Methods for plant transformation

Abstract

The methods disclosed herein use a morphogenetic gene or a repressor gene to provide a transgenic plant comprising a single copy of a transgenic event, wherein the morphogenetic gene or the repressor gene is not integrated into the genome of the plant and the plant does not contain a vector (plasmid) backbone.

Description

Technical Field

[0001] The present disclosure relates generally to the field of plant molecular biology, including genetic manipulation of plants. More specifically, the present disclosure relates to rapid, efficient methods for generating transformed plants.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 641,725, filed on March 12, 2018, and U.S. Provisional Patent Application Serial No. 62 / 641,733, filed on March 12, 2018, each of which is incorporated herein by reference in its entirety.

[0004] Citations of sequence listings submitted as text files via EFS-WEB

[0005] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing, with the file name 20190309_7486WOPCT_SeqList.TXT, created on March 9, 2019, and having a size of 803,180 bytes, and submitted simultaneously with this specification. The sequence listing contained in the ASCII formatted file is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0006] The use of standard transformation and regeneration schemes is both time-consuming and inefficient, and has a negative impact on the development schedule of transgenic products, assuming that there is usually a "priority development window" of seasonal restrictions to determine which gene constructs are preferentially used for large-scale field work based on the results obtained during preliminary studies. Available standard transformation and regeneration methods have multiple shortcomings, which limit the speed and efficiency of producing and screening transgenic plants. For example, many standard transformation and regeneration methods need to use high-level auxins or cytokinins, and need to relate to the steps of embryonic callus formation or organogenesis, resulting in the need of several weeks of procedures before plant growth in greenhouse environments after transformation. Still need to use a variety of starting tissue types in multiple inbred lines to produce significantly higher conversion frequencies and significantly higher quality events (including a copy of the trait gene expression cassette without the event of a carrier (plasmid) skeleton) transformation method, the inbred line includes a series of genetic diversity and has an important commercial conversion inbred line. Summary of the invention

[0007] The present disclosure includes methods and compositions for producing transgenic events comprising one copy of a trait gene.In another aspect, the present disclosure provides seeds of a plant produced by the methods disclosed herein.

[0008] On the one hand, the present disclosure provides a method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: contacting a plant cell with a T-DNA containing a trait gene expression cassette and a T-DNA containing a morphogenic gene expression cassette; selecting a plant cell containing a trait gene expression cassette and not containing a morphogenic gene expression cassette; and regenerating a transgenic plant from the plant cell. On the other hand, contacting comprises contacting the plant cell with a T-DNA containing a trait gene expression cassette in a first bacterial strain and a T-DNA containing a morphogenic gene expression cassette in a second bacterial strain at the same time. On yet another hand, contacting comprises contacting the plant cell with a bacterial strain containing a plasmid, the plasmid comprising a trait gene expression cassette within the T-DNA and a morphogenic gene expression cassette outside the T-DNA. On yet another hand, contacting comprises contacting the plant cell with a T-DNA containing a trait gene expression cassette in a bacterial strain and a T-DNA containing a morphogenic gene expression cassette. In another aspect, the morphogenic gene expression cassette comprises: (i) a nucleotide sequence encoding a WUS / WOX homeobox 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 another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide. In another aspect, the nucleotide sequence encoding a WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9. In another aspect, the nucleotide sequence encodes a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding a Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, and the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3 or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, the trait gene expression cassette comprises a trait gene selected from the group of genes that confer pest resistance, genes that confer herbicide resistance, genes that confer stress tolerance, genes that confer drought resistance, genes that confer nitrogen utilization efficiency (NUE), genes that confer disease resistance, and genes that confer the ability to alter metabolic pathways. In another aspect, the first bacterial strain and the second bacterial strain are the same bacterial strain.On the other hand, the same bacterial strain is selected from the group of disarmed Agrobacteria, Ochrobactrum bacteria and Rhizobiaceae bacteria. On the other hand, disarmed Agrobacteria is selected from the group of AGL-1, EHA105, GV3101, LBA4404 and LBA4404 THY-. On the other hand, Ochrobactrum bacteria are selected from Table 2. On the other hand, Rhizobiaceae bacteria are selected from Table 3. On yet another hand, the first bacterial strain and the second bacterial strain are different bacterial strains. On the other hand, different bacterial strains are selected from: (i) disarmed Agrobacteria and Ochrobactrum bacteria; (ii) disarmed Agrobacteria and Rhizobiaceae bacteria; and (iii) Rhizobiaceae bacteria and Ochrobactrum bacteria. On the other hand, disarmed Agrobacteria is selected from the group of AGL-1, EHA105, GV3101, LBA4404 and LBA4404 THY-. On the other hand, the bacterium of the genus Ochrobacter is selected from Table 2. On the other hand, the bacterium of the family Rhizobiaceae is selected from Table 3. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 50:50. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 75:25. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 90:10. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 95:5. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 99:1. On the other hand, the morphogenic gene expression cassette further comprises a promoter selected from Table 4 or Table 5. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene. On the other hand, the morphogenic gene expression cassette comprises an HBSTART3 promoter operably linked to a WUS gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene and an ubiquitin promoter operably linked to a LEC1 gene. In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene and a PLTP promoter operably linked to a LEC1 gene. In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene, and the WUS gene is fused to a T2A viral peptide LEC1 gene fusion. In another aspect, the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence. In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene, and the WUS gene is fused to a T2A viral peptide BBM gene fusion.In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene fused to a T2A viral peptide RepA gene fusion. In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene and a PLTP promoter operably linked to a PKLamiRNA fusion. In another aspect, plant seeds comprising a trait gene expression cassette are provided.

[0009] In one aspect, the present disclosure provides a method for producing a transgenic plant having one copy of a trait gene expression cassette and one copy of a selection marker expression cassette, comprising: contacting a plant cell with a bacterial strain containing a plasmid, the plasmid comprising a T-DNA having a right border and a left border flanking the trait gene expression cassette and the selection marker expression cassette, and a repressor gene expression cassette outside the T-DNA, wherein the selection marker expression cassette comprises a promoter repressed by a repressor polypeptide expressed by the repressor gene; selecting a plant cell containing a trait gene expression cassette and a selection marker expression cassette without a repressor gene expression cassette; and regenerating a transgenic plant from a plant cell. On the other hand, the repressor gene expression cassette comprises a nucleotide sequence encoding a tetracycline repressor (TETR), an ethametsulfuron repressor (ESR), a chlorsulfuron repressor (CSR), and a repressor-dCAS9 fusion with a guide RNA targeting a promoter in the T-DNA. On the other hand, the nucleotide sequence encodes a tetracycline repressor (TETR) that represses a promoter operably linked to a selection marker in the T-DNA. On the other hand, the nucleotide sequence encodes an ethametsulfuron repressor (ESR) of a promoter operably connected to a selection marker in T-DNA. On the other hand, the nucleotide sequence encodes a chlorsulfuron repressor (CSR) of a promoter operably connected to a selection marker in T-DNA. On the other hand, the nucleotide sequence encodes a repressor-dCAS9 fusion with a guide RNA that represses a promoter operably connected to a selection marker in T-DNA. On the other hand, the nucleotide sequence encodes a repressor-dCAS9 fusion with a guide RNA that represses a promoter operably connected to a selection marker in T-DNA. On the other hand, the trait gene expression cassette comprises: a trait gene selected from a gene that grants pest resistance, a gene that grants herbicide resistance, a gene that grants stress tolerance, a gene that grants drought resistance, a gene that grants nitrogen utilization efficiency (NUE), a gene that grants disease resistance, and a gene that grants the ability to change metabolic pathways. On the other hand, the bacterial strain is a rhizobiales selected from the group of disarmed Agrobacterium, Ochrobacterium bacteria, or Rhizobiumaceae bacteria. On the other hand, disarmed Agrobacterium is selected from the group of AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-. In another aspect, the Ochrobacterium bacterium is selected from Table 2. In another aspect, the Rhizobiaceae bacterium is selected from Table 3. In another aspect, a plant seed comprising a trait gene expression cassette is provided.

[0010] In one aspect, the present disclosure provides a method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: contacting a plant cell with a T-DNA containing a trait gene expression cassette in a first bacterial strain and a T-DNA containing a morphogenetic gene expression cassette in a second bacterial strain simultaneously; wherein the morphogenetic gene expression cassette comprises: (i) a nucleotide sequence encoding a WUS / WOX homeobox 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); wherein the trait gene expression cassette comprises: a trait gene selected from the group consisting of a gene conferring pest resistance, a gene conferring herbicide resistance, a gene conferring stress tolerance, a gene conferring drought resistance, a gene conferring nitrogen use efficiency (NUE), a gene conferring disease resistance, and a gene conferring the ability to alter a metabolic pathway; selecting a plant cell containing a trait gene expression cassette and not containing a morphogenetic gene expression cassette; and regenerating a transgenic plant from the plant cell. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9. In another aspect, the nucleotide sequence encodes a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, and the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3 or the ovule development protein 2 (ODP2) polypeptide is ODP2. On the other hand, the first bacterial strain and the second bacterial strain are the same bacterial strain. On the other hand, the same bacterial strain is selected from the group of Agrobacterium disarmament, Ochrobacterium bacteria and Rhizobium family bacteria. On the other hand, Agrobacterium disarmament is selected from the group of AGL-1, EHA105, GV3101, LBA4404 and LBA4404 THY-. On the other hand, Ochrobacterium bacteria are selected from Table 2. On the other hand, Rhizobium family bacteria are selected from Table 3. On the other hand, the first bacterial strain and the second bacterial strain are different bacterial strains. On the other hand, the different bacterial strains are selected from: (i) Agrobacterium disarmament and Ochrobacter bacteria; (ii) Agrobacterium disarmament and Rhizobium family bacteria; and (iii) Rhizobium family bacteria and Ochrobacter bacteria.On the other hand, the disarmed Agrobacterium is selected from the group of AGL-1, EHA105, GV3101, LBA4404 and LBA4404 THY-. On the other hand, the Ochrobacterium bacteria are selected from Table 2. On the other hand, the Rhizobiumaceae bacteria are selected from Table 3. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 50:50. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 75:25. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 90:10. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 95:5. On the other hand, the first bacterial strain and the second bacterial strain are present in a ratio of 99:1. On the other hand, the morphogenic gene expression cassette further comprises a promoter selected from Table 4 or Table 5. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene. On the other hand, the morphogenic gene expression cassette comprises an HBSTART3 promoter operably linked to a WUS gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a ubiquitin promoter operably connected to the LEC1 gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a PLTP promoter operably connected to the LEC1 gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide LEC1 gene fusion. On the other hand, the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide BBM gene fusion. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide RepA gene fusion. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a PLTP promoter operably connected to the PKLamiRNA fusion. In another aspect, a plant seed comprising a trait gene expression cassette is provided.

[0011] In one aspect, the present disclosure provides a method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: contacting a plant cell with a bacterial strain containing a plasmid, the plasmid comprising a trait gene expression cassette within a T-DNA and a morphogenic gene expression cassette outside the T-DNA; wherein the morphogenic gene expression cassette comprises: (i) a nucleotide sequence encoding a WUS / WOX homeobox 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); wherein the trait gene expression cassette comprises: a trait gene selected from the group consisting of a gene conferring pest resistance, a gene conferring herbicide resistance, a gene conferring stress tolerance, a gene conferring drought resistance, a gene conferring nitrogen use efficiency (NUE), a gene conferring disease resistance, and a gene conferring the ability to alter a metabolic pathway; selecting a plant cell containing a trait gene expression cassette and not containing a morphogenic gene expression cassette; and regenerating a transgenic plant from the plant cell. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9. In another aspect, the nucleotide sequence encodes a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, and the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3 or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, the bacterial strain is a Rhizobiales selected from the group of Agrobacterium disarmamenti, Ochrobacterium bacteria, and Rhizobiaceae bacteria. In another aspect, Agrobacterium disarmamenti is selected from the group of AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-. In another aspect, Ochrobacterium bacteria are selected from Table 2. In another aspect, Rhizobiaceae bacteria are selected from Table 3. In another aspect, the morphogenic gene expression cassette further comprises a promoter selected from Table 4 or Table 5. In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene. In another aspect, the morphogenic gene expression cassette comprises an HBSTART3 promoter operably linked to a WUS gene.On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a ubiquitin promoter operably connected to the LEC1 gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a PLTP promoter operably connected to the LEC1 gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide LEC1 gene fusion. On the other hand, the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide BBM gene fusion. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide RepA gene fusion. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a PLTP promoter operably connected to the PKLamiRNA fusion. In another aspect, a plant seed comprising a trait gene expression cassette is provided.

[0012] In one aspect, the present disclosure provides a method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: contacting a plant cell with a bacterial strain comprising a T-DNA comprising a trait gene expression cassette and a T-DNA comprising a morphogenic gene expression cassette; wherein the morphogenic gene expression cassette comprises: (i) a nucleotide sequence encoding a WUS / WOX homeobox 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); wherein the trait gene expression cassette comprises: a trait gene selected from the group consisting of a gene conferring pest resistance, a gene conferring herbicide resistance, a gene conferring stress tolerance, a gene conferring drought resistance, a gene conferring nitrogen use efficiency (NUE), a gene conferring disease resistance, and a gene conferring the ability to alter a metabolic pathway; selecting a plant cell containing a trait gene expression cassette and not containing a morphogenic gene expression cassette; and regenerating a transgenic plant from the plant cell. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9. In another aspect, the nucleotide sequence encodes a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3, or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, the nucleotide sequence encodes a WUS / WOX homeobox polypeptide and a Babyboom (BBM) polypeptide or an ovule development protein 2 (ODP2) polypeptide. In another aspect, the nucleotide sequence encoding the WUS / WOX homeobox polypeptide is selected from WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5 and WOX9, and the Babyboom (BBM) polypeptide is selected from BBM2, BMN2 and BMN3 or the ovule development protein 2 (ODP2) polypeptide is ODP2. In another aspect, a bacterial strain is a Rhizobiales selected from the group of Agrobacterium disarmamenti, Ochrobacterium bacteria, and Rhizobiaceae bacteria. In another aspect, Agrobacterium disarmamenti is selected from the group of AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-. In another aspect, Ochrobacterium bacteria are selected from Table 2. In another aspect, Rhizobiaceae bacteria are selected from Table 3. In another aspect, the morphogenic gene expression cassette further comprises a promoter selected from Table 4 or Table 5. In another aspect, the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene. In another aspect, the morphogenic gene expression cassette comprises an HBSTART3 promoter operably linked to a WUS gene.On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a ubiquitin promoter operably connected to the LEC1 gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a PLTP promoter operably connected to the LEC1 gene. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide LEC1 gene fusion. On the other hand, the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide BBM gene fusion. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene, and the WUS gene is fused to the T2A viral peptide RepA gene fusion. On the other hand, the morphogenic gene expression cassette comprises a PLTP promoter operably connected to the WUS gene and a PLTP promoter operably connected to the PKLamiRNA fusion. In another aspect, a plant seed comprising a trait gene expression cassette is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The efficiency of somatic embryogenesis for plasmids PHP81718 (WUS treatment) and PHP80728 (TAG-RFP treatment) as further described in Example 11 is shown. DETAILED DESCRIPTION

[0014] The disclosure herein will be described more fully hereinafter with reference to the accompanying drawings, in which some but not all possible aspects are shown. Indeed, the disclosure can be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure can satisfy applicable legal requirements.

[0015] Those skilled in the art to which the disclosed method relates will consider that many modifications and other aspects disclosed herein have the benefit of the teachings presented in the following description and the associated drawings. Therefore, it should be understood that the disclosure is not limited to the specific aspects disclosed, and modifications and other aspects are intended to be included within the scope of the appended claims. Although specific terms are employed herein, these terms are used only in a general and descriptive sense and not for purposes of limitation.

[0016] The terminology used herein is for the purpose of describing specific aspects only and is not intended to be limiting. As used in the specification and claims, the term "comprising" may include aspects of "consisting of". Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods belong. In this specification and the claims that follow, reference will be made to several terms defined herein.

[0017] The present disclosure includes methods for producing transgenic plants using morphogenetic genes. As used herein, the term "morphogenetic gene" refers to a gene that, when ectopically expressed, stimulates the formation of somatic cell-derived structures that can produce plants. More precisely, the ectopic expression of morphogenetic genes stimulates the de novo formation of somatic embryos or organogenic structures (e.g., shoot meristems) that can produce plants. This stimulated de novo formation occurs in cells expressing morphogenetic genes or in neighboring cells. Morphogenetic genes can be transcription factors that regulate the expression of other genes, or they can be genes that affect hormone levels in plant tissues, both of which can stimulate morphological changes. As used herein, the term "morphogenetic factor" refers to morphogenetic genes and / or proteins expressed by morphogenetic genes.

[0018] Morphogenic genes are involved in plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, initiation of somatic embryogenesis, accelerated somatic embryo maturation, initiation and / or development of apical meristem, initiation and / or development of shoot meristem, or a combination thereof, such as WUS / WOX genes (WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, or WOX9), see U.S. Patents 7,348,468 and 7,256,322 and U.S. Patent Application Publication Nos. 2017 / 0121722 and 2007 / 0271628, the entireties of which are incorporated herein by reference; Laux et al. (1996) Development 122:87-96; and Mayer et al. (1998) Cell 95:805-815; van der Graaff et al., 2009, Genome Biology [Genome Biology] 10: 248; Dolzblasz et al., 2016, Mol. Plant [Molecular Plant] 19: 1028-39. It is expected that regulation of WUS / WOX can regulate plant and / or plant tissue phenotypes, including plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, initiation of somatic embryogenesis, acceleration of somatic embryo maturation, initiation and / or development of apical meristem, initiation and / or development of shoot meristem, or a combination thereof. Expression of Arabidopsis WUS can induce stem cells in vegetative tissues, which can differentiate into somatic embryos (Zuo, et al. (2002) Plant J [Plant Journal] 30: 349-359). Also of interest in this regard are the MYB118 gene (see U.S. Pat. No. 7,148,402), the MYB115 gene (see Wang et al. (2008) Cell Research 224-235), the BABYBOOM gene (BBM; see Boutilier et al. (2002) Plant Cell 14: 1737-1749) or the CLAVATA gene (see, for example, U.S. Pat. No. 7,179,963).

[0019] The morphogenic polynucleotide sequences and amino acid sequences of WUS / WOX homeobox polypeptides can be used in the disclosed methods. The Wuschel protein (hereinafter referred to as WUS) plays a key role in the initiation and maintenance of the apical meristem, which contains a pool of pluripotent stem cells (Endrizzi et al., (1996) Plant Journal 10:967-979; Laux et al., (1996) Development 122:87-96; and Mayer et al., (1998) Cell 95:805-815). Arabidopsis plant mutants of the WUS gene contain stem cells that are misspecified and appear to undergo differentiation. WUS encodes a novel homeodomain protein that may act as a transcriptional regulator (Mayer et al., (1998) Cell 95:805-815). The stem cell population of the Arabidopsis shoot meristem is thought to be maintained by a regulatory loop between the CLAVATA (CLV) gene, which promotes organ initiation, and the WUS gene, which is required for stem cell identity, wherein the CLV gene represses WUS at the transcriptional level, and WUS expression is sufficient to induce meristem cell identity and expression of the stem cell marker CLV3 (Brand et al., (2000) Science 289:617-619; Schoof et al., (2000) Cell 100:635-644). Constitutive expression of WUS in Arabidopsis has been shown to result in adventitious bud proliferation in leaves (in planta) (Laux, T., Talk Presented at the XVI International Botanical Congress Meeting, August 1-7, 1999, St. Louis, Mo.).

[0020] In one aspect, the WUS / WOX homeobox polypeptide useful in the methods of the present disclosure is a 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, the entireties of which are incorporated herein by reference, and van der Graaff et al., 2009, Genome Biology 10: 248). The WUS / WOX homeobox polypeptide useful in the methods of the present disclosure may be obtained or derived from any plant described herein. Other WUS / WOX genes useful in the methods of the present disclosure are listed in Table 1 below.

[0021] Table 1.

[0022]

[0023]

[0024] Other morphogenic genes that can be used in the present disclosure include, but are not limited to, LEC1 (U.S. Pat. No. 6,825,397, which is incorporated herein 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 gene 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), the Agrobacterium AV-6b gene (Wabiko and Minemura 1996, Plant Physiol. 112:939-951), a combination of the Agrobacterium IAA-h and IAA-m genes (Endo et al. 2002, Plant Cell Rep. 20:923-928), the Arabidopsis SERK gene (Hecht et al. 2001, Plant Physiol. 127:803-816), the Arabidopsis AGL15 gene (Harding et al. 2003, Plant Physiol. 133:653-663), the FUSCA gene (Castle and Meinke, Plant Cell 6:25-41), and the PICKLE gene (Ogas et al. 1999, PNAS 96:13839-13844).

[0025] As used herein, the term "expression cassette" refers to a unique component of a vector DNA consisting of coding and non-coding sequences including 5' and 3' regulatory sequences that control expression in transformed / transfected cells.

[0026] As used herein, the term "coding sequence" refers to the portion of a DNA sequence defined by the start and stop codons that encode the amino acids of a protein.

[0027] 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 encode amino acids for a protein, such as a 5' untranslated region, introns, and a 3' untranslated region. Non-coding sequences may also refer to RNA molecules, such as microRNAs, interfering RNAs, or RNA hairpins, which, when expressed, can downregulate the expression of endogenous genes or other transgenics.

[0028] As used herein, the term "regulatory sequence" refers to a nucleic acid molecule segment that can increase or decrease gene expression. Regulatory sequences include promoters, terminators, enhancer elements, silencing elements, 5'UTR and 3'UTR (untranslated region).

[0029] As used herein, the term "Agro1" refers to Agrobacterium carrying a plasmid having a T-DNA containing a "trait gene" expression cassette.

[0030] As used herein, the term "Agro2" refers to Agrobacterium carrying a plasmid having a T-DNA containing a "morphogenic gene" expression cassette, such as but not limited to BBM and / or WUS2.

[0031] As used herein, the term "transfer cassette" refers to a T-DNA comprising one or more expression cassettes flanked by a right border and a left border.

[0032] As used herein, T-DNA refers to the portion of the Ti plasmid that is inserted into the genome of the host plant cell.

[0033] As used herein, the term "selectable marker" refers to a transgene that, when expressed in a transformed / transfected cell, confers resistance to a selective agent such as antibiotics, herbicides, and other compounds that are toxic to non-transformed / non-transfected cells.

[0034] As used herein, the term "EAR" refers to an ethylene response element binding factor-related amphipathic repression motif having a general consensus sequence of LLxLxL, DNLxxP, LxLxPP, R / KLFGV or TLLLFR as a transcriptional repression signal within a transcription factor. Adding an EAR-type repressor element to a DNA binding protein (e.g., a transcription factor, dCAS9 or LEXA (as an example)) confers a transcriptional repression function to the fusion protein (Kagale, S., and Rozwadowski, K. 2010. Plant Signaling and Behavior 5: 691-694).

[0035] As used herein, the term "transcription factor" means a protein that controls the transcription rate of a specific gene by binding to the DNA sequence of the promoter and up-regulating or down-regulating expression. Examples of transcription factors (also morphogenic genes) include members of the AP2 / EREBP family (including BBM (ODP2), plethora and aintegumenta subfamilies, CAAT-box binding proteins (such as LEC1 and HAP3) and members of the MYB, bHLH, NAC, MADS, bZIP and WRKY families.

[0036] Ovule development protein 2 (ODP2) polypeptides and related polypeptides such as the morphogenic polynucleotide sequences and amino acid sequences of Babyboom (BBM) protein family proteins can be used in the methods of the present disclosure. In one aspect, a polypeptide comprising two AP2-DNA binding domains is an ODP2, BBM2, BMN2 or BMN3 polypeptide, see U.S. Patent Application Publication No. 2017 / 0121722, which is incorporated herein by reference in its entirety. The ODP2 polypeptides that can be used in the methods of the present disclosure comprise two predicted APETALA2 (AP2) domains and are members of the AP2 protein family (PFAM Accession No. PF00847). The AP2 family of putative transcription factors has been shown to regulate a wide range of developmental processes, and family members are characterized by the presence of an AP2 DNA binding domain. The conserved core is predicted to form an amphipathic alpha helix that binds DNA. The AP2 domain was first identified in APETALA2, an Arabidopsis protein that regulates meristem identity, floral organ specification, seed coat development, and floral homeotic gene expression. AP2 domains have now been found in a variety of proteins.

[0037] The ODP2 polypeptides useful in the methods of the present disclosure have homology to several polypeptides within the AP2 family, see, for example, US 8420893, which is incorporated herein by reference in its entirety. Figure 1 , provides an alignment of maize and rice ODP2 polypeptides with eight other proteins that have two AP2 domains. Figure 1 The consensus sequence of all proteins appearing in the alignment of US 8420893 is also provided in. The polypeptide comprising two AP2-DNA binding domains that can be used in the disclosed methods can be obtained or derived from any plant described herein. In one aspect, the polypeptide comprising two AP2-DNA binding domains that can be used in the disclosed methods is an ODP2 polypeptide. In one aspect, the polypeptide comprising two AP2-DNA binding domains that can be used in the disclosed methods is a BBM2 polypeptide. The ODP2 polypeptide and BBM2 polypeptide that can be used in the disclosed methods can be obtained or derived from any plant described herein.

[0038] The morphogenetic gene can be stably incorporated into the genome of the plant or can be transiently expressed. On the one hand, the expression of the morphogenetic gene is controlled. The controlled expression can be a pulse expression of the morphogenetic gene in a specific period. Alternatively, the morphogenetic gene can be expressed only in certain transformed cells and not in other cells. The expression of the morphogenetic gene can be controlled by a variety of methods disclosed below. The morphogenetic gene that can be used for the disclosed method can be obtained from or derived from any plant species described herein.

[0039] 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 progeny thereof. Plant cells may 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 cell includes but is not limited to from following cell: seed, suspension culture, explant, immature embryo, embryo, zygotic embryo, somatic embryo, embryogenic callus, meristem, somatic meristem, organogenic callus, protoplast, embryo derived from mature ear derived seed, leaf base, leaf of mature plant, leaf tip, immature inflorescence, ear, immature ear, long whisker, cotyledon, immature cotyledon, hypocotyl, meristem region, callus, leaf cell, stem cell, root cell, bud cell, gametophyte, sporophyte, pollen and microspore.Plant part comprises differentiation and undifferentiated tissue, includes but is not limited to the cell (for example unicellular, protoplast, embryo and callus) in the cultivation of root, stem, bud, leaf, pollen, seed, tumor tissue and various forms.Plant tissue can be in plant or in plant organ, tissue or cell culture.Grain means the mature seed produced by commercial grower for the purpose outside cultivation or breeding species. Progeny, variants and mutants of the regenerated plants are also included within the scope of the present disclosure, provided that such progeny, variants and mutants contain the introduced polynucleotide.

[0040] The present disclosure can be used for transformation of any plant species, including but not limited to monocots and dicots. Monocots include, but are not limited to, barley, maize (corn), millet (e.g., pearl millet (Echinochloa crusgalli), pig millet (Panicum miliaceum), foxtail millet (Setaria italica), longshan millet (Eleusinecoracana), teff (Eragrostis tef), oats, rice, rye, Setaria species, sorghum, triticale, or wheat, or leaf and stem crops, including, but not limited to, bamboo, beach grass, bluegrass, reed, ryegrass, sugarcane; lawn grass, ornamental grasses and other grasses such as switchgrass and turf grass. Alternatively, dicots used in the present disclosure include, but are not limited to, kale, cauliflower, broccoli, mustard plants, cabbage, peas, clover, alfalfa, beans, tomatoes, peanuts, cassava, soybeans, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or cotton.

[0041] Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica species (e.g., Brassica napus, Brassica rapa, Brassica juncea), particularly those Brassica species that can be used as a source of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), foxtail millet (Setaria italica), millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max, tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananascomosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Betavulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals and conifers.

[0042] The present disclosure can use higher plants, such as the classes of angiosperms and gymnosperms. Plants suitable for suitable species of the present disclosure can be from Acanthaceae, Alliumceae, Alstroemeriaceae, Amaryllisceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Carnosaceae, Cruciferae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicumaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Cocaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanophyllaceae, Musaceae, Myrtaceae, Blue Fruit Tree Family, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae and Vitaceae. Plants from members of the following genera can be used in the methods of the present disclosure: okra, abies, maple, bentgrass, allium, alstroemeria, pineapple, andrographis, andrographis, artemisia, arundo, belladonna, berberis, beta, redwood, brassica, calendula, camellia, camptotheca, cannabis, capsicum, carthamus, vinca, cephalotaxus, chrysanthemum, cinchona, citrus, coffee, colchicum, coleus, cucumis, pumpkin, bermudagrass, datura, dianthus, digitalis, yam, oil palm, ephedra, saccharum, coca, eucalyptus, fescue, strawberry, snowdrop, soybean, cotton, , sunflower, rubber tree, barley, henbane, jatropha, lettuce, flax, tarragon, lupin, tomato, lycopodium, cassava, alfalfa, mint, miscanthus, banana, tobacco, rice, millet, poppy, guayule, pennisetum, petunia, phalaenopsis, timothy, pine, bluegrass, poinsettia, poplar, rauwolfia, castor, rose, sugarcane, willow, bloodroot, hyssop, secale, solanum, sorghum, rice grass, spinach, chrysanthemum, yew, theobroma, triticale, wheat, spike grass, Veratrum, periwinkle, grape and maize.

[0043] Plants that are 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, e.g., Panicum virgatum (switchgrass), Miscanthus giganteus (miscanthus), Saccharum spp. (sugarcane, energycane), Populus balsamifera (poplar), cotton (Gossypium barbadense, Gossypium hirsutum), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), sorghum (Sorghum bicolor, Sorghum vulgare), Erianthus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.spp.) (Eucalyptus, including E. grandis (and its hybrids, known as "urograndis"), E. globulus, E. camaldulensis, E. tereticornis, E. viminalis, E. nitens, E. saligna and E. urophylla), Triticosecales spp. (Triticum - wheat x rye), Teff (Eragrostis tef), bamboo, Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax), Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Phaseolus vulgaris (green beans), Phaseolus limensis (lima beans), Lathyrus spp. (peas), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica spp. (B. napus (canola), B. rapa, B. juncea), Brassica oleracea)(broccoli, cauliflower, brussel sprouts), Camellia sinensis(tea), Fragaria ananassa(strawberry), Theobroma cacao(cocoa), Coffeaarabica)(coffee), Vitis vinifera)(grape), Ananas comosus)(pineapple), Capsicum annum)(hot & sweet pepper), Arachis hypogaea)(peanuts), Ipomoea batatus)(sweet potato), Coconut (Cocos nucifera)(coconut), Citrus spp.(citrus trees), Avocado (Persea americana)(avocado), Fig (Ficus casica), Guava (Psidium guajava), Mango (Mangiferaindica), Olive (Olea europaea), Papaya (Carica papaya)(papaya), Cashew (Anacardium occidentale (cashew), Macadamia integrifolia (macadamia tree), Prunus amygdalus (almond), Allium cepa (onion), Cucumis melo (musk melon), Cucumis sativus (cucumber), Cucumis cantalupensis (cantaloupe), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Cyamopsistetragonoloba (guar bean), Ceratoniasiliqua)(locust bean), Trigonella foenum-graecum(fenugreek), Vigna radiata(mung bean), Vigna unguiculata(cowpea), Vicia faba(fava bean), Cicer arietinum(chickpea), Lensculinaris(lentil), Papaver somniferum(opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis sativa, Camptotheca acuminate, Catharanthus roseus roseus, Vinca rosea, Cinchona officinalis, Colchicum autumnale, Veratrum califomica., Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedrasinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum) (Huperzia serrata, Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinariacanadensis), Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Menthapiperita (mint), Bixa orellana (achiote), Alstroemeria spp., Rosa spp. (rose), Rhododendron spp. (azalea), Macrophylla hydrangea (hydrangea), Hibiscus rosasanensis (hibiscus), Tulipa spp. (tulips), Narcissus spp. (daffodils), Petunia hybrida (petunias), Dianthus caryophyllus (carnation), Euphorbia pulcherrima (poinsettia), chrysanthemum, Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa annua (Poa spp.),pratensis) (bluegrass), Lolium spp. (ryegrass), Phleum pratense (timothy), and conifers.

[0044] Conifers can be used in the present disclosure and include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), jack pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Eastern or Canadian hemlock (Tsuga canadensis); Western hemlock (Tsuga heterophylla); Mountain hemlock (Tsuga mertensiana); American larch or larch (Larch); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); firs such as silver fir (Abies avellana); amabilis) and balsam fir (Abies balsamea); and cedars, such as western arborvitae (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0045] Turfgrasses may be used in the present disclosure and include, but are not limited to: annual bluegrass (Poa annua), annual ryegrass (Lolium multiflorum), Canada bluegrass (Poa canadensis), colonial bentgrass (Agrostistenuis), creeping bentgrass (Agrostis palustris), wheatgrass (Agrostis serrata), flat-eared wheatgrass (Agrostis palustris), hard fescue (Festuca longifolia), Kentucky bluegrass (Poa canadensis), orchardgrass (Timothy), perennial ryegrass (Lolium perenne), red foxgrass (Festuca australis), small brangrass (White bentgrass), rough-stemmed bluegrass (Poa serrata), sheep fescue (Ghee), smooth brome (Bromegrass serrata), timothy (Phleumpratense), velvet bentgrass (Agrostis serrata), canina), weeping alkaligrass (Puccinellia distans), western wheatgrass (bluestem wheatgrass), St. Augustine grass (Puccinellia distans), Zoysia grass (Zodiaceae species), Bahia grass (Paspalum notatum), Ice grass (carpetgrass), Centipede grass (Earthquake grass), Pennisetum crypticum (ground grass), Seashore paspalum (Sea paspalum), blue gramma (Grammar grass), buffalo grass (Buchloe dactyloids), sideoatsgramma (sideoatsgramma).

[0046] In particular aspects, the plants transformed by the methods of the present disclosure are crop plants (e.g., corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, rice, sorghum, wheat, millet, tobacco, etc.). Special target plants include cereal plants, oilseed plants, and leguminous plants that provide target seeds. Target seeds include cereal seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oilseed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include, but are not limited to, beans and peas. Beans include, but are not limited to, guar beans, locust beans, fenugreek, soybeans, French beans, cowpeas, mung beans, lima beans, broad beans, lentils, and chickpeas.

[0047] 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. A transgenic plant is defined as a mature fertile plant containing a transgene.

[0048] In disclosed method, various plant-derived explants can be used, including immature embryos, 1-5mm zygotic embryos, 3-5mm embryos, and embryos derived from seeds, leaf bases, leaves, leaf tips, immature flowers, tassels, immature fringe, and filaments in mature fringe sources. On the one hand, the explants used in the disclosed method can be derived from seeds, leaf bases, leaves, leaf tips, immature flowers, tassels, immature fringe, and filaments in mature fringe sources. The explants used in the disclosed method can be derived from any plant described herein.

[0049] The present disclosure encompasses isolated or substantially purified nucleic acid compositions. An "isolated" or "purified" nucleic acid molecule or protein or its biologically active portion is substantially free of other cellular materials or components that are usually associated or interacted with the nucleic acid molecule or protein as found in its naturally occurring environment, or substantially free of culture medium when produced by recombinant technology or substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" nucleic acid is substantially free of sequences that are naturally located at the flanks of the nucleic acid (i.e., sequences at the 5' and 3' ends of the nucleic acid) (including protein coding sequences) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various aspects, 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 are naturally located at the flanks of the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. Proteins that are substantially free of cellular material include protein preparations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the protein or biologically active portion thereof used in the methods of the present disclosure is recombinantly produced, optimally, the culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or chemicals other than the protein of interest. The sequences used in the methods of the present disclosure can be isolated from the 5' untranslated regions that flank their respective transcription start sites. The present disclosure includes isolated or substantially purified nucleic acid or protein compositions useful in the methods of the present disclosure.

[0050] As used herein, the term "fragment" refers to a portion of a nucleic acid sequence. The fragment of the sequence used in the method of the present disclosure retains the biological activity of the nucleic acid sequence. Alternatively, the fragment of the nucleotide sequence that can be used as a hybridization probe may not necessarily retain biological activity. A fragment of the nucleotide sequences disclosed herein can 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, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1480, 1490, 1500, 1510, 1525, 1530, 1540, 1551 0, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275, 2300, 2325, 2350, 2375, 24 00, 2425, 2450, 2475, 2500, 2525, 2550, 2575, 2600, 2625, 2650, 2675, 2700, 2725, 2750, 2775, 2800, 2825, 2850, 2875, 2900, 2925, 2950, ​​2975, 3000, 3025, 3050, 3075, 3100, 3125, 3150, 3175, 3200, 3225, 3250, 3275, 3300, 3325, 3350, 3375, 3400, 3425, 3450, 3475, 3500, 3525, 3 550、3575、3600、3625、3650、3675、3700、3725、3750、3775、3800、3825、3850、3875、3900、3925、3950、3975、4000、4025、4050、4075、4100、4125、4150、4175、4200、4225、4250、4275、4300、4325、4350、4375、4400、4425、4450、4475、4500、4525、4550、4575、4600、4625、4650、4675、4700、4725、4750、4775、4800、4825、4850、4875、4900、4925、4950、4975、5000、5025、5050、5075、5100、5125、5150、5175、5200、5225、5250、5275、5300、5325、5350、5375、5400、5425、5450、5475、550 0, 5525, 5550, 5575, 5600, 5625, 5650, 5675, 5700, 5725, 5750, 5775, 5800, 5825, 5850, 5875, 5900, 5925, 5950, 5975, 6000, 6025, 6050, 6075, 6100, 6125, 6150, 6175, 6200 or 6225 nucleotides, and 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 evaluating that portion for activity.

[0051] Also encompassed are fragments and variants of the nucleotide sequences used for the disclosed methods and proteins encoded thereby. As used herein, the term "fragment" refers to a portion of a nucleotide sequence, a portion of a protein or amino acid sequence encoded thereby. A fragment of a nucleotide sequence can encode a protein fragment that retains the biological activity of a native protein. Alternatively, a fragment of a nucleotide sequence as a hybridization probe does not typically encode a fragment protein that retains biological activity. Therefore, the fragment range of a nucleotide sequence can be from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to a full-length nucleotide sequence encoding a protein used for the disclosed methods.

[0052] As used herein, the term "variant" refers to a sequence having substantial similarity to a sequence disclosed herein. The variant is included in the deletion and / or addition of one or more nucleotides or peptides at one or more internal sites in a natural polynucleotide, and / or the replacement of one or more nucleotides or peptides at one or more sites in a natural polynucleotide or polypeptide. As used herein, "natural" nucleotide or peptide sequences include naturally occurring nucleotides or peptide sequences, respectively. With respect to nucleotide sequences, molecular biology techniques well known in the art can be used to identify naturally occurring variants, such as polymerase chain reaction (PCR) and hybridization techniques outlined herein to identify. The biologically active variants of the protein used in the disclosed method can differ from the native protein by as little as 1-15 amino acid residues, as little as 1-10, for example 6-10, as little as 5, as little as 4, 3, 2 or even 1 amino acid residue.

[0053] Nucleotide sequence variants also include artificially synthesized nucleotide sequences, such as those produced by, for example, site-directed mutagenesis techniques. Typically, the variants of the nucleotide sequences disclosed herein will have at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, to 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleotide sequence, as determined by the sequence alignment programs described elsewhere herein using default parameters. Also encompassed are biologically active variants of the nucleotide sequences disclosed herein. Biological activity can be measured using techniques such as northern blot analysis, reporter gene activity measurements obtained from transcriptional fusions, etc. See, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), hereinafter "Sambrook", which is 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), etc., produced under the control of a promoter operably linked to the nucleotide fragment or variant may be measured. See, e.g., Matz et al. (1999) Nature Biotechnology 17:969-973; U.S. Pat. No. 6,072,050, which is incorporated herein by reference in its entirety; Nagai et al., (2002) Nature Biotechnology 20(1):87-90. Nucleotide sequence variants also encompass sequences produced by mutagenesis and recombinogenic procedures such as DNA shuffling. By this program, one or more different nucleotide sequences can be manipulated to produce new nucleotide sequences. In this way, a recombinant polynucleotide library is produced by a colony of related sequence polynucleotides, which comprises sequence regions with basic sequence identity and capable of homologous recombination in vitro or in vivo. The strategy for this DNA reorganization is 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.

[0054] Methods for mutagenesis and nucleotide sequence changes 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. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (Macmillan Publishing Company, New York) and references cited therein, which are incorporated herein by reference in their entirety. Guidance on 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). Conservative substitutions, such as exchanging one amino acid for another with similar properties, will be optimal.

[0055] The nucleotide sequences disclosed herein can be used to isolate corresponding sequences from other organisms (particularly other plants, more particularly other monocots or dicots). In this manner, methods such as PCR, hybridization, etc. can be used to identify such sequences (based on their sequence homology to the sequences set forth herein). Sequences isolated based on sequence identity to the complete sequences set forth herein or to fragments of the complete sequences are encompassed by the present disclosure.

[0056] In the PCR method, oligonucleotide primers can be designed for PCR reactions to amplify the corresponding DNA sequence from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning 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), which are incorporated herein by reference in their entirety. 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, and the like.

[0057] 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 group of cloned genomic DNA fragments or cDNA fragments (i.e., a genome or cDNA library) from a selected organism. These 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, probes for hybridization can be made by synthesizing oligonucleotides based on sequence labels disclosed herein. Methods for preparing hybridization probes and for constructing genomic libraries are generally known in the art and are disclosed in Sambrook (supra).

[0058] For example, the complete sequence disclosed herein, or one or more parts thereof, can be used as probes capable of specific hybridization with corresponding sequences and messenger RNA. To achieve specific hybridization under a variety of conditions, the sequence comprised by such probes is unique in the sequence, and generally its length is at least about 10 nucleotides or its length is at least about 20 nucleotides. Such probes can be used to amplify the corresponding sequence by PCR by the selected plant. This technology can be used to separate other coding sequences from the desired organism, or as a diagnostic assay for determining the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (plaques or colonies, see, for example, Sambrook, supra).

[0059] Hybridization of such sequences can be carried out under stringent conditions. The term "stringent conditions" or "stringent hybridization conditions" means conditions in which the degree of hybridization of a probe to its target sequence is detectably higher (e.g., at least 2 times higher than background) than the degree of hybridization of the probe to other sequences. Stringent conditions are sequence-dependent and will be different in different situations. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequence so that a lower degree of similarity is detected (heterologous detection). The probe length is typically less than about 1000 nucleotides, and optimally, it is less than 500 nucleotides in length.

[0060] Typically, stringent conditions are those under which the salt concentration is less than about 1.5 M sodium ions, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides), and at least about 60° C. for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37° C. using a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1 to 2 times SSC (20 times SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary medium stringency conditions include hybridization at 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and washing in 0.5x to 1x SSC at 55°C to 60°C. Exemplary high stringency conditions include hybridization at 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a final wash at 0.1x SSC at 60°C to 65°C for a duration of at least 30 minutes. The duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours. The duration of washing will be at least a length of time sufficient to reach equilibrium.

[0061] Specificity is typically determined as a function of post-hybridization washes, the key factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the thermal melting point (Tm) can be estimated approximately from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5°C+16.6(log M)+0.41(%GC)-0.61(%form)-500 / L; where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm decreases by approximately 1°C for each 1% of mismatch; therefore, Tm, hybridization, and / or wash conditions can be adjusted to allow hybridization to sequences of the desired identity. For example, if a sequence with 90% identity is sought, the Tm can be reduced by 10°C. Typically, stringent conditions are selected to be about 5°C lower than the Tm of the specific sequence and its complement at a defined ionic strength and pH. However, very stringent conditions can utilize hybridization and / or washing at 1°C, 2°C, 3°C, or 4°C lower than the Tm; moderately stringent conditions can utilize hybridization and / or washing at 6°C, 7°C, 8°C, 9°C, or 10°C lower than the Tm; and low stringency conditions can utilize hybridization and / or washing at 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C lower than the Tm. Using the equation, hybridization and wash compositions, and the desired Tm, one of ordinary skill in the art will understand that variations in the stringency of the hybridization and / or wash solutions are essentially described. If the desired degree of mismatching results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. A comprehensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds., (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York), which are incorporated herein by reference in their entireties. See also, Sambrook supra.Thus, the present disclosure encompasses isolated sequences that are active and that hybridize under stringent conditions to the sequences disclosed herein or fragments thereof.

[0062] In general, sequences that are active and hybridize to the sequences disclosed herein will have at least 40% to 50% homology, about 60%, 70%, 80%, 85%, 90%, 95% to 98% or higher homology to the disclosed sequences. That is, the sequence similarity of the sequences can be within a certain range, sharing at least about 40% to 50%, about 60% to 70%, and about 80%, 85%, 90%, 95% to 98% sequence similarity.

[0063] The following terms are used to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity."

[0064] As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence.

[0065] As used herein, a "comparison window" refers to a continuous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., spaces) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Typically, the length of the comparison window is at least 20 consecutive nucleotides, and optionally can be 30, 40, 50, 100 or longer. It will be appreciated by those skilled in the art that, due to gaps in the polynucleotide sequence, in order to avoid high similarity with the reference sequence, a gap penalty is typically introduced and subtracted from the number of matches.

[0066] Methods of alignment of sequences for comparison are well known in the art. Thus, determination of percent sequence identity between any two sequences can be performed using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17; the algorithm of Smith et al., (1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2:482; the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453; the algorithm of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 85:2444-2448; the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 872:264, modified version Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:5873-5877, which are incorporated herein by reference in their entirety.

[0067] Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. These implementations include, but are not limited to, the CLUSTAL program in the PC / Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (version 2.0); and the GCG Wisconsin Genetics Software Package. ) version 10 (obtained from Accelrys, Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignment using these programs can be performed using default parameters. The CLUSTAL program is fully described in Higgins et al. (1988) Gene 73:237-244; Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331; the entire contents of which are incorporated herein by reference. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. When comparing amino acid sequences, the ALIGN program can use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The BLAST program of Altschul et al., (1990) J. Mol. Biol. 215:403, incorporated herein by reference in its entirety, is based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleotide sequences encoding proteins of the present disclosure. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to proteins or polypeptides of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389 (incorporated herein by reference in its entirety). Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997), supra. When using BLAST, Gapped BLAST, PSI-BLAST, the default parameters of each program (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the National Center for Biotechnology Information website at www.ncbi.nlmnih.gov.Comparison can also be done by manual inspection.

[0068] Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the values ​​obtained using GAP version 10 using the following parameters: for % identity and % similarity of nucleotide sequences, GAP weight 50 and length weight 3 and nwsgapdna.cmp scoring matrix are used; for % identity and % similarity of amino acid sequences, GAP weight 8 and length weight 2 and BLOSUM62 scoring matrix are used; or any equivalent programs thereof. As used herein, an "equivalent program" is any sequence comparison program that produces an alignment for any two sequences in question that has identical nucleotide or amino acid residue pairings and identical percentage sequence identity when compared to the corresponding alignment produced by GAP version 10.

[0069] The GAP program uses the algorithm of Needleman and Wunsch (supra) to find the comparison of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions, and produces an alignment with the maximum number of matched bases and the least gaps. It allows providing gap generation penalties and gap extension penalties in units of matched bases. GAP must obtain the benefit of the number of gap generation penalty matches for each gap it inserts. If a gap extension penalty greater than zero is selected, GAP must additionally obtain the benefit of the gap length multiplied by the gap extension penalty for each inserted gap. In the GCG Wisconsin Genetics Software Package In version 10 of , the default gap creation penalty values ​​and gap extension penalty values ​​for protein sequences are 8 and 2, respectively. For nucleotide sequences, the default gap creation penalty is 50, while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as integers selected from the group consisting of 0 to 200. Thus, for example, the gap creation penalty and the gap extension penalty can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more.

[0070] GAP represents one member of the best alignment family. There may be many members of this family, but the others are not of better quality. GAP displays four performance factors for alignment: quality, ratio, identity and similarity. For aligning sequences, quality is the metric that is maximized. Ratio is the quality divided by the number of bases in the shorter segment. Percent identity is the percentage of symbols that actually match. Percent similarity is the percentage of similar symbols. Symbols opposite gaps are ignored. Similarity is scored when the score matrix value for a pair of symbols is greater than or equal to the similarity threshold of 0.50. GCG Wisconsin Genetics Package The scoring matrix used in Version 10 is BLOSUM62 (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915, incorporated herein by reference in its entirety).

[0071] As used herein, "sequence identity" or "identity" in the context of two nucleic acids or polypeptide sequences refers to the same residues in the two sequences when the maximum correspondence is aligned over a specified comparison window. When using the percentage of sequence identity for proteins, it is recognized that non-identical residue positions generally differ by conservative amino acid substitutions, in which amino acid residues are substituted by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and thus do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upward to correct the conservative nature of the substitution. Sequences differing from these conservative substitutions are referred to as having "sequence similarity" or "similarity". Methods for performing this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby improving percentage sequence identity. Therefore, for example, when the same amino acid score is 1, and the score of non-conservative substitutions is zero, the score of conservative substitutions is between zero and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, CA).

[0072] As used herein, "sequence identity percentage" means a value determined by comparing two optimally aligned sequences over a comparison window, wherein the polynucleotide sequence portion in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) to perform optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to produce the sequence identity percentage.

[0073] The term "substantial identity" of a polynucleotide sequence means that, when compared to a reference sequence using an alignment program using standard parameters, the polynucleotide comprises a sequence having at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity. One skilled in the art will recognize that these values ​​can 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, etc. Substantial identity of amino acid sequences for these purposes generally means at least 60%, 70%, 80%, 90%, and at least 95% sequence identity.

[0074] Another indication that nucleotide sequence is substantially identical is whether two molecules hybridize to each other under stringent conditions.Usually, stringent conditions are selected to be about 5 ℃ lower than the Tm of the specified sequence under defined ionic strength and pH.However, stringent conditions encompass temperatures about 1 ℃ to about 20 ℃ of scopes lower than Tm, depending on the required stringency as determined in other parts of this paper.If the polypeptides encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially identical, these nucleic acids remain substantially identical.For example, when the maximum codon degeneracy allowed using genetic coding produces a copy of nucleic acid, this may occur.An indication that two nucleotide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid has immunological cross-reactivity with the polypeptide encoded by the second nucleic acid.

[0075] "Variant" means a substantially similar sequence. For polynucleotides, conservative variants include those sequences that encode an amino acid sequence of one of the target morphogenetic genes and / or genes / polynucleotides disclosed herein due to the degeneracy of the genetic code. Variant polynucleotides also include synthetically derived polynucleotides, such as those produced, for example, by using site-directed mutagenesis but still encoding proteins of the target morphogenetic genes and / or genes / polynucleotides disclosed herein. In general, variants of specific target morphogenetic genes and / or genes / polynucleotides disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the specific target morphogenetic genes and / or genes / polynucleotides, as determined by the sequence alignment programs and parameters described elsewhere herein.

[0076] "Variant" protein means a protein derived from a natural protein by the deletion or addition of one or more amino acids at one or more internal sites in the natural protein, and / or the substitution of one or more amino acids at one or more sites in the natural protein. The variant proteins encompassed by the present disclosure have biological activity, that is, they continue to have the desired biological activity of the natural protein, that is, the polypeptide has a morphogenetic gene and / or gene / polynucleotide of the target activity. Such variants can be produced by, for example, genetic polymorphism or artificial manipulation. The biologically active variants of the target natural morphogenetic gene and / or gene / polynucleotide of the protein disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of the natural protein, as determined by the sequence alignment program and parameters described elsewhere in the text. Biologically active variants of the proteins of the present disclosure may differ from the protein by as few as 1-15 amino acid residues, as few as 1-10, e.g., 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0077] Sequences and genes disclosed herein and variants and fragments thereof can be used for genetic engineering of plants, for example, for producing transformed or transgenic plants to express a target phenotype. As used herein, the terms "transformed plants" and "transgenic plants" refer to plants comprising heterologous polynucleotides in their genomes. Typically, heterologous polynucleotides are stably integrated into the transgenic or transformed plant genome so that the polynucleotides are passed on to successive generations. Heterologous polynucleotides can be integrated into the genome individually or as part of a recombinant DNA construct. It should be understood that, as used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been changed by the presence of heterologous nucleic acids, including those transgenics initially so changed and those produced from the initial transgenic by sexual hybridization or asexual propagation.

[0078] A transgenic "event" is produced by transforming a plant cell with a heterologous DNA construct comprising a nucleic acid expression cassette comprising a gene of interest; regenerating a population of plants resulting from the insertion of the transferred gene into the genome of the plant; and selecting plants characterized by insertion at a specific genomic location. An event is phenotypically characterized by the expression of the inserted gene. At the genetic level, an event is part of the genetic makeup of a plant. The term "event" also refers to offspring resulting from a sexual cross between a transformant and another plant, wherein the offspring comprises the heterologous DNA.

[0079] Transformation protocols and protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocot or dicot). Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include: microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. [European Molecular Biology Association Journal] 3: 2717-2722), and ballistic particle acceleration methods (see, e.g., Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, Gamborg and Phillips, eds. (Springer-Verlag, Berlin) (maize); McCabe et al. (1988) Biotechnology [Biotechnology] 6: 923-926); and Lecl transformation method (WO 00 / 28058). See also, Weissinger et al., (1988) Ann. Rev. Genet. [Annual Review of Genetics] 22: 421-477; Sanford, et al., (1987) Particulate Science and Technology [Microparticle Science and Technology] 5: 27-37 (onion); Christou et al. (1988) Plant Physiol.Plant Physiology 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. [In vitro Cell Developmental Biology] 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. [Theoretical and Applied Genetics] 96: 319-324 (soybean); Datta et al. (1990) Biotechnology [Biotechnology] 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85: 4305-4309 (maize); Klein et al. (1988) Biotechnology [Biotechnology] 6: 559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, Chapman et al., eds. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Biology 5:117-119 (pollen); Reports [Plant Cell Reports] 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.Theoretical and Applied Genetics 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., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); and U.S. Patent Application Publication No. 2017 / 0121722 (rapid plant transformation), all of which are incorporated herein by reference in their entirety. .

[0080] The methods provided herein rely on the use of bacteria-mediated and / or biolistic-mediated gene transfer to produce regenerable plant cells with incorporated target nucleotide sequences. Bacterial strains that can be used in the methods of the present disclosure include, but are not limited to, disarmed Agrobacterium, Ochrobactrum bacteria, or Rhizobiaceae bacteria.

[0081] Detoxified Agrobacterium useful in the methods of the present invention include, but are not limited to, AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-.

[0082] Ochrobacterium bacterial strains that can be used in the methods of the present invention include, but are not limited to, those listed in Table 2.

[0083] Table 2.

[0084]

[0085]

[0086] Rhizobiaceae bacterial strains useful in the methods of the present invention include, but are not limited to, those listed in Table 3 (see also US 9,365,859, which is incorporated herein by reference in its entirety).

[0087] Table 3.

[0088]

[0089]

[0090]

[0091]

[0092] The polynucleotides of the present disclosure can be introduced into plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve incorporating the nucleotide constructs of the present disclosure into viral DNA or RNA molecules. Methods involving viral DNA or RNA molecules for introducing polynucleotides into plants and expressing proteins encoded therein are known in the art. See, for example, 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 entirety.

[0093] The methods of the present disclosure involve introducing a polypeptide or polynucleotide into a plant. As used herein, "introducing" means providing a polynucleotide or polypeptide into a plant in such a way that the sequence is able to enter the interior of a cell of the plant. The methods of the present disclosure do not depend on a specific method for introducing a sequence into a plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing a polynucleotide or polypeptide into a plant are known in the art, including but not limited to stable transformation methods, transient transformation methods, and virus-mediated methods.

[0094] "Stable transformation" is a transformation in which a nucleotide construct or expression cassette introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that a polynucleotide or expression cassette is introduced into the plant and does not integrate into the genome of the plant, or that a polypeptide is introduced into the plant.

[0095] Reporter genes or selectable marker genes can also be included in the expression cassettes disclosed herein and used in the disclosed methods. Examples of suitable reporters known in the art can be found in, for example, Jefferson, et al., (1991) 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) Bio Techniques 19: 650-655 and Chiu et al., (1996) Current Biology 6: 325-330, incorporated herein by reference in their entirety.

[0096] Selectable markers include DNA segments that allow people to identify or select molecules or cells containing it, usually under specific conditions, or to select for it. These markers can encode activities such as, but not limited to, the production of RNA, peptides or proteins, or can provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, etc. Examples of selectable markers include, but are not limited to, DNA segments comprising restriction endonuclease sites; DNA segments encoding products that confer resistance to other toxic compounds (e.g., antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT)); DNA segments encoding products that are otherwise lacking in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that can be easily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cell surface proteins); generating new primer sites for PCR (e.g., juxtaposition of two DNA sequences that were not previously juxtaposed), comprising DNA sequences that are inactivated or activated by restriction endonucleases or other DNA modifying enzymes, chemicals, etc.; and comprising DNA sequences required for specific modifications (e.g., methylation) that allow their identification.

[0097] Selectable marker genes for selecting transformed cells or tissues may include genes that confer antibiotic resistance or herbicide resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al., (1983) EMBO J. [Journal of the European Molecular Biology Association] 2: 987-992); methotrexate (Herrera Estrella et al., (1983) Nature [Nature] 303: 209-213; Meijer et al., (1991) Plant Mol. Biol. [Plant Molecular Biology] 16: 807-820); hygromycin (Waldron et al., (1985) Plant Mol. Biol. [Plant Molecular Biology] 5: 103-108 and Zhijian et al., (1995) Plant Mol. Biol. [Plant Molecular Biology] 5: 103-108); Science 108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176); sulfonamides (Guerineau et al. (1990) Plant Mol. Biol. 7:171-176); Mol. Biol. [Plant Molecular Biology] 15: 127-36); bromoxynil (Stalker et al., (1988) Science [Science] 242: 419-423); glyphosate (Shaw et al., (1986) Science [Science] 233: 478-481 and U.S. Patent Application Serial Nos. 10 / 004,357 and 10 / 427,692); phosphinothricin (DeBlock et al., (1987) EMBO J. [Journal of the European Molecular Biology Association] 6: 2513-2518), which are incorporated herein by reference in their entirety.

[0098] Selectable markers that confer resistance to herbicidal compounds include genes encoding resistance and / or tolerance to herbicidal compounds, such as glyphosate, sulfonylureas, glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). See generally Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) PhD thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. [Molecular and Cellular Biology] 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. [Nucleic Acids Research] 19: 4647-4653; Hillen and Wissman (1989) Topics Mol. Struc. Biol. [Topics in Molecular and Structural Biology] 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.Antimicrob Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) PhD thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334: 721-724. Such disclosures are incorporated herein by reference.

[0099] Certain selectable markers useful in the methods of the invention include, but are not limited to, the maize HRA gene that confers resistance to sulfonylureas and imidazolinones (Lee et al., 1988, EMBO J [Journal of the European Molecular Biology Association] 7: 1241-1248), the GAT gene that confers resistance to glyphosate (Castle et al., 2004, Science [Science] 304: 1151-1154), genes that confer resistance to spectinomycin, such as the aadA gene (Svab et al., 1990, Plant Mol Biol. [Plant Molecular Biology] 14: 197-205), and the bar gene that confers resistance to glufosinate (White et al., 1990, Nucl. Acids Res. [Nucleic Acids Research] 25: 1062), as well as PAT (or moPAT for maize, see Rasco-Gaunt et al., 2003, Plant Cell Biol. [Nucleic Acids Research] 25: 1062). [Plant Cell Reports] 21: 569-76) and the PMI gene that allows growth on mannose-containing media (Negrotto et al., 2000, Plant Cell Rep. [Plant Cell Reports] 22: 684-690) are very useful for rapid selection during the short elapsed time covered by somatic embryogenesis and embryo maturation in this method. However, the percentage of wild-type escapees may vary depending on the selection marker used and the crop, inbred line or variety being transformed. In corn and sorghum, the HRA gene is effective in reducing the frequency of wild-type escapees.

[0100] Other genes that can be used to restore transgenic events would include, but are not limited to, examples such as GUS (β-glucuronidase; Jefferson, (1987) Plant Mol. Biol. Rep. 5:387), GFP (green fluorescent protein; Chalfie et al., (1994) Science 263:802), luciferase (Riggs et al., (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen et al., (1992) Methods Enzymol. 216:397-414), various fluorescent proteins with alternating emission optima spanning far red, red, orange, yellow, green, cyan and blue (Shaner et al., 2005, Nature 55:114), and fluorescent proteins with alternating emission optima spanning far red, red, orange, yellow, green, cyan and blue (Shaner et al., 2005, Nature 55:115). Examples include genes encoding anthocyanin production in maize (Ludwig et al., (1990) Science 247:449), which are incorporated herein by reference in their entirety.

[0101] The above list of selectable markers is not intended to be limiting.Any selectable marker can be used in the methods of the present disclosure.

[0102] On the one hand, the disclosed method provides a transformation method that allows positive growth selection. It will be appreciated by those skilled in the art that conventional plant transformation methods rely mainly on negative selection schemes as described above, in which antibiotics or herbicides (negative selection agents) are used to inhibit or kill untransformed cells or tissues, and transgenic cells or tissues continue to grow due to the expression of resistance genes. In contrast, the disclosed method can be used without applying a negative selection agent. Therefore, although wild-type cells can grow unhindered, by comparison, cells affected by the controlled expression of morphogenic genes can be easily identified because their growth rate relative to the surrounding wild-type tissues is accelerated. In addition to simply observing faster growth, the disclosed method also provides transgenic cells that exhibit faster morphogenesis relative to non-transformed cells. Therefore, this differential growth and morphogenesis development can be used to easily distinguish transgenic plant structures from surrounding non-transformed tissues, and the process is referred to as "positive growth selection" in this article.

[0103] The present disclosure provides methods for producing transgenic plants with increased efficiency and speed using a variety of starting tissue types in multiple inbred lines and providing significantly higher transformation frequencies and significantly higher quality events (events containing one copy of a trait gene expression cassette without a vector (plasmid) backbone), the inbred lines including transformed inbred lines representing a range of genetic diversity and having important commercial uses. The disclosed methods may further include polynucleotides that provide improved traits and characteristics. The disclosed methods may further include polynucleotides that provide improved traits and characteristics.

[0104] As used herein, "trait" refers to the physiological, morphological, biochemical, or physical characteristics of a plant or a particular plant material or cell. In some cases, this feature is visible to the human eye, such as seed or plant size; or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of a seed or leaf; or by observing metabolic or physiological processes, such as by measuring the uptake of carbon dioxide; or by observing the expression level of one or more genes, such as by adopting Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems; or by agronomic observations such as stress tolerance, yield, or pathogen tolerance.

[0105] In addition to using traditional breeding methods, agronomically important traits (e.g., oil, starch, and protein content) can also be altered genetically. Modifications include increasing the content of oleic acid, saturated and unsaturated oils, increasing the levels of lysine and sulfur, providing essential amino acids, and also modifications to starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, which are incorporated herein by reference. Another example is a lysine- and / or sulfur-rich seed protein encoded by soybean 2S albumin described in U.S. Pat. No. 5,850,016, and a chymotrypsin inhibitor from barley described by Williamson et al. (1987) Eur. J. Biochem. [European Journal of Biochemistry] 165: 99-106, the disclosures of which are incorporated herein by reference.

[0106] Derivatives of the coding sequence can be made by site-directed mutagenesis to increase the level of a preselected amino acid in the encoded polypeptide. For example, methionine-rich plant proteins such as those from sunflower seeds (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, Applewhite, ed. (American Oil Chemists Society, Champaign, Ill., pp. 497-502; incorporated herein by reference); corn (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both incorporated herein by reference); and rice (Musumura et al. (1989) Plant Mol. Biol. 12:123, incorporated herein by reference) can be used. Other agriculturally important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

[0107] Many agronomic traits can affect "yield", including but not limited to plant height, number of peas, position of peas on the plant, number of internodes, incidence of pea crushing, particle size, efficiency of node and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stresses, carbon assimilation, plant structure, resistance to lodging, percentage of seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include: germination efficiency (including germination under stress conditions), growth rate (including growth rate under stress conditions), number of spikes, number of seeds per spike, seed size, composition of seeds (starch, oil, protein), and characteristics of seed filling. Also of interest is the generation of transgenic plants that exhibit desirable phenotypic characteristics that may or may not confer an increase in total plant yield. Such characteristics include enhanced plant morphology, plant physiology, or improved components of mature seeds harvested from transgenic plants.

[0108] The "increased yield" of the transgenic plants of the present disclosure can be demonstrated and measured in many ways, including bulk density, number of seeds per plant, seed weight, number of seeds per unit area (i.e., seeds per acre, or seed weight), bushels / acre, tons / acre, kilograms / hectare. For example, maize yield can be measured as the yield of shelled corn kernels per unit production area, typically reported based on moisture adjustment (e.g., 15.5% moisture), for example, in bushels / acre or metric tons / hectare. The increased yield can be due to increased utilization of key biochemical compounds (e.g., nitrogen, phosphorus, and carbohydrates), or due to increased tolerance to environmental stresses (e.g., cold, heat, drought, salt, and pest or pathogen attack). Trait-enhanced recombinant DNA can also be used to provide transgenic plants with improved growth and development and ultimately increased yield as a result of altered expression of plant growth regulators or changes in the cell cycle or photosynthesis pathways.

[0109] "Enhanced traits" as used to describe various aspects of the present disclosure include improved or enhanced water use efficiency or drought tolerance, osmotic stress tolerance, high salinity stress tolerance, heat stress tolerance, enhanced cold tolerance (including germination cold tolerance), increased yield, improved seed quality, increased nitrogen use efficiency, earlier plant growth and development, delayed plant growth and development, enhanced seed protein, and enhanced seed oil production.

[0110] Any target polynucleotide or trait gene can be used in the method of the present disclosure. Various changes of the phenotype conferred by the target gene or trait gene include changes for changing the fatty acid composition in the plant, changing the amino acid content, starch content or carbohydrate content of the plant, changing the pathogen defense mechanism of the plant, changing the grain size, changing the sucrose loading, etc. The target gene or trait gene can also participate in regulating the influx of nutrients, and participate in regulating the expression of phytate genes, particularly reducing the phytic acid level in the seed. These results can be achieved by providing the expression of heterologous products or increasing the expression of endogenous products in the plant. Alternatively, the results can be achieved by providing one or more endogenous products, particularly the expression of enzymes or coenzyme factors in the plant is reduced. These changes lead to changes in the phenotype of the transformed plant.

[0111] As used herein, "trait gene" refers to a gene of interest that is integrated into a plant genome to produce a fertile transgenic TO plant. Selectable markers such as high-resistance acetolactate synthase (HRA) and visual markers such as fluorescent protein genes are used herein by way of example. Trait genes can be any gene (or combination of genes) that imparts an agronomic, physiological, biochemical or physical phenotype that increases value.

[0112] The target gene / trait gene reflects the commercial market and benefits of those genes involved in crop development. Target crops and markets change, and as developing countries open international markets, new crops and technologies will also appear. In addition, as the understanding of agronomic traits and characteristics (such as yield and hybrid vigor) increases, the selection of genes and traits for conversion will also change accordingly. The general category of the target nucleotide sequence or gene or trait gene for the disclosed method includes, for example, those genes related to information (such as zinc fingers), those genes related to communication (such as kinases), and those genes related to housekeeping (such as heat shock proteins). The more specific category of transgenic, for example, includes genes encoding important traits of agronomy, insect resistance, disease resistance, herbicide resistance, sterility, environmental stress resistance (tolerance changes to cold, salt, drought, etc.), cereal characteristics and commercial products.

[0113] Heterologous coding sequences, heterologous polynucleotides and target polynucleotides expressed by promoter sequences transformed by the methods disclosed herein can be used to change the phenotype of plants. Various changes in phenotype are of interest, including expression of modified genes in plants, changing plant defense mechanisms to pathogens or insects, increasing plant tolerance to herbicides, changing plant development in response to environmental stresses, regulating plant responses to salt, temperature (heat and cold), drought, etc. These results can be obtained by expressing a target heterologous nucleotide sequence comprising an appropriate gene product. In a particular aspect, the target heterologous nucleotide sequence is a plant endogenous sequence whose expression level is increased in a plant or plant part. Results can be obtained by providing expression of one or more endogenous gene products (particularly hormones, receptors, signaling molecules, enzymes, transporters or cofactors) that are changed or by affecting nutrient uptake in plants. These changes result in changes in the phenotype of the transformed plant. Still other transgenic species include genes that induce the expression of exogenous products (such as enzymes, cofactors and hormones) from plants and other eukaryotic organisms and prokaryotes.

[0114] It is recognized that any gene of interest, polynucleotide of interest, trait gene of interest, or multiple genes / polynucleotides / traits can be operably linked to one or more promoters and expressed in plants transformed by the methods disclosed herein, such as an insect resistance trait gene that can be stacked with one or more other input trait genes (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, etc.) or output trait genes (e.g., increased yield, modified starch, improved oil profile, amino acid balance, high lysine or methionine, improved digestibility, improved fiber quality, drought resistance, etc.).

[0115] The promoter can be operably linked to an agronomically important trait gene for expression in a plant transformed by the methods disclosed herein that affects the quality of grains, such as the level and type of saturated and unsaturated oils (increased oleic acid content), the quality and quantity of essential amino acids, increased levels of lysine and sulfur, levels of cellulose, and starch and protein content. The promoter can be operably linked to a trait gene that provides for modification of the hordothionin protein for expression in a plant transformed by the methods disclosed herein, which are described in U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and 5,703,049; these patents are incorporated herein by reference in their entirety. Another example of a trait gene that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein is the soybean 2S albumin encoding a lysine- and / or sulfur-rich seed protein described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, Williamson et al., (1987) Eur. J. Biochem 165:99-106, the disclosure of which is incorporated herein by reference in its entirety.

[0116] The promoter can be operably linked to an insect resistance trait gene for expression in plants transformed by the methods disclosed herein, the insect resistance trait gene encoding resistance to yield-affecting pests such as rootworms, cutworms, European corn borer, etc. Such genes 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 entirety. Genes encoding disease resistance traits that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein include, for example, detoxification genes, such as those 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 herein by reference in their entirety.

[0117] Herbicide resistance trait genes that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein include genes encoding resistance to herbicides that inhibit acetolactate synthase (ALS), particularly sulfonylurea herbicides (e.g., acetolactate synthase (ALS) genes containing mutations that confer such resistance (particularly S4 and / or Hra mutations)), genes encoding resistance to herbicides that inhibit glutamine synthase (e.g., gluconate or basta (e.g., bar gene)), genes encoding resistance to glyphosate (e.g., EPSPS genes and GAT genes; see, e.g., U.S. Patent Application Publication Nos. 2004 / 0082770, WO 03 / 092360, and WO 05 / 012515, which are incorporated herein by reference in their entireties), or other such genes known in the art. The bar gene encodes resistance to the herbicide basast, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene mutant encodes resistance to the herbicide chlorsulfuron, any and all of which can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein.

[0118] Glyphosate resistance is conferred by mutant 5-enolpyruvate-3-phosphate synthase (EPSPS) and aroA genes, which can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein. See, for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses nucleotide sequences in the form of EPSPS that confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry et al. also describes genes encoding EPSPS enzymes, which can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein. See also, 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; 5,633,448; 5,510,471; Re. 36,449; RE 37,287E and 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 entirety. Glyphosate resistance is also conferred to plants expressing a gene that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein, the gene encoding a glyphosate oxidoreductase as described more fully in U.S. Patent Nos. 5,776,760 and 5,463,175 (which are incorporated herein by reference in their entirety). Glyphosate resistance can also be conferred to plants by overexpression of a gene encoding a glyphosate N-acetyltransferase that is operably linked to a promoter for expression in plants transformed by the methods disclosed herein. See, for example, U.S. Patent Application Publication Nos. 2004 / 0082770, WO 03 / 092360 and WO05 / 012515, which are incorporated herein by reference in their entirety.

[0119] The sterile gene operably connected to a promoter to be expressed in a plant transformed by the method disclosed herein can also be encoded in a DNA construct and provide an alternative method for physical emasculation. Examples of genes used in this manner include male tissue preferred genes and genes (such as QM) with male sterile phenotypes described in U.S. Patent No. 5,583,210 (incorporated herein by reference in their entirety). Other genes operably connected to a promoter to be expressed in a plant transformed by the method disclosed herein include kinases and those encoding compounds toxic to male or female gametophyte development.

[0120] Commercial traits can also be encoded by one or more genes operably linked to a promoter for expression in plants transformed by the methods disclosed herein, which can increase the expression of starch for ethanol production or provide proteins, for example. Another important commercial use of transformed plants is the production of polymers and bioplastics, such as described in U.S. Pat. No. 5,602,321 (incorporated herein by reference in its entirety). Genes that promote the expression of polyhydroxyalkanoates (PHAs), such as beta ketothiolase, PHB enzyme (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase, can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein (see Schubert et al., (1988) J. Bacteriol. [Journal of Bacteriology] 170: 5837-5847, which is incorporated herein by reference in its entirety).

[0121] Examples of other suitable trait genes and their associated phenotypes that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein include genes encoding viral coat proteins and / or RNA or other viral genes or plant genes that confer viral resistance; genes that confer fungal resistance; genes that promote increased yield; and genes that provide resistance to stresses such as cold, dehydration due to drought, heat and salinity, toxic metals or trace elements, and the like.

[0122] By way of illustration, and not intended to be limiting, the following is a list of additional examples of the types of trait genes that can be operably linked to a promoter for expression in plants transformed by the methods disclosed herein.

[0123] 1. Trait genes that confer resistance to insects or diseases and encode:

[0124] (A) Plant disease resistance genes. Plant defenses are generally activated by specific interactions between the products of disease resistance genes (R) in plants and the products of corresponding avirulence (Avr) genes in pathogens. Plant varieties can be transformed with cloned resistance genes to engineer plants that are resistant to specific pathogen strains. See, e.g., Jones et al., (1994) Science 266:789 (cloning of the tomato Cf-9 gene for resistance to Cradosporium fulvum); Martin et al., (1993) Science 262:1432 (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encoding a protein kinase); Mindrinos et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae); McDowell and Woffenden, (2003) Trends Biotechnol. 21(4):178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82, each of which is incorporated by reference in its entirety. Disease-resistant plants are more resistant to pathogens than wild-type plants.

[0125] (B) Bacillus thuringiensis proteins, derivatives thereof, or synthetic polypeptides modeled thereon. See, e.g., Geiser et al., (1986) Gene 48:109, which discloses the cloning and nucleotide sequence of a Bt delta-endotoxin gene. In addition, DNA molecules encoding delta-endotoxin genes can be purchased from the American Type Culture Collection (Rockville, Md., USA), e.g., under ATCC Accession Nos. 40098, 67136, 31995, and 31998. Other examples of genetically engineered Bacillus thuringiensis transgenes are given in the following patents and patent applications and are incorporated herein by reference for this purpose: U.S. Patent Nos. 5,188,960; 5,689,052; 5,880,275; WO 91 / 14778; WO 99 / 31248; WO 01 / 12731; WO 99 / 24581; WO 97 / 40162 and U.S. Application Serial Nos. 10 / 032,717; 10 / 414,637 and 10 / 606,320, which are incorporated herein by reference in their entirety.

[0126] (C) Insect-specific hormones or pheromones, such as ecdysone and juvenile hormone, variants thereof, mimetics based thereon, or antagonists or agonists thereof. See, e.g., Hammock et al., (1990) Nature 344:458, which discloses baculovirus expression of cloned juvenile hormone esterase (an inactivator of juvenile hormone), which is incorporated herein by reference in its entirety.

[0127] (D) Insect-specific peptides that, when expressed, disrupt the physiology of the affected pest. See, for example, the following disclosures: Regan, (1994) J. Biol. Chem. 269:9 (expression cloning yields DNA coding for insect diuretic hormone receptor); Pratt et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin is identified in Diploptera puntata); Chattopadhyay et al., (2004) Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J Nat Rev. 55(1):33-54. Prod [Journal of Natural Products] 67(2): 300-310; Carlini and Grossi-de-Sa, (2002) Toxicon [Toxin] 40(11): 1515-1539; Ussuf et al., (2001) Curr Sci. [Current Science] 80(7): 847-853; and Vasconcelos and Oliveira, (2004) Toxicon [Toxin] 44(4): 385-403; the entireties of which are incorporated herein by reference. See also U.S. Pat. No. 5,266,317 to Tomalski et al., which discloses genes encoding insect-specific toxins, the entirety of which is incorporated herein by reference.

[0128] (E) An enzyme that causes hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoid derivative, or another non-protein molecule having insecticidal activity.

[0129] (F) enzymes involved in the modification (including post-translational modification) of biologically active molecules; for example, glycolytic enzymes, proteolytic enzymes, lipolytic enzymes, nucleases, cyclases, transaminases, esterases, hydrolases, phosphatases, kinases, phosphorylases, polymerases, elastases, chitinases and glucanases, whether natural or synthetic. See, PCT Application No. WO 93 / 02197 to Scott et al., which discloses the nucleotide sequence of a callase gene, which document is incorporated herein by reference in its entirety. DNA molecules containing chitinase coding sequences can be obtained, for example, from ATCC Accession Nos. 39637 and 67152. See also Kramer et al., (1993) Insect Biochem. Molec. Biol. 23:691, which teaches the nucleotide sequence of a cDNA encoding a tobacco hookworm chitinase, and Kawalleck et al., (1993) Plant Molec. Biol. 21:673, which provides the nucleotide sequence of a parsley ubi4-2 polyubiquitin gene, U.S. patent application serial numbers 10 / 389,432, 10 / 692,367, and U.S. Patent No. 6,563,020, which are incorporated herein by reference in their entireties.

[0130] (G) Molecules that stimulate signal transduction. See, for example, Botella et al., (1994) Plant Molec. Biol. 24:757, which discloses the nucleotide sequence of a mung bean calmodulin cDNA clone; and Griess et al., (1994) Plant Physiol. 104:1467, which provides the nucleotide sequence of a maize calmodulin cDNA clone; the entire contents of which are incorporated herein by reference.

[0131] (H) Hydrophobic transient peptides. See, PCT Application No. WO 95 / 16776 and U.S. Pat. No. 5,580,852 (disclosing peptide derivatives of supu peptides (which inhibit fungal plant pathogens)) and PCT Application No. WO 95 / 18855 and U.S. Pat. No. 5,607,914 (teaching synthetic antimicrobial peptides that confer disease resistance); the documents are incorporated herein by reference in their entirety.

[0132] (I) a membrane permease, a channel former or channel blocker. See, for example, Jaynes et al., (1993) Plant Sci. 89:43, which discloses heterologous expression of a cecropin-β cleavage peptide analog to provide transgenic tobacco plants resistant to Pseudomonas solanacearum; the document is incorporated herein by reference in its entirety.

[0133] (J) Viral invasive proteins or complex toxins derived therefrom. For example, accumulation of viral coat proteins in transformed plant cells confers resistance to viral infection and / or disease development caused by viruses derived from foreign protein genes and by related viruses. See, Beachy et al., (1990) Ann. Rev. Phytopathol. 28:451, which is incorporated herein by reference in its entirety. Coat protein-mediated resistance has conferred transformed plants resistance to alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus, and tobacco mosaic virus. Same as above.

[0134] (K) Insect-specific antibodies or immunotoxins derived therefrom. Thus, antibodies targeting key metabolic functions in the insect gut will inactivate the affected enzymes, killing the insects. See Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzyme inactivation in transgenic tobacco by production of single-chain antibody fragments); which document is incorporated herein by reference in its entirety.

[0135] (L) Virus-specific antibodies. See, e.g., Tavladoraki et al., (1993) Nature 366:469, which shows that transgenic plants expressing recombinant antibody genes are not attacked by viruses, and which is incorporated herein by reference in its entirety.

[0136] (M) A developmental-arrestive protein produced in nature by a pathogen or parasite. Thus, fungal α-1,4-D-polygalacturonase promotes fungal colonization and plant nutrient release by dissolving plant cell walls. See, Lamb et al., (1992) Bio / Technology 10:1436, which is incorporated herein by reference in its entirety. Toubar et al., (1992) Plant J. 2:367, which describes the cloning and characterization of a gene encoding a bean polygalacturonase inhibitory protein, which is incorporated herein by reference in its entirety.

[0137] (N) A developmental arrest protein produced by a plant in nature. For example, Logemann et al. (1992) Bio / Technology 10:305, which is incorporated herein by reference in its entirety, have shown that transgenic plants expressing a barley ribosome inactivating gene have increased resistance to fungal diseases.

[0138] (O) Genes involved in the systemic acquired resistance (SAR) response and / or pathogenesis-related genes. Briggs, (1995) Current Biology 5(2): 128-131, Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4): 456-64 and Somssich, (2003) Cell 113(7): 815-6, which are incorporated herein by reference in their entirety.

[0139] (P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol. 101: 709-712 and Parijs et al., (1991) Planta 183: 258-264 and Bushnell et al., (1998) Can. J. of Plant Path. 20(2): 137-149. See also U.S. Patent Application No. 09 / 950,933, which is incorporated herein by reference in its entirety.

[0140] (Q) Detoxification genes, such as genes for fumonisin, beauvericin, candida albicans, and zearalenone and structurally related derivatives thereof. See, e.g., U.S. Pat. No. 5,792,931, which is incorporated herein by reference in its entirety.

[0141] (R) Cystatins and cysteine ​​protease inhibitors. See U.S. Application Serial No. 10 / 947,979, which is incorporated herein by reference in its entirety.

[0142] (S) Defensin genes. See WO 03 / 000863 and US Application Serial No. 10 / 178,213, which are incorporated herein by reference in their entirety.

[0143] (T) Genes that confer resistance to nematodes. See WO 03 / 033651, and Urwin et al., (1998) Planta 204:472-479, and Williamson, (1999) Curr Opin Plant Bio. 2(4):327-31; each of which is incorporated herein by reference in its entirety.

[0144] (U) Genes that confer resistance to anthracnose stalk rot (caused by the fungus Colletotrichum graminearum), such as rcg1. See Jung et al., Generation-means analysis and quantitative trait locus mapping of Anthracnose Stalk Rot genes in Maize, Theor. Appl. Genet. (1994) 89: 413-418, and U.S. Provisional Patent Application No. 60 / 675,664, which are incorporated herein by reference in their entirety.

[0145] 2. Trait genes that confer resistance to herbicides, such as:

[0146] (A) Herbicides that inhibit growing points or meristems, such as imidazolinones or sulfonylureas. Exemplary genes of this class encode mutant ALS and AHAS enzymes, for example, as described in Lee et al. (1988) EMBO J. 7:1241 and Miki et al. (1990) Theor. Appl. Genet. 80:449, respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824 and International Publication WO 96 / 33270, which are incorporated herein by reference in their entirety.

[0147] (B) Glyphosate (resistance conferred by mutant 5-enolpyruvyl-3-phosphoshikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar) genes), as well as pyridyloxy or phenoxypropionic acid and cyclohexanone (ACCase inhibitor encoding genes). See, e.g., U.S. Pat. No. 4,940,835 to Shah et al., which discloses nucleotide sequences in the form of EPSPS that confer resistance to glyphosate. U.S. Pat. No. 5,627,061 to Barry et al. also describes genes encoding EPSPS enzymes. See also, U.S. Patent Nos. 6,566,587; 6,338,961; 6,248,876 B1; 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; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287E and 5,491,288 and International Publication Nos. EP 1173580; WO 01 / 66704; EP 1173581 and EP 1173582, which are incorporated herein by reference in their entirety. Glyphosate resistance is also conferred to plants expressing genes encoding glyphosate oxidoreductase, which is more fully described in U.S. Patents 5,776,760 and 5,463,175 (which are incorporated herein by reference in their entirety). In addition, glyphosate resistance can be conferred to plants by overexpressing genes encoding glyphosate N-acetyltransferase. See, for example, U.S. Patent Application Publication No. 2004 / 0082770, WO 03 / 092360 and WO 05 / 012515, which are incorporated herein by reference in their entirety. DNA molecules encoding mutant aroA genes can be obtained under ATCC Accession No. 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Patent No. 4,769,061 of Comai, which is incorporated herein by reference in its entirety. European Patent Application No. 0333033 to Kumada et al. and US Patent No. 4,975,374 to Goodman et al., which are incorporated herein by reference in their entirety, disclose nucleotide sequences of glutamine synthetase genes that confer resistance to herbicides such as L-phosphinothricin.Leemans et al. European Patent Nos. 0 242 246 and 0 242 236, and De Greef et al., (1989) Bio / Technology 7:61 (which describes the production of transgenic plants expressing a chimeric bar gene encoding phosphinothricin acetyltransferase activity), provide nucleotide sequences of phosphinothricin-acetyl-transferase genes, which are incorporated herein by reference in their entirety. See also, U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 B1 and 5,879,903, which are incorporated herein by reference in their entirety. Exemplary genes that confer resistance to phenoxypropionic acids and cyclohexanones (e.g., sethoxydim and haloxyfop-butyl) are the Acc1-S1, Acc1-S2, and Acc1-S3 genes, which are described in Marshall et al. (1992) Theor. Appl. Genet. 83:435, which is incorporated herein by reference in its entirety.

[0148] (C) Herbicides that inhibit photosynthesis, such as triazines (psbA gene and gs+ gene) and benzonitrile (nitrilase gene). Transformation of Chlamydomonas with a plasmid encoding a mutant psbA gene is described in Przibilla et al., (1991) Plant Cell 3:169, which is incorporated herein by reference in its entirety. The nucleotide sequences of nitrilase genes are disclosed in Stalker, U.S. Pat. No. 4,810,648, which is incorporated herein by reference in its entirety, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441 and 67442. The cloning and expression of DNA encoding glutathione S-transferase is described in Hayes et al., (1992) Biochem. J. 285:173, which is incorporated herein by reference in its entirety.

[0149] (D) Acetohydroxyacid synthase, which has been found to confer resistance to a variety of herbicides on plants expressing the enzyme, has been introduced into various plants (see, e.g., Hattori et al., (1995) Mol Gen Genet. 246:419, which is incorporated herein by reference in its entirety). Other genes that confer herbicide resistance include those encoding rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase chimeric proteins (Shiota et al., (1994) Plant Physiol. 106(1): 17-23), glutathione reductase and superoxide dismutase (Aono et al., (1995) Plant Cell Physiol. 36: 1687), and various phosphotransferase genes (Datta et al., (1992) Plant Mol Biol. 20: 619), which are incorporated herein by reference in their entirety.

[0150] (E) Protoporphyrinogen oxidase (protox) is necessary for the production of chlorophyll, which is necessary for the survival of all plants. Protoporphyrinogen oxidase is used as a target for a variety of herbicide compounds. These herbicides also inhibit the growth of all different types of plants that exist, leading to their complete destruction. The development of plants resistant to these herbicides and containing altered protoporphyrinogen oxidase activity is described in the following documents: U.S. Patent Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373; and International Publication No. WO 01 / 12825, which are incorporated herein by reference in their entirety.

[0151] 3. Trait genes that confer or contribute to altered grain characteristics, such as:

[0152] (A) fatty acids that are altered, for example, by:

[0153] (1) Down-regulation of stearoyl-ACP desaturase to increase the stearic acid content of plants. See, Knultzon et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 99 / 64579 (Genes for Desaturases to Alter Lipid Profiles in Corn), which are incorporated herein by reference in their entirety.

[0154] (2) increasing oleic acid by FAD-2 gene modification and / or decreasing linolenic acid by FAD-3 gene modification (see, U.S. Pat. Nos. 6,063,947, 6,323,392, 6,372,965, and WO 93 / 11245, which are incorporated herein by reference in their entireties);

[0155] (3) changing the conjugated linolenic acid or linoleic acid content, as described in WO 01 / 12800, which is incorporated herein by reference in its entirety;

[0156] (4) Altering LEC1, AGP, Dek1, Superal1, milps, various Ipa genes (such as lpa1, lpa3, hpt or hggt). For example, see WO 02 / 42424, WO 98 / 22604, WO 03 / 011015, U.S. Pat. No. 6,423,886, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,825,397, U.S. Patent Application Publication Nos. 2003 / 0079247, 2003 / 0204870, WO 02 / 057439, WO 03 / 011015 and Rivera-Madrid et al., (1995) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 92: 5620-5624, which are incorporated herein by reference in their entirety.

[0157] (B) Changing the phosphorus content, e.g. by

[0158] (1) The introduction of a phytase encoding gene will promote the decomposition of phytate and add more free phosphate to the transformed plants. For example, see Van Hartingsveldt et al., (1993) Gene 127:87, which discloses the nucleotide sequence of the Aspergillus niger phytase gene, which is incorporated herein by reference in its entirety.

[0159] (2) Upregulation of genes that reduce phytate content. In maize, this can be accomplished, for example, by cloning and reintroducing DNA associated with one or more alleles, such as the LPA allele identified in a maize mutant characterized by low levels of phytic acid, as described in Raboy et al., (1990) Maydica 35:383, and / or by altering inositol kinase activity, as described in WO 02 / 059324, U.S. Patent Application Publication No. 2003 / 0009011, WO 03 / 027243, U.S. Patent Application Publication No. 2003 / 0079247, WO 99 / 05298, U.S. Patent No. 6,197,561, U.S. Patent No. 6,291,224, U.S. Patent No. 6,391,348, WO 2002 / 059324, U.S. Patent Application Publication No. 2003 / 0079247, WO 98 / 45448, WO 99 / 55882, WO 01 / 04147, which are incorporated herein by reference in their entirety.

[0160] (C) For example, altered carbohydrates are produced by altering genes for enzymes that affect starch branching patterns, or altering genes for thioredoxins such as NTR and / or TRX (see, U.S. Pat. No. 6,531,648, which is incorporated herein by reference in its entirety) and / or gamma zein knockouts or mutants such as cs27 or TUSC27 or en27 (see, U.S. Pat. No. 6,858,778 and U.S. Patent Application Publication Nos. 2005 / 0160488 and 2005 / 0204418, which are incorporated herein by reference in their entireties). See, Shiroza et al., (1988) J. Bacteriol. 170:810 (nucleotide sequence of a mutant fructosyltransferase gene of Streptococcus), Steinmetz et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide sequence of a levansucrase gene of Bacillus subtilis), Pen et al., (1992) Bio / Technology 10:292 (production of transgenic plants expressing Bacillus licheniformis α-amylase), Elliot et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequence of a tomato invertase gene), et al., (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of barley α-amylase gene) and Fisher et al., (1993) Plant Physiol. 102:1045 (corn endosperm starch branching enzyme II), WO 99 / 10498 (improving digestibility and / or starch extraction by modifying UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method for producing high oil seed by altering starch level (AGP)), which are incorporated herein by reference in their entirety. The fatty acid modification genes mentioned above can also be used to affect starch content and / or composition through the interaction of the starch pathway and the oil pathway.

[0161] (D) Altered antioxidant content or composition, such as altered tocopherols or tocotrienols. See, for example, U.S. Pat. No. 6,787,683, U.S. Patent Application Publication Nos. 2004 / 0034886 and WO 00 / 68393, directed to manipulation of antioxidant levels by altering phytyl isopentenyl transferase (ppt), WO 03 / 082899, directed to manipulation of antioxidant levels by altering homogentisate geranylgeranyl transferase (hggt), which are incorporated herein by reference in their entirety.

[0162] (E) Altered essential seed amino acids. See, for example, U.S. Pat. No. 6,127,600 (method for increasing the accumulation of essential amino acids in seeds), U.S. Pat. No. 6,080,913 (dual method for increasing the accumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), WO 99 / 40209 (altering the amino acid composition in seeds), WO 99 / 29882 (method for altering the amino acid content of proteins), U.S. Pat. No. 5,850,016 (altering the amino acid composition in seeds), WO 98 / 20133 (proteins with elevated levels of essential amino acids), U.S. Patent No. 5,885,802 (high methionine), U.S. Patent No. 5,885,801 (high threonine), U.S. Patent No. 6,664,445 (plant amino acid biosynthetic enzymes), U.S. Patent No. 6,459,019 (increased lysine and threonine), U.S. Patent No. 6,441,274 (plant tryptophan synthase beta subunit), U.S. Patent No. 6,346,403 (methionine metabolizing enzymes), U.S. Patent No. 5,939,599 (high sulfur), U.S. Patent No. 5,912,414 (increased methionine), WO 98 / 56935 (plant amino acid biosynthetic enzymes), WO 98 / 45458 (engineered seed proteins with a higher percentage of essential amino acids), WO 98 / 42831 (lysine increase), U.S. Patent No. 5,633,436 (increase in sulfur-containing amino acid content), U.S. Patent No. 5,559,223 (synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improving the nutritional value of plants), WO 96 / 01905 (threonine increase), WO 95 / 15392 (lysine increase), U.S. Patent Application Publication No. 2003 / 0163838, U.S. Patent Application Publication No. 2003 / 0150014, U.S. Patent Application Publication No. 2004 / 0068767, U.S. Patent No. 6,803,498, WO 01 / 79516 and WO 00 / 09706 (Ces A: cellulose synthase), U.S. Patent No. 6,194,638 (hemicellulose), U.S. Patent No. 6,399,859 and U.S. Patent Application Publication No. 2004 / 0025203 (UDPGdH), U.S. Patent No. 6,194,638 (RGP), which are incorporated herein by reference in their entirety.

[0163] Additional genes may be included in the expression cassettes useful in the methods disclosed herein. Non-limiting examples include:

[0164] A. Genes that create sites for site-specific DNA integration

[0165] This includes the introduction of FRT sites that can be used in the FLP / FRT system and / or Lox sites that can be used in the Cre / Loxp system. For example, see Lyznik et al., (2003) Plant Cell Rep 21:925-932 and WO 99 / 25821, which are incorporated herein by reference in their entirety. Other systems that can be used include the Gin recombinase of bacteriophage Mu (Maeser et al., 1991; Vicki Chandler, The Maize Handbook Chapter 118 (Springer, 1994)), the Pin recombinase of Escherichia coli (Enomoto et al., 1983) and the R / RS system of the pSR1 plasmid (Araki et al., 1992), which are incorporated herein by reference in their entirety.

[0166] B. Genes that affect abiotic stress resistance (including but not limited to flowering, ear and seed development, improved nitrogen use efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, salt resistance or tolerance) and increased yield under stress. See, for example, WO 00 / 73475, in which water use efficiency is altered by altering malic acid; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705, U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No. 6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat. No. 6,717,034, WO2000060089, WO 2001026459, WO 2001035725, WO 2001034726, WO 2001035727, WO2001036444, WO 2001036597, WO 2001036598, WO 2002015675, WO 2002017430, WO2002077185, WO 2002079403, WO 2003013227, WO 2003013228, WO 2003014327, WO2004031349, WO 2004076638, WO 9809521 and WO 9938977, which describe genes, including CBF genes and transcription factors, that are effective in alleviating the negative effects of freezing, high salinity and drought on plants, as well as conferring other positive effects on plant phenotype; U.S. Patent Application Publication Nos. 2004 / 0148654 and WO 01 / 36596, in which abscisic acid is altered in plants, resulting in improved plant phenotypes, such as increased yield and / or increased tolerance to abiotic stress; WO 2000 / 006341, WO 04 / 090143, U.S. Patent Application Serial No. 10 / 817483 and U.S. Patent No. 6,992,237, wherein cytokinin expression is modified, causes plant to have the stress tolerance of increase, for example drought tolerance and / or the yield of increase, it is incorporated into the text by reference in its entirety. Also referring to WO 0202776, WO 2003052063, JP 2002281975, U.S. Patent No. 6,084,153, WO 0164898, U.S. Patent No. 6,177,275 and U.S. Patent No. 6,107,547 (the raising of nitrogen utilization rate and the change of nitrogen responsiveness), described document is incorporated into the text by reference in its entirety. About ethylene change, referring to U.S. Patent Application Publication No. 2004 / 0128719, U.S. Patent Application Publication No. 2003 / 0166197 and WO 200032761, described document is incorporated into the text by reference in its entirety.For plant transcription factors or transcriptional regulators of abiotic stress, see, for example, US Patent Application Publication No. 2004 / 0098764 or US Patent Application Publication No. 2004 / 0078852, which are incorporated herein by reference in their entireties.

[0167] C. Other genes and transcription factors that affect plant growth and agronomic traits (such as yield, flowering, plant growth and / or plant architecture) can be introduced or introgressed into plants, see, for example, WO 97 / 49811 (LHY), WO 98 / 56918 (ESD4), WO 97 / 10339 and U.S. Pat. No. 6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 96 / 14414 (CON), WO 96 / 38560, WO 01 / 21822 (VRN1), WO 00 / 44918 (VRN2), WO 99 / 49064 (GI), WO 00 / 46358 (FRI), WO 97 / 29123, U.S. Pat. No. 6,794,560, U.S. Pat. No. 6,307,126 (GAI), WO 99 / 09174 (D8 and Rht) and WO 2004076638 and WO 2004031349 (transcription factors), which are incorporated herein by reference in their entirety.

[0168] As used herein, "antisense orientation" includes a polynucleotide sequence that is operably linked to a promoter in the direction of transcription of the antisense strand. The antisense strand is fully complementary to the endogenous transcription product so that the translation of the endogenous transcription product is often inhibited. "Operably linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one nucleic acid fragment is affected by another. For example, when a promoter is able to affect the expression of a coding sequence, it is operably linked to a 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 a sense or antisense direction.

[0169] The heterologous nucleotide sequence operably linked to the promoter and its related biologically active fragment or variant for the method disclosed herein can be the antisense sequence of the target gene. The term "antisense DNA nucleotide sequence" refers to a sequence in the opposite direction of the 5' to 3' normal direction of the nucleotide sequence. When the antisense DNA sequence is delivered to the plant cell, it prevents the normal expression of the DNA nucleotide sequence of the targeted gene. The antisense nucleotide sequence encodes an RNA transcript that is complementary to and hybridizes with the endogenous messenger RNA (mRNA) produced by the transcription of the DNA nucleotide sequence of the targeted gene. In this case, the production of the natural protein encoded by the targeted gene is suppressed to achieve the desired phenotypic response. The antisense sequence can be modified as long as the sequence hybridizes with the corresponding mRNA and interferes with the expression of the corresponding mRNA. In this way, antisense constructs with 70%, 80%, 85% sequence identity to the corresponding antisense sequence can be used. In addition, part of the antisense nucleotide can be used to disrupt the expression of the target gene. Generally, a sequence of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or more nucleotides can be used. Thus, a promoter can be operably linked to an antisense DNA sequence to reduce or inhibit expression of a native protein in a plant transformed by the methods disclosed herein.

[0170] "RNAi" refers to a series of related technologies for reducing gene expression (see, e.g., U.S. Patent No. 6,506,559, which is incorporated herein by reference in its entirety). Older technologies referred to by other names are now believed to be based on the same mechanism, but are given different names in the literature. These names include "antisense inhibition", which produces antisense RNA transcripts that can inhibit the expression of a target protein, and "cosuppression" or "justice inhibition", which refers to the production of justice RNA transcripts that can inhibit the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020, which is incorporated herein by reference in its entirety). Such technologies rely on the use of constructs that can lead to the accumulation of double-stranded RNA, one of which is complementary to the target gene to be silenced.

[0171] As used herein, the term "promoter" or "transcription initiation region" means a DNA regulatory region that generally comprises a TATA box or a DNA sequence capable of directing RNA polymerase II to initiate RNA synthesis at an appropriate transcription initiation site of a specific coding sequence. The promoter may additionally comprise other recognition sequences generally located upstream or 5' of the TATA box or a DNA sequence capable of directing RNA polymerase II to initiate RNA synthesis, referred to as an upstream promoter element, which affects the transcription initiation rate.

[0172] The transcription initiation region, i.e., the promoter, may be native, homologous, foreign, or heterologous to the host, or may be a native sequence or a synthetic sequence. Foreign means that the transcription initiation region is not found in the wild-type host into which it is introduced. With respect to the target coding sequence, either native or heterologous promoters may be used.

[0173] The transcription (expression) cassette will contain a transcription and translation initiation region, a target DNA sequence / trait gene, and a transcription and translation termination region that functions in plants in the 5'-3' direction of transcription. The termination region may be natural with the transcription initiation region, may be natural with the target DNA sequence, or may be derived from another source. Convenient termination regions may be obtained from the potato proteinase inhibitor (PinII) gene or from the sequence of the Ti-plasmid of Agrobacterium tumefaciens, such as the nopaline synthase, octopine synthase, and opaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 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; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

[0174] The expression cassette may additionally include a 5' leader sequence in the expression cassette construct. Such leader sequences may function to enhance translation. Translation leader sequences are known in the art and include: microRNA virus leaders, such as the EMCV leader (encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, TR, and Moss, B. (1989) PNAS USA, 86: 6126-6130); Potyvirus leaders, such as the TEV leader (tobacco etch virus) (Allison et al. (1986); MDMV leader (maize dwarf mosaic virus); Virology, 154: 9-20) and human immunoglobulin heavy chain binding protein (BiP) (Macejak, DG, and P. Sarnow (1991) Nature, 353: 90-94); alfalfa mosaic virus coat protein MARNA (AMV RNA 4) (Jobling, SA, and Gehrke, L., (1987) Nature, 325: 622-625); tobacco mosaic virus leader sequence (TMV) (Gallie et al. (1989) Molecular Biology of RNA, pp. 237-256, Gallie et al. (1987) Nucl. Acids Res. 15: 3257-3273); maize chlorotic mottle virus leader sequence (MCMV) (Lornmel, SA et al. (1991) Virology, 81: 382-385). See also, Della-Cioppa et al. (1987) Plant Physiology, 84: 965-968; and endogenous maize 5' untranslated sequence. Other methods known to enhance translation and enhance mRNA stability, such as introns, such as 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), etc., which are incorporated herein by reference in their entirety, may also be used.

[0175] The expression cassette may contain one or more than one gene or nucleic acid sequence to be transferred and expressed in the transformed plant. Thus, each nucleic acid sequence will be operably linked to the 5' and 3' regulatory sequences. Alternatively, multiple expression cassettes may be provided.

[0176] In preparing expression cassettes for use in the disclosed methods, various DNA fragments may be manipulated to provide DNA sequences in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or joints may be employed to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., conversion and transversion) may be involved.

[0177] The purpose morphogenetic gene and / or trait gene / polynucleotide introduced into the explant by the disclosed method can be operably connected to a suitable promoter." Plant promoter " is a promoter (whether its origin is a plant cell) that can start transcription in a plant cell. Exemplary plant promoters include but are not limited to those obtained from plants, plant viruses, and bacteria (such as from Agrobacterium or Rhizobium) that are included in the gene expressed in plant cells. The example of a promoter under developmental control includes a promoter that preferentially starts transcription in certain tissues (such as leaves, roots or seeds). Such a promoter is referred to as "tissue preferred". The promoter that only starts transcription in certain tissues is referred to as "tissue-specific". "Cell type" specific promoters mainly drive expression in certain cell types (for example, vascular cells in roots or leaves) in one or more organs. Tissue-specific promoters, tissue-preferred promoters, cell type-specific promoters and inducible promoters constitute the category of "non-constitutive" promoters.

[0178] An "inducible" or "repressible" promoter can be a promoter under environmental or exogenous control. Examples of environmental conditions that can affect transcription by an inducible promoter include anaerobic conditions, or certain chemicals, or the presence of light. Alternatively, exogenous control of an inducible or repressible promoter can be affected by providing suitable chemicals or other agents that cause induction or inhibition of the promoter via interaction with the target polypeptide. Inducible promoters include heat-inducible promoters, estradiol-responsive promoters, chemical-inducible promoters, and the like. Pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced after infection by pathogens; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitinase, and the like. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. [Journal of Plant Pathology] 89: 245-254; Uknes et al., (1992) Plant Cell [Plant Cell] 4: 645-656 and Van Loon, (1985) Plant Mol. Virol. [Plant Molecular Virology] 4: 111-116. Inducible promoters that can be used in the methods of the present invention include GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A and XVE promoters.

[0179] Chemically inducible promoters can be repressed by tetracycline repressor (TETR), ethylbensulfuron repressor (ESR) or chlorsulfuron repressor (CSR), and derepression occurs upon addition of a tetracycline-related ligand or a sulfonylurea ligand. The repressor can be TETR, and the tetracycline-related ligand is doxycycline or anhydrotetracycline. (Gatz, C., Frohberg, C. and Wendenburg, R. (1992) Strict repression and homogeneous derepression of a modified CaMV 35S promoter by tetracycline in intact transgenic tobacco plants, Plant J. 2, 397-404). Alternatively, the repressor may be an ESR, and the sulfonylurea ligand is etsulfuron-methyl, chlorsulfuron-methyl, metsulfuron-methyl, sulfosulfuron-methyl, chlorimuron-methyl, nicosulfuron-methyl, primisulfuron-methyl, bensulfuron-methyl, sulfosulfuron-methyl, trifloxysulfuron-methyl, foramsulfuron-methyl, iodosulfuron-methyl, prosulfuron-methyl, thifensulfuron-methyl, sulfosulfuron-methyl, mesosulfuron-methyl or chlorpyrisulfuron-methyl (US 20110287936 is incorporated herein by reference in its entirety). If the repressor is a CSR, the CSR ligand is chlorsulfuron-methyl. See U.S. Patent No. 8,580,556, which is incorporated herein by reference in its entirety.

[0180] A "constitutive" promoter is a promoter that is active under most conditions. Promoters used in the present disclosure include those disclosed in WO 2017 / 112006 and those disclosed in U.S. Provisional Application 62 / 562,663. Constitutive promoters for expressing genes in plants are known in the art. These include, but are not limited to, the 35S promoter of cauliflower mosaic virus (Depicker et al. (1982) Mol. Appl. Genet. 1:561-573; Odell et al. (1985) Nature 313:810-812), the ubiquitin promoter (Christensen et al. (1992) Plant Mol. Biol. 18:675-689), promoters from genes such as ribulose bisphosphate carboxylase (De Almeida et al. (1989) Mol. Gen. Genet. 218:78-98), actin (McElroy et al. (1990) Plant Mol. Biol. 219:90-91), mitochondrial promoter (Crockett et al. (1991) Plant Mol. Biol. 314:91-93), mitochondrial promoter (Crockett et al. (1992) Plant Mol. Biol. 315:91-93), mitochondrial promoter (Crockett et al. (1993) Plant Mol. Biol. 316:92-94), mitochondrial promoter (Crockett et al. (1993) Plant Mol. Biol. 317:93-95), mitochondrial promoter (Crockett et al. (1994) Plant Mol. Biol. 318:92-93), mitochondrial promoter (Crockett et al. (1994) Plant Mol. Biol. 319:93-94), mitochondrial promoter (Crockett et al. (1994) Plant Mol. Biol. 319:93-95), mitochondrial promoter (Crockett et al. (1994) Plant Mol. Biol. 311:93-93), mitochondrial promoter (Crockett et al. ( J. [Plant Journal] 2: 163-171), histone, DnaJ (Baszczynski et al. (1997) Maydica [Medical Journal] 42: 189-201), etc. In various aspects, constitutive promoters that can be used in the disclosed methods 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 and ZM-ADF PRO (ALT2) promoters.

[0181] The promoters listed in Table 4 below that are useful in the present disclosure include those disclosed in U.S. Patent Application Publication No. 2017 / 0121722 and U.S. Patent No. 8,710,206, which are incorporated herein by reference in their entirety.

[0182] Table 4.

[0183] name describe ZM-PLTP Maize PLTP promoter SB-PLTP1 Sorghum bicolor PLTP1 promoter ZM-FBP1 Maize promoter of fructose-1,6-bisphosphatase ZM-RFP NAD(P)-binding Rossmann-fold protein from maize promoter ZM-APMP Maize promoter of adipocyte plasma membrane associated protein-like protein ZM-RfeSP Maize promoter of Rieske[2Fe-2S] iron-sulfur domain protein ZM-CRR6 Maize promoter of chlorine respiration reduction 6 gene ZM-GLYK D-Glycerate 3-kinase, maize promoter of chloroplast-like protein gene ZM-CAB7 Chlorophyll ab-binding protein 7, a maize promoter of chloroplast-like protein ZM-UBR Maize promoter of ultraviolet B repressor gene ZM-HBP Maize promoter of Soul heme-binding family protein ZM-PSAN Maize promoter of photosystem I reaction center subunit psi-N ZM-SDR Maize promoter for short-chain dehydrogenase / reductase AXIG1 AXIG1 promoter DR5 DR5 promoter ZM-PLTP1 Maize PLTP1 promoter ZM-PLTP2 Maize PLTP2 promoter SB-PLTP2 Sorghum bicolor PLTP2 promoter SB-PLTP3 Sorghum bicolor PLTP3 promoter SI-PLTP1 Xiaomi PLTP promoter OS-PLTP1 Rice PLTP promoter OS-PLTP2 Rice PLTP2 promoter ZM-LGL PRO Maize promoter of lactoylglutathione lyase gene ZM-LEA14-A PRO Maize promoter of the gene encoding late embryonic abundant protein Lea-14-A ZM-LEA34-D PRO Maize promoter of the gene encoding late embryonic abundant protein Lea34-D ZM-SDRPRO(Long) Maize promoter for short-chain dehydrogenase / reductase (long) OS-SDR PRO Rice promoter for short-chain dehydrogenase / reductase SB-SDR PRO Two-color sorghum promoter for short-chain dehydrogenase / reductase GM-EF1A PRO Soybean EF1A promoter

[0184] Other promoters useful in the disclosed methods are listed in Table 5 below.

[0185] Table 5.

[0186]

[0187]

[0188]

[0189]

[0190] As used herein, the term "regulatory element" also refers to a DNA sequence that is usually, but not always, upstream (5') of the coding sequence of a structural gene, including sequences that control the expression of the coding region by providing recognition of RNA polymerase and / or other factors required to initiate transcription from a specific site. An example of a regulatory element that provides recognition of RNA polymerase or other transcription factors to ensure initiation at a specific site is a promoter element. Promoter elements include core promoter elements responsible for transcription initiation, as well as other regulatory elements that modify gene expression. It should be understood that nucleotide sequences located within introns of the coding region sequence or 3' of the coding region sequence may also help regulate the expression of the target coding region. Examples of suitable introns include, but are not limited to, maize IVS6 introns or maize actin introns. Regulatory elements may also include those located downstream (3') of the transcription start site, or within the transcribed region, or both. In the context of the disclosed method, post-transcriptional regulatory elements may include elements that are active after transcription initiation, such as translation and transcription enhancers, translation and transcription repressors, and mRNA stability determinants.

[0191] As used throughout this disclosure, a "heterologous nucleotide sequence," "heterologous polynucleotide of interest," "heterologous polynucleotide," or "heterologous trait gene" is a sequence that does not naturally occur with the promoter sequence of the present invention or is operably linked to the promoter. Although such a nucleotide sequence or trait gene is foreign to the promoter sequence, it may be homologous or native, or heterologous, or foreign to the plant host. Similarly, the promoter sequence may be homologous or native, or heterologous or foreign to the plant host and / or the polynucleotide of interest.

[0192] The DNA construct / expression cassette used in the disclosed method may also include other enhancers (translation or transcription enhancers) as needed. These enhancer regions are well known to those skilled in the art and may include the ATG start codon and adjacent sequences. The start codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. Translation control signals and start codons can come from a variety of natural and synthetic sources. The translation start region can be provided from the source of the transcription start region or from a structural gene. The sequence may also be derived from a regulatory element selected to express the gene and may be specifically modified to increase the translation of the mRNA. It should be recognized that enhancers can be used in combination with promoter regions to increase transcription levels. It should be recognized that enhancers can be used in combination with promoter regions to increase transcription levels. Enhancers are nucleotide sequences that function to increase the expression of promoter regions. Enhancers are known in the art and include SV40 enhancer regions, 35S enhancer elements, etc. It is also known that some enhancers can also change normal promoter expression patterns, such as by causing promoter constitutive expression, when the same promoter is only expressed in a specific tissue or some specific tissues without an enhancer. Enhancers useful in the disclosed methods are listed in Table 6 below.

[0193] Table 6.

[0194]

[0195] 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. In contrast, a strong promoter drives expression of a coding sequence at a high level or at a level of about 1 / 10 transcripts to about 1 / 100 transcripts to about 1 / 1,000 transcripts.

[0196] It is recognized that the sequences used in the disclosed methods can be used with their native coding sequences, resulting in phenotypic changes in transformed plants. The morphogenetic genes and target genes disclosed herein, as well as variants and fragments thereof, can be used in the disclosed methods for genetic manipulation of any plant. The term "operably linked" means that the transcription or translation of the heterologous nucleotide sequence is affected by the promoter sequence.

[0197] In one aspect of the present disclosure, the expression cassette comprises a transcription initiation region or variants thereof or fragments thereof operably linked to a morphogenetic gene and / or a heterologous nucleotide sequence. Such an expression cassette may have a plurality of restriction sites for subjecting the insertion of the nucleotide sequence to transcriptional regulation by the regulatory region. The expression cassette may additionally contain a selectable marker gene and a 3' termination region.

[0198] The expression cassette may comprise a transcription initiation region (i.e., a promoter or a variant or fragment thereof), a translation initiation region, a morphogenic gene and / or a heterologous nucleotide sequence of interest, a translation termination region in the 5′-3′ direction of transcription, and optionally a transcription termination region functional in the host organism. The regulatory regions (i.e., promoters, transcription regulatory regions, and translation termination regions), morphogenic genes, and / or polynucleotides of interest used in the disclosed methods may be native / similar to the host cell or to each other. Alternatively, these regulatory regions, morphogenic genes, and / or polynucleotides of interest may be heterologous to the host cell or to each other. As used herein, "heterologous" with respect to a sequence refers to a sequence that is derived from an alien species, or, if derived from the same species, a sequence that is substantially modified from its natural form in a composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from that from which the polynucleotide is derived, or, if from the same / similar species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter of the operably linked polynucleotide.

[0199] The termination region may be native to the transcriptional initiation region, may be native to the operably linked morphogenetic gene and / or may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, morphogenetic gene and / or expressed DNA sequence, plant host, or any combination thereof). Convenient termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. 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; the entireties of which are incorporated herein by reference.

[0200] An expression cassette comprising a promoter operably connected to a morphogenetic gene and / or optionally further operably connected to a heterologous nucleotide sequence, a heterologous polynucleotide of interest, a heterologous polynucleotide nucleotide, a target sequence or a trait gene can be used to transform any plant. Alternatively, the heterologous polynucleotide of interest, the heterologous polynucleotide nucleotide, the target sequence or the trait gene operably connected to a promoter can be located on a separate expression cassette outside a transfer DNA (T-DNA). In this way, genetically modified plants, plant cells, plant tissues, seeds, roots, etc. can be obtained. An expression cassette comprising a sequence of the present disclosure can also contain at least one additional nucleotide sequence for a gene, a heterologous nucleotide sequence, a heterologous polynucleotide of interest or a heterologous polynucleotide to be co-transformed into an organism. Alternatively, the one or more additional nucleotide sequences can be provided in another expression cassette.

[0201] Where appropriate, the nucleotide sequence / trait gene whose expression is to be under the control of the promoter sequence and any other nucleotide sequence can be optimized to improve expression in the transformed plant. That is, these nucleotide sequences can be synthesized using plant-preferred codons to improve expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92: 1-11, which is incorporated herein by reference in its entirety for a discussion of host-preferred codon usage. Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Pat. 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 entirety.

[0202] It is known that additional sequence modifications can enhance gene expression in a cellular host. These include elimination of sequences encoding false polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the heterologous nucleotide sequence can be adjusted to an average level for a given cellular host, calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid the presence of predictable hairpin secondary mRNA structures.

[0203] As used herein, "vector" refers to a DNA molecule, such as a plasmid, cosmid or bacteriophage, used to introduce a nucleotide construct, such as an expression cassette, into a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites, into which foreign DNA sequences can be inserted in a determinable manner without losing the basic biological function of the vector, as well as marker genes suitable for identifying and selecting cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.

[0204] The transformed cells can be grown into plants in a conventional manner. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84, which is 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 with expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited, and then the seeds are harvested to ensure that the desired phenotypic characteristic has been expressed. In this way, the present disclosure provides transformed seeds (also referred to as "transgenic seeds") having a nucleotide construct stably incorporated into its genome that can be used in the disclosed methods, for example, an expression cassette that can be used in the disclosed methods.

[0205] There are various methods for regenerating plants from plant tissues. Specific regeneration methods will depend on the initial plant tissue and the specific plant species to be regenerated. It is well known in the art (Weissbach and Weissbach, (1988) in: Methods for Plant Molecular Biology [Plant Molecular Biology Method], (ed.), Academic Press, Inc., San Diego, California, incorporated herein by reference in its entirety) to regenerate, grow and cultivate plants from single plant protoplast transformants or from various converted explants. This regeneration and growth process generally include the steps of: selecting transformed cells, cultivating those individualized cells through the usual stages of embryonic development, through the rooting seedling stage. Regenerate transgenic embryos and seeds in the same manner. Then the transgenic rooted shoots of the gained are planted in a suitable plant growth medium (such as soil). Preferably, the regenerated plant is self-pollinated to provide a homozygous transgenic plant. Alternatively, the pollen obtained from the regenerated plant is hybridized with the plant producing seeds of the agronomically important strain. Instead, pollen from plants of these important lines is used to pollinate the regenerated plants.Transgenic plants containing the desired polynucleotide of interest produced by the disclosed methods are grown using methods well known to those skilled in the art.

[0206] The method for inserting polynucleotides in the specific position of plant genome is known in the art. The insertion of polynucleotides at the desired genome position is realized by using site-specific recombination system. See, for example, WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855 and WO 99 / 25853, which are incorporated herein by reference in their entirety. In short, the target polynucleotides flanked by two different recombination sites may be included in the T-DNA transfer box. The T-DNA transfer box is introduced into the plant, and the plant has stably incorporated the target site into its genome, and the target site flanks two different recombination sites corresponding to the site of the transfer box. Suitable recombinase is provided, and the transfer box is integrated into the target site. Thus, the target polynucleotide is integrated into the specific chromosome position in the plant genome.

[0207] The disclosed method can be used to introduce polynucleotides into explants, and the polynucleotides are used to target specific sites in the genome of plants derived from plant cells for modification. Site-specific modifications that can be introduced with the disclosed method include modifications produced using any method for introducing site-specific modifications, including but not limited to using gene repair oligonucleotides (e.g., U.S. Publication 2013 / 0019349), or by using double-strand break technology, such as TALEN, large-range nucleases, zinc finger nucleases, CRISPR-Cas, etc. For example, the disclosed method can be used to introduce the CRISPR-Cas system into plant cells or plants for the following purposes: genome modification of target sequences in the genome of plants or plant cells, selection of plants, deletion of bases or sequences, gene editing, and insertion of target polynucleotides into the genome of plants or plant cells. Therefore, the disclosed method can be used together with the CRISPR-Cas system to provide an effective system for modifying or changing target sites and target nucleotides in the genome of plants, plant cells or seeds. The Cas endonuclease gene is a plant-optimized Cas9 endonuclease, wherein the plant-optimized Cas9 endonuclease is capable of binding to a genomic target sequence of a plant genome and generating a double-strand break therein.

[0208] The Cas endonuclease is guided by a guide nucleotide to identify double-strand breaks at a specific target site and optionally introduce the double-strand breaks into the genome of the cell. The CRISPR-Cas system provides an effective system for modifying the target site in the genome of a plant, a plant cell or a seed. Further provided is a method for using a guide polynucleotide / Cas endonuclease system to provide an effective system for modifying the target site in the cell genome and editing the nucleotide sequence in the cell genome. Once the genomic target site is identified, a variety of methods can be used to further modify the target site so that they include a variety of purpose polynucleotides. The disclosed method can be used to introduce a CRISPR-Cas system, and the system is used to edit the nucleotide sequence in the cell genome. The nucleotide sequence to be edited (purpose nucleotide sequence) can be located inside or outside the target site identified by the Cas endonuclease.

[0209] CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) (also known as SPIDR-interspersed direct repeats) constitute a family of recently described DNA loci. CRISPR loci consist of short, highly conserved DNA repeats (usually 24 to 40 bp, repeated from 1 to 140 times, also called CRISPR repeats) that are partially palindromic. The repeats (usually species-specific) are spaced by variable sequences of constant length (usually 20 to 58, depending on the CRISPR locus (WO2007 / 025097, published on March 1, 2007)).

[0210] The CRISPR locus was first identified in Escherichia coli (Ishino et al. (1987) J. Bacterial. 169:5429-5433; Nakata et al. (1989) J. Bacterial. 171:3553-3556). Similar interspersed short sequence repeats have been identified in Halobacterium thaliana, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (Groenen et al. (1993) Mol. Microbiol. 10:1057-1065; Hoe et al. (1999) Emerg. Infect. Dis. 5:254-263; Masepohl et al. (1996) Biochim. Biophys. Acta 1307:26-30; Mojica et al. (1995) Mol. Microbiol. 17:85-93). The CRISPR locus differs from other SSRs in the structure of the repeat sequences, which have been termed short regularly interspaced repeats (SRSRs) (Janssen et al. (2002) OMICS J. Integ. Biol. 6:23-33; Mojica et al. (2000) Mol. Microbiol. 36:244-246). The repeat sequences are short elements that occur in clusters that are always regularly spaced apart by variable sequences of constant length (Mojica et al. (2000) Mol. Microbiol. 36:244-246).

[0211] Cas genes include genes that are typically coupled, associated, or proximal or adjacent to flanking CRISPR loci. The terms "Cas gene" and "CRISPR-associated (Cas) gene" are used interchangeably herein. A comprehensive review of the Cas protein family is presented in the following literature: Haft et al. (2005) Computational Biology, PLoS ComputBiol 1(6): e60.doi: 10.1371 / journal.pcbi.0010060.

[0212] In addition to the four gene families initially described, another 41 CRISPR-related (Cas) gene families have been described in U.S. Patent Application Publication No. 2015 / 0059010, which is incorporated herein by reference. This reference shows that CRISPR systems belong to different categories, with different repetition patterns, groups of genes and species ranges. The number of Cas genes at a given CRISPR locus can be different between species. Cas endonucleases relate to Cas proteins encoded by Cas genes, wherein the Cas proteins can introduce double-strand breaks into a DNA target sequence. Cas endonucleases are guided by guidance polynucleotides to identify double-strand breaks at specific target sites and optionally introduce the double-strand breaks into the genome of cells. As used herein, the term "guiding polynucleotides / Cas endonuclease system" includes a Cas endonuclease capable of introducing double-strand breaks into a DNA target sequence and a complex of guidance polynucleotides. When the target sequence is recognized by the guide nucleotide, the Cas endonuclease unwinds the DNA duplex immediately adjacent to the genomic target site and cleaves both DNA strands, provided that the correct protospacer adjacent motif (PAM) is oriented approximately 3' to the target sequence (see Figures 2A and 2B of U.S. Patent Application Publication No. 2015 / 0059010).

[0213] In one aspect, the Cas endonuclease gene is a Cas9 endonuclease, such as, but not limited to, the Cas9 genes listed in SEQ ID NOs: 462, 474, 489, 494, 499, 505, and 518 in WO 2007 / 025097 published on March 1, 2007 (incorporated herein by reference). In another aspect, the Cas endonuclease gene is a plant, maize or soybean optimized Cas9 endonuclease, such as, but not limited to, U.S. Patent Application Publication No. 2015 / 0059010. Figure 1 Those shown in A.

[0214] In another aspect, the Cas endonuclease gene is operably linked to an SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal downstream of the Cas codon region (Tinland et al., (1992) Proc. Natl. Acad. Sci. USA 89:7442-6).

[0215] In one aspect, the Cas endonuclease gene is the Cas9 endonuclease gene of nucleotides 2037-6329 of SEQ ID NO: 1, 124, 212, 213, 214, 215, 216, 193 or SEQ ID NO: 5 of U.S. Patent Application Publication No. 2015 / 0059010, or any functional fragment or variant thereof.

[0216] With respect to Cas endonucleases, the terms "functional fragment", "functionally equivalent fragment" and "functionally equivalent fragment" are used interchangeably herein. These terms refer to a portion or subsequence of a Cas endonuclease sequence in which the ability to generate double-strand breaks is retained.

[0217] With respect to Cas endonucleases, the terms "functional variant", "functionally equivalent variant" and "functionally equivalent variant" are used interchangeably herein. These terms refer to variants of Cas endonucleases in which the ability to generate double-strand breaks is retained. These fragments and variants can be obtained via methods such as site-directed mutagenesis and synthetic construction.

[0218] In one aspect, the Cas endonuclease gene is a plant codon-optimized Streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the type N(12-30)NGG and that can in principle be targeted.

[0219] Endonucleases are enzymes that cut the phosphodiester bonds in polynucleotide chains, and are included in restriction endonucleases that cut DNA at specific sites without damaging bases. Restriction endonucleases include type I, type II, type III, and type IV endonucleases, and these restriction endonucleases further include subtypes. In type I and type III systems, both methylase and restriction enzyme activity are included in a single complex. Endonucleases also include meganucleases, also referred to as homing endonucleases (HE enzymes), which are similar to restriction endonucleases, combine and cut at specific recognition sites, but for meganucleases, these recognition sites are usually longer, about 18bp or longer (patent application PCT / US 12 / 30061 filed on March 22, 2012). Based on conservative sequence motifs, meganucleases are classified into four families, and the family is LAGLIDADG, GIY-YIG, HNH, and His-Cys box family. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. Meganucleases are notable for their long recognition sites and also for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganucleases is similar to that for other restriction endonucleases. Meganucleases are also characterized by the prefixes F-, I-, or PI- for enzymes encoded by independent ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. This cleavage activity can be used to produce double-strand breaks. For a review of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol [New Views in Biotechnology] 5: 521-7; and Sadowski (1993) FASEB [Journal of the Federation of American Societies for Experimental Biology] 7: 760-7. In some instances, the recombinase is from the Integrase or Resolvase family. TAL effector nucleases are a new class of sequence-specific nucleases that can be used to cause double-strand breaks at specific target sequences in the genome of plants or other organisms. (Miller, et al. (2011) Nature Biotechnology 29: 143-148). Zinc finger nucleases (ZFNs) are engineered double-strand break inducers consisting of a zinc finger DNA binding domain and a double-strand-break-inducing domain. Recognition site specificity is conferred by the zinc finger domain, which typically contains two, three, or four zinc fingers, for example with a C2H2 structure, however other zinc finger structures are known and have been engineered. The zinc finger domain is suitable for designing polypeptides that specifically bind to a selected polynucleotide recognition sequence.ZFNs include an engineered DNA binding zinc finger domain connected to a non-specific endonuclease domain (e.g., a nuclease domain from an Ms-type endonuclease, such as Fok1). Additional functionality can be fused to the zinc finger binding domain, including a transcription activator domain, a transcription 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 3-finger domain recognizes a sequence of 9 consecutive nucleotides, and since dimerization of the nuclease requires, two sets of zinc finger triplets are used to bind to a recognition sequence of 18 nucleotides.

[0220] As used herein, "Dead-CAS9" (dCAS9) is used to provide a transcriptional repressor domain. dCAS9 has been mutated so that it can no longer cut DNA. dCAS0 can still bind when guided to the sequence by gRNA, and can also be fused to a repressor element (see Gilbert et al., Cell 2013 July 18; 154(2): 442-451, Kiani et al., 2015 November Nature Methods Vol. 12 No. 11: 1051-1054). As described herein, dCAS9 fused to a repressor element is abbreviated as dCAS9-REP, where the repressor element (REP) can be any known repressor motif that has been characterized in plants (for review, see Kagale and Rozxadowski, 20010 Plant Signaling & Behavior 5: 6, 691-694). The expressed guide RNA (gRNA) binds to the dCAS9-REP protein and targets the binding of the dCAS9-REP fusion protein to a specific predetermined nucleotide sequence within the promoter (promoter within the T-DNA). For example, if it is expressed outside the borders using the ZM-UBIPRO::dCAS9-REP::PINII TERM box and the U6-POL PRO::gRNA::U6 TERM box, and the gRNA is designed to direct the dCAS9-REP protein to bind to the SB-UBI promoter in the expression box SB-UBI PRO::moPAT::PINII TERM within the T-DNA, any event that integrates the sequence outside the borders will be sensitive to bialaphos. Transgenic events that only integrate the T-DNA will express moPAT and be resistant to bialaphos. The advantage of using a dCAS9 protein fused to a repressor (as opposed to TETR or ESR) is the ability to target these repressors to any promoter within the T-DNA. TETR and ESR are limited to cognate operator binding sequences. Alternatively, a synthetic zinc finger nuclease fused to a repressor domain can be used instead of gRNA and dCAS9-REP (Urritia et al., 2003, Genome Biol. 4:231), as described above.

[0221] Bacteria and archaea have developed adaptive immune defenses called clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems that use short RNA to directly degrade foreign nucleic acids (WO 2007 / 025097, published on March 1, 2007). The type II CRISPR / Cas system from bacteria uses crRNA and tracrRNA to guide the Cas endonuclease to its DNA target. The crRNA (CRISPR RNA) contains a region that is complementary to one strand of the double-stranded DNA target and base-pairs with tracrRNA (trans-activating CRISPR RNA) to form an RNA duplex that guides the Cas endonuclease to cut the DNA target.

[0222] As used herein, the term "guide nucleotide" refers to a synthetic fusion of two RNA molecules - crRNA (CRISPR RNA) and tracrRNA comprising a variable targeting domain. On the one hand, the guide nucleotide comprises a variable targeting domain of 12 to 30 nucleotide sequences and an RNA fragment that can interact with a Cas endonuclease.

[0223] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease, and enables the Cas endonuclease to recognize and optionally cut a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide can include at least one nucleotide, a phosphodiester bond, or a connection modification, such as, but not limited to, a locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, a thiophosphate bond, a connection to a cholesterol molecule, a connection to a polyethylene glycol molecule, a connection to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent connection resulting in cyclization. The guide polynucleotide comprising only ribonucleic acid is also referred to as a "guide nucleotide".

[0224] The guiding polynucleotide can be a double molecule (also referred to as a duplex guiding polynucleotide) comprising a first nucleotide sequence domain complementary to a nucleotide sequence in a target DNA (referred to as a variable targeting domain or VT domain) and a second nucleotide sequence domain (referred to as a Cas endonuclease recognition domain or CER domain) that interacts with a Cas endonuclease polypeptide. The CER domain of the double-molecule guiding polynucleotide comprises two separate molecules that hybridize along the complementary region. The two separate molecules can be RNA, DNA, and / or RNA-DNA combination sequences. On the one hand, the first molecule of a duplex guiding polynucleotide comprising a VT domain connected to a CER domain is referred to as "crDNA" (when composed of a continuous extension of DNA nucleotides) or "crRNA" (when composed of a continuous extension of RNA nucleotides) or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The cr nucleotides may comprise fragments of cRNA naturally present in bacteria and archaea. In one aspect, the size of naturally occurring cRNA fragments in bacteria and archaea present in the cr nucleotides disclosed herein can be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.

[0225] In one aspect, the second molecule of the duplex guide polynucleotide comprising a CER domain is referred to as "tracrRNA" (when composed of a continuous stretch of RNA nucleotides) or "tracrDNA" (when composed of a continuous stretch of DNA nucleotides) or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides. In one aspect, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA.

[0226] The guide polynucleotide can also be a single molecule, comprising a first nucleotide sequence domain complementary to a nucleotide sequence in a target DNA (referred to as a variable targeting domain or VT domain) and a second nucleotide domain (referred to as a Cas endonuclease recognition domain or CER domain) that interacts with a Cas endonuclease polypeptide. "Domain" means a continuous extension of nucleotides that can be RNA, DNA, and / or RNA-DNA combination sequences. The VT domain and / or CER domain of a single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. On the one hand, a single guide polynucleotide comprises a cr nucleotide (comprising a VT domain connected to a CER domain) connected to a tracr nucleotide (comprising a CER domain), wherein the connection is a nucleotide sequence comprising an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. A single guide polynucleotide comprising a sequence from a cr nucleotide and a tracr nucleotide can be referred to as a "single guide nucleotide" (when composed of a continuous extension of RNA nucleotides) or a "single guide DNA" (when composed of a continuous extension of DNA nucleotides) or a "single guide nucleotide-DNA" (when composed of a combination of RNA and DNA nucleotides). In one aspect of the present disclosure, a single 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, wherein the guide nucleotide / Cas endonuclease complex can guide the Cas endonuclease to a plant genomic target site such that the Cas endonuclease can introduce a double-strand break into the genomic target site. One aspect of using a single guide polynucleotide versus a duplex guide polynucleotide is that only one expression cassette needs to be made in order to express the single guide polynucleotide.

[0227] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and include a nucleotide sequence that is complementary to one strand (nucleotide sequence) of a double-stranded DNA target site. The % complementarity between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In one aspect, the variable targeting domain comprises a continuous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0228] The terms "Cas endonuclease recognition domain" or "CER domain" of a guide polynucleotide are used interchangeably herein and include a nucleotide sequence (e.g., a second nucleotide sequence domain of a guide polynucleotide) that interacts with a Cas endonuclease polypeptide. The CER domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., modifications described herein), or any combination thereof.

[0229] The nucleotide sequence connecting the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. In one aspect, the nucleotide sequence connecting the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 98, 99, or 100 nucleotides in length. In another aspect, the nucleotide sequence linking the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can comprise a tetranucleotide loop sequence, such as, but not limited to, a GAAA tetranucleotide loop sequence.

[0230] The nucleotide sequence modifications of the guide polynucleotide, VT domain and / or CER domain can be selected from, but not limited to, the group consisting of: a 5′ cap, a 3′ poly(A) tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a protein binding site, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2′-fluoro A nucleotides, 2′-fluoro U nucleotides, 2′-O-methyl RNA nucleotides, a phosphorothioate bond, a linkage to a cholesterol molecule, a linkage to a polyethylene glycol molecule, a linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage, or any combination thereof. The modifications can produce at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group consisting of: modified or regulated stability, subcellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity to complementary target sequences, modified resistance to cellular degradation and increased cellular permeability.

[0231] In one aspect, the guide nucleotide and the Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double-strand break at a DNA target site.

[0232] In one aspect of the disclosure, the variable target domain may be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0233] In one aspect of the present disclosure, the guide nucleotide comprises a cRNA (or cRNA fragment) and a tracrRNA (or tracrRNA fragment) that can form a complex with a type II Cas endonuclease in a type II CRISPR / Cas system, wherein the guide nucleotide / Cas endonuclease complex can guide the Cas endonuclease to a plant genome target site, such that the Cas endonuclease can introduce a double-strand break into the genome target site. The guide nucleotide can be directly introduced into a plant or plant cell using any method known in the art (e.g., but not limited to, particle bombardment or topical application).

[0234] In one aspect, the guide nucleotide can also be introduced indirectly by introducing a recombinant DNA molecule comprising a corresponding guide DNA sequence operably linked to a plant-specific promoter capable of transcribing the guide nucleotide in a plant cell. The term "corresponding guide DNA" includes a DNA molecule that is identical to an RNA molecule, but in which a "T" replaces each "U" of the RNA molecule.

[0235] In one aspect, the guide nucleotides are introduced via particle bombardment or using the disclosed methods for Agrobacterium transformation of a recombinant DNA construct comprising the corresponding guide DNA operably linked to a plant U6 polymerase III promoter.

[0236] On the one hand, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. An advantage of using a guide nucleotide versus a duplex crRNA-tracrRNA is that only one expression cassette needs to be made in order to express the fused guide nucleotide.

[0237] The terms "target site", "target sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", and "genomic target locus" are used interchangeably herein and refer to a polynucleotide sequence in the genome of a plant cell (including chloroplast DNA and mitochondrial DNA) at which a double-strand break is induced in the genome of the plant cell by a Cas endonuclease. The target site may be an endogenous site in the plant genome, or alternatively, the target site may be heterologous to the plant, so that it does not naturally occur in the genome, or the target site may be found in a heterologous genomic location compared to where it occurs in nature.

[0238] As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of a plant and is at the endogenous or native location of the target sequence in the genome of a plant. In one aspect, the target site can be similar to a DNA recognition site or target site that is specifically recognized and / or bound by a double-strand break inducing agent, such as a LIG3-4 endonuclease (U.S. Patent Application Publication No. 2009 / 0133152) or a MS26++ meganuclease (U.S. Patent Application Publication No. 2014 / 0020131).

[0239] "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a plant. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the plant, but located at a different location in the genome of the plant (i.e., a non-endogenous or non-native location).

[0240] "Altered target site", "altered target sequence", "modified target site" and "modified target sequence" are used interchangeably herein and refer to a target sequence as disclosed herein: the target sequence comprises at least one change when compared to the unaltered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0241] In one aspect, the disclosed methods can be used to introduce into a plant a polynucleotide for gene suppression of a target gene in the plant. For several aspects of plant genetic engineering, it is desirable to reduce the activity of a specific gene (also known as gene silencing or gene suppression). Many techniques for gene silencing are well known to those skilled in the art, including but not limited to antisense technology (see, e.g., Sheehy et al. (1988) Proc. Natl. Acad. Sci. USA 85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566; and 5,759,829); cosuppression (e.g., Taylor (1997) Plant Cell 9:1245; Jorgensen (1990) Trends 50:1166; and U.S. Pat. Nos. 5,127,679; and 5,759,829); =Biotech. [Trends in Biotechnology] 8(12): 340-344; Flavell (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91: 3490-3496; Finnegan et al. (1994) Bio / Technology [Biotechnology] 12: 883-888; and Neuhuber et al. (1994) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 244: 230-241); RNA interference (Napoli et al. (1990) Plant Cell [Plant Cell] 2: 279-289; U.S. Patent No. 5,034,323; Sharp (1999) Genes Dev. [Genes and Development] 13: 139-141; Zamore et al. (2000) Cell [Cell] 101: 25-33; Javier (2003) Nature [Nature] 425: 257-263; and Montgomery et al. (1998) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 95: 15502-15507); virus-induced gene silencing (Burton et al. (2000) Plant Cell [Plant Cell] 12: 691-705; and Baulcombe (1999) Curr. Op. Plant Bio. Current Status in Plant Biology 2: 109-113); target RNA-specific ribozymes (Haseloff et al. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000) Nature 407: 319-320; WO 99 / 53050; WO 02 / 00904; and WO 98 / 53083); ribozymes (Steinecke et al. (1992) EMBO J.[EMBO Journal] 11: 1525; U.S. Pat. No. 4,987,071; and Perriman et al. (1993) Antisense Res. Dev. [Antisense Research and Development] 3: 253); oligonucleotide-mediated targeted modification (e.g., WO 03 / 076574 and WO 99 / 25853); zinc finger targeting molecules (e.g., WO 01 / 52620; WO03 / 048345; and WO 00 / 42219); artificial microRNA (US 8106180; Schwab et al. (2006) Plant Cell [Plant Cell] 18: 1121-1133); and other methods known to those skilled in the art or combinations of the above methods. .

[0242] On the one hand, the disclosed method can be used for introducing the polynucleotides used for the targeted integration of nucleotide sequences into plants. For example, the disclosed method can be used for introducing a T-DNA expression cassette to transform a plant comprising a target site, wherein the transfer cassette comprises the target nucleotide sequence flanking different recombination sites. On the one hand, the target site comprises at least one group of different recombination sites corresponding to those on the T-DNA expression cassette. The exchange of the nucleotide sequence flanking the recombination site is carried out by a recombinase. Therefore, the disclosed method can be used for introducing a T-DNA expression cassette to target and integrate a nucleotide sequence, wherein the flank of the T-DNA expression cassette is a different recombination site identified by a recombinase, and the recombinase identifies and realizes the reorganization at different recombination sites. Therefore, the disclosed method and composition can be used for improving the development efficiency and the development speed of the plant containing different recombination sites.

[0243] Thus, the disclosed methods can further include methods for directed, targeted integration of exogenous nucleotides into transformed plants. In one aspect, the disclosed methods use novel recombination sites in a gene targeting system that facilitates directed targeting of desired genes and nucleotide sequences to corresponding recombination sites previously introduced into the target plant genome.

[0244] On the one hand, the nucleotide sequence of two non-identical recombination sites is introduced into one or more cells of the explant derived from the target organism genome, thereby setting up the target site for inserting the target nucleotide sequence. Once a stable plant or cultured tissue has been established, a second construct or the target nucleotide sequence with recombination sites corresponding to those recombination sites of the flank target site is introduced into the plant or tissue of stable transformation in the presence of the recombinase protein. This process results in the exchange of the nucleotide sequence between the non-identical recombination sites of the target site and the T-DNA expression cassette.

[0245] It should be appreciated that the transformed plants prepared in this manner may contain multiple target sites; i.e., multiple sets of non-identical recombination sites. In this manner, multiple manipulations of target sites in the transformed plants are available. The target site in the transformed plant refers to a DNA sequence that has been inserted into the transformed plant genome and contains non-identical recombination sites.

[0246] Examples of recombination sites for use in the disclosed methods are known in the art and include FRT sites (see, e.g., Schlake and Bode (1994) Biochemistry 33:12746-12751; Huang et al. (1991) Nucleic Acids Research 19:443-448; Paul D. Sadowski (1995) Progress in Nucleic Acid Research and Molecular Biology 51, 53-91; Michael M. Cox (1989) Mobile DNA, Berg and Howe (eds.) American Society of Microbiology, Washington, D.C., 116-670; Dixon et al. (1995) 18:449-458; Umlauf and Cox (1988) The EMBO Journal of the European Molecular Biology Association 7: 1845-1852; Buchholz et al. (1996) Nucleic Acids Research 24: 3118-3119; Kilby et al. (1993) Trends Genet. 9: 413-421: Rossant and Geagy (1995) Nat. Med. 1: 592-594; Albert et al. (1995) The Plant J. 7: 649-659: Bayley et al. (1992) Plant Mol. Biol. [Plant Molecular Biology] 18: 353-361; Odell et al. (1990) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 223: 369-378; and Dale and Ow (1991) Proc. Natl. Aead. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 88: 10558-105620; all of which are incorporated herein by reference); Lox (Albert et al. (1995) Plant J. [Plant Journal] 7: 649-659; Qui et al. (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91: 1706-1710; Stuurman et al. (1996) Plant Mol. Biol. [Plant Molecular Biology] 32: 901-913; Odell et al. (1990) Mol. Gen. Gevet.[Molecular and General Genetics] 223: 369-378; Dale et al. (1990) Gene [Gene] 91: 79-85; and Bayley et al. (1992) Plant Mol. Biol. [Plant Molecular Biology] 18: 353-361.) The two-micron plasmid found in most naturally occurring strains of Saccharomyces cerevisiae encodes a site-specific recombinase that promotes the inversion of DNA between the two inverted repeats. This inversion plays a central role in the expansion of plasmid copy number. .

[0247] A protein named FLP protein catalyzes site-specific recombination events. The minimal recombination site (FRT) has been defined and contains two reverse 13 base pair (bp) repeats around an asymmetric 8-bp spacer. The FLP protein cuts the site at the junction of the repeat and the spacer and covalently connects to the DNA via the 3' phosphate. Site-specific recombinases (such as FLP) cut and reconnect DNA at specific target sequences, which leads to precisely defined recombination between two identical sites. In order to work, the system requires recombination sites and recombinases. No auxiliary factors are required. Therefore, the entire system can be inserted into plant cells and work therein. The yeast FLP\FRT site-specific recombination system has been shown to work in plants. So far, the system has been used to remove unwanted DNA. See Lyznik et al. (1993) Nucleic Acids Res. [Nucleic Acids Research] 21: 969-975. In contrast, the present disclosure utilizes non-identical FRTs for the control of exchange, targeting, arrangement, insertion and expression of nucleotide sequences in plant genomes.

[0248] On the one hand, it is necessary to transform a target organism, such as an explant from a plant, containing a target site that is integrated into its genome. The target site is characterized in that the flanks have non-identical recombination sites. In addition, a targeting box is needed, which contains a nucleotide sequence that is flanked by non-identical recombination sites corresponding to those sites included in the target site of the transformed organism. It is necessary to recognize a recombinase that recognizes non-identical recombination sites and catalyzes site-specific recombination.

[0249] It should be appreciated that the recombinase can be provided by any means known in the art. That is, by transient expression or by providing a messenger RNA (mRNA) of the recombinase or recombinase protein, it can be provided in an organism or plant cell by transforming an organism with an expression cassette capable of expressing the recombinase in the organism.

[0250] "Non-identical recombination sites" means that the flanking recombination sites are not identical in sequence and will not recombine, or the recombination between the sites will be minimized. That is, one flanking recombination site can be a FRT site, and the second recombination site can be a mutated FRT site. The non-identical recombination sites used in the method of the present disclosure prevent or greatly inhibit the reorganization between the two flanking recombination sites and the excision of the nucleotide sequence contained therein. Therefore, it should be recognized that any suitable non-identical recombination sites can be used in the present disclosure, including FRT and mutated FRT sites, FRT and lox sites, lox and mutated lox sites, and other recombination sites known in the art.

[0251] By suitable non-identical recombination sites is meant that, in the presence of an active recombinase, excision of the sequence between the two non-identical recombination sites (if any) occurs at a significantly lower efficiency than the targeted arrangement of the nucleotide sequence recombination-mediated exchange into the plant genome. Thus, suitable non-identical sites for use in the present disclosure include those sites where the recombination efficiency between the sites is low; for example, where the efficiency is less than about 30% to about 50%, preferably less than about 10% to about 30%, more preferably less than about 5% to about 10%.

[0252] As noted above, the recombination sites in the targeting cassette correspond to the recombination sites in the target site of the transformed plant. That is, if the target site of the transformed plant contains non-identical recombination sites flanked by FRT and mutant FRT, the targeting cassette will contain non-identical recombination sites for the same FRT and mutant FRT.

[0253] It should also be recognized that the recombinase used in the disclosed method will depend on the recombination sites in the target site of the transformed plant and in the targeting box. That is, if the FRT site is used, the FLP recombinase is required. In the same way, in the case of using lox sites, the Cre recombinase is required. If the different recombination sites contain both FRT and lox sites, both FLP and Cre recombinases will be required in the plant cell.

[0254] The FLP recombinase is a protein that catalyzes a site-specific reaction that is involved in amplifying the copy number of the two-micron plasmid of Saccharomyces cerevisiae during DNA replication. The FLP protein has been cloned and expressed. See, e.g., Cox (1993) Proc. Natl. Acad. Sci. USA 80:4223-4227. The FLP recombinase used in the present disclosure may be an enzyme derived from Saccharomyces. It may be preferred to synthesize the recombinase using plant-preferred codons for optimal expression in the plant of interest. See, e.g., U.S. Application Serial No. 08 / 972,258, filed on November 18, 1997, entitled "Novel Nucleic Acid Sequence Encoding FLP Recombinase," which is incorporated herein by reference.

[0255] The bacteriophage recombinase Cre catalyzes site-specific recombination between two lox sites. The Cre recombinase is known in the art. See, for example, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J. Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet. 22:477-488; and Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702. All of which are incorporated herein by reference. Such Cre sequences can also be synthesized using plant-preferred codons.

[0256] Where appropriate, the nucleotide sequence to be inserted into the plant genome can be optimized to increase expression in the transformed plant. In the case of mammalian, yeast or bacterial genes used in the present disclosure, they can be synthesized using plant preferred codons to improve expression. It should be recognized that for expression in monocots, dicot genes can also be synthesized using monocot preferred codons. Methods for synthesizing plant preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 477-498, which are incorporated herein by reference. Plant preferred codons can be determined from the codons used more frequently in the proteins expressed in the target plant. It should be recognized that monocot or dicot preferred sequences as well as plant preferred sequences of specific plant species can be constructed. See, e.g., EPA 0359472; EPA 0385962; WO 91 / 16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research, 17:477-498. U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; etc., which are incorporated herein by reference. It is further recognized that all or any portion of the gene sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences may also be used.

[0257] Additional sequence modifications are known to enhance gene expression in cellular hosts and can be used in the present disclosure. These include: elimination of sequences encoding spurious polyadenylation signals and exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to an average level for a given cellular host calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid the presence of foreseeable hairpin secondary RNA structures.

[0258] The present disclosure also includes a new FLP recombination target site (FRT). FRT has been identified as a minimal sequence containing two 13 base pair repeats separated by an 8 base spacer, as shown below: 5′-GAAGTTCCTATTC[TCTAGAAA]GTATAGGAACTTC3′, where the nucleotides in brackets represent spacers. As long as the two 13 base repeats are separated by eight nucleotides, the nucleotides in the spacer can be replaced by a combination of nucleotides. It seems that the actual nucleotide sequence of the spacer is not critical; however, for the practice of the present disclosure, certain substitutions of nucleotides in the spacer region may work better than other substitutions. During strand exchange, the eight base pair spacer participates in DNA-DNA pairing. The asymmetry of this region determines the direction of site alignment in the recombination event, which will subsequently lead to inversion or excision. As shown above, most spacers can be mutated without loss of function. See, for example, Schlake and Bode (1994) Biochemistry [Biochemistry] 33: 12746-12751, incorporated herein by reference.

[0259] The new FRT mutation site can be used to practice the disclosed method. Such mutation site can be constructed by PCR-based mutagenesis. Although the mutation FRT site is known (see SEQ ID No 2,3,4 and 5 of WO 1999 / 025821), it is recognized that other mutation FRT sites can be used to practice the disclosure. The disclosure does not use specific FRT or recombination sites, but can use different recombination sites or FRT sites with nucleotide sequence targeted insertion and expression in plant genome. Therefore, other mutation FRT sites can be constructed and used based on the disclosure.

[0260] As discussed above, in the presence of a recombinase, a genomic DNA containing a target site with non-identical recombination sites causes recombination together with a carrier containing a T-DNA expression cassette with corresponding non-identical recombination sites. The nucleotide sequence of the T-DNA expression cassette between the flanking recombination sites is exchanged with the nucleotide sequence of the target site between the flanking recombination sites. In this way, the target nucleotide sequence can be accurately incorporated into the host's genome.

[0261] It is recognized that many variations of the present disclosure can be implemented. For example, a target site with a plurality of different recombination sites can be constructed. Therefore, a plurality of genes or nucleotide sequences can be stacked or sorted at the precise position in the plant genome. Similarly, once a target site has been established in the genome, other recombination sites can be introduced by incorporating such sites into the nucleotide sequence of the T-DNA expression cassette and transferring the sites to the target sequence. Therefore, once a target site has been established, it is possible to add sites or change sites subsequently by restructuring.

[0262] Another variation includes providing a promoter or transcription initiation region operably linked to a target site in an organism. Preferably, the promoter will be located 5' of the first recombination site. By transforming an organism with a T-DNA expression cassette comprising a coding region, expression of the coding region will occur when the T-DNA expression cassette is integrated into the target site. This aspect provides a method for selecting transformed cells (particularly plant cells) by providing a selectable marker sequence as a coding sequence.

[0263] Other advantages of this system include the ability to reduce the complexity of the integration of the transgenic or transferred DNA in an organism by utilizing the T-DNA expression cassette as discussed above and selecting the organism with a simple integration pattern. In the same manner, the preferred sites in the genome can be identified by comparing several transformation events. The preferred sites in the genome include sites that do not destroy the expression of the essential sequence and provide sufficient expression of the transgenic sequence.

[0264] The disclosed methods also provide methods for combining multiple expression cassettes at one location within the genome. Recombination sites can be added or deleted at a target site within the genome.

[0265] Any means known in the art for bringing the three components of this system together can be used in the present disclosure. For example, a plant can be stably transformed to have a target site in its genome. The recombinase can be transiently expressed or provided. Alternatively, a nucleotide sequence capable of expressing the recombinase can be stably integrated into the genome of the plant. In the presence of the corresponding target site and the recombinase, a T-DNA transfer cassette flanked by corresponding non-identical recombination sites is inserted into the genome of the transformed plant.

[0266] Alternatively, the components of this system can be put together by sexual hybridization of transformed plants. In this respect, the transformed plant (parent one) containing the target site that is integrated into its genome can be sexually hybridized with the second plant (parent two) genetically transformed with the T-DNA expression cassette containing the different recombination sites of the flanks (which corresponds to the recombination sites in plant one). Plant one or plant two contain the nucleotide sequence expressing the recombinase in its genome. The recombinase can be under the control of a constitutive or inducible promoter. In this way, the expression of the recombinase and the subsequent activity at the recombination site can be controlled.

[0267] The disclosed method can be used for the targeted integration of the nucleotide sequence transferred into a specific chromosome site. The nucleotide sequence can encode any target nucleotide sequence. Specific target genes include those that provide easy analysis of functional characteristics for host cells and / or organisms, such as marker genes and other genes that change the phenotype of receptor cells, etc. Therefore, genes that affect plant growth, height, susceptibility to diseases and insects, nutritional value, etc. can be used in the present disclosure. Nucleotide sequences can also encode "antisense" sequences to turn off or modify gene expression.

[0268] It should be appreciated that the nucleotide sequence can be used in a functional expression unit or a T-DNA expression cassette. A functional expression unit or a T-DNA expression cassette means a target nucleotide sequence with a functional promoter, and in most cases a terminator. In the practice of the present disclosure, there are various ways to obtain a functional expression unit. In one aspect of the present disclosure, the target nucleic acid is transferred or inserted into the genome as a functional expression unit.

[0269] Alternatively, the nucleotide sequence can be inserted into the site 3' of the promoter region in the genome. In the latter case, the coding sequence is inserted into the promoter region 3' so that a functional expression unit is achieved when integrated. The T-DNA expression cassette will contain a transcription initiation region or a promoter operably connected to the nucleic acid encoding the target peptide. This type of expression cassette is provided with a plurality of restriction sites for one or more target genes so that the gene is located under the transcriptional regulation of the regulatory region.

[0270] experiment

[0271] Example 1: Plasmid

[0272] For a description of plasmids containing the specified components mentioned in the examples, see Table 7. Plasmid IDs followed by "+" include T-DNA containing the specified components and other specified components located outside the T-DNA left border (LB). As known to those skilled in the art, other components can alternatively be located outside the T-DNA right border (RB).

[0273] Table 7.

[0274]

[0275]

[0276]

[0277]

[0278] Example 2: Culture medium

[0279] See Tables 8, 9 and 10 for a description of the media formation used for transformation, selection and regeneration mentioned in the Examples.

[0280] Table 8.

[0281]

[0282]

[0283]

[0284] Table 9.

[0285]

[0286]

[0287] Table 10 has been summarized the composition of various substrata for soybean transformation, tissue culture and regeneration.In this table, if this is the starting material for transformation, substratum M1 is used for suspension culture.Substratum M2 and M3 represent the typical co-culture medium that can be used for the Agrobacterium transformation of the explant of above-mentioned listed whole range.Substratum M4 can be used for selection (using appropriate selection agent), M5 is used for somatic embryo maturation, and substratum M6 is used for sprouting to produce T0 plantlet.

[0288] Table 10.

[0289]

[0290]

[0291]

[0292] Example 3: Particle bombardment

[0293] The pioneer inbred line PH184C (disclosed in US8445763) was used, which contains a pre-integrated site-specific integration target site (Chrom-1 target site) consisting of UBI PRO: FRT1: NPTII:: PINII TERM + FRT87 in chromosome-1. Prior to bombardment, 10-12 DAP (days after pollination) immature embryos were isolated from ears of the pioneer inbred line PH184C and placed in 605J medium and 16% sucrose for 3 hours to allow the scutellum cells to plasmolyze.

[0294] Four plasmids are typically used per particle bombardment:

[0295] 1) Donor plasmid (100 ng / μl) containing a FRT-flanked donor cassette for recombinase-mediated cassette exchange, e.g., a plasmid containing FRT1:PMI::PINII TERM+UBI1ZM PRO::DS-RED2::PINII TERM+FRT87 (PHP0004);

[0296] 2) plasmid containing the expression cassette UBI1ZM PRO::FLPm::PINII TERM (PHP5096) (2.5 ng / μl);

[0297] 3) Plasmid containing the expression cassette ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM+FMV&PCSV enhancer (10 ng / μl) (PHP89030); and

[0298] 4) Plasmid containing the expression cassette ZM-PLTP PRO::ZM-WUS2::IN2-1TERM (5 ng / ul) (PHP89179).

[0299] To attach the DNA to the 0.6 μm gold particles, the four plasmids were mixed together by adding 10 μl of each plasmid together for a total of 40 μl in a low binding microcentrifuge tube (Sorenson Bioscience 39640T). To this suspension was added 50 μl of 0.6 μm gold particles (30 μg / μl) and 1.0 μl of Transit 20 / 20 (Catalog No. MIR5404, MirusBio LLC), and the suspension was placed on a rotary shaker for 10 minutes. The suspension was centrifuged at 10,000 RPM (approximately 9400 x g) and the supernatant was discarded. The gold particles were resuspended in 120 μl of 100% ethanol, sonicated briefly at low power, and 10 μl was pipetted onto each carrier disk. The carrier disks were then air dried to remove any residual ethanol. Particle bombardment was performed using a Biolistics PDF-1000 at 28 inches of mercury using a 200 PSI rupture disk. After particle bombardment, immature embryos were selected on 605J medium modified to contain 12.5 g / l mannose and 5 g / l maltose and without sucrose. After 10-12 weeks of selection, plantlets were regenerated and analyzed using qPCR. It is expected that the co-delivery of PLTP::ODP2 (PHP89030) and PLTP::WUS2 (PHP89179) together with SSI components (donor DNA (PHP0004) + UBI::FLP (PHP5096)) will produce a high frequency (i.e., a ratio of 4-7% relative to the number of bombarded immature embryos) of donor fragment site-specific integration into the Chrom-1 target site.

[0300] Example 4: Agrobacterium-mediated transformation of maize

[0301] A. Preparation of Agrobacterium master plates.

[0302] Master plates were prepared by streaking A. tumefaciens carrying the binary donor vector from -80 °C frozen aliquots onto solid 12R medium and growing at 28 °C in the dark for 2-3 days.

[0303] B. Growing Agrobacterium on solid medium.

[0304] Single or multiple colonies of Agrobacterium were picked from the master plate and streaked onto a second plate containing 810K medium and incubated overnight at 28°C in the dark.

[0305] Agrobacterium infection medium (700A; 5 ml) and 100 mM 3′-5′-dimethoxy-4′-hydroxyacetophenone (acetosyringone; 5 μL) were added to a 14 mL conical tube in a fume hood. Approximately 3 loopfuls of Agrobacterium from the second plate were suspended in the test tube, which was then vortexed to form a uniform suspension. The suspension (1 ml) was transferred to a spectrophotometer tube, and the optical density (550 nm) of the suspension was adjusted to a reading of approximately 0.35-1.0. The Agrobacterium concentration was approximately 0.5 to 2.0×10 9 cfu / mL. The final Agrobacterium suspension was aliquoted into 2 mL microcentrifuge tubes, each containing approximately 1 mL of the suspension. The suspension was then used as quickly as possible.

[0306] C. Growing Agrobacterium in liquid culture.

[0307] Alternatively, Agrobacterium can be prepared for transformation by growing in liquid medium. The day before infection, 125ml flask was made with 30ml 557A culture medium (10.5g / l dipotassium hydrogen phosphate, 4.5g / l potassium dihydrogen phosphate anhydrous, 1g / l ammonium sulfate, 0.5g / l sodium citrate dehydrate, 10g / l sucrose, 1mM magnesium sulfate) and 30 μL spectinomycin (50mg / mL) and 30 μL acetosyringone (20mg / mL). The semi-ring Agrobacterium from the second flat plate was suspended in a flask, and placed on an orbital shaker set at 200rpm, and incubated overnight at 28°C. Agrobacterium culture was centrifuged at 5000rpm for 10 minutes. Remove supernatant and add the Agrobacterium infection culture medium (700A) with acetosyringone solution. Bacteria are resuspended by vortexing, and the optical density (550nm) of Agrobacterium suspension is adjusted to a reading of about 0.35 to 2.0.

[0308] D. Maize transformation.

[0309] The ear of maize (Zea mays L.) cultivar is surface sterilized in 20% (v / v) bleach (5.25% sodium hypochlorite) plus 1 drop of Tween 20 for 15-20 minutes, then washed 3 times in sterile water. Immature embryos (IE) are separated from the ear and placed in 2ml agrobacterium infection medium (700A) with acetosyringone solution. The optimal size of the embryo varies with inbred lines, but for transformation with WUS2 and ODP2, a wide range of immature embryo sizes can be used. Take out agrobacterium infection medium (810K), 1ml agrobacterium suspension is added to the embryo, and the test tube is vortexed for 5-10 seconds. Allow the microcentrifuge tube to stand for 5 minutes in a fume hood. Agrobacterium suspension and embryo are poured on 710I (or 562V) co-culture medium (see Table 8). Any embryo remaining in the tube is transferred to a flat plate using a sterile scraper. Extract the agrobacterium suspension and place the embryo on the axial side of the culture medium. The plates were incubated in the dark at 21°C for 1-3 days for co-cultivation.

[0310] The embryos are transferred to resting medium (605T medium) without selection. After 3 to 7 days, the embryos are transferred to maturation medium (289Q medium) supplemented with a selection agent.

[0311] Example 5: Agrobacterium mixture for recovery of maize transgenic events containing only trait expression cassette T-DNA

[0312] The following experiments demonstrate that delivery of a plasmid containing WUS2 together with a plasmid containing a trait expression cassette improves the recovery of transgenic TO plants containing only the trait T-DNA but no vector (plasmid) backbone.

[0313] Immature embryos of maize non-hard stem inbred HC69 were transformed with Agrobacterium strain LBA4404THY- (see U.S. Pat. No. 8,334,429, incorporated herein by reference in its entirety) (Agro1), which contained PHP86491, a T-DNA with two "traits" (ZS-GREEN and ZM-HRA) expression cassettes (Table 7). This Agrobacterium (Agro1) was used alone (control treatment) to infect immature embryos or as part of a mixture with another Agrobacterium (Agro2) containing a WUS expression cassette (PHP87598, PHP88156, PHP88157 or PHP88158, see Table 7). For treatments (Trts) 1-4, two Agrobacteria were mixed at 95% of the PHP86491 plasmid (Agro1) containing the trait and 5% of the plasmid (Agro2) containing WUS (PHP87598, PHP88156, PHP88157 or PHP88158, see Table 7). As shown in Table 11 below, when only Agrobacterium (Agro1) containing the trait T-DNA (PHP86491) plasmid was used for transformation, the frequency of recovering T0 plants with a single copy of T-DNA and no plasmid backbone (sequences outside the T-DNA borders) was low (Trt C = 1.8%). However, when a second Agrobacterium (Agro2) containing a T-DNA with a WUS expression cassette (PHP87598, PHP88156, PHP88157 or PHP88158, see Table 7) was mixed with Agro1 at a low titer, it was observed that the recovery frequency of transgenic T0 plants containing only a single copy of the trait T-DNA (PHP86491-ZS-GREEN+ZM-HRA) without integrating the WUS-containing T-DNA (PHP87598, PHP88156, PHP88157 or PHP88158, see Table 7) or the plasmid backbone was significantly increased (Trts 1-4 in Table 11 was 6.4% to 16.2%). For Trt1, in which the T-DNA in Agro2 (PHP87598) contains three viral enhancer elements (ENH) at the 5′ end of the ZM-PLTP promoter driving WUS expression and a heat shock promoter driving CRE expression, a constitutively expressed anthocyanin expression cassette (NOS PRO::CRC::SB-GKAF TERM) and loxP sites flanking all three expression cassettes, the integration frequency of the single copy trait T-DNA and the plasmid-free backbone was 6.5%.For PHP88156, PHP88157, and PHP88158, all of which contained a 3xENH:PLTP promoter driving WUS with or without a maize-derived enhancer element (EME) located near the TATA box of the PLTP promoter (triplicate (3xEME in PHP88156), single copy (EME in PHP88157), or without EME (EME- in PHP88158)) and a constitutively expressed anthocyanin expression cassette, the frequency of T0 plants containing only a single copy of the trait T-DNA and no plasmid backbone was 6.4 (PHP88156), 16.2 (PHP88157), and 12.1% (PHP88158). Escapes (regenerated non-transgenic T0 plants) were 4% when Agro2 contained PHP88157 and 1% when Agro2 contained PHP88158. 3X ENH had no significant effect on NOS PRO::CRC::SB-GKAF The TERM expression cassette was effective, and if a T-DNA containing the ENH and NOS PRO::CRC::SB-GKAF TERM expression cassettes was integrated, the embryos were dark red and failed to produce plantlets. Treatments 2-4 (Table 11) showed that mixing a second Agrobacterium containing the WUS expression cassette at a low titer (5% relative to the Agro1 containing the trait-T-DNA) provided an effective alternative for improving the recovery of TO plants containing only a single copy of the trait T-DNA and no plasmid backbone, without the need to track the transformation process by excising the WUS expression cassette.

[0314] Table 11.

[0315]

[0316] Immature embryos of maize hard stem inbred PH1V69 disclosed in U.S. Pat. No. 8,907,160 and incorporated herein by reference in its entirety were transformed with Agrobacterium strain LBA4404 THY-(Agro1) containing PHP86491 (Table 7). The Agrobacterium (Agro1) was used alone (control treatment) to infect immature embryos or as part of a mixture (95% Agro1 and 5% Agro2) with another Agrobacterium (Agro2) containing a WUS expression cassette (PHP87598 or PHP88156 or PHP88157 or PHP88157).

[0317] In the control treatment (Agro1 alone), no escapes (regenerated non-transgenic T0 plants) were recovered, nor were T0 plants containing only a single copy of the trait T-DNA and no plasmid backbone recovered. In contrast, when a second Agrobacterium (Agro2) containing PHP87598 (Table 7) was mixed with Agro1 and the mixture was used for transformation, no escapes were recovered, and the frequency of T0 plants containing only a single copy of the trait T-DNA and no plasmid backbone was 16.5%. When PHP87078 (Table 7) was used to replace PHP87598 in Agro2, no escapes were observed in the regenerated T0 plants, and the frequency of T0 plants containing only a single copy of the trait T-DNA and no plasmid backbone was 7.1%. These frequencies represent a significant improvement in our ability to recover high-quality T0 plants (single copy traits without integrated exogenous sequences (no plasmid backbone or any T-DNA sequences from Agro2)) using morphogenic gene expression cassettes that do not integrate the WUS-containing expression cassette (e.g., 3xENH:PLTP::WUS2).

[0318] The immature embryos of maize hard stem inbred PH1V69 were transformed with Agrobacterium strain LBA4404THY- containing plasmid PHP86491 (Table 7). This Agrobacterium (Agro1) was used as part of a mixture with another Agrobacterium (Agro2) containing plasmid PHP87598 (Table 7), and the plasmid contained a WUS expression cassette. Two different ratios of Agro1:Agro2 were tested, i.e. 50:50 or 90:10. When the 50:50 mixture was used for transformation, no escape was observed in the resulting T0 plant, and the frequency of the T0 plant containing only a single copy of the trait T-DNA and no plasmid backbone was 8.2%. When the 90:10 mixture was used for transformation, no escape was observed in the resulting T0 plant, and the frequency of the T0 plant containing only a single copy of the trait T-DNA and no plasmid backbone was 26%. This result demonstrates that the relative concentrations of Agro1 and Agro2 in the transformation mixture influence the frequency of TO plants containing only a single copy of the trait T-DNA and no plasmid backbone.

[0319] Immature embryos from four different tropical maize inbred lines were harvested and transformed with one of the two "trait" expression cassettes using PHP81561 (ZS-YELLOW+NPTII) or PHP86491 (ZS-GREEN and ZM-HRA). For all four inbred lines, PH2KD1, PH44MH, PH4BAH, and PH28SV, the transformation efficiency was effectively 0% using either G-418 selection or imazapyr selection (for the NPTII or HRA gene, respectively). However, when a second Agrobacterium containing (PHP88158, see Table 7) was added at a titer of 10% relative to the trait-containing Agro1 (containing PHP81561, see Table 7) and G-418 selection was used throughout somatic embryo maturation and germination, the frequency of recovered T0 plants containing only a single copy of T-DNA and no vector (plasmid) backbone was 30% for PH2KD1, 38% for PH44MH, 43% for PH4BAH, and 3.2% for PH28SV, relative to the number of starting embryos. When the second Agrobacterium containing PHP88158 was added at a titer of 10% relative to Agro1 (containing PHP86491) containing ZS-GREEN and ZM-HRA- and selected with 0.1 mg / l of methamphetamine throughout somatic embryo maturation and germination, the frequency of recovered TO plants containing only a single copy of T-DNA and no vector (plasmid) backbone was 15% for PH2KD1, 18% for PH44MH, 9.6% for PH4BAH, and 0% for PH28SV relative to the number of starting embryos. These results indicate that the use of a WUS expression cassette within the T-DNA of Agro2 and mixing Agro2 at 10% relative to the trait-containing Agro1 (90%), has produced an efficient method for integrating the trait-T-DNA, with a very efficient frequency of recovering TO plants containing only a single copy of T-DNA and no vector (plasmid) backbone.

[0320] Example 6: Use of a WUS2 expression cassette outside of the T-DNA borders containing a trait to stimulate transformation and selection of single copy TO maize plants that do not contain the WUS2 expression cassette integrated into the chromosomal DNA

[0321] For these experiments, a set of three plasmid designs were tested. All three plasmids contained the same T-DNA (RB+LOXP+SI-UBI3 PRO::ZS-GREEN1::PINII TERM+SB-ALS PRO::ZM-HRA::SB-PEPC1 TERM+LB) but differed in the WUS2 or WUS2 / ODP2 expression cassette outside the left border. Specifically, PHP86295 contains two expression cassettes ZM-PLTP PRO:: ZM-WUS2:: IN2-1 TERM + ZM-PLTP PRO:: ZM-ODP2:: OS-T28TERM (see Table 7) just outside the left border, PHP86296 contains a single expression cassette ZM-PLTP PRO:: ZM-WUS2:: IN2-1TERM just outside the left border, and PHP86297 contains a single expression cassette ZM-PLTP PRO:: ZM-WUS2~T2A~ZM-ODP2:: OS-T28TERM just outside the left border. For Pioneer hard stem inbred PH1V69, after Agrobacterium-mediated transformation with these respective T-DNAs, 0.05 to 0.1 mg / l of methamphetamine was selected during somatic embryo maturation and regeneration, and T0 plants were analyzed by qPCR. According to qPCR analysis, using PHP86295, 10.2% of the recovered T0 plants were single copies of the trait genes within the T-DNA, lacking integrated WUS and / or ODP2 without plasmid backbone contamination, and these values ​​were 12.2% and 12.3% for PHP86296 and PHP86297, respectively. These results show that placing the WUS2 and / or ODP2 expression cassette outside the left border of the T-DNA provides the necessary stimulation for transformation without the integration of these morphogenetic gene expression cassettes. This result further proves that WUS and / or ODP2 can be expressed transiently after "outside the border" delivery to stimulate maize transformation. On the contrary, using these same vectors in the inbred HC69 did not increase the recovery of single-copy trait T-DNA events without a vector (plasmid) backbone, which is likely due to the inbreeding tendency to produce multi-copy integration events with a high degree of border reading.

[0322] Example 7: Use of multiple left borders between T-DNA and morphogenetic gene expression cassettes increases the frequency of single copy trait T-DNA integration events

[0323] Maize inbred lines differ in many morphological and physiological characteristics. One manifestation of this variability is the sensitivity of immature embryo scutellum of different inbred lines to T-DNA delivery and integration during Agrobacterium infection and co-cultivation. For example, when Agrobacterium strain LBA4404THY- was used to transform Pioneer Inbred HC69, transgenic T0 plants recovered at high frequency contained multiple copies of T-DNA and read at the T-DNA left border (LB), resulting in the integration of the vector (plasmid) backbone. The introduction of multiple left borders will alleviate this trend. For example, the introduction of multiple left borders in PHP86296 between the T-DNA containing the trait and the flanking WUS2 expression cassette resulted in the following configuration: RB+LOXP+SI-UBI3 PRO::ZS-GREEN1::PINII TERM+SB-ALSPRO::ZM-HRA::SB-PEPC1 TERM+LB+LB+LB+ZM-PLTP PRO::ZM-WUS2::IN2-1 TERM. When LBA4404 containing the above-described multiple LB T-DNAs and flanking WUS2 expression cassettes is used to transform HC69, it is expected that a significantly higher frequency of recovered TO plants will be single copy T-DNA and have no integrated WUS2 expression cassette and will not contain the plasmid backbone.

[0324] Example 8: Mutagenesis of the left border sequence increases the frequency of recovering transgenic events

[0325] Maize inbred lines are different in many forms and physiological characteristics.A kind of performance of this variability is that the immature embryo scutellum of different inbred lines is sent to T-DNA and integrated sensitivity during agrobacterium infection and co-cultivation.For example, when using Agrobacterium strain LBA4404THY- to transform pioneer inbred PH28SV, almost no cell accepts LB flanking sequence in the scutellum, such as flank WUS2 expression cassette, is therefore not stimulated to form new somatic embryo rapidly.Through the selective mutagenesis LB sequence, the effect of LB can be changed, to allow higher frequency LB to read.When used for transformation, it is expected that the somatic embryo of higher frequency rapid formation and the gained T0 plant that comprises single copy trait gene and does not have carrier (plasmid) skeleton will be produced.

[0326] Agrobacterium co-cultivation of varieties that are more resistant to infection may make expression of morphogenetic genes beyond the border delivery impractical. It is expected that the use of more aggressive Agrobacterium strains (such as AGL1, EHA101, EHA105 or any strain with the super-toxic Ti plasmid pTiBo54) can address this potential disadvantage. In addition, the use of binary vectors with higher copy numbers can be used to deliver an increased number of T-DNA molecules per cell, thereby amplifying beyond the border reads and subsequent morphogenetic gene expression.

[0327] Example 9: Plasmid Mixtures for Recovery of Canola Transgenic Events Containing Only Trait T-DNA

[0328] Rapeseed seeds were surface sterilized and germinated on half-strength MS medium to produce sterile one-week-old plants. Hypocotyls were placed in 10 ml 20A medium (Table 9) and cut into 2 mm fragments. Agrobacterium strain LBA4404 THY- was suspended in 20A medium and adjusted to 0.6 OD550, and 10-200 μl of this suspension was added to each flat plate containing hypocotyls.

[0329] Hypocotyl fragments were transformed with Agrobacterium strain LBA4404THY-(Agro1) containing PHP88193, which is a T-DNA with two "traits" (SPCN and ZS-YELLOW1 N1) expression cassettes (Table 7). This Agrobacterium (Agro1) was used alone (control treatment) to infect hypocotyl fragments, or as part of a mixture with another Agrobacterium (Agro2) containing PHP90094 (SEQ ID NO: 116) (HA-WUS expression cassette (Table 7)). The mixture contained 95% Agro1 and 5% Agro2. After co-cultivation with Agrobacterium in 20A medium (Table 9) at low light and 21°C, the hypocotyl fragments were transferred to selection medium, starting with 70A medium (Table 9) for two weeks, 70B medium (Table 9) for another two weeks, and then transferred to 70C medium (Table 9) for two weeks. For continued germination and rooting, the shoots were transferred to a solid medium consisting of half-strength MS salts, 10 g / l sucrose, 0.5 mg / l IBA and 5 g / l TC agar adjusted to pH 5.7. Once sufficient roots were formed, the plantlets were transferred to soil in the greenhouse.

[0330] In the control treatment with Agro1 alone, due to the stringent selection of spectinomycin, it is expected that no escapees (regenerated non-transgenic T0 plants) will be recovered, the frequency of recovering transgenic T0 plants will be low (i.e., <1.5%), the time from Agrobacterium infection to recovery of T0 plantlets usually takes 3-4 months, and the frequency of T0 plants containing only a single copy of the trait T-DNA and no vector (plasmid) backbone will be low. In contrast, when a second Agrobacterium (Agro2) containing PHP90094 is mixed with Agro1 and the mixture is used for transformation, it is expected that i) no escapees will be recovered, ii) the frequency of recovering transgenic T0 plants will increase, iii) the time frame from Agrobacterium infection to T0 plant recovery will be greatly reduced, and iv) the frequency of T0 plants containing only a single copy of the trait T-DNA and no vector (plasmid) backbone will be greatly increased.

[0331] Example 10: Use of a WUS2 expression cassette outside of a trait-containing T-DNA border to stimulate transformation and selection of single copy TO canola plants that do not contain the WUS2 expression cassette

[0332] A single Agrobacterium strain containing a single T-DNA is used for transformation, wherein the "trait" expression cassette is located within the left and right borders of the T-DNA and the WUS expression cassette is just outside the left border. For example, a plasmid (PHP0001, see Table 7) containing an enhanced 35S PRO::WUS expression cassette having the following configuration can be used: RB+AT-UBQ10 PRO::CTP::SPCN::UBQ14 TERM+GM-UBQ PRO::ZS-YELLOW N1::NOS TERM+LB+FMV ENH:PSCV ENH:MMV ENH:CAMV35S PRO::HA-WUS::OS-T28 TERM.

[0333] For the control treatment, PHP88193, in which the T-DNA contains SPCN and ZS-YELLOW N1 expression cassettes but no WUS expression cassette, due to the stringent selection of spectinomycin, it is expected that no escapees will be recovered (regenerated non-transgenic T0 plants), the frequency of recovering transgenic T0 plants will be low (i.e., <1.5%), the time frame from Agrobacterium infection to recovery of T0 plantlets usually takes 3-4 months, and the frequency of T0 plants containing only a single copy of the trait T-DNA and no vector (plasmid) backbone will be low. In contrast, when the enhanced 35S PRO::WUS expression cassette described above is added to the plasmid immediately outside the LB of the T-DNA, after Agrobacterium-mediated transformation, it is expected that i) no escapees will be recovered, ii) the frequency of recovering transgenic T0 plants will increase, iii) the time frame from Agrobacterium infection to T0 plant recovery will be greatly reduced, and iv) the frequency of T0 plants containing only a single copy of the trait T-DNA and no integrated WUS or vector (plasmid) backbone will be greatly increased.

[0334] Example 11: WUS expression cassette located at the 3′ end of the left border improves the recovery of transgenic soybean events containing components within the T-DNA

[0335] For this experiment, a binary vector containing the PVS1 replication origin and a single T-DNA was constructed. A control vector (PHP80728) with two expression cassettes within the left and right borders of the T-DNA was constructed. The first expression cassette consisted of the soybean UBQ promoter driving the expression of TAG-RFP, followed by the Arabidopsis UBQ3 terminator. The second expression cassette consisted of the soybean SAMS promoter driving the expression of the soybean herbicide resistance ALS gene (HRA) and the soybean ALS terminator.

[0336] A second vector (PHP81718) was constructed containing the same herbicide resistance ALS gene and the same TAG-RFP expression cassette within the T-DNA and additionally containing the expression cassette outside the left border: GM-EF1A PRO::AT-WUS::UBQ14TERM. These two plasmids were introduced into Agrobacterium strain AGL1 for comparison.

[0337] After the immature cotyledons of Pioneer soybean variety 93Y21 were transformed with a control T-DNA plasmid (PHP80728) or WUS (PHP81718) outside the left border T-DNA plasmid, the cotyledons were cultured for two weeks, moved to M5 maturation medium supplemented with the selective herbicide 10 μg / l chlorsulfuron, and then the T0 plantlets were sent to the greenhouse. T0 plants were sampled and PCR screened for the presence of ALS, TAG-RFP and WUS expression cassettes.

[0338] WUS (PHP81718) outside the left border T-DNA plasmid greatly improves the efficiency of somatic embryogenesis (see Figure 1 ), increased the efficiency of embryogenesis from a median of less than 5% in the TAG-RFP control (PHP80728) to more than 25% in the WUS treatment (PHP81718). This directly translated into an increase in the frequency of transgenic mature somatic embryos that were able to germinate and form transgenic T0 plants.

[0339] Transformation using the TAG-RFP control vector (PHP80728) did not produce T0 plants. In contrast, WUS treatment (PHP81718) produced some T0 plants. Six T0 plants were analyzed by qPCR, and four T0 plants contained ALS and TAG-RFP expression cassettes, but lacked integrated WUS and had no plasmid backbone contamination. This result shows that WUS can be transiently expressed at a high enough level to stimulate the formation of somatic embryos together with the integrated T-DNA after "out-of-bounds" delivery without integrating into the genome. This provides an effective method for quickly producing transgenic soybean events (carrying the traits ALS and TAG-RFP) using WUS without having to excise the WUS expression cassette because it is only transiently expressed and not integrated.

[0340] Example 12: Use of a WUS2 Expression Cassette and a Wheat Dwarf Virus RepA Expression Cassette Outside of a Trait-Containing T-DNA Border to Stimulate Transformation and Selection of Single Copy TO Maize Plants Containing No WUS2 or RepA Expression Cassettes

[0341] For these experiments, two plasmids were compared. The first contained a T-DNA comprising the following components; RB+LOXP+SI-UBI3 PRO::ZS-GREEN1::PINII TERM+SB-ALS PRO::ZM-HRA::SB-PEPC1 TERM+LB with an additional expression cassette just outside the left border; ZM-PLTP PRO::ZM-WUS2::IN2-1TERM (see PHP86296 in Table 7). The second plasmid contained a T-DNA comprising the following components; RB+LOXP+SI-UBI PRO::ZS-GREEN1::PINII TERM+SB-ALS PRO::ZM-HRA::SB-PEPCI TERM+LB, with two additional expression cassettes just outside the left border; ZM-PLTP PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTP PRO::WDV-RepA::OS-T28 TERM (see PHP0002 in Table 7) (WDV-RepA is wheat dwarf virus RepA, see U.S. Pat. No. 6,284,947, which is incorporated herein by reference in its entirety). For Pioneer hardstem inbred PH1V69, after Agrobacterium-mediated transformation with these respective T-DNAs, 0.05 to 0.1 mg / l imazapyr selection was performed during somatic embryo maturation and regeneration, and TO plants were analyzed by qPCR. When using PHP86296, it is expected that the recovery frequency (relative to the number of initial immature embryos) of the TO plant that is a single copy trait gene and does not have a carrier (plasmid) backbone in T-DNA is about 10%. When using PHP0002 in Agrobacterium, it is expected that the conversion frequency will be higher than the control (PHP86296), the growth of transgenic somatic embryos will be accelerated, and the recovery frequency of the TO plant that is a single copy trait gene and lacks integrated WUS and RepA simultaneously and does not have a carrier (plasmid) backbone in T-DNA increases relative to the control (PHP86296) processing. It is expected that WUS2 and RepA expression cassettes will be placed outside the T-DNA left border to provide improved conversion stimulation, and will not be accompanied by the integration of these morphogenetic gene expression cassettes.

[0342] Alternatively, it is expected that T-DNA (PHP0005) containing a single expression cassette comprising RB+SI-UBI PRO::ZS-GREEN::PINII TERM+SB-ALSPRO::ZM-HRA::SB-PEPC1 TERM+LB+ZM-PLTP PRO::WUS2~T2A~WDV-RepA::IN2-1 TERM outside of LB is equivalent to the control, but has higher transformation efficiency, accelerates the growth of transgenic somatic embryos relative to the control treatment, and increases the recovery frequency of T0 plants that are single copy trait genes within the T-DNA and lack integrated WUS and RepA and have no vector (plasmid) backbone.

[0343] Example 13: Use of WUS2 and amiRNA expression cassettes targeting the maize PICKLE (PKL) transcript outside the T-DNA boundaries containing the trait to stimulate transformation and selection of single copy TO maize plants without WUS2 or RepA expression cassettes

[0344] For these experiments, two plasmids were compared. The first, PHP86296, contained a T-DNA comprising; RB+LOXP+SI-UBI3 PRO::ZS-GREEN1::PINII TERM+SB-ALS PRO::ZM-HRA::SB-PEPC1 TERM+LB with an additional expression cassette just outside the left border; ZM-PLTP PRO::ZM-WUS2::IN2-1TERM.

[0345] The second plasmid contains a modified PHP86296 (PHP0003 in Table 7) with an additional expression cassette ZM-PLTP PRO::PKLamiRNA::OS-T28 TERM outside the left border. The PKLamiRNA component is an artificial microRNA that targets a short sequence in the maize PICKLE (PKL) transcript that destabilizes the PKL transcript and causes degradation. For the pioneer hard stem inbred PH1V5T, after Agrobacterium-mediated transformation with these respective T-DNAs, 0.05 to 0.1 mg / l cypermethrin selection was performed during somatic embryo maturation and regeneration, and T0 plants were analyzed by qPCR. It is expected that the use of PHP86296 will result in a recovery frequency (relative to the number of starting immature embryos) of T0 plants that are single copy trait genes within the T-DNA and lack integrated WUS and PKLamiRNA and do not have a plasmid backbone of about 1%. When PHP0003 is used in Agrobacterium, it is expected that the transformation frequency will be higher than PHP86296, the growth of transgenic somatic embryos will be accelerated, and the recovery frequency of T0 plants with a single copy of the trait gene within the T-DNA and lacking integrated WUS and RepA and without a plasmid backbone will be increased relative to the PHP86296 treatment.

[0346] When using the Agrobacterium comprising these two plasmids to transform maize leaf tissue from inbreeding PH1V5T, then carry out 0.05 to 0.1mg / l methamphetamine selection during somatic embryo maturation and regeneration, analyze T0 plant by qPCR.Expected use PHP86296 will cause the recovery of transgenic callus, and after two to three weeks, somatic embryo will form (relative to the number of initial immature embryo) with a frequency of about 1%.When using PHP0003 in Agrobacterium, expected conversion frequency will be higher than PHP86296, somatic embryo will directly form (without intermediate callus stage) from leaf cell fast, and in T-DNA, be single copy trait gene lacking WUS and PKLamiRNA integrated simultaneously and do not have the recovery frequency of T0 plant of carrier (plasmid) skeleton to increase relative to PHP86296 processing.

[0347] Example 14: Use of a constitutively expressed tetracycline repressor outside the borders of the trait-containing T-DNA and a repressible promoter driving expression of moPAT~DsRED within the T-DNA to facilitate recovery of a single copy bialaphos resistance event that does not contain a repressor expression cassette immediately outside the LB.

[0348] For these experiments, plasmid PHP38042 (see Table 7) contained a T-DNA comprising the following components; RB+CAMV35SPRO:3xTET-OP:ADH1 INTRON1::MO-PAT~DS-RED EXPRESS::PINII TERM+LB+UBI1ZM PRO::TET REPRESSOR::PINII TERM.

[0349] For the pioneer hard stem inbred PH1V5T, after Agrobacterium-mediated transformation with this T-DNA, 3.0 mg / l bialaphos selection was performed during somatic embryo maturation and regeneration, and T0 plants were analyzed by qPCR. It is expected that the use of PHP38042 will result in a recovery frequency (relative to the number of starting immature embryos) of about 3-5% of T0 plants that are single-copy trait genes within the T-DNA and lack an integrated TET REPRESSOR and do not have a plasmid backbone, and T0 plants that lack an integrated TET REPRESSOR will have herbicide tolerance and express DS-RED.

[0350] Example 15: Use of a WUS2 expression cassette and a constitutively expressed dCAS-repressor (REP) fusion expression cassette outside of a T-DNA border containing a trait gene to stimulate transformation and selection of single copy TO maize plants that do not contain a WUS2 or RepA expression cassette

[0351] For these experiments, two plasmids were compared. The first contained a T-DNA containing the following components: RB+SI-UBI3PRO::ZS-GREEN1::PINII TERM+SB-ALS PRO::ZM-HRA::SB-PEPC1 TERM+LB (control plasmid). The second plasmid contained the same components (RB+SI-UBI3 PRO::ZS-GREEN1::PINII TERM+SB-ALSPRO::ZM-HRA::SB-PEPC1 TERM+LB) within the T-DNA plus three additional expression cassettes immediately outside the left border: i) an expression cassette containing ZM-U6 PRO::gRNA::ZM-U6 TERM; ii) a ZM-UBI PRO::dCAS9~REP::PINII TERM expression cassette; iii) a PLTP PRO::ZM-WUS2::IN2-1TERM expression cassette (test plasmid). The gRNA within the first expression cassette has been designed to target dCAS9~REP to the SB-ALS PRO sequence, which transcriptionally silences HRA expression and sensitizes cells to imazapyr.

[0352] For Pioneer hard stem inbred HC69, after Agrobacterium-mediated transformation with Agrobacterium strain LBA4404 THY- containing a control plasmid, T0 plants were analyzed by qPCR during somatic embryo maturation and regeneration. Using the control plasmid in Agrobacterium, the recovery frequency (relative to the number of immature embryos infected by Agrobacterium) of T0 plants with a single copy trait gene in T-DNA and no vector (plasmid) backbone is expected to be about 5%. When the test plasmid is used in Agrobacterium, the expected transformation frequency is higher than the control, accelerating the growth of transgenic somatic embryos relative to the control treatment, and increasing the recovery frequency of T0 plants with a single copy trait gene in T-DNA while lacking integrated WUS and dCAS9-REP and no vector (plasmid) backbone. It is expected that placing the dCAS9-REP and WUS2 expression cassettes outside the left border of the T-DNA will provide improved transformation stimulation without the integration of these morphogenetic gene expression cassettes.

[0353] Alternatively, in the above example, a synthetic zinc finger nuclease fused to a repressor domain can be used instead of gRNA and dCAS9-REP (Urritia et al., 2003, Genome Biol. 4: 231).

[0354] Example 16: Transformation of cotton

[0355] Gossypium hirsutum cultivars Coker 310 and 312 were surface sterilized using 40% (v / v; 80 ml bleach and 120 ml water) commercial bleach and 1 drop of Triton X-100 for 30 minutes and rinsed three times with sterile water at 2-minute intervals. After bleach treatment, the seeds were rinsed with 95% ethanol for 1 minute and then rinsed three times with sterile water. The seeds were then placed on sterile paper towels to blot dry.

[0356] Twenty surface sterilized seeds were germinated, 5 seeds per phytic acid tray, at 28°C in 'cottonseed medium' (CSM; (GH3210)) containing 1 / 2 strength MS salts (2.2 g / L), 1 / 2 strength B5 vitamins, 0.9 g / L magnesium chloride hexahydrate, 15 g / L glucose, 3.6 g / L Phytagel (Sigma), pH adjusted to 5.8, and incubated in the dark for approximately 7-9 days.

[0357] After about 3 days, when the seed coat cracks and the radicle sprouts. Remove the seed coat by using sterile tweezers, gently pinch the rounded tips (chalazas) of all seeds on the surface of the culture medium to expose the folded cotyledons, and insert the radicle into the culture medium. The phytic acid plate is incubated again at 28°C to complete germination. After 8 days, the hypocotyl and cotyledon explants are removed from the seedlings with scissors. The hypocotyl and cotyledon explants are placed in a sterile culture dish and cut into 0.9 cm fragments (for the hypocotyl) and 0.5 cm with a scalpel blade. 2 The fragments were then placed in sterile 100x25 mm (width x depth) petri dishes with 35 ml of liquid co-culture medium (MS salts, B5 vitamins, 30 g / L glucose, 0.9 g / L magnesium chloride hexahydrate, 1.9 g / L potassium nitrate, 0.1 g / L inositol, pH 5.8) in preparation for Agrobacterium treatment.

[0358] In this experiment, plasmid PHP89484 contained T-DNA with the following components: RB+AT-UBQ10 PRO::CTP::SPCN::UBQ14 TERM+GM-UBQ PRO::DS-RED2::UBQ3 TERM+LB+CAMV35S PRO::HA-WUS::OS-T28 TERM+FMC ENH::PCSV ENH::MMV ENH. The control plasmid (PHP88193) without the WUS expression cassette contained T-DNA with the following components; RB+AT-UBQ10 PRO::CTP::SPCN::UBQ14 TERM+GM-UBQ PRO::ZS-YELLOW1 N1::NOS TERM+LB.

[0359] Agrobacterium strain LBA4404 containing PHP89484 was prepared in 810K minimal medium plus 100ug / ml spectinomycin and 200μM acetosyringone. The Agrobacterium culture was grown overnight in an incubator shaker at 28°C and 280rpm until the OD at 600nm reached 0.6-0.8. The bacterial cells were precipitated by centrifugation at 4000rpm for 15 minutes and resuspended in 700A medium with a final OD600 of 0.6. 40ml of Agrobacterium solution was added to each petri dish containing the cut segments and incubated for 30 minutes under intermittent shaking.

[0360] After infection, about 50 hypocotyl segments or 50 cotyledon explants are placed in a petri dish with solid 710I co-culture medium and covered with sterile filter paper. Co-cultivate 4 days under weak light (5 μE) at 28 ° C under a photoperiod of 16 h. Then the explants are directly transferred to the surface of 'callus induction medium' (CIM) (MS salts, B5 vitamins, 30 g / L glucose, 0.9 g / L magnesium chloride hexahydrate, 1.9 g / L potassium nitrate, 0.1 g / L inositol, 0.1 mg / L kinetin, 0.1 mg / L (0.45 μM) 2,4-D, 500 mg / L carbenicillin, 3.6 g / L Phytagel (pH 5.8)) without selection agent. After 14 days, the explants are transferred to CIM together with 60 mg / l spectinomycin.

[0361] After 7 days of Agrobacterium treatment, transient RFP expression was observed under a surface fluorescence stereomicroscope. Strongly expressed DS-RED foci were observed around the cut surface in cotyledon and hypocotyl explants. DS-RED expression was observed in stable callus, which showed multicellular DS-RED expression structures after 3 weeks. In contrast, when control T-DNA (in PHP88193) was used in the same strain and under the same experimental conditions, based on fluorescence, T-DNA delivery was similar, but three weeks after Agrobacterium treatment, the growth of multicellular structures was not observed.

[0362] Example 17: Use of viral enhancers and barley LTP2 PRO to drive WUS2 expression further improved recovery of T-DNA single copy only transgenic events for "trait".

[0363] Agrobacterium strain LBA4404 THY- containing the virulence plasmid PHP71539 (SEQ ID NO: 132) was used in all treatments. PHP86491 (SEQ ID NO: 110) containing T-DNA with a "trait" expression cassette (RB+SB-UBI PRO::ZS-GREEN1::OS-UBI TERM+SB-ALS PRO::ZM-HRA::PINII TERM+LB) was also used for all treatments. Treatment 1 (Trt. No. 1) served as a negative control using a single Agrobacterium strain (Agro1) containing only PHP86491. Treatment 2 (Trt. No. 2) was used as a positive control, and a second Agrobacterium (Agro2) containing PHP88158 (SEQ ID NO: 109) (RB + FMV ENH: PSCV ENH: MMVENH: ZM-PLTP PRO:: ZM-WUS2:: IN2-1 TERM + NOS PRO:: CRC:: SB-GKAF TERM + LB) was mixed with Agro 1. Both Agrobacterium suspensions were adjusted to 0.7 OD550 and then mixed at a ratio of 90% Agro 1 to 10% Agro 2. In the third treatment, Treatment 3 (Trt. No. 3), PHP93161 (SEQ ID NO: 130) (RB+LOXP+FMV ENH:PCSV ENH:MMV ENH::LTP2 PRO::ZM-WUS2::IN2-1 TERM+NOS PRO::CRC::SB-GKAF TERM+LB) replaced PHP88158 in Agro2, and the ratio of Agro1 / Agro2 in Trt. No. 2 and Trt. No. 3 remained the same (90% / 10%).

[0364] In Trt.No.1, three hundred and fifty-eight (358) immature embryos of inbred HC69 were infected with the Agrobacterium mixture and ninety-one (91) transgenic T0 plants were produced (transformation frequency of 25%), of which 11% of those plants were single copy trait (relative to the starting number of embryos). In Trt.No.2, both transformation frequency and single copy frequency were improved relative to Trt.No.1 (from 25% to 31% and from 11% to 16%, respectively). The improvement in the final "% Single Copy of Trait" value is the ultimate measure of successful delivery of high quality fertile transgenic plants to the greenhouse. This value is affected by the results of multiple individual steps in the process, including the initial transformation frequency (production of transgenic cells), the regeneration frequency (production of plants from transgenic cells), and the final molecular analysis. Molecular analysis confirmed that the transgenic plants i) were independent integration events, ii) contained a single copy of the desired T-DNA and its complex trait expression cassette, iii) did not contain the T-DNA with the morphogenic gene expression cassette, and iv) did not contain the Agrobacterium plasmid backbone sequence. Each of these outcomes (transformation frequency, regeneration frequency, and molecular quality control frequency) contributed to the attrition in the overall process and are summarized as the "Trait Single Copy %" frequencies shown in Table 12. Due to the importance of this final outcome - the frequency of producing high quality fertile T0 plants - the improvement from 11% in Trt. No. 1 to 16% in Trt. No. 2 represents an important and substantial improvement in the overall transformation efficiency of this valuable pioneer inbred line.

[0365] As described above, in PHP93161 (SEQ ID NO: 130), the promoter used to express the ZM-WUS2 gene was switched from the ZM-PLTP promoter in Trt. No. 2 to the LTP2 promoter in Trt. No. 3 (Kalla et al., 1994, The Plant J. 6: 849-860). The two plant promoters in Trt. No. 2 and Trt. No. 3 were preceded by three promoter enhancer elements from three different viruses (i.e., Scrophularia mosaic virus, Peanut chlorotic streak virus, and Mirabilis mosaic virus), and these three enhancer element sequences are abbreviated as "3xENH" in this order in Table 12. When the LTP2 promoter was used in Trt. No. 3, both the transformation frequency and the "trait single-copy %" frequency increased relative to Trt. No. 2 (from 31% to 45% and from 16% to 24%, respectively). This indicates that different promoters can be used for this strategy. This also strengthens the conclusion drawn from comparing Trt. No. 1 and Trt. No. 2; by mixing one Agrobacterium (containing a trait expression cassette within the T-DNA) with another Agrobacterium (containing a morphogenic gene expression cassette within the T-DNA), the total frequency of generating high-quality fertile T0 plants (independent events based on PCR characterization) increased compared to using a single Agrobacterium containing only the T-DNA with the trait.

[0366] Table 12.

[0367]

[0368] Table 12 shows the transformation efficiency when Agro2 (morphogenic gene expression cassette) was absent in Trt. No. 1, or when using Agro2 (Trt. No. 2 and Trt. No. 3) containing a WUS2 gene expression cassette with two different promoters. The transformation frequency was calculated based on the number of transgenic (Tnx) T0 plants recovered relative to the starting number of immature embryos. Quantitative PCR was used to determine the single-copy analysis of the trait genes (ZS-GREEN1 AND HRA) and the morphogenic gene (WUS2) to determine the gene presence and copy number. T0 plants containing only a single-copy trait gene without the WUS2 gene were used to calculate the "trait single-copy %" value relative to the starting number of immature embryos.

[0369] Example 18: Improving sunflower hypocotyl transformation using a mixture of Agrobacterium containing a trait and Agrobacterium containing a morphogenic gene

[0370] Agrobacterium strain LBA4404 THY- was used for transformation of sunflower (Helianthus annuus variety F1503LG). Mature, dried sunflower seeds were sterilized in a bleach solution (10-15% v / v water with one drop of Tween detergent) for fifteen (15) minutes and then rinsed three (3) times in sterile water. The seeds were imbibed overnight. The embryos were removed from the softened shells. Once the embryos were isolated, cuts were made at the base of the cotyledons to facilitate embryo separation, exposing the leaf primordia covering the apical meristem. The root tip was left on the embryo. After isolation, the embryo axis (EA) was transferred to a petri dish for infection. Two Agrobacterium, each containing a different T-DNA plasmid, were suspended in liquid infection medium. The first Agrobacterium carries plasmid PHP92349 (SEQ ID NO: 131), which has a T-DNA containing a "trait" expression cassette of SPCN (spectinomycin resistance) and DS-RED2 (fluorescence) genes, specifically containing RB+LOXP+GM-QBU PRO::CTP:SPCN::UBQ14 TERM+GM-EF1A2PRO::DS-RED2::UBQ3 TERM+LOXP+LB. The second Agrobacterium carries PHP90094, which has a T-DNA containing a morphogenetic gene expression cassette of HA-WUS, specifically containing RB+CAMV35S PRO::HA-WUS::OS-T28 TERM+FMVENHANCER:PCSV ENHANCER:MMV ENHANCER+LB. Each Agrobacterium strain was suspended in 20A medium, and the concentration of the bacterial suspension was adjusted to 0.5 OD550. The Agrobacterium suspensions were mixed together in a 1:1 ratio and five (5) ml of the suspension was added to the EA to cover the explants. The EA was then placed under vacuum with gentle agitation for twenty (20) minutes. The EA was removed from the Agrobacterium and the radicle end was inserted down into 272AC medium (standard MS salts and vitamin levels (Murashige and Skoog, 1962, Physiol. Plant 15: 473-497), 0.1 g / l inositol, 50 mg / l thymidine, 100 uM acetosyringone, 0.1 mg / l BAP, 40 g / l sucrose, 6 g / l Bacto agar, pH 5.6), leaving the apical meristem on top of the 272AC medium and placed for co-cultivation at 21°C under low light for three days.After co-cultivation, EA was transferred to 272AB spectinomycin selection medium (standard MS salt and vitamin levels (Murashige and Skoog, 1962, Physiol. Plant 15: 473-497), 0.1 g / l inositol, 10 mg / l meropenem, 0.1 mg / l metatope, 30 mg / l spectinomycin dihydrochloride, 0.1 ug / l, 40 g / l sucrose, 6 g / l Bacto agar, pH 5.6) at 28°C in full light.

[0371] EA is allowed to grow, and the bleached leaves are pruned regularly. After being exposed to spectinomycin, green parts or whole green leaves are observed in about three weeks. The green tissue also expresses DS-RED, confirming that the T-DNA from PHP92349 has been integrated. In order to take root, the transgenic event is transferred to Bio-Dome Sponge rooting material (Park Seed Co., 3507 Cokesbury Road, Hodges, SC). After being transferred to Bio-Dome Sponge rooting material, roots are formed in 1 to 3 weeks, and the plant is potted and transferred to a greenhouse or growth chamber.

[0372] In the control experiment, only a single Agrobacterium containing the "trait" plasmid PHP92349 was used, and only three (3) transgenic T0 plants were recovered from eight hundred and twenty-three (823) Agrobacterium-infected EAs, with a transformation frequency of 0.4%. When a 1:1 mixture of Agrobacterium containing PHP92349 (the "trait" expression cassette) and Agrobacterium containing PHP90094 (the morphogenetic gene (WUS) expression cassette) was used, six transgenic T0 plants were recovered from three hundred and seventy (370) Agrobacterium-infected EAs, with a transformation frequency of 1.6%. In addition, all transgenic T0 plants containing the "trait" did not contain the morphogenetic gene (WUS) expression cassette. This demonstrates the advantage of using a mixture of two Agrobacteria to impart morphogenic stimulation without integrating the morphogenetic gene expression cassette, improving the transformation efficiency of sunflower.

[0373] Example 19: Co-delivery of two T-DNA plasmids from one bacterial strain

[0374] An alternative to using a mixture of two Agrobacterium (each containing one of the two plasmids) to deliver a trait gene expression cassette and a morphogenic gene expression cassette is to use a single Agrobacterium containing two different T-DNAs. This is achieved by constructing a single plasmid containing two T-DNAs or by using two separate plasmids, one of which contains the trait gene expression cassette T-DNA and the other plasmid contains the morphogenic gene expression cassette T-DNA. Maize inbred lines differ in many morphological and physiological characteristics. One manifestation of this variability is the sensitivity of immature embryo scutellum of different inbred lines to T-DNA delivery and integration during Agrobacterium infection and co-cultivation. For example, when a mixture of plasmids contained in two bacterial strains is used to transform non-hard stem inbred HC69, many cells only receive one of the plasmids and therefore will not be stimulated to quickly form new somatic embryos and produce transgenic events with traits. Instead of Agro1:Agro2, which is used to deliver different ratios of plasmid mixtures, delivering a plasmid mixture from the same bacterial cell can result in a higher frequency of cells receiving two T-DNAs and allow a higher frequency of transgenic events to be produced. When used for transformation, a higher frequency of rapidly forming somatic embryos is expected and the resulting TO plants containing a single copy of the trait gene expression cassette T-DNA without vector (plasmid) DNA and without integration of the morphogenetic gene expression cassette T-DNA will be produced.

[0375] Example 20: Regulation of plasmid copy number using T-DNA plasmids with different replication origins

[0376] For these experiments, two different T-DNAs were delivered from a single bacterial cell in different ratios (relative to each other) by carrying these T-DNAs on two plasmids with different replication origins (ORIs). Different ORIs maintain different numbers of plasmids in bacterial cells. Different ORIs can be used to titrate plasmid copy number to obtain different ratios of plasmids in the same bacterial cell. In this experiment, a plasmid was designed that had one or more ORIs selected from the following ORIs in the same bacterial cell: BBR1 ORI; PSA ORI; PSA+PARDE ORI; PVS1 ORI; repABC ORI; RK2 ORI; RK2+PARDE ORI; or RSF1010 ORI (U.S. Patent Publication 20180216123 and WO 2017078836, incorporated herein by reference in their entirety). Among these different ORIs, it has been previously determined that in Agrobacterium, pVS1 confers high plasmid copy number (approximately 20 copies / cell) while repABC confers low copy number (approximately 1-2 copies / cell) (Zhi et al., 2015, Plant Cell Rep. [Plant Cell Reports] 34:745-754).

[0377] Table 13.

[0378]

[0379] To verify this concept, the first plasmid was constructed with an ORI that resulted in a high plasmid copy number (pVS1) and a T-DNA containing a trait gene expression cassette such as ZS-GREEN and HRA. The second plasmid was constructed with an ORI that resulted in a low copy number (repABC) and a T-DNA that contained a morphogenetic gene expression cassette (WUS2). Both plasmids were introduced into a single Agrobacterium. When the plasmids in the same bacterial cell were co-delivered into the same plant cell, the relative ratio of the T-DNA delivered would reflect the stable ratio of these plasmids in the Agrobacterium.

[0380] In this way, compared with the method of mixing two different Agrobacterium containing trait gene expression cassette T-DNA or morphogenesis gene expression cassette T-DNA (i.e., with 90% / 10% Agro1 and Agro2 mixture), a similar effect is produced. Similar to the Agrobacterium mixing method, delivering a plasmid mixture from the same bacterial strain can lead to a higher frequency of cells that accept two T-DNAs and allow a higher transformation frequency. When used for transformation, a higher frequency of rapidly formed somatic embryos is expected, and a resulting TO plant containing a single copy of the trait gene expression cassette T-DNA without a vector (plasmid) DNA and without integrating the morphogenesis gene expression cassette T-DNA will be produced.

[0381] Example 21. Using a repressor gene expression cassette outside the T-DNA to repress the expression of a selectable marker within the T-DNA

[0382] The Agrobacterium strain LBA4404 THY- used carries a T-DNA carried on a plasmid containing a repression / de-repression selection marker in addition to a cognate repressor expression gene cassette outside the T-DNA. For example, the Agrobacterium plasmid contains the following T-DNA: RB+CAMV35S PRO:TOP3::NPTII::PINII TERM+LB, where TOP3 indicates that the CAMV35S promoter has three tetracycline repressor binding sequences (operator sequences) overlapping the transcription start site around the TATA box, and UBI1ZM PRO::TETR::PINII located outside the left border. In addition to the expression cassettes described in the above configuration, a trait gene expression cassette within the T-DNA (using the exemplary trait ZS-GREEN1) and a morphogenesis gene expression cassette outside the T-DNA (using WUS2) can also be added to obtain the following Agrobacterium plasmid: RB+SB-UBI PRO::ZS-GREEN1::OS-UBI TERM+CAMV35S PRO::TOP3::NPTII::PINII TERM+LB+UBI1ZM PRO::TETR::PINII+PLTP::WUS2::In2-1TERM (abbreviated RB+ZS-GREEN+REPRESSIBLE NPTII+LB+TETR+WUS2).

[0383] The second configuration (RB+ZS-GREEN+REPRESSIBLE NPTII+LB+TETR+WUS2) was introduced into maize immature embryos from inbred HC69 by agrobacterium infection. In most of the transgenic events recovered, it is expected that only T-DNA (comprising trait gene expression cassette and selectable marker gene expression cassette) is integrated, while TETR and WUS2 gene expression cassettes are not integrated, resulting in transient expression of TETR and WUS2 genes. Therefore, somatic embryo formation stimulated by WUS2 increases the frequency of transformation, while transient TETR expression causes NPTII expression to be temporarily suppressed. After one week of post-infection cultivation, transgenic cells are transferred to a culture medium containing 150mg / lG418. It is expected that the somatic embryo formed by the T-DNA (having trait gene expression cassette and selectable marker gene expression cassette) formed comprising integration is easily selected, and a high frequency of fertile transgenic plants is produced.

[0384] Example 22. Use of a dCAS9:repressor fusion expression cassette outside of T-DNA to repress expression of a selectable marker within T-DNA

[0385] The Agrobacterium strain LBA4404 THY- used carries a T-DNA carried on a plasmid containing a constitutive promoter driving expression of a selectable marker in addition to the dCAS9-repressor expression cassette in addition to the T-DNA. For example, the Agrobacterium plasmid contains the following T-DNA: RB+UBI1ZM PRO::NPTII::PINII TERM+LB and UBI1ZM PRO::dCAS:EAR::PINII outside the left border, where EAR represents the sequence of the repressor peptide fused in frame with dCAS9. In addition to the expression cassettes described in the above configuration, a trait gene expression cassette within the T-DNA (using the exemplary trait ZS-GREEN1) and a morphogenesis gene expression cassette outside the T-DNA (using WUS2) can also be added to obtain the following Agrobacterium plasmid: RB+SB-UBI PRO::ZS-GREEN1::OS-UBI TERM+CAMV35S PRO::TOP3::NPTII::PINII TERM+LB+UBI1ZM PRO::dCAS9:EAR::PINII+zmu6pro::gRNA::ZMU6 TERM+PLTP::WUS2::In2-1 TERM (abbreviated RB+ZS-GREEN+REPRESSIBLE NPTII+LB+dCAS9:EAR+gRNA+WUS2).

[0386] The second configuration (RB+ZS-GREEN+REPRESSIBLE NPTII+LB+dCAS9:EAR+gRNA+WUS2) was introduced into maize immature embryos from inbred HC69 by Agrobacterium infection. In most of the transgenic events recovered, only T-DNA (comprising a trait gene expression cassette and a selectable marker gene expression cassette) was expected to be integrated, while dCAS9:EAR and WUS2 gene expression cassettes were not integrated, resulting in transient expression of dCAS9:EAR and WUS2 genes. Therefore, somatic embryo formation stimulated by WUS2 increased the frequency of transformation, while transient dCAS9:EAR expression caused NPTII expression to be temporarily suppressed. After one week of culture after infection, transgenic cells can be transferred to a culture medium containing 150mg / l G418. It is expected that somatic embryos formed containing integrated T-DNA (having a trait gene expression cassette and a selectable marker gene expression cassette) are easily selected, and a high frequency of fertile transgenic plants are produced.

[0387] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a protein" includes one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless expressly stated otherwise.

[0388] All patents, publications, and patent applications mentioned in this specification are indicative of the levels of those skilled in the art to which the disclosure pertains. All patents, publications, and patent applications are incorporated herein by reference in their entirety to the same extent as if each individual patent, publication, or patent application was expressly and individually indicated to be incorporated by reference in its entirety.

[0389] 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 may be practiced within the scope of the appended claims.

Claims

1. A method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: (a) Plant cells (i) contacting with a T-DNA comprising a trait gene expression cassette in a first plurality of bacteria and a T-DNA comprising a morphogenetic gene expression cassette in a second plurality of bacteria, wherein the first and second plurality of bacteria comprise bacteria of the genus Agrobacterium and the first plurality of bacteria and the second plurality of bacteria are present in a ratio of 50:50, a ratio of 75:25, a ratio of 90:10, or a ratio of 95:5, and wherein the morphogenetic gene expression cassette comprises a nucleotide sequence encoding a WUS2 gene; or (ii) contacting with a plurality of bacteria containing a plasmid, wherein the plasmid comprises a trait gene expression cassette within a T-DNA and a morphogenetic gene expression cassette outside the T-DNA, wherein the plurality of bacteria comprises bacteria of the genus Agrobacterium, and wherein the morphogenetic gene expression cassette comprises a nucleotide sequence encoding (1) a WUS2 gene or (2) a WUS2 gene and an ODP2 gene; (b) selecting a plant cell containing the trait gene expression cassette and not containing a morphogenetic gene expression cassette; and (c) regenerating a transgenic plant from the selected plant cell, wherein the morphogenic gene expression cassette is not excised from the genome of the selected plant cell.

2. The method of claim 1, wherein contacting comprises contacting the plant cell with a T-DNA comprising a trait gene expression cassette in a first plurality of bacteria and a T-DNA comprising a morphogenetic gene expression cassette in a second plurality of bacteria, wherein the first and second plurality of bacteria comprise bacteria of the genus Agrobacterium, and wherein the morphogenetic gene expression cassette comprises a WUS2 gene.

3. The method of claim 1 , wherein the contacting comprises contacting the plant cell with a plurality of bacteria containing a plasmid, the plasmid comprising a trait gene expression cassette within a T-DNA and a morphogenic gene expression cassette outside the T-DNA, wherein the plurality of bacteria comprises bacteria of the genus Agrobacterium, and wherein the morphogenic gene expression cassette comprises a gene encoding (1) a WUS2 gene or (2) a WUS2 gene and an ODP2 gene.

4. The method according to any one of claims 1 to 3, wherein the trait gene expression cassette comprises: A trait gene selected from the group consisting of a gene that confers pest resistance, a gene that confers herbicide resistance, a gene that confers stress tolerance, a gene that confers drought resistance, a gene that confers nitrogen use efficiency (NUE), a gene that confers disease resistance, and a gene that confers the ability to alter a metabolic pathway.

5. The method according to any one of claims 1 to 3, wherein the Agrobacterium bacterium comprises a disarmed Agrobacterium selected from the group consisting of AGL-1, EHA105, GV3101, LBA4404 and LBA4404 THY-.

6. The method of any one of claims 1 or 2, wherein the first plurality of bacteria and the second plurality of bacteria are present in a 50:50 ratio.

7. The method of any one of claims 1 or 2, wherein the first plurality of bacteria and the second plurality of bacteria are present in a ratio of 75:

25.

8. The method of any one of claims 1 or 2, wherein the first plurality of bacteria and the second plurality of bacteria are present in a ratio of 90:

10.

9. The method of any one of claims 1 or 2, wherein the first plurality of bacteria and the second plurality of bacteria are present in a ratio of 95:

5.

10. The method of any one of claims 1-3, wherein the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS2 gene.

11. The method of any one of claims 1-3, wherein the morphogenic gene expression cassette comprises the HBSTART3 promoter operably linked to the WUS2 gene.

12. The method of any one of claims 1-3, wherein the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence.

13. A method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: contacting the plant cell with a T-DNA containing a trait gene expression cassette in a first plurality of bacteria and a T-DNA containing a morphogenesis gene expression cassette in a second plurality of bacteria, wherein the first and second plurality of bacteria comprise bacteria of the genus Agrobacterium and the first plurality of bacteria and the second plurality of bacteria are present in a ratio of 50:50, a ratio of 75:25, a ratio of 90:10, or a ratio of 95:5; wherein the morphogenic gene expression cassette comprises a nucleotide sequence encoding the WUS2 gene, and The trait gene expression cassette comprises: A trait gene selected from the group consisting of a gene that confers pest resistance, a gene that confers herbicide resistance, a gene that confers stress tolerance, a gene that confers drought resistance, a gene that confers nitrogen use efficiency (NUE), a gene that confers disease resistance, and a gene that confers the ability to alter a metabolic pathway; Selecting a plant cell containing the trait gene expression cassette and not containing a morphogenetic gene expression cassette; as well as regenerating a transgenic plant from the plant cell, wherein the morphogenic gene expression cassette is not excised from the genome of the selected plant cell.

14. The method of claim 13, wherein the Agrobacterium bacterium comprises a disarmed Agrobacterium selected from the group consisting of AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-.

15. The method according to claim 13 or 14, wherein the first bacterial strain and the second bacterial strain are present in a ratio of 50:

50.

16. The method according to claim 13 or 14, wherein the first bacterial strain and the second bacterial strain are present in a ratio of 75:

25.

17. The method of claim 13 or 14, wherein the first bacterial strain and the second bacterial strain are present in a ratio of 90:

10.

18. The method of claim 13 or 14, wherein the first bacterial strain and the second bacterial strain are present in a ratio of 95:

5.

19. The method of claim 13, wherein the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS2 gene.

20. The method of claim 13, wherein the morphogenic gene expression cassette comprises the HBSTART3 promoter operably linked to the WUS2 gene.

21. The method of claim 13, wherein the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence.

22. A method for producing a transgenic plant having one copy of a trait gene expression cassette, the method comprising: contacting the plant cells with a bacterial strain containing a plasmid comprising a trait gene expression cassette within the T-DNA and a morphogenetic gene expression cassette outside the T-DNA, wherein the plurality of bacteria comprises bacteria of the genus Agrobacterium, wherein the morphogenic gene expression cassette comprises (1) a nucleotide sequence encoding the WUS2 gene or (2) a nucleotide sequence encoding the WUS2 gene and a nucleotide sequence encoding the ODP2 gene, and The trait gene expression cassette comprises: A trait gene selected from the group consisting of a gene that confers pest resistance, a gene that confers herbicide resistance, a gene that confers stress tolerance, a gene that confers drought resistance, a gene that confers nitrogen use efficiency (NUE), a gene that confers disease resistance, and a gene that confers the ability to alter a metabolic pathway; Selecting a plant cell containing the trait gene expression cassette and not containing a morphogenetic gene expression cassette; as well as regenerating a transgenic plant from the plant cell, wherein the morphogenic gene expression cassette is not excised from the genome of the selected plant cell.

23. The method of claim 22, wherein the Agrobacterium bacterium comprises a disarmed Agrobacterium strain selected from the group consisting of AGL-1, EHA105, GV3101, LBA4404, and LBA4404 THY-.

24. The method of claim 22, wherein the morphogenic gene expression cassette comprises a PLTP promoter operably linked to a WUS gene.

25. The method of claim 22, wherein the morphogenic gene expression cassette comprises the HBSTART3 promoter operably linked to the WUS gene.

26. The method of claim 22, wherein the morphogenic gene expression cassette further comprises a viral or plant enhancer sequence.

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