-preferred codons can be determined from the codons more frequently used in proteins expressed in the plant of interest. It is recognized that monocotyledonous or dicotyledonous plant-preferred sequences can be constructed for a particular plant species, as well as sequences preferred for that plant. See, for example, European Patent Application Publication Nos. A-0359472, A-0385962, International Patent Application Publication No. WO 91 / 16432, Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328, and Murray et al. (1989) Nucleic Acids Research, 17:477-498. U.S. Patent Nos. 5,380,831; 5,436,391, etc. (incorporated herein by reference). Furthermore, it is recognized that all or any portion of the gene sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences may also be used.

[0156] Further modifications of the sequence are known to enhance gene expression in cellular hosts and can be used in the present disclosure. Such modifications include removing sequences encoding pseudo polyadenylation signals, sequences encoding exon-intron splice site signals, sequences encoding transposon-like repeats, and other such well-characterized sequences that may be harmful to gene expression. The GC content of the sequence, calculated by reference to known genes expressed in host cells, can be adjusted to the average level for a given cellular host. If possible, the sequence is modified to avoid predicted hairpin secondary RNA structures.

[0157] The present disclosure also encompasses novel FLP recombination target sites (FRTs). The FRT has been identified as a minimal sequence containing two 13-base pair repeats separated by an 8-base spacer. Nucleotides in the spacer region can be substituted with any combination of nucleotides, as long as the two 13-base repeats are separated by 8 nucleotides. The actual nucleotide sequence of the spacer is not critical; however, for the practice of the present disclosure, some substitutions of nucleotides in the spacer region may work better than others. The 8-base pair spacer is involved in DNA-DNA pairing during strand displacement. The asymmetry of this region determines the direction of site alignment in the recombination event, which then leads to reversal or excision. As noted above, most of the spacer can be mutated without loss of function. See, e.g., Schlake and Bode (1994) Biochemistry 33:12746-12751 (incorporated herein by reference).

[0158] Novel FRT mutant sites can be used in practicing the methods of the present disclosure. Such mutant sites can be constructed by PCR-based mutagenesis. Mutant FRT sites are known (see SEQ ID NOS: 2, 3, 4, and 5 in WO 1999 / 025821), but it is recognized that other mutant FRT sites can be used in practicing the present disclosure. The present disclosure is not limited to the use of a particular FRT or recombination site; rather, non-identical recombination or FRT sites can be used for targeted insertion and expression of nucleotide sequences in a plant genome. Thus, other mutant FRT sites can be constructed and used based on the present disclosure.

[0159] As discussed above, when genomic DNA containing a target site with non-identical recombination sites is combined with a vector containing a T-DNA expression cassette with corresponding non-identical recombination sites in the presence of a recombinase, recombination occurs. The nucleotide sequence of the T-DNA expression cassette located between the flanking recombination sites is replaced with the nucleotide sequence of the target site located between the flanking recombination sites. In this way, the desired nucleotide sequence can be precisely integrated into the host genome.

[0160] It will be appreciated that many variations of the present disclosure can be implemented. For example, a target site can be constructed with multiple, non-identical recombination sites. In this manner, multiple genes or nucleotide sequences can be stacked or aligned at precise locations in the plant genome. Similarly, once a target site has been established in the genome, additional recombination sites can be introduced by incorporating additional recombination sites into the nucleotide sequence of the T-DNA expression cassette and transferring the sites to the target sequence. In this manner, once a target site has been established, additional sites can be added or altered by subsequent recombination.

[0161] Another variation involves providing a promoter or transcription initiation region operably linked to the target site in the organism. The promoter is preferably located 5' to the initial recombination site. By transforming the organism with a T-DNA expression cassette containing the coding region, expression of the coding region will occur when the T-DNA expression cassette is integrated into the target site. This embodiment provides a method for selecting transformed cells, particularly plant cells, by providing a selectable marker sequence as the coding sequence.

[0162] Other advantages of this system include the ability to reduce the complexity of integrating transgenes or transDNA into organisms by using T-DNA expression cassettes as discussed above, and the selection of organisms with simple integration patterns. Similarly, by comparing several transformation events, preferred sites within the genome can be identified. Preferred sites within the genome include those that do not interfere with the expression of essential sequences and that allow sufficient expression of the transgene sequence.

[0163] The disclosed methods also provide a means to combine multiple expression cassettes at a single location within a genome, allowing for the addition or excision of recombination sites at target sites within the genome.

[0164] In the present disclosure, any means known in the art for bringing together the three components of the system can be used. For example, a plant can be stably transformed to harbor a target site within its genome. A recombinase can be transiently expressed or provided. Alternatively, a nucleotide sequence capable of expressing a recombinase can be stably integrated into the plant genome. A T-DNA expression cassette flanked by corresponding non-identical recombination sites is inserted into the genome of a transformed plant in the presence of the corresponding target site and recombinase.

[0165] Alternatively, the components of this system can be combined by sexually crossing transformed plants. In this embodiment, a transformed plant containing a target site integrated into its genome, i.e., the first parent, can be sexually crossed with a second plant, i.e., the second parent, that has been genetically transformed with a T-DNA expression cassette containing flanking, non-identical recombination sites corresponding to the first plant. The first plant or the second plant contains a nucleotide sequence in its genome that expresses a recombinase. The recombinase can be under the control of a constitutive or inducible promoter. In this way, the expression of the recombinase and its subsequent activity at the recombination site can be controlled.

[0166] The disclosed methods are useful for targeting the integration of an introduced nucleotide sequence into a specific chromosomal site. The nucleotide sequence can encode any nucleotide sequence of interest. Specific genes of interest include those that provide a host cell and / or organism with an easily analyzable functional characteristic, such as a marker gene or other gene that alters the phenotype of the recipient cell. Thus, the present disclosure can be used with genes that affect plant growth, height, susceptibility to disease, insects, nutritional value, etc. The nucleotide sequence can also encode an "antisense" sequence that silences or alters gene expression.

[0167] It is recognized that the nucleotide sequence can be used in a functional expression unit or T-DNA expression cassette. A functional expression unit or T-DNA expression cassette refers to a nucleotide sequence of interest having a functional promoter and, in most cases, a termination region. There are various ways to achieve a functional expression unit in the practice of this disclosure. In one aspect of this disclosure, the nucleic acid of interest is introduced or inserted into the genome as a functional expression unit.

[0168] Alternatively, the nucleotide sequence can be inserted into the genome at a site 3' to the promoter region. In this latter case, insertion of the coding sequence 3' to the promoter region will result in a functional expression unit upon integration. The T-DNA expression cassette will contain a transcription initiation region, or promoter, operably linked to a nucleic acid encoding the peptide of interest. Such an expression cassette contains multiple restriction sites for insertion of a gene or genes of interest under the transcriptional control of the regulatory region.

[0169] A summary of SEQ ID NOs: 1-198 useful in the methods of the present disclosure is provided in Table 3.

[0170] [Table 1]

[0171] [Table 2]

[0172] [Table 3]

[0173] [Table 4]

[0174] [Table 5]

[0175] [Table 6]

[0176] [Table 7]

[0177] [Table 8]

[0178] [Table 9]

[0179] [Table 10]

[0180] [Table 11]

[0181] [Table 12]

[0182] [Table 13]

[0183] [Table 14]

[0184] [Table 15]

[0185] [Table 16]

[0186] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0187] Aspects of the present disclosure will be further clarified in the following examples, in which parts and percentages are by weight and degrees are degrees Celsius unless otherwise specified. These examples illustrate aspects of the present disclosure, but are given by way of illustration only. From the above description and these examples, one skilled in the art will be able to ascertain the essential characteristics of aspects of the present disclosure, and can make various changes and modifications thereto to adapt them to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be encompassed within the scope of the appended claims.

[0188] Example 1: Plant material and medium composition A wide variety of tissues or explants can be used in the current method, including vegetative plant organs and their composite tissues, such as, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral meristems). Various media compositions used for soybean transformation, tissue culture, and regeneration are outlined in Table 4. In this table, medium M1 is used for initiating suspension cultures when this is the starting material for transformation. Media M2 and M3 represent representative co-cultivation media useful for Agrobacterium transformation of the full range of explants listed above. Medium M4 is useful for selection (with appropriate selective agents), M5 is used for somatic embryo maturation, and medium M6 is used for germination to generate TO plantlets.

[0189] [Table 17]

[0190] [Table 18]

[0191] After 1–5 days of co-cultivation, the tissue is cultured in M3 medium without selection for 1 week (recovery period) and then transferred to selection. During selection, antibiotics or herbicides are added to the M3 medium to select for stable transformants. To initiate counterselection against Agrobacterium, 300 mg / L Timentin® (sterile ticarcillin disodium mixed with potassium clavulanate, PlantMedia, Dublin, OH, USA) is also added, and both the selection agent and Timentin® are maintained in the medium throughout the selection period (up to a total of 8 weeks). The selection medium is changed weekly. After 6–8 weeks on selection medium, the transformed tissue becomes visible as green tissue against a background of bleached (or necrotic), unhealthy tissue. These tissue explants are cultured for an additional 4–8 weeks.

[0192] Healthy green somatic embryos were then transferred to M5 medium containing 100 mg / L Timentin®. After a total of 4 weeks of maturation in M5 medium, the mature somatic embryos were placed in sterile, empty Petri dishes and sealed with Micropore™ tape (3M Health Care, St. Paul, MN, USA) or placed in plastic boxes (without fiber tape) at room temperature for 4-7 days.

[0193] The dried embryos were planted on M6 medium and incubated at 26°C with an 18-h photoperiod and 60–100 μE / m 2 After 4–6 weeks on germination medium, plantlets were transferred to moistened Jiffy-7 peat pellets (Jiffy Products Ltd, Shippagan, Canada) and grown under the following conditions: 60–100 μE / m 2 They are left sealed in clear plastic tray boxes until acclimated in a Percival incubator at 24°C / 26°C day / night temperature with a 16-hour photoperiod. Finally, hardened plantlets are planted in 2-gallon pots containing moistened SunGro 702 and grown to seed maturity in the greenhouse.

[0194] Example 2: Soybean transformation In soybean, standard protocols for particle bombardment (Finer and McMullen, 1991, In Vitro Cell Dev. Biol.-Plant 27:175-182), Agrobacterium-mediated transformation (Jia et al., 2015, Int J. Mol. Sci. 16:18552-18543, U.S. Patent Application Publication No. 2017 / 0121722, incorporated herein by reference in its entirety), Ochrobactrum-mediated transformation (U.S. Patent Application Publication No. 2018 / 0216123, incorporated herein by reference in its entirety), or Rhizobiaceae-mediated transformation (U.S. Patent Application No. 9,365,859, incorporated herein by reference in its entirety) can be used with the methods of the present disclosure.

[0195] Media useful for soybean transformation are listed in Table 5.

[0196] [Table 19]

[0197] Example 3: GM-HBSTART3 or GM-LTP3 promoters controlling expression of morphogenetic genes improve transformation The use of the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or other promoters disclosed herein to drive expression of Arabidopsis LEC1, LEC2, KN1, STM, or LEC1-like (Kwong et al., (2003) The Plant Cell, Vol. 15, 5-18) genes in expression cassettes containing fluorescent markers has been found to increase the frequency of somatic embryogenesis and the recovery of transgenic TO plants. Agrobacterium strain LBA4404 was used to transform various vegetative plant organs and their complex tissues of Pioneer soybean cultivar PHY21, including, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (apical meristem, root meristem, secondary meristem, axillary meristem, and floral meristem). Four days after the initiation of Agrobacterium infection, the tissues were washed with sterile culture medium to remove excess bacteria. After approximately nine days, the tissues were transferred to somatic embryo maturation medium, and transgenic somatic embryos were expected to be ready for drydown approximately 22 days later. At this point, fully formed mature somatic embryos fluoresce under an epifluorescence stereomicroscope equipped with appropriate filter sets. The developed somatic embryos are functional and germinate into healthy plants in the greenhouse. This rapid method for generating somatic embryos and germinating them to form plants is expected to shorten the typical time frame from Agrobacterium infection to transfer of transgenic T0 plants to the greenhouse from four months (conventional soybean transformation) to approximately two to three months.

[0198] Example 4: Use of the GM-HBSTART3 or GM-LTP3 promoter to control expression of Agrobacterium IPT genes promotes direct shoot formation and improves transformation The use of the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or any of the other promoters disclosed herein to drive expression of the Agrobacterium IPT gene in an expression cassette containing a fluorescent marker has been found to increase the frequency of forming multiple shoots and the recovery of transgenic TO plants. Agrobacterium strain LBA4404 is used to transform various vegetative plant organs and complex tissues thereof of Pioneer soybean cultivar PHY21, including, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem). Four days after the initiation of Agrobacterium infection, the tissue is washed with sterile culture medium to remove excess bacteria and transferred to a medium that promotes numerous shoot outgrowth. After nine days, the tissue is transferred to a medium that favors shoot development, and after 22 days, the transgenic shoots are expected to be transferred to a medium that promotes rooting. At this point, the initial plantlets fluoresce under an epifluorescence stereomicroscope equipped with the appropriate filter set. Functional plantlets develop rapidly and continue to grow in the greenhouse, producing healthy plants. This rapid, direct method for transgenic plant formation is expected to shorten the typical time frame from Agrobacterium infection to transfer of transgenic T0 plants to the greenhouse from four months (conventional soybean transformation) to approximately two to three months.

[0199] Example 5: The GM-HBSTART3 or GM-LTP3 promoter controlling expression of the Arabidopsis monopterus-delta gene promotes direct shoot formation and improves transformation The use of the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or any of the other promoters disclosed herein to drive expression of the Arabidopsis monopteros-delta gene in an expression cassette containing a fluorescent marker has been found to increase the frequency of multiple shoot formation and the recovery of transgenic TO plants. Agrobacterium strain LBA4404 is used to transform various vegetative plant organs and complex tissues of Pioneer soybean cultivar PHY21, including, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem). Four days after the initiation of Agrobacterium infection, the tissue is washed with sterile culture medium to remove excess bacteria and transferred to a medium that promotes numerous shoot outgrowth. After nine days, the tissue is transferred to a medium that favors shoot development, and after 22 days, the transgenic shoots are expected to be transferred to a medium that promotes rooting. At this point, the initial plantlets fluoresce under an epifluorescence stereomicroscope equipped with the appropriate filter set. Functional plantlets develop rapidly and continue to grow in the greenhouse, producing healthy plants. This rapid, direct method for transgenic plant formation is expected to shorten the typical time frame from Agrobacterium infection to transfer of transgenic T0 plants to the greenhouse from four months (conventional soybean transformation) to approximately two to three months.

[0200] Example 6: GM-HBSTART3 or GM-LTP3 promoters controlling expression of growth-promoting genes improve transformation Use of the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or any of the other promoters disclosed herein to drive expression of the Agrobacterium AV-6B gene, the Agrobacterium IAA-h gene, the Agrobacterium IAA-m gene, the Arabidopsis SERK or the Arabidopsis AGL15 gene in an expression cassette containing a fluorescent marker is found to increase the frequency of somatic embryogenesis and the recovery of transgenic TO plants. Agrobacterium strain LBA4404 was used to transform various vegetative plant organs and their complex tissues of Pioneer soybean cultivar PHY21, including, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (apical meristem, root meristem, secondary meristem, axillary meristem, and floral meristem). Four days after the initiation of Agrobacterium infection, the tissues were washed with sterile culture medium to remove excess bacteria. After nine days, the tissues were transferred to somatic embryo maturation medium, and after 22 days, transgenic somatic embryos were expected to be ready for drying down. At this point, fully formed mature somatic embryos fluoresce under an epifluorescence stereomicroscope equipped with appropriate filter sets. The developed somatic embryos are functional and germinate into healthy plants in the greenhouse. This rapid method for generating somatic embryos and germinating them to form plants is expected to shorten the typical time frame from Agrobacterium infection to transfer of transgenic T0 plants to the greenhouse from four months (conventional soybean transformation) to approximately two to three months.

[0201] Example 7: Combining a viral enhancer element with the GM-HBSTART3 promoter driving expression of WUS further improves soybean transformation The use of a viral enhancer element, such as the 35S enhancer, adjacent to the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or any of the other promoters disclosed herein to drive WUS expression in an expression cassette containing a fluorescent marker results in a further increase in the frequency of somatic embryo formation and somatic embryo maturation compared to the use of the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or any of the other promoters disclosed herein alone, resulting in an overall increase in the recovery of transgenic TO plants. Agrobacterium strain LBA4404 was used to transform various vegetative plant organs and their complex tissues of Pioneer soybean cultivar PHY21, including, but not limited to, leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (apical meristem, root meristem, secondary meristem, axillary meristem, and floral meristem). Four days after the initiation of Agrobacterium infection, the tissues were washed with sterile culture medium to remove excess bacteria. After nine days, the tissues were transferred to somatic embryo maturation medium, and after 22 days, the transgenic somatic embryos were ready for drying down. At this point, fully formed mature somatic embryos fluoresce under an epifluorescence stereomicroscope equipped with appropriate filter sets. The developed somatic embryos are functional and germinate into healthy plants in the greenhouse. This rapid method of generating somatic embryos and germinating them to form plants shortens the typical time frame from Agrobacterium infection to transfer of transgenic T0 plants to the greenhouse from four months (traditional soybean transformation) to approximately two to three months.

[0202] Other enhancer elements have been tested in a similar manner and are shown to also result in increased transformation compared to the use alone of the GM-HBSTART3 promoter (SEQ ID NO: 1), the GM-LTP3 promoter (SEQ ID NO: 124), or any of the other promoters disclosed herein. Such enhancers include viral enhancers such as Cauliflower Mosaic Virus 35S and Mirabilis Mosaic Virus 2xMMV, as well as endogenous plant enhancer elements.

[0203] Example 8: Orthologues of the Arabidopsis WUS gene function to promote somatic embryo formation in Brassica The following particle bombardment transformations are compared, all containing plasmid QC318 (SEQ ID NO: 117) with GM-EF1A PRO::GM-EF1A INTRON1::ZS-YELLOW::NOS TERM + GM-SAMS PRO::GM-SAMS INTRON1::GM-ALS::GM-ALS TERM. Treatments included: 1) control (no added gene), 2) pVER9662 (SEQ ID NO: 118) with an AT-UBI PRO driving expression of the Arabidopsis WUS gene, 3) UBIGMWUS (SEQ ID NO: 119) with an AT-UBI PRO driving expression of the Glycine max WUS gene, 4) UBIMTWUS (SEQ ID NO: 120) with an AT-UBI PRO driving expression of the Medicago truncatula WUS gene, 5) UBILJWUS (SEQ ID NO: 121) with an AT-UBI PRO driving expression of the Lotus japonica WUS gene, 6) UBIPVWUS (SEQ ID NO: 122) with an AT-UBI PRO driving expression of the Phaseolus vulgaris WUS gene, and 7) AT-UBI UBIPHWUS (SEQ ID NO: 123) with PRO is included.

[0204] Various vegetative plant organs and their complex tissues of Brassica, including but not limited to leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including but not limited to apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem), are isolated for transformation by particle bombardment. A mixture of two plasmids was co-transfected; the first contained an expression cassette consisting of AT-UBI PRO driving the expression of the cDNA sequences of each of the WUS orthologs (pVER9662 (SEQ ID NO: 118), UBIGMWUS (SEQ ID NO: 119), UBIMTWUS (SEQ ID NO: 120), UBILJWUS (SEQ ID NO: 121), UBIPVWUS (SEQ ID NO: 122), and UBIPHWUS (SEQ ID NO: 123)) and an expression cassette for ZS-YELLOW (QC318 (SEQ ID NO: 117)). Explants were cultured for two weeks. After two weeks, the number of fluorescing spheroid embryos was counted and tabulated for each treatment.

[0205] Two weeks after particle bombardment, few fluorescent globular somatic embryos were observed in the bombarded control plant organs and their composite tissues, such as immature cotyledons, split seeds, isolated embryonic axes, mature cotyledonary nodes, hypocotyls, epicotyls, or leaf tissues of Brassica, whereas in all other treatments (containing WUS genes from different dicotyledonous plant species driven by the AT-UBI promoter), numerous fluorescent somatic embryos were observed in the bombarded plant organs and their composite tissues, such as leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem).

[0206] Example 9: Orthologues of the Arabidopsis WUS gene function to promote somatic embryo formation in sunflower The following particle bombardment transformations are compared, all containing plasmid QC318 (SEQ ID NO: 117) with GM-EF1A PRO::GM-EF1A INTRON1::ZS-YELLOW::NOS TERM + GM-SAMS PRO::GM-SAMS INTRON1::GM-ALS::GM-ALS TERM. Treatments included: 1) control (no added gene), 2) pVER9662 (SEQ ID NO: 118) with an AT-UBI PRO driving expression of the Arabidopsis WUS gene, 3) UBIGMWUS (SEQ ID NO: 119) with an AT-UBI PRO driving expression of the Glycine max WUS gene, 4) UBIMTWUS (SEQ ID NO: 120) with an AT-UBI PRO driving expression of the Medicago truncatula WUS gene, 5) UBILJWUS (SEQ ID NO: 121) with an AT-UBI PRO driving expression of the Lotus japonica WUS gene, 6) UBIPVWUS (SEQ ID NO: 122) with an AT-UBI PRO driving expression of the Phaseolus vulgaris WUS gene, and 7) AT-UBI UBIPHWUS (SEQ ID NO: 123) with PRO is included.

[0207] Various vegetative plant organs of sunflower and their complex tissues, including but not limited to leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including but not limited to apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem), are isolated for transformation by particle bombardment. A mixture of two plasmids was co-transfected; the first contained an expression cassette consisting of AT-UBI PRO driving the expression of the cDNA sequences of each of the WUS orthologs (pVER9662 (SEQ ID NO: 118), UBIGMWUS (SEQ ID NO: 119), UBIMTWUS (SEQ ID NO: 120), UBILJWUS (SEQ ID NO: 121), UBIPVWUS (SEQ ID NO: 122), and UBIPHWUS (SEQ ID NO: 123)) and an expression cassette for ZS-YELLOW (QC318 (SEQ ID NO: 117)). Explants were cultured for two weeks. After two weeks, the number of fluorescing spheroid embryos was counted and tabulated for each treatment.

[0208] Two weeks after particle bombardment transformation, few fluorescent globular somatic embryos were observed in the bombarded control sunflower plant organs and their composite tissues, whereas in all other treatments (containing WUS genes from different dicotyledonous plant species driven by the AT-UBI promoter), numerous fluorescent somatic embryos were observed in the bombarded sunflower plant organs and their composite tissues, including but not limited to leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including but not limited to apical meristem, root meristem, secondary meristem, axillary meristem, and floral bud meristem).

[0209] Example 10: Use of WUS orthologs from four different species to promote direct shoot formation after Agrobacterium-mediated transformation of soybean leaf segments Agrobacterium strain LBA4404 THY- containing the poplar WUS (RV026520 (SEQ ID NO: 164) (POPTR-WUS)), Amaranthus WUS (RV026533 (SEQ ID NO: 165) (AMAHY-WUS)), WUS gene from apple (RV026531 (SEQ ID NO: 157) (MALDO-WUS)), or gene from Gnetum (RV026522 (SEQ ID NO: 154) (GNEGN-WUS)) was used to transform tissue sections cut from sterile immature leaves of in vitro-grown Pioneer soybean cultivar PHY21. Agrobacterium strain LBA4404THY- containing vectors for the genes listed above and in Table 3 was used for infection, and all bacterial cultures were adjusted to an OD of 0.5 for infection. All vectors contained the selectable marker gene SPCN (spectinomycin). Leaf explants were infected for 30 minutes and placed on co-cultivation medium in the dark at 21°C for 3 days. After co-cultivation, the explants were transferred to shoot regeneration medium for 3-4 weeks. Elongated shoots were transferred to rooting medium. In the control treatment (transformed with RV022814 (SEQ ID NO: 168) (NO WUS)) containing the SPCN and ZS-YELLOW1 N1 expression cassette, no direct shoot formation from the leaf segments was observed. However, when transformed with T-DNA containing the three expression cassettes (WUS, SPC, and ZS-YELLOW1 N1), green, healthy shoots were produced from poplar WUS (RV026520 (SEQ ID NO: 164) (POPTR-WUS)), Amaranthus WUS (RV026533 (SEQ ID NO: 165) (AMAHY-WUS)), Gnetum WUS (RV026522 (SEQ ID NO: 154) (GNEGN-WUS)), and apple WUS (RV026531 (SEQ ID NO: 157) (MALDO-WUS)) at frequencies of 23.6%, 38.4%, 52%, and 52.5%, respectively.

[0210] Example 11: Regeneration from soybean leaf and stem explants infected with vectors containing developmental genes and the selectable marker gene SPCN This method can be used with soybean lines, including elite lines. Leaf and stem explants of 93Y21 were harvested. Leaves were cut into uniformly sized sections, approximately 30–60 mm. Stem internodes were cut into sections approximately 0.3–0.8 cm long. Agrobacterium strain LBA4404THY- containing the vectors listed in Table 6 was used for infection. All bacterial cultures were adjusted to an OD of 0.5 for infection. All vectors contained the selectable marker gene SPCN (spectinomycin). Leaf and stem internode explants were infected for 30 min and then placed on co-cultivation medium in the dark at 21°C for 3 days. After co-cultivation, the explants were transferred to shoot regeneration medium. Infection frequency was assessed by screening for transient expression of the selectable marker gene 5 and 20 days after transformation. Shoot regeneration was observed approximately 30 days after infection (Table 6). Transgenic shoots were evaluated for the presence of the SPCN marker gene. As shown in Table 6, expression of WUS genes from phylogenetically distinct dicotyledonous or gymnosperm species promoted direct shoot formation from soybean leaf or stem explants. When transformed with T-DNA containing Amaranthus WUS (RV026533 (SEQ ID NO: 165) (AMAHY-WUS)), poplar WUS (RV026520 (SEQ ID NO: 164) (POPTR-WUS)), apple WUS (RV026531 (SEQ ID NO: 157) (MALDO-WUS)), and Gnetum WUS (RV026522 (SEQ ID NO: 154) (GNEGN-WUS)), green, healthy shoots were produced from soybean leaf or stem explants (at frequencies of 20%, 15%, 5%, and 5.5%, respectively).

[0211] As shown in Table 6, no shoot induction was observed when shoot induction 199A medium was used. These results suggest that the recovery of green, healthy shoots can be further improved by improving the shoot induction medium formulation.

[0212] [Table 20]

[0213] [Table 21]

[0214] [Table 22]

[0215] Example 12: Orthologues of the Arabidopsis WUS gene promote leaf morphogenetic development in Brassica WUS genes from 12 different dicotyledonous, two gymnosperm, and one monocotyledonous species were tested for efficacy by assessing their ability to promote the growth of transgenic green shoot responses in Brassica under selection on spectinomycin-containing media. All treatments containing a WUS expression cassette had identical T-DNA architectures, with the exception of the WUS gene used in the construct. The T-DNA configuration was RB+CAMV35S PRO::WUS::OS-T28 TERM+GM-UBQ PRO::GM-UBQ5UTR::GM-UBQ INTRON1::ZS-YELLOW1 N1::NOS TERM+AT-UBIQ10 PRO::AT-UBIQ10 5UTR::AT-UBIQ10 INTRON1::CTP::SPCN::UBQ14 TERM+GM-EF1A2 PRO::GM-EF1A2 5'UTR::GM-EF1A2 INTRON1::DS-RED2::UBQ TERM+LB (the variable WUS gene is shown in bold and italics).

[0216] Brassica napus seeds were surface sterilized with 50% Clorox solution and germinated on solid medium containing MS basal salts and vitamins. Seedlings were grown in the light at 28°C, and leaves were collected. The leaves were transferred to 100 x 25 mm Petri dishes containing 10 ml of 20A medium (Table 7) containing 200 mM acetosyringone and then sliced ​​into 3-5 mm long pieces. After slicing, 40 μl of Agrobacterium solution (Agrobacterium strain LBA4404 THY-, OD 200) containing the above expression cassette was added. 550 0.50) was added to the dish, and the Petri dishes containing the leaf / Agrobacterium mixture were either vacuum infiltrated or wounded. The vacuum-placed leaves were exposed to 15 PSI for 1 minute. The wounded leaf segments were each pierced 10 times with a needle. The wounded and vacuum-infiltrated dishes were transferred to dim light and co-cultivated at 21°C for 3 days.

[0217] After co-cultivation, the leaf segments were removed from the Agrobacterium solution, lightly wiped on sterile filter paper, and then placed on 70D medium (Table 7). 2 The leaf segments were then transferred to 70D medium (70C shoot elongation medium, Table 7) under a 16-hour photoperiod at 6000 rpm for 1 week. The segments were then transferred to 70A medium without spectinomycin. The newly developed shoots were transferred to 70C shoot elongation medium (Table 7) and grown in a bright room (60-100 μE / m 2 The plants were then returned to a 16-hour photoperiod (at 1000 x 1000 Hz, 16 hours photoperiod) for one month. Seven weeks after the initial transformation, shoots were counted.

[0218] [Table 23]

[0219] As shown in Tables 8 and 9, expression of WUS genes from phylogenetically distinct dicotyledonous, gymnosperm, or monocotyledonous species promoted direct shoot formation in canola leaf segments. This promotion of shoot development and the ability to restore spectinomycin-resistant shoots varied depending on the source of the WUS gene. In pine (RV026525 (SEQ ID NO: 167) (PINTA-WUS)), cucumber (RV026521 (SEQ ID NO: 166) (CUCSA-WUS)), Amborella (RV026529 (SEQ ID NO: 162) (AMBTR-WUS)), poplar (RV026520 (SEQ ID NO: 164) (POPTR-WUS)), and tomato (RV026592 (SEQ ID NO: 196) (SL-WUS)), this transgenic shoot promotion was similar to that seen in the negative control treatment. As shown in Tables 8 and 9 below, WUS genes from other species showed 29% in cassava (de novo shoot RV026524 (SEQ ID NO: 158) (MANES-WUS)), 25% in both apple (RV026531 (SEQ ID NO: 157) (MALDO-WUS)) and columbine (RV026528 (SEQ ID NO: 163) (AQUCO-WUS)), 25% in grapevine (RV026526 (SEQ ID NO: 155) (VITVI-WUS)) and Gnetum gnemone (Gnetum gnemon (gymnosperm) (RV026522 (SEQ ID NO: 154) (GNEGN-WUS)), 21%, Petunia (RV026534 (SEQ ID NO: 156) (PETHY-WUS)), 17%, Eelgrass (monocotyledonous) (RV026527 (SEQ ID NO: 161) (ZOSMA-WUS)), 8%, and Kalanchoe (RV026523 (SEQ ID NO: 159) (KALFE-WUS)), 4%.

[0220] [Table 24]

[0221] [Table 25]

[0222] [Table 26]

[0223] [Table 27]

[0224] Example 13: Orthologues of the Arabidopsis WUS gene promote morphogenetic development in cowpea leaves The efficacy of WUS genes from 12 different dicotyledonous, two gymnosperm, and one monocotyledonous species was tested by assessing their ability to promote the growth of transgenic green shoot responses in cowpea leaves. In all treatments containing a WUS expression cassette, the T-DNA architecture was identical except for the WUS gene used in the construct. The T-DNA configuration was RB+CAMV35S PRO::WUS::OS-T28 TERM+GM-UBQ PRO::GM-UBQ 5UTR::GM-UBQ INTRON1::ZS-YELLOW1 N1::NOS TERM+AT-UBIQ10 PRO::AT-UBIQ10 5UTR::AT-UBIQ10 INTRON1::CTP::SPCN::UBQ14 TERM+GM-EF1A2 PRO::GM-EF1A2 5'UTR::GM-EF1A2 INTRON1::DS-RED2::UBQ TERM+LB (the variable WUS gene is shown in bold and italics).

[0225] Seeds of Vigna unguiculata IT86D-1010 were sterilized with chlorine gas and germinated on solid medium containing MS basal salts and vitamins. Seedlings were grown in the light at 28°C, and leaves were collected. Leaf tissue was transferred to 100 x 25 mm Petri dishes containing 10 ml of 20A medium (Table 10) containing 200 mM acetosyringone and sliced ​​into 3-5 mm long pieces. After slicing, the 20A solution was removed from each Petri plate and replaced with 5 ml of a different Agrobacterium solution (Agrobacterium strain LBA4404 THY-, OD 0.50 at 550 nM) containing an expression cassette containing the different WUS genes listed above. The dishes were agitated at room temperature for 30 minutes. The treated leaf tissue was then transferred to 562V solid medium (Table 10) and co-cultivated in the dark at 21°C. After co-cultivation, the leaf segments were removed from the Agrobacterium solution, gently blotted on sterile filter paper, placed on 70D medium (Table 10), and moved to a bright room (26°C and bright light). After 3 days, the tissue was placed on 13113F resting medium (Table 10). After 8 days, the tissue on the resting medium was photographed. Transgenic regenerable plant structures (RPS) were tabulated for each infected leaf explant (see Table 11 below).

[0226] [Table 28]

[0227] Expression of WUS genes from phylogenetically different dicotyledonous, gymnosperm, or monocotyledonous species enhanced the growth response of transgenic regenerable plant structures (RPS) in cowpea leaf explants, as shown in Table 11. The average response of the WUS genes per explant ranged from 0.5 to 5.0 RPS, while the "No WUS" and "No Agro" negative controls showed no transgenic regenerable plant structures (RPS).

[0228] [Table 29]

[0229] Example 14: WUS promotes more rapid formation of green shoots from transformed tobacco leaf segments Tobacco (Nicotiana benthamiana) seedlings were germinated and grown under sterile light conditions on 90A medium (Table 7). Leaves were excised and cut into 2 cm x 0.5 cm strips while immersed in 20A liquid medium (Table 7). Approximately seven leaf segments were obtained from each leaf. Segments from four leaves were used per Agrobacterium infection. Agrobacterium strain LBA4404 THY- contained a binary vector carrying PHP71539 (a plasmid containing virulence genes as disclosed in U.S. Patent Application Publication No. 2019 / 0078106, incorporated herein by reference in its entirety) and T-DNA. In one treatment, the T-DNA contained a WUS-free (NO WUS) expression cassette. All treatments containing the WUS expression cassette had the same T-DNA configuration, except for the WUS gene used in the construct. The T-DNA configuration was RB+CAMV35S PRO::WUS::OS-T28 TERM+GM-UBQ PRO::GM-UBQ5UTR::GM-UBQ INTRON1::ZS-YELLOW1 N1::NOS TERM+AT-UBIQ10 PRO::AT-UBIQ10 5UTR::AT-UBIQ10 INTRON1::CTP::SPCN::UBQ14 TERM+GM-EF1A2 PRO::GM-EF1A2 5'UTR::GM-EF1A2 INTRON1::DS-RED2::UBQ TERM+LB (variable WUS gene is shown in bold and italics). The T-DNA was grown in 15 ml Falcon tubes in 20 A liquid medium until OD . 550An Agrobacterium suspension was prepared to achieve a pH of 0.5. 20 μl of the Agrobacterium suspension was diluted into 10 ml of 20A medium per well (in a multi-well plate) containing the excised leaf tissue. The plate was gently shaken for 10 min and then incubated under dim light (approximately 25-30 μE m) for 3 days of co-cultivation with Agrobacterium in 20A medium. -2 s -1 ) placed underneath.

[0230] After 3 days of co-cultivation, the leaf segments were transferred to 70D medium (see Table 7) and cultured at 27°C in the dark. Ten days after transformation, the tissues were subcultured onto fresh 70D medium. Eleven days after transformation, when distinct shoots had not yet emerged or were just beginning to emerge, primary shoots (green, regenerable plant structures) were counted for all tissues. Tables 12, 13, and 14 show the number of primary shoots per tobacco leaf segment 11 days after Agrobacterium infection. As shown in Tables 12, 13, and 14, the average number of early shoots observed per initially infected leaf segment was increased for all WUS alleles tested (compared to the no WUS control value of 2.2 early shoots / segment), ranging from 4.2 early shoots / segment for Manihot esculenta (RV026524 (SEQ ID NO: 158) (MANES-WUS)) to 16.5 early shoots / segment for Kalanchoe fedtschenkoi (RV026523 (SEQ ID NO: 159) (KALFE-WUS)) and Petunia hybrid (RV026534 (SEQ ID NO: 156) (PETHY-WUS)).

[0231] [Table 30]

[0232] [Table 31]

[0233] [Table 32]

[0234] Seventeen days after Agrobacterium infection, the number of leaf segments that formed green shoots was counted and tabulated as shown in Tables 15, 16, and 17. The frequency at which de novo green shoot formation was observed on Agrobacterium-infected leaf segments was calculated as a percentage, as shown in Tables 15, 16, and 17, and approximately 11% of the "NO WUS" treated leaf segments formed shoots. As shown in Tables 15, 16, and 17, all treatments in which a WUS expression cassette was present (all WUS orthologs tested) resulted in shoot formation frequencies above the control treatment (11%), ranging from 25% for Vitis vinifera (grapevine) WUS (RV026526 (SEQ ID NO: 155) (VITVI-WUS)) to 100% for Populus trichocarpa (poplar) WUS (RV026520 (SEQ ID NO: 164) (POPTR-WUS)). Figure 1 is a graphical representation of the percent (%) of treated leaf segments that formed de novo green shoots.

[0235] [Table 33]

[0236] [Table 34]

[0237] [Table 35]

[0238] Example 15 - Canola stem transformation Stem explants ranging in length from 0.3 to 0.8 cm were sectioned and co-cultivated with LBA4404THY-Agrobacterium containing construct RV029481 ((SEQ ID NO: 191) (GG-WUS+HSP:CRE+IPT)). After 2 days of co-cultivation, the explants were transferred to 70A medium and grown at 26°C and under bright light (60-100 μE / m 2 The explants were incubated under a 16-hour photoperiod (45°C, 1000 / s). After 7 or 14 days of incubation, the dishes containing the explants were transferred to 45°C and 70% relative humidity for a 2-hour heat shock, after which the dishes were returned to 26°C. The number of explants that formed chloroplast meristems was counted 18 days after co-cultivation (Table 18). The explants continued to form somatic meristems after excision of the GG-WUS and IPT genes.

[0239] [Table 36]

[0240] Example 16: Use of WUS orthologs from four different species to promote direct shoot formation after Agrobacterium-mediated transformation of soybean leaf segments The Agrobacterium strain LBA4404 THY- carrying a T-DNA carrying a WUS expression cassette, a heat-inducible CRE cassette, and an SPCN expression cassette and a DS-RED2 expression cassette is used. The Agrobacterium strains contained the poplar WUS gene (RV029630 (SEQ ID NO: 193) (PT-WUS+HSP:CRE), the poplar WUS gene fused to a nuclear localization sequence (RV029480) (SEQ ID NO: 194) (ALA-NLS-PT-WUS+HSP:CRE), or the poplar WUS gene and IPT gene (RV029479) (SEQ ID NO: 195) (PT-WUS+HSP:CRE+IPT). Another set of Agrobacterium strains contained the Gnetum WUS gene (RV029631 (SEQ ID NO: 192) (GG-WUS+HSP:CRE)), the Gnetum WUS gene fused to a nuclear localization sequence (RV029480) (SEQ ID NO: 194) (ALA-NLS-PT-WUS+HSP:CRE), or the poplar WUS gene and IPT gene (RV029479) (SEQ ID NO: 195) (PT-WUS+HSP:CRE+IPT). The six Agrobacterium strains were used to transform tissue sections cut from sterile immature leaves grown in vitro. The Agrobacterium method, transformation, and media series through co-cultivation and shoot elongation were as previously described.

[0241] Soybean lines, such as elite lines, for example, but not limited to, 93Y21, can be used for this process. Leaf and stem explants are harvested, with the leaves cut into 3-8 mm sections and the internodes (stem explants) cut into approximately 0.5 cm long sections. Agrobacterium strain LBA4404 containing the above vectors is used for infection, and all bacterial cultures are grown at OD 200 for infection. 550The ratio is adjusted to 0.5. All vectors contain the selectable marker gene SPCN (spectinomycin). Leaf and internode explants are infected for 30 minutes and placed on co-cultivation medium in the dark at 21°C for 3 days. After co-cultivation, the explants are transferred to shoot regeneration medium. Infection frequency is assessed by screening for transient expression of the selectable marker gene 5 and 20 days after transformation. Heat-shock-driven expression of the CRE gene induces excision of the WUS, CRE, or IPT gene stably integrated into the genome. Shoot and root morphology is improved by excision of the IPT and WUS genes. Shoot regeneration is observed approximately 20 days after infection. Transgenic shoots are further assessed for the presence of the marker gene.

[0242] In control treatments (transformed with NO WUS or IPT in the T-DNA) containing the SPCN and ZS-YELLOW1 N1 expression cassette (RV022814 (SEQ ID NO: 168) (NO WUS)), no shoot formation directly from leaf segments is expected to be observed. However, when transformation is performed to introduce T-DNA containing the Gnetum WUS gene and the poplar WUS gene, green, healthy shoots are expected to form. Agrobacterium infection of leaf or stem tissue with all six WUS constructs is expected to produce healthy, fertile plants in which the WUS, IPT, and CRE genes have been excised.

[0243] Example 17 - Stem transformation of dicotyledonous plants Dicotyledonous stem explants ranging in length from 0.1 to 1.0 cm were sectioned and co-cultured with LBA4404THY-Agrobacterium containing a construct containing a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2), as well as an IPT gene and HSP:CRE. After two days of co-culture, the explants were transferred to 70A medium and grown at 26°C under bright light (60-100 μE / m). 2 After 7 or 14 days of incubation, the dishes containing the explants are transferred to 45°C and 70% relative humidity for a 2-hour heat shock, after which the dishes are returned to 26°C. Somatic meristems and / or somatic embryos are expected to form 10 to 21 days after co-cultivation. After excision of the WUS, BBM / ODP2, and IPT genes, the explants are expected to continue to form somatic meristems and / or somatic embryos.

[0244] Example 18 - Dicotyledonous Leaf Transformation Dicotyledonous leaf explants are cut into uniformly sized sections, approximately 10-80 mm in size, and co-cultured with LBA4404 THY-Agrobacterium containing a construct containing a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2), as well as an IPT gene and an HSP:CRE. After two days of co-culture, the explants are transferred to 70A medium and grown at 26°C under bright light (60-100 μE / m 2After 7 or 14 days of incubation, the dishes containing the explants are transferred to 45°C and 70% relative humidity for a 2-hour heat shock, after which the dishes are returned to 26°C. Somatic meristems and / or somatic embryos are expected to form 10 to 21 days after co-cultivation. After excision of the WUS, BBM / ODP2, and IPT genes, the explants are expected to continue to form somatic meristems and / or somatic embryos.

[0245] Example 19: Nuclease-mediated genome modification of dicotyledonous plant leaves Methods for performing CRISPR-Cas nuclease-mediated genome modifications are described in U.S. Pat. No. 9,637,739, U.S. Patent Application Publication No. 2014 / 0068797, U.S. Patent Application Publication No. 2019 / 0040405, and U.S. Pat. No. 10,510,457, each of which is incorporated by reference in its entirety.

[0246] Dicotyledonous leaf explants are cut into uniformly sized sections, approximately 10-80 mm in size, and co-cultured with LBA4404 THY-Agrobacterium containing a construct comprising a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2), and providing a polynucleotide encoding a site-specific polypeptide or a site-specific polypeptide. After co-culture, the leaf explants are transferred to fresh medium and grown at 26°C and under bright light (60-100 μE / m 2The plants are then incubated under a photoperiod of 1000 x 1000 / s and 16 hours at 2000 x 1000 x 1000 light. After 10 to 21 days of co-cultivation, genome-edited regenerable plant constructs containing the genome edited product but not the morphogenetic gene expression cassette are expected to be formed. Genome-edited regenerable plant constructs containing the genome edited product but not the morphogenetic gene expression cassette are formed at a frequency that is increased by about 0.1% to about 1.0%, about 1.1% to about 10%, about 10.1% to about 20%, about 20.1% to about 30%, about 30.1% to about 40%, about 40.1% to about 50%, about 50.1% to about 60%, about 60.1% to about 70%, about 70.1% to about 80%, about 80.1% to about 90%, and about 90.1% to about 100% compared to the frequency of formation of genome-edited regenerable plant constructs when dicotyledonous plant organs or composite tissues thereof are not contacted with the morphogenetic gene expression cassette.

[0247] Example 20: Nuclease-mediated genome modification of dicotyledonous plant stems Methods for performing CRISPR-Cas nuclease-mediated genome modifications are described in U.S. Pat. No. 9,637,739, U.S. Patent Application Publication No. 2014 / 0068797, U.S. Patent Application Publication No. 2019 / 0040405, and U.S. Pat. No. 10,510,457, each of which is incorporated by reference in its entirety.

[0248] Dicotyledonous stem explants ranging in length from 0.1 to 1.0 cm are sectioned and co-cultured with LBA4404 THY-Agrobacterium, which contains a construct containing a nucleotide sequence encoding a functional WUS / WOX polypeptide, or a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide, or a combination of a nucleotide sequence encoding a functional WUS / WOX polypeptide and a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2), and provides a polynucleotide encoding a site-specific polypeptide or a site-specific polypeptide. After co-culture, the stem explants are transferred to fresh medium and grown at 26°C under bright light (60-100 μE / m 2The plants are then incubated under a photoperiod of 1000 x 1000 / s and 16 hours at 2000 x 1000 x 1000 light. After 10 to 21 days of co-cultivation, genome-edited regenerable plant constructs containing the genome edited product but not the morphogenetic gene expression cassette are expected to be formed. Genome-edited regenerable plant constructs containing the genome edited product but not the morphogenetic gene expression cassette are formed at a frequency that is increased by about 0.1% to about 1.0%, about 1.1% to about 10%, about 10.1% to about 20%, about 20.1% to about 30%, about 30.1% to about 40%, about 40.1% to about 50%, about 50.1% to about 60%, about 60.1% to about 70%, about 70.1% to about 80%, about 80.1% to about 90%, and about 90.1% to about 100% compared to the frequency of formation of genome-edited regenerable plant constructs when dicotyledonous plant organs or composite tissues thereof are not contacted with the morphogenetic gene expression cassette.

[0249] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "protein" includes one or more proteins and equivalents thereof known to those of skill in the art. Unless clearly indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0250] All patents, publications, and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents, publications, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0251] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications can be practiced within the scope of the appended claims. Furthermore, the present application relates to the invention described in the claims, but may also include the following as other aspects. (1) A method for producing a transgenic dicotyledonous plant containing a heterologous polynucleotide, comprising: contacting a vegetative plant organ of a dicotyledonous plant or a complex tissue thereof with a T-DNA containing the heterologous polynucleotide and a morphogenetic gene expression cassette; selecting plant cells that contain the heterologous polynucleotide and that do not contain a morphogenetic gene expression cassette, wherein the plant cells form regenerable plant structures that contain the heterologous polynucleotide and that do not contain a morphogenetic gene expression cassette; and regenerating a transgenic plant from said regenerable plant structure containing said heterologous polynucleotide and not containing a morphogenetic gene expression cassette. (2) The morphogenetic gene expression cassette is (i) a nucleotide sequence encoding a functional WUS / WOX polypeptide; or (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide; or (iii) The method according to (1) above, comprising a combination of (i) and (ii). (3) The method described in (2) above, wherein the nucleotide sequence encodes the functional WUS / WOX polypeptide. (4) The method described in (3) above, wherein the nucleotide sequence encoding the functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9. (5) The method described in (2) above, wherein the nucleotide sequence encodes the Babyboom (BBM) polypeptide or the ovule development protein 2 (ODP2) polypeptide. (6) The method described in (5) above, wherein the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the nucleotide sequence encoding the ovule development protein 2 (ODP2) polypeptide is ODP2. (7) The method described in (2) above, wherein the nucleotide sequence encodes the functional WUS / WOX polypeptide, and the Babyboom (BBM) polypeptide or the ovule development protein 2 (ODP2) polypeptide. (8) The method described in (7) above, wherein the nucleotide sequence encoding the functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the nucleotide sequence encoding the ovule development protein 2 (ODP2) polypeptide is ODP2. (9) The heterologous polynucleotide is The heterologous polynucleotide is selected from the group consisting of a heterologous polynucleotide that confers nutritional enhancement, a heterologous polynucleotide that confers modification of oil content, a heterologous polynucleotide that confers modification of protein content, a heterologous polynucleotide that confers modification of metabolite content, a heterologous polynucleotide that confers increased yield, a heterologous polynucleotide that confers abiotic stress tolerance, a heterologous polynucleotide that confers drought tolerance, a heterologous polynucleotide that confers cold tolerance, a heterologous polynucleotide that confers herbicide tolerance, a heterologous polynucleotide that confers disease and pest resistance, a heterologous polynucleotide that confers pathogen resistance, a heterologous polynucleotide that confers insect resistance, a heterologous polynucleotide that confers nitrogen use efficiency (NUE), a heterologous polynucleotide that confers disease resistance, a heterologous polynucleotide that confers increased biomass, a heterologous polynucleotide that confers the ability to alter a metabolic pathway, and combinations thereof. (10) The method according to (1) above, wherein the growing plant organ of a dicotyledonous plant or a composite tissue thereof is selected from the group consisting of leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral bud meristems), and combinations thereof. (11) The method according to (10), wherein the leaf explant is selected from the group consisting of leaves, basal leaves, cauline leaves, scattered leaves and opposite leaves, cruciform opposite leaves, opposite superposed leaves, whorl leaves, stalk leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, compound leaves, and combinations thereof. (12) The method according to (10), wherein the stem explant is selected from the group consisting of a stem node region, a stem internode region, a petiole, a hypocotyl, an epicotyl, a stolon, a rhizome, a tuber, a corm, and combinations thereof. (13) The method according to (1) or (10) above, wherein the dicotyledonous plant is selected from the group consisting of soybean, cotton, sunflower, cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus, citrus, papaya, cacao, cucumber, apple, Capsicum, melon, or Brassica. (14) The morphogenetic gene expression cassette comprises a polynucleotide encoding a functional WUS / WOX polypeptide; the functional WUS / WOX polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, or 148; or the functional WUS / WOX polypeptide The method according to any one of (1) to (4), (7) and (8) above, wherein the nucleotide sequence is encoded by any one of the nucleotide sequences of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, or 147. (15) The method according to (1) or (2), wherein the morphogenetic gene expression cassette further comprises a polynucleotide sequence encoding a site-specific recombinase selected from the group consisting of FLP, FLPe, KD, Cre, SSV1, Lambda Int, PhiC31 Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153, and the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally regulated promoter. (16) The method according to (15) above, further comprising excising the morphogenetic gene expression cassette. (17) A transgenic plant produced by the method described in (1) or (16) above. (18) Seeds of the transgenic plant according to (1) or (17) above, which contain the heterologous polynucleotide. (19) The method according to (13) or (14) above, wherein the regenerable plant structure is formed at a frequency that is increased by about 0.1% to about 1.0%, about 1.1% to about 10%, about 10.1% to about 20%, about 20.1% to about 30%, about 30.1% to about 40%, about 40.1% to about 50%, about 50.1% to about 60%, about 60.1% to about 70%, about 70.1% to about 80%, about 80.1% to about 90%, and about 90.1% to about 100% compared to the frequency of formation of a regenerable plant structure when the dicotyledonous plant organ or its composite tissue is not contacted with the morphogenetic gene expression cassette. (20) A method for producing a genome-edited dicotyledonous plant, comprising: contacting a dicotyledonous growing plant organ or a complex tissue thereof with a T-DNA containing a morphogenetic gene expression cassette to provide a polynucleotide encoding a site-specific polypeptide or a site-specific polypeptide; selecting plant cells that contain the genome edit and that do not contain a morphogenetic gene expression cassette, wherein the plant cells form regenerable plant structures that contain the genome edit and that do not contain a morphogenetic gene expression cassette; and regenerating a genome-edited plant from said regenerable plant structure containing said genome edits and no morphogenetic gene expression cassette. (21) The morphogenetic gene expression cassette is (i) a nucleotide sequence encoding a functional WUS / WOX polypeptide; or (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide; or (iii) The method according to (20) above, comprising a combination of (i) and (ii). (22) The method according to (21) above, wherein the nucleotide sequence encodes the functional WUS / WOX polypeptide. (23) The method according to (22) above, wherein the nucleotide sequence encoding the functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9. (24) The method according to (21) above, wherein the nucleotide sequence encodes the Babyboom (BBM) polypeptide or the ovule development protein 2 (ODP2) polypeptide. (25) The method described in (24) above, wherein the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2, and BMN3, or the nucleotide sequence encoding the ovule development protein 2 (ODP2) polypeptide is ODP2. (26) The method according to (21) above, wherein the nucleotide sequence encodes the functional WUS / WOX polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. (27) The method described in (26) above, wherein the nucleotide sequence encoding the functional WUS / WOX polypeptide is selected from WUS, WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the nucleotide sequence encoding the ovule development protein 2 (ODP2) polypeptide is ODP2. (28) The method according to (21) above, wherein the site-specific polypeptide is selected from the group consisting of zinc finger nucleases, meganucleases, TALENs, and CRISPR-Cas nucleases. (29) The method according to (28), wherein the CRISPR-Cas nuclease is Cas9 or Cpfl nuclease, and further comprises providing a guide RNA. (30) The method according to any one of (20), (28), and (29) above, wherein the site-specific nuclease induces an insertion, deletion, or substitution mutation. (31) The method according to (29) above, wherein the guide RNA and CRISPR-Cas nuclease are a ribonucleoprotein complex. (32) The method according to (20), wherein the growing plant organ of a dicotyledonous plant or a composite tissue thereof is selected from the group consisting of a leaf explant, a leaf primordium, a stipule, a cotyledon, a cotyledonary node, a mesocotyl, a stem explant, a primary root, a secondary lateral root, a root segment, a shoot, and a meristem (including, but not limited to, an apical meristem, a root meristem, a secondary meristem, an axillary meristem, and a floral bud meristem), and a combination thereof. (33) The method according to (32), wherein the leaf explant is selected from the group consisting of leaves, basal leaves, cauline leaves, scattered leaves and opposite leaves, cruciform opposite leaves, opposite superposed leaves, whorl leaves, stalk leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, compound leaves, and combinations thereof. (34) The method according to (32), wherein the stem explant is selected from the group consisting of a stem node region, a stem internode region, a petiole, a hypocotyl, an epicotyl, a stolon, a rhizome, a tuber, a corm, and combinations thereof. (35) The method according to (20) or (32), wherein the dicotyledonous plant is selected from the group consisting of soybean, cotton, sunflower, cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus, citrus, papaya, cacao, cucumber, apple, Capsicum, melon, or Brassica. (36) The morphogenetic gene expression cassette comprises a polynucleotide encoding a functional WUS / WOX polypeptide, wherein the functional WUS / WOX polypeptide is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, and 148. or the functional WUS / WOX polypeptide is encoded by a nucleotide sequence of any of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, or 147. (37) The method according to (20) or (21), wherein the morphogenetic gene expression cassette further comprises a polynucleotide sequence encoding a site-specific recombinase selected from the group consisting of FLP, FLPe, KD, Cre, SSV1, Lambda Int, PhiC31 Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153, and the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally regulated promoter. (38) The method according to (37) above, further comprising excising the morphogenetic gene expression cassette. (39) A genome-edited plant produced by the method described in (20) or (38) above. (40) Seeds of the genome-edited plant according to (20) or (39), comprising the genome edited product. (41) The method according to (35) or (36), wherein the regenerable plant structure is formed at a frequency that is increased by about 0.1% to about 1.0%, about 1.1% to about 10%, about 10.1% to about 20%, about 20.1% to about 30%, about 30.1% to about 40%, about 40.1% to about 50%, about 50.1% to about 60%, about 60.1% to about 70%, about 70.1% to about 80%, about 80.1% to about 90%, and about 90.1% to about 100% compared to the frequency of formation of a genome-edited regenerable plant structure when the dicotyledonous plant organ or composite tissue thereof is not contacted with the morphogenetic gene expression cassette.

Claims

1. 1. A method for producing a transgenic dicotyledonous plant containing a heterologous polynucleotide, comprising: contacting a vegetative plant organ of a dicotyledonous plant or a complex tissue thereof with a T-DNA containing the heterologous polynucleotide and a morphogenetic gene expression cassette; selecting plant cells containing the heterologous polynucleotide and not containing a morphogenetic gene expression cassette, wherein the plant cells form regenerable plant structures containing the heterologous polynucleotide and not containing a morphogenetic gene expression cassette; and regenerating a transgenic plant from said regenerable plant structure containing said heterologous polynucleotide and not containing a morphogenetic gene expression cassette. Including, (1) the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a functional WUS polypeptide, the functional WUS polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 83, 91, 128, 130, 132, 134, 136, 138, or 142, and the dicotyledonous plant comprises a plant of the genus Brassica; or (2) The method, wherein the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a functional WUS polypeptide, the functional WUS polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 81, 128, 130 or 144, and the dicotyledonous plant comprises a soybean plant.

2. The heterologous polynucleotide is 2. The method of claim 1, wherein the heterologous polynucleotide is selected from the group consisting of a heterologous polynucleotide that confers nutritional enhancement, a heterologous polynucleotide that confers modified oil content, a heterologous polynucleotide that confers modified protein content, a heterologous polynucleotide that confers modified metabolite content, a heterologous polynucleotide that confers increased yield, a heterologous polynucleotide that confers abiotic stress tolerance, a heterologous polynucleotide that confers drought tolerance, a heterologous polynucleotide that confers cold tolerance, a heterologous polynucleotide that confers herbicide tolerance, a heterologous polynucleotide that confers pest resistance, a heterologous polynucleotide that confers pathogen resistance, a heterologous polynucleotide that confers insect resistance, a heterologous polynucleotide that confers nitrogen utilization efficiency (NUE), a heterologous polynucleotide that confers disease resistance, a heterologous polynucleotide that confers increased biomass, a heterologous polynucleotide that confers the ability to alter metabolic pathways, and combinations thereof.

3. 2. The method of claim 1, wherein the growing plant organ of a dicotyledonous plant or a composite tissue thereof is selected from the group consisting of a leaf explant, a leaf primordium, a stipule, a cotyledon, a cotyledonary node, a mesocotyl, a stem explant, a primary root, a secondary lateral root, a root segment, a shoot, and a meristem (including, but not limited to, an apical meristem, a root meristem, a secondary meristem, an axillary meristem, and a floral bud meristem), and combinations thereof.

4. 4. The method of claim 3, wherein the leaf explant is selected from the group consisting of leaves, basal leaves, cauline leaves, scattered and opposite leaves, cross-pair leaves, opposite superposed leaves, whorl leaves, stalk leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, compound leaves, and combinations thereof.

5. 4. The method of claim 3, wherein the stem explant is selected from the group consisting of a stem node region, a stem internode region, a petiole, a hypocotyl, an epicotyl, a stolon, a rhizome, a tuber, a corm, and combinations thereof.

6. (a) the functional WUS polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 83, 91, 128, 130, 132, 134, 136, 138, or 142, and the dicotyledonous plant comprises a plant of the genus Brassica; The functional WUS polypeptide is encoded by a nucleotide sequence comprising any of SEQ ID NOs: 82, 90, 127, 129, 131, 133, 135, 137, or 141; Or, (b) the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a functional WUS polypeptide, wherein the functional WUS polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 81, 128, 130, or 144; and the dicotyledonous plant comprises a soybean plant; 2. The method of claim 1, wherein the functional WUS polypeptide is encoded by the nucleotide sequence of any of SEQ ID NOs: 80, 127, 129, or 143.

7. 2. The method of claim 1, wherein the morphogenetic gene expression cassette further comprises a polynucleotide sequence encoding a site-specific recombinase selected from the group consisting of FLP, FLPe, KD, Cre, SSV1, Lambda Int, Phi C31 Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153, wherein the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally-regulated promoter.

8. The method of claim 7 further comprising excising the morphogenetic gene expression cassette.

9. The method of claim 1 or 6, wherein the genome-edited regenerable plant structure is formed at a frequency that is increased by 0.1% to 1.0%, 1.1% to 10%, 10.1% to 20%, 20.1% to 30%, 30.1% to 40%, 40.1% to 50%, 50.1% to 60%, 60.1% to 70%, 70.1% to 80%, 80.1% to 90%, and 90.1% to 100% compared to the frequency of formation of regenerable plant structures when the dicotyledonous plant organ or composite tissue thereof is not contacted with the morphogenetic gene expression cassette.

10. A method for producing a genome-edited dicotyledonous plant, comprising: contacting a vegetative plant organ of a dicotyledonous plant or a complex tissue thereof with a T-DNA containing a morphogenetic gene expression cassette to provide (A) a polynucleotide encoding a site-specific polypeptide or (B) a site-specific polypeptide; selecting plant cells that contain the genome edit and that do not contain a morphogenetic gene expression cassette, wherein the plant cells form regenerable plant structures that contain the genome edit and that do not contain a morphogenetic gene expression cassette; and and regenerating a genome-edited plant from the regenerable plant structure containing the genome edit and not containing a morphogenetic gene expression cassette. Including, (1) the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a functional WUS polypeptide, the functional WUS polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 83, 91, 128, 130, 132, 134, 136, 138, or 142, and the dicotyledonous plant comprises a plant of the genus Brassica; or (2) The method, wherein the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a functional WUS polypeptide, the functional WUS polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 81, 128, 130 or 144, and the dicotyledonous plant comprises a soybean plant.

11. 11. The method of claim 10, wherein the site-directed polypeptide is selected from the group consisting of a zinc finger nuclease, a meganuclease, a TALEN, and a CRISPR-Cas nuclease.

12. 12. The method of claim 11, wherein the CRISPR-Cas nuclease is Cas9 or Cpfl nuclease, and further comprising providing a guide RNA.

13. 13. The method of any one of claims 10, 11 and 12, wherein the site-directed polypeptide results in an insertion, deletion or substitution mutation.

14. 13. The method of claim 12, wherein the guide RNA and CRISPR-Cas nuclease are a ribonucleoprotein complex.

15. 11. The method of claim 10, wherein the dicotyledonous growing plant organ or composite tissue thereof is selected from the group consisting of leaf explants, leaf primordia, stipules, cotyledons, cotyledonary nodes, mesocotyls, stem explants, primary roots, secondary lateral roots, root segments, shoots, and meristems (including, but not limited to, apical meristems, root meristems, secondary meristems, axillary meristems, and floral bud meristems), and combinations thereof.

16. 16. The method of claim 15, wherein the leaf explant is selected from the group consisting of leaves, basal leaves, cauline leaves, scattered and opposite leaves, cross-opposite leaves, opposite superposed leaves, whorl leaves, stalk leaves, sessile leaves, sessile leaves, subsessile leaves, leaves with stipules, stipule-less leaves, simple leaves, compound leaves, and combinations thereof.

17. 16. The method of claim 15, wherein the stem explant is selected from the group consisting of a stem node region, a stem internode region, a petiole, a hypocotyl, an epicotyl, a stolon, a rhizome, a tuber, a corm, and combinations thereof.

18. (a) the functional WUS polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 83, 91, 128, 130, 132, 134, 136, 138, or 142, and the dicotyledonous plant comprises a plant of the genus Brassica; The functional WUS polypeptide is encoded by a nucleotide sequence comprising any of SEQ ID NOs: 82, 90, 127, 129, 131, 133, 135, 137, or 141; Or, (b) the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a functional WUS polypeptide, wherein the functional WUS polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 81, 128, 130, or 144; and the dicotyledonous plant comprises a soybean plant; 11. The method of claim 10, wherein the functional WUS polypeptide is encoded by the nucleotide sequence of any of SEQ ID NOs: 80, 127, 129, or 143.

19. 11. The method of claim 10, wherein the morphogenetic gene expression cassette further comprises a polynucleotide sequence encoding a site-specific recombinase selected from the group consisting of FLP, FLPe, KD, Cre, SSV1, Lambda Int, Phi C31 Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153, wherein the site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally-regulated promoter.

20. 20. The method of claim 19, further comprising excising the morphogenetic gene expression cassette.

21. The regenerable plant structure is formed at a frequency that is increased by 0.1% to 1.0%, 1.1% to 10%, 10.1% to 20%, 20.1% to 30%, 30.1% to 40%, 40.1% to 50%, 50.1% to 60%, 60.1% to 70%, 70.1% to 80%, 80.1% to 90%, and 90.1% to 100% compared to the frequency of formation of a genome-edited regenerable plant structure when the dicotyledonous plant organ or composite tissue thereof is not contacted with the morphogenetic gene expression cassette. The method of claim 10 or 18.

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