Compositions and methods for somatic embryogenesis in dicotyledons

Abstract

Methods for producing dicotyledonous somatic embryos are provided, including methods for transforming and regenerating transgenic plants from these somatic embryos.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 335,488, filed on April 27, 2022, which is hereby incorporated by reference in its entirety.

[0003] References to sequence listings submitted electronically

[0004] An official copy of the sequence listing is submitted electronically with this specification in the form of a sequence listing file in xml format, named 8761-WO-PCT.ST26, created on April 24, 2023, and 379,276 bytes in size, which is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to the field of plant molecular biology, including tissue culture and genetic techniques in plants. The present disclosure also provides compositions and methods for rapidly and efficiently producing dicotyledonous somatic embryos and transforming dicotyledonous plants. Background Art

[0006] The method that is used to produce somatic embryo in dicotyledon may be difficult, laborious and time-consuming.In addition, many commercially relevant kinds of dicotyledon and inbred line are resistant for the traditional culture method that is used for somatic embryo generation.Therefore, use traditional culture method, only the dicotyledon genotype of limited range can produce somatic embryo.

[0007] Despite its limitation, the transformation mediated by Agrobacterium remains a widely used method in the experimental development of dicotyledonous plant species (such as cotton, canola, alfalfa, tomato and cucumber) and in some cases commercial development. Up to now, the most common method for contacting cells with Agrobacterium comprises: cultivate explant tissue, such as immature embryos (" co-culture "), may include "delay " or "quiescent" (non-selective) steps, cultivate on the selection medium containing one or more growth hormones subsequently, thereby allow cell dedifferentiation to form callus. During this callus formation phase, select the resistant callus of conversion on the selection medium in the presence of suitable selection agent. This is followed by cell growth on regeneration and rooting medium under the condition of promoting callus differentiation and callus regeneration as plant. However, these traditional methods are subject to a series of challenges in practice.

[0008] For example, in cotton, traditional methods of producing somatic embryonic tissue, transforming the tissue, and regenerating transgenic plants that can be transferred to soil for further growth can take 4-5 months and are often quite inefficient. In another example, canola transformation protocols often regenerate plants that are deformed due to hydrophobicity and poor quality bud formation. Chu et al. (2020) Frontiers in Plant Sciences 11(579524): 1-11. In addition, these methods tend to be very labor-intensive.

[0009] Need to prepare dicotyledonous somatic embryo for the improved, simpler, faster and more effective method of research and product development application.Also need to be able to transform the plant variety and the novel method of inbred line that the transformation scheme of having established is had resistance.This type of alternative method can save time, and provides greater flexibility aspect making research and product development decision.Such method can also be used for realizing the generation of transgenic plant of greater quantity and / or wider range for gene detection and / or product development in the mode of reducing material and labor cost, promotes the transformation of higher throughput and / or expands the plant material pool that can be used for transformation. Summary of the invention

[0010] Compositions and methods disclosed herein are based at least in part on the following unexpected discovery: the combination of two or more heterologous morphogenetic genes can be used to induce somatic embryo formation in dicot explants. In some instances, disclosed compositions and methods induce the formation of somatic embryos in dicot species and / or dicot explant types that are resistant to the somatic embryo formation of the method used. In some instances, relative to the method used, disclosed compositions and methods induce the formation of dicot somatic embryos in a simpler, faster, more cost-effective and / or easier scalable high-throughput manner. In some instances, disclosed compositions and methods induce the formation of dicot somatic embryos, and these dicot somatic embryos are used to produce transgenic plants in dicot species, dicot lines and / or dicot explant types that are resistant to the somatic embryo formation of the method used (or regeneration of transgenic plants from somatic embryo tissue).

[0011] In one aspect, provided herein is a method for producing dicot somatic embryos, the method comprising expressing in a dicot explant: (i) a first heterologous nucleotide sequence encoding a Babyboom (BBM) or RWP-RK domain (RKD) polypeptide and (ii) a second heterologous nucleotide sequence encoding a leafy cotyledon (LEC) polypeptide. As shown in the examples herein, it was unexpectedly found that the combined expression of these morphogenic polypeptides induces somatic embryo formation in a representative number of dicot species, including some dicots that do not respond or respond much less efficiently to established techniques for inducing somatic embryogenesis. In one example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide. In another example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide. In an additional example of this method, the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide. In yet another example of this method, the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide.

[0012] The dicot somatic embryos produced by the methods disclosed herein can be used in transformation methods to produce transgenic plants or plant tissues. The somatic embryos can be used for transient or stable transformation. Transient or stable transformation can be performed by methods based on Agrobacterium or biolistics.

[0013] In a second aspect, provided herein is a method for producing a transgenic dicot. This method comprises: (i) transforming a dicot explant with a first heterologous nucleotide sequence encoding a BBM polypeptide or a RKD polypeptide and a second heterologous nucleotide sequence encoding a LEC polypeptide; (ii) transforming the explant with a transgene; (iii) inducing somatic embryo formation in the explant to produce a transgenic somatic embryo; and (iv) culturing the transgenic somatic embryo tissue under germination, rooting and sprouting conditions to form a transgenic dicot plant comprising the transgene. In one example of this transformation method, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide. In another example of this transformation method, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide. In yet another example of this transformation method, the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide. In another example of this transformation method, the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide. Additionally, in each of the aforementioned examples of the transformation method, the method can further include excising the first heterologous nucleotide sequence and the second heterologous nucleotide sequence encoding the morphogenic polypeptides (BBM or RKD and LEC1 or LEC2) after somatic embryo induction.

[0014] For example, the first heterologous nucleotide sequence encoding the morphogenic polypeptides discussed herein (BBM, BBM AP2 domain, RKD, LEC1, LEC2 or FUS3) and the second heterologous nucleotide sequence can be flanked by recognition sequences for a site-specific recombinase, and the disclosed transformation methods can include providing explants with a site-specific recombinase that recognizes these recognition sites and can excise the flanking coding sequences for these morphogenic polypeptides after embryo induction. In a specific example, one or more morphogenic polypeptides flanked by a site-specific recombinase (e.g., BBM, BBM AP2 domain, RKD, LEC1, LEC2 or FUS3) are coupled to a second polypeptide domain (e.g., CAAT binding factor 1A activation domain (CBF1A) or glucocorticoid receptor (GR)).

[0015] In some instances, the disclosed dicotyledon transformation method advantageously shortens the time usually required to produce the transgenic dicotyledon. After transformation with the first heterologous nucleotide sequence encoding the morphogenetic polypeptides and the second heterologous nucleotide sequence, the time to produce the TO plant can be shortened by several weeks or months. For example, relative to the established method for transformation of the same dicotyledonous plant species, the time to regenerate the transgenic TO plant can be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks (or longer).

[0016] In a third aspect, provided herein is a method for producing dicot somatic embryos, the method comprising expressing a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide, a LEC2 polypeptide, or both in a dicot explant. The method comprises expressing (i) a first heterologous nucleotide sequence encoding a BBM polypeptide and (ii) a second heterologous nucleotide sequence encoding a LEC2 polypeptide in a dicot explant, wherein the BBM polypeptide, the LEC2 polypeptide, or both the BBM polypeptide and the LEC2 polypeptide are coupled to CBF1A. In one example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide coupled to CBF1A (e.g., via a linker domain) (BBM-CBF1A) and the second heterologous nucleotide sequence encodes a LEC2 polypeptide. In a second example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide coupled to CBF1A (e.g., via a linker domain) (LEC2-CBF1A). In a third example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide coupled to CBF1A (eg, via a linker domain) and the second heterologous nucleotide sequence encodes a LEC2 polypeptide coupled to CBF1A (eg, via a linker domain).

[0017] In a fourth aspect, the present invention provides a method for producing dicot somatic embryos, the method comprising expressing CBF1A coupled to a truncated form of a BBM polypeptide in a dicot explant, wherein the truncated BBM comprises a BBM AP2 domain and a C-terminus, but lacks a BBM N-terminus. The method comprises expressing a first heterologous nucleotide sequence encoding CBF1A coupled to the truncated BBM polypeptide in a dicot explant.

[0018] In a fifth aspect, the present invention provides a method for producing dicot somatic embryos, the method comprising expressing a FUSCA3 (FUS3) gene in a dicot explant. The method comprises expressing (i) a first heterologous nucleotide sequence encoding a BBM polypeptide and (ii) a second heterologous nucleotide sequence encoding a FUS3 polypeptide in a dicot explant.

[0019] In a sixth aspect, provided herein is a method for producing dicot somatic embryos, the method comprising expressing a glucocorticoid receptor (GR) coupled to a BBM polypeptide, a LEC2 polypeptide, or both in a dicot explant. The method comprises expressing in a dicot explant (i) a first heterologous nucleotide sequence encoding a BBM polypeptide and (ii) a second heterologous nucleotide sequence encoding a LEC2 polypeptide, wherein the BBM polypeptide, the LEC2 polypeptide, or both the BBM polypeptide and the LEC2 polypeptide are coupled to GR. In one example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide coupled to GR (BBM-GR) and the second heterologous nucleotide sequence encodes a LEC2 polypeptide. In a second example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide coupled to GR (LEC2-GR). In a third example of this method, the first heterologous nucleotide sequence encodes a BBM polypeptide coupled to GR and the second heterologous nucleotide sequence encodes a LEC2 polypeptide coupled to GR. The dicot somatic embryos produced by any of the methods of the third, fourth, fifth or sixth aspects can be used in a transformation method to produce transgenic plants or plant tissues. The somatic embryos can be used for transient or stable transformation. Transient or stable transformation can be performed by methods based on Agrobacterium or biolistics. In a specific example of this transformation method, after somatic embryo induction, the first heterologous nucleotide sequence encoding a morphogenic polypeptide (i.e., a combination of the disclosed BBM, LEC2, BBM-CBF1A, LEC2-CBF1A, BBM-GR, LEC2-GR and / or FUS3 polypeptides) and the second heterologous nucleotide sequence.

[0020] The explant tissue used in any of the aforementioned methods can be a hypocotyl, epicotyl, cotyledon, leaf or root. Explant can be from a plant in the dicotyledonous plants of Ranunculales, Fabales, Cucurbitales, Malvaceae, Brassicales, Solanales, Asterales or Apiales as exemplified herein. Methods described herein, expression cassettes and carriers can be used to produce or derive somatic embryos in the true dicotyledonous plant explants in Platycodon grandiflorus branch, labial branch, mallow branch or bean branch and in Papaveraceae, Leguminosae, Cucurbitaceae, Malvaceae, Cruciferae, Solanaceae, Compositae or Umbelliferae.

[0021] Also disclosed herein are expression constructs suitable for use in the methods described herein for producing somatic embryos and / or transformation. Thus, in one aspect, the present disclosure provides an expression construct comprising (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBM or RWP-RK domain (RKD) polypeptide; and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding a leafy leaf (LEC) polypeptide. For example, the first heterologous nucleotide sequence may be SEQ ID NO:4 encoding a BBM, and the second polypeptide may be SEQ ID NO:6 encoding a LEC2 polypeptide. In another example, the first heterologous nucleotide sequence may be SEQ ID NO:8 encoding a RKD polypeptide, and the second polypeptide may be SEQ ID NO:6 encoding a LEC polypeptide. The first promoter and the second promoter may be inducible promoters, developmentally regulated promoters, or constitutive promoters. The first promoter and the second promoter may be any promoter suitable for expressing the morphogenetic proteins disclosed herein.

[0022] In another aspect, the present disclosure provides an expression construct comprising a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide, a LEC2 polypeptide, or both. For example, the construct may comprise (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBM polypeptide (BBM-CBF1A) coupled to CBF1A (e.g., via a linker domain); and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding a LEC2 polypeptide. In another example, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide (LEC2-CBF1A) coupled to CBF1A (e.g., via a linker domain). In yet another example, the first heterologous nucleotide sequence encodes BBM-CBF1A and the second heterologous nucleotide sequence encodes LEC2-CBF1A. The first promoter and the second promoter may be inducible promoters, developmentally regulated promoters, or constitutive promoters. The first promoter and the second promoter can be any promoter suitable for expressing the morphogenic proteins disclosed herein.

[0023] Another aspect of the present disclosure provides an expression construct comprising a truncated form of a BBM containing the BBM AP2 domain. For example, the construct may comprise (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBM AP2 domain; and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding a LEC2 polypeptide. In another example, the construct comprises (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBMAP2 domain (BBM AP2-CBF1A) coupled to CBF1A (e.g., via a linker domain); and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding a LEC2 polypeptide.

[0024] Yet another aspect of the present disclosure provides an expression construct comprising (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBM polypeptide; and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding FUS3.

[0025] In still another aspect, the present disclosure provides an expression construct comprising a glucocorticoid receptor (GR) coupled to a BBM polypeptide, a LEC2 polypeptide, or both. For example, the construct may comprise (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBM polypeptide (BBM-GR) coupled to a GR; and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding a LEC2 polypeptide. In another example, the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide (LEC2-GR) coupled to a GR. In yet another example, the first heterologous nucleotide sequence encodes a BBM-GR and the second heterologous nucleotide sequence encodes a LEC2-GR. The first promoter and the second promoter may be an inducible promoter, a developmentally regulated promoter, or a constitutive promoter. The first promoter and the second promoter may be any promoter suitable for expressing the morphogenetic protein disclosed herein.

[0026] Each of the aforementioned expression constructs may further comprise recognition sites for a site-specific recombinase, which are flanked by the 5' and 3' ends of the sequence comprising the first heterologous nucleotide sequence and the second heterologous nucleotide sequence (e.g., the recognition sites may flank the sequence comprising an expression cassette containing (a) the first promoter and the first heterologous nucleotide sequence and (b) the second promoter and the second heterologous nucleotide sequence). In a specific example, each of the aforementioned expression constructs may further comprise a nucleotide sequence encoding the site-specific recombinase, which recognizes and excises the intervening sequences flanked by the recognition sites. The site-specific recombinase sequence may optionally be operably linked to an inducible promoter, a developmentally regulated promoter, a constitutive promoter, or any promoter suitable for expressing the morphogenetic protein disclosed herein.

[0027] DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0028] Figure 1 is a phylogenetic diagram showing the relative position of the various dicot species used for transformation in the Examples described herein.

[0029] Figure 2 is a phylogenetic diagram showing the relative positions of different dicot species used as sources of the LEC2 genes used in the Examples described herein.

[0030] Figure 3 is a phylogenetic diagram showing the relative positions of different dicot species used as the source of the BBM1 genes used in the Examples described herein.

[0031] The nucleotide sequences listed in the accompanying sequence table and quoted in this article are displayed using standard letter abbreviations for nucleotide bases. Only one chain of each nucleotide sequence is shown, but any reference to the chain shown is understood to include the complementary chain. The description of the sequence table submitted herein is provided in Table 1.

[0032] Table 1

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[0042] DETAILED DESCRIPTION

[0043] 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.

[0044] Those skilled in the art to which the disclosed compositions and methods relate will consider that many modifications and other aspects disclosed herein have the benefit of the teachings presented in the following description and the associated drawings. Therefore, it should be understood that the disclosure is not limited to the specific aspects disclosed, and modifications and other aspects are intended to be included within the scope of the appended claims. Although specific terms are employed herein, these terms are used in a general and descriptive sense only and not for purposes of limitation.

[0045] It should also be understood that 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 compositions and methods belong. In this specification and the claims that follow, reference will be made to several terms defined herein.

[0046] 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."

[0047] 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.

[0048] 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.

[0049] 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. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS [Computer Applications in Biological Sciences] 4:11-17; Smith, et al., (1981) Adv. Appl. Math. [Advanced Applied Mathematics] 2:482; Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453; Pearson and Lipman (1988) Proc. Natl. Am. Soc. Am., 1996:101-113; Smith et al., (1981) Adv. Appl. Math. [Advanced Applied Mathematics] 2:482; Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453; Pearson and Lipman (1988) Proc. Natl. Am. Soc ... l.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 according to 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.

[0050] Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (version 2.0) and the GCG Wisconsin Genetics Software GAP, BESTFIT, BLAST, FASTA, and TFASTA in version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the 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, which are incorporated herein by reference in their entirety. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. When comparing amino acid sequences, the ALIGN program can use the 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, which is 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 the nucleotide sequence encoding the proteins of the disclosure. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the proteins or polypeptides of the 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, which is incorporated herein by reference in its entirety. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterative search, which detects long-range relationships between molecules. See Altschul et al., (1997), supra. When using BLAST, Gap 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.nlm.nih.gov. Alignments can also be made by manual inspection.

[0051] 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.

[0052] The GAP program uses the algorithm of Needleman and Wunsch (supra) to find the alignment 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 gap creation penalties and gap extension penalties to be provided in units of matched bases. GAP must obtain the benefit of the number of gap creation 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 GCG Wisconsin Genetics Software 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, and the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group consisting of an integer from 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.

[0053] GAP gives one member of the family with the best alignment. There may be many members of this family, but the others are not of better quality. GAP displays four merit factors for alignment: quality, ratio, identity, and similarity. Quality is the metric that is maximized in order to align the sequences. 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 symbols that are similar. Symbols corresponding to 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 Software 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).

[0054] 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).

[0055] 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.

[0056] 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.

[0057] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.Usually, stringent conditions are selected to be about 5°C lower than the Tm of the specified sequence under defined ionic strength and pH.However, stringent conditions encompass temperatures of about 1°C to about 20°C 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, this may occur when the maximum codon degeneracy allowed by the genetic codon is used to produce a nucleic acid copy.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.

[0058] A. Expression cassettes and constructs for dicot transformation

[0059] In a first aspect, the disclosure provides a morphogenetic gene cassette comprising at least two morphogenetic genes that, when expressed, promote transformation and somatic embryogenesis in dicotyledons. As used herein, "morphogenetic gene cassette" and "morphogenetic gene expression cassette" refer to a nucleotide construct expressing (i.e., encoding) one or both of the two morphogenetic genes.

[0060] As used herein, "morphogenetic gene" refers to a gene that stimulates the formation of a somatic cell-derived structure that can produce a plant when expressed. For example, the ectopic expression of a morphogenetic gene stimulates the de novo formation of a somatic embryo that can produce a plant. This stimulated de novo formation occurs in cells expressing morphogenetic genes or in adjacent cells. Morphogenetic genes can be transcription factors that regulate the expression of other genes, or genes that affect hormone levels in plant tissues, both of which can stimulate morphology to change. As used herein, "transcription factor" refers to a protein that controls the transcription rate of a specific gene by binding to the DNA sequence of a promoter and up-regulating or down-regulating expression.

[0061] The disclosure also provides expression constructs comprising morphogenetic gene cassettes as described herein. "Expression construct," "expression vector," and "vector" can be used interchangeably and refer to DNA molecules, such as plasmids, cosmids, or bacteriophages, used to introduce nucleotide constructs, such as expression cassettes, into host cells. The cloning vector can include one or a small amount of restriction endonuclease recognition sites, into which foreign DNA sequences can be inserted in a determinable manner without losing the basic biological functions of the vector, and marker genes suitable for identifying and selecting cells transformed by the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance, or ampicillin resistance.

[0062] The morphogenic genes disclosed herein include, but are not limited to, genes of the leafy leaf (LEC) family (e.g., LEC1 (Lotan et al., 1998, Cell 93: 1195-1205) and LEC2 (Stone et al., 2008, Proc. Nat'l. Acad. Sci. USA 105: 3151-3156; Belide et al., 2013, Plant Cell Tiss. Organ Cult. 113: 543-553)), genes of the RWP-RK domain family (e.g., RKD4), and genes of the AP2 / EREBP family (e.g., BBM (also known as ODP2)).

[0063] In some aspects, the morphogenetic gene cassette comprises a first heterologous nucleotide sequence encoding a first morphogenetic gene and a second heterologous nucleotide sequence encoding a second morphogenetic gene. As used herein, the term "heterologous" used in conjunction with "nucleotide sequence," "polynucleotide," "coding sequence," "gene," "protein," or "polypeptide" refers to a sequence that is not naturally present in the context disclosed herein or is not operably linked to a promoter sequence disclosed herein. Although such a nucleotide sequence is heterologous to the context or promoter sequence presented herein, it may be homologous or native, or heterologous, or foreign to the plant host.

[0064] In some aspects, the morphogenetic gene cassette used in the methods and constructs disclosed herein comprises a first heterologous nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second heterologous nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). For example, the first heterologous nucleotide sequence and the second heterologous nucleotide sequence can encode a BBM1 and a LEC1 polypeptide, respectively, a BBM1 and a LEC2 polypeptide, respectively, a BBM2 and a LEC1 polypeptide, respectively, or a BBM2 and a LEC2 polypeptide, respectively.

[0065] In some aspects, the morphogenic gene cassette used in the methods and constructs disclosed herein comprises a first heterologous nucleotide sequence encoding a RKD polypeptide and a second heterologous nucleotide sequence encoding a LEC polypeptide. For example, the first heterologous nucleotide sequence can encode RKD4 and the second heterologous nucleotide sequence can encode a LEC1 or LEC2 polypeptide.

[0066] The term "Babyboom1" or "BBM1" is used herein to refer to a gene, coding sequence, protein or variant thereof. Babyboom and BBM1 may refer to a BBM1 gene, coding sequence, protein or variant thereof from Brassica napus or from Arabidopsis thaliana, sunflower, elm, cucumber, Cleome, Walnut, Populus trichocarpa, pigeon pea, cotton, cassava, sweet orange, grape, apple or tomato. For example, the BBM1 nucleotide sequence may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with any one of SEQ ID NO: 4, 28, 30, 32, 34, 36, 38, 40, 42, 43, 45, 48, 50, 52, 54, 75, 77 or 79. Alternatively, for example, the BBM1 protein sequence can exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any of SEQ ID NO:5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78 or 80.

[0067] The terms "Leaf2" and "LEC2" are used herein to refer to a gene, coding sequence, protein, or variant thereof.Leaf2 and LEC2 may refer to a LEC2 gene from Arabidopsis thaliana or from Halophora salina, Gossypium hirsutum, Cassava, Cleome, Cleome, Juglans regia, Populus trichocarpa, or Aquilegia caerulea, or variant thereof. For example, the LEC2 nucleotide sequence may exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity to any one of SEQ ID NO: 6, 12, 14, 16, 18, 20, 22, 24, or 26. Alternatively, for example, the LEC2 protein sequence can exhibit at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0068] In one aspect, a morphogenetic gene cassette is provided, comprising a first heterologous nucleotide sequence encoding a BBM1 polypeptide from Brassica napus or a variant thereof and a second heterologous nucleotide sequence encoding a LEC2 polypeptide from Arabidopsis thaliana or a variant thereof. In another aspect, a morphogenetic gene cassette is provided herein, comprising a BBM1 coding sequence from Brassica napus or a variant thereof and a LEC1 coding sequence from Arabidopsis thaliana or a variant thereof.

[0069] In one aspect, the present invention provides a morphogenetic gene cassette, comprising a first heterologous nucleotide sequence encoding a RWP-RK domain-containing protein 4 (RKD4) polypeptide or a variant thereof from Arabidopsis thaliana and a second heterologous nucleotide sequence encoding a LEC2 polypeptide or a variant thereof from Arabidopsis thaliana. In another aspect, the present invention provides a morphogenetic gene cassette, comprising a RKD4 coding sequence or a variant thereof from Arabidopsis thaliana and a LEC1 coding sequence or a variant thereof from Arabidopsis thaliana.

[0070] In one aspect, provided herein is a morphogenetic gene cassette comprising an RKD4 coding sequence from Arabidopsis thaliana or a variant thereof and an LEC2 coding sequence from Arabidopsis thaliana or a variant thereof.

[0071] In one aspect, provided herein is a morphogenetic gene cassette comprising a BBM nucleotide sequence encoding SEQ ID NO: 5 and a LEC nucleotide sequence encoding any one of SEQ ID NO: 7, 11, 13, 15, 17, 19, 21, 23, 25 or 27.

[0072] In another aspect, provided herein is a morphogenetic gene cassette comprising a BBM nucleotide sequence encoding any one of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80 and a LEC1 or LEC2 nucleotide sequence. The LEC1 nucleotide sequence may encode SEQ ID NO: 11. The LEC2 nucleotide sequence may encode any one of SEQ ID NO: 7, 13, 15, 17, 19, 21, 23, 25, or 27.

[0073] In another aspect, the present invention provides a morphogenetic gene cassette comprising an RKD nucleotide sequence (e.g., a sequence encoding SEQ ID NO: 9) and a LEC1 or LEC2 nucleotide sequence. The LEC2 nucleotide sequence may encode any one of SEQ ID NO: 7, 13, 15, 17, 19, 21, 23, 25, or 27. The LEC1 nucleotide sequence may encode SEQ ID NO: 11.

[0074] Also provided herein is a morphogenic gene cassette comprising a first nucleotide sequence encoding a BBM polypeptide (BBM-CBF1A polypeptide) coupled to a CCAAT binding factor 1A activation domain (CBF1A) and a second nucleotide encoding a LEC1 or LEC2 polypeptide. The BBM protein sequence may be any one of SEQ ID NOs: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80, the CBF1A domain may be SEQ ID NO: 71, and the BBM and CBF1A may be coupled via any suitable linker sequence. The LEC2 nucleotide sequence may encode any one of SEQ ID NOs: 7, 13, 15, 17, 19, 21, 23, 25, or 27. The LEC1 nucleotide sequence may encode SEQ ID NO: 11. The BBM and CBF1A may be coupled via any suitable linker sequence.

[0075] In various aspects, provided herein is a morphogenetic gene cassette comprising a first nucleotide sequence encoding a BBM polypeptide and a second nucleotide encoding a LEC1 or LEC2 polypeptide coupled to a CBF1A domain (e.g., SEQ ID NO: 71). The first BBM nucleotide sequence may encode any one of SEQ ID NOs: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80. The second nucleotide sequence may encode a LEC2 polypeptide of SEQ ID NOs: 7, 13, 15, 17, 19, 21, 23, 25, or 27 coupled to a CBF1A domain; or the second nucleotide sequence may encode a LEC1 polypeptide of SEQ ID NO: 11 coupled to a CBF1A domain. The LEC polypeptide and CBF1A may be coupled via any suitable linker sequence.

[0076] In a separate aspect, a morphogenetic gene cassette is provided herein, comprising a first nucleotide sequence encoding a truncated BBM (BBM AP2) retaining its AP2 domain and a second nucleotide sequence encoding a LEC1 or LEC2 polypeptide. In some examples, the AP2 domain can be coupled to a CCAAT binding factor 1A activation domain (CBF1A) to produce a BBM AP2-CBF1A. Thus, the morphogenetic gene cassette provided may comprise a first nucleotide sequence encoding a BBM AP2 (e.g., SEQ ID NO: 69) and a LEC2 nucleotide sequence encoding any one of SEQ ID NO: 7, 13, 15, 17, 19, 21, 23, 25 or 27. Alternatively, the provided morphogenetic gene cassette may include a first nucleotide sequence encoding a BBM AP2 (e.g., SEQ ID NO: 69) linked to a CBF1A (e.g., SEQ ID NO: 71) and a LEC2 sequence encoding any one of SEQ ID NOs: 7, 13, 15, 17, 19, 21, 23, 25, or 27. In different examples, the morphogenetic gene cassette may include a first nucleotide sequence encoding a BBM AP2 (e.g., SEQ ID NO: 68) and a LEC1 nucleotide sequence encoding SEQ ID NO: 11; or the morphogenetic gene cassette may include a first nucleotide sequence encoding a BBM AP2 (e.g., SEQ ID NO: 68) linked to a CBF1A (SEQ ID NO: 70) and a second LEC1 nucleotide sequence encoding SEQ ID NO: 11. The BBM AP2 and CBF1A may be linked by any suitable linker sequence.

[0077] In yet another aspect, the present invention provides a morphogenetic gene cassette comprising a first nucleotide sequence encoding a BBM polypeptide and a second nucleotide sequence encoding a FUS3 polypeptide (e.g., SEQ ID NO: 57). Thus, in this gene cassette, the first nucleotide sequence can encode a BBM polypeptide of SEQ ID NO: 5, and the second nucleotide sequence can encode a FUS3 polypeptide. Alternatively, in another example, the morphogenetic gene cassette can comprise a BBM nucleotide sequence encoding any one of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80 and a second nucleotide sequence encoding a FUS3 polypeptide.

[0078] Each nucleotide sequence encoding a morphogenic gene may be operably linked to a constitutive promoter, an inducible promoter, a developmentally regulated promoter, or a tissue-specific promoter that allows initiation of transcription in plant tissues. Promoters suitable for expressing the morphogenic genes disclosed herein include: UBI, actin, EF1A, RUBACT, EFTU2, CAB, CSVMV, oxygen-evolving enhancer (OEE), aquaporin (AQP), photosystem I subunit D-2 (PSID), SCBV, LLDAV, EVCV, DMMV, BSV(AY)PRO, CYMV PRO FL, IN2-2, NOS, -135 version of 35S, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, AT-HSP811, AT-HSP811L, GM-HSP173B, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron, PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A or LEA-D34.

[0079] In some aspects, after inducing somatic embryo formation in dicot explants, the first heterologous nucleotide sequence and the second heterologous nucleotide sequence encoding the morphogenic gene are excised. In this way, the expression of the morphogenic gene can be controlled by excision at a desired time after transformation. For example, the expression cassette can be targeted for excision by a site-specific recombinase. When a site-specific recombinase is used to control the expression of the morphogenic gene, the morphogenic gene cassette further comprises an appropriate site-specific excision site flanking the nucleotide sequence to be excised. In some cases, the expression construct comprising the morphogenic gene cassette further comprises a nucleotide sequence encoding a site-specific recombinase. Alternatively, the site-specific recombinase does not need to be co-located on the same expression construct comprising the morphogenic gene cassette, and on the contrary, the recombinase can be expressed from different constructs. The site-specific recombinases that can be used in the methods and constructs disclosed herein include FLP, FLPe, KD, Cre, SSV1, λInt, Int, HK022, R, B2, B3, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1 or U153. The site-specific recombinase can be operably linked to a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmentally regulated promoter, such as UBI, actin, EF1A, RUBACT, EFTU2, CAB, CSVMV, oxygen-evolving enhancer (OEE), aquaporin (AQP), photosystem I subunit D-2 (PSID), SCBV, LLDAV, EVCV, DMMV, BSV(AY)PRO, CYMV PRO FL, IN2-2, NOS, -135 version of 35S, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, AT-HSP811, AT-HSP811L, GM-HSP173B, or a promoter activated by tetracycline, ethametsulfuron or chlorsulfuron, PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A or LEA-D34.

[0080] B. Methods for Somatic Embryogenesis and Regeneration of Transgenic Dicotyledonous Plants

[0081] On the other hand, the disclosure provides a method for inducing somatic embryo production in dicot explants by expressing a morphogenetic gene cassette as described herein to promote somatic embryo formation. More specifically, methods disclosed herein make it possible to achieve direct somatic embryogenesis (DSE) of explant cells. Somatic embryogenesis is a form of induced plant cell totipotency, in which embryos develop from somatic cells without fertilization. As used herein, "direct somatic embryogenesis" refers to the formation of somatic embryos directly from the explant surface, without dedifferentiation phase or callus formation.

[0082] As used herein, "somatic embryo" refers to a multicellular structure that progresses through a developmental stage similar to that of a zygotic embryo. In some embodiments disclosed herein, somatic embryos are tissues that can regenerate transgenic plants after transformation. The characteristics of somatic embryos can be the formation of globular and transitional stage embryos, the formation of hypocotyls and scutellum, and the accumulation of lipids and starch. In some cases, by the relatively high nucleus and cytoplasm ratio of somatic embryos, the presence of a single large nucleolus and a relatively low heterochromatin level, the presence of a fragmented vacuole, and the cell wall containing callose of the somatic embryo, somatic embryos can be identified histologically and distinguished from surrounding cells.

[0083] In some aspects, the method for producing dicot somatic embryos comprises expressing the morphogenetic gene cassette disclosed herein in a dicot explant, and culturing the explant under conditions suitable for somatic embryo formation. The morphogenetic gene cassette may be any of those disclosed herein, the morphogenetic gene cassette comprising a first nucleotide sequence encoding a first morphogenetic polypeptide and a second nucleotide sequence encoding a second morphogenetic polypeptide. Thus, the method for producing dicot somatic embryos comprises expressing a first heterologous nucleotide sequence encoding a BBM or RKD polypeptide and a second heterologous nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2) in a dicot explant.

[0084] Provided herein is a method for producing dicot somatic embryos, the method comprising expressing in a dicot explant: a first heterologous nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second heterologous nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2) 1. For example, the method can include expressing in a dicot explant a first heterologous nucleotide sequence encoding a BBM1 nucleotide sequence (which encodes SEQ ID NO: 5) and a second heterologous LEC nucleotide sequence encoding any one of SEQ ID NO: 7, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In other examples, the method can include expressing in a dicot explant a first heterologous BBM nucleotide sequence encoding any one of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80 and a LEC1 or LEC2 nucleotide sequence. Thus, the method may comprise expressing in a dicot explant a first heterologous BBM nucleotide sequence encoding any one of SEQ ID NOs: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80 and a LEC2 nucleotide sequence encoding SEQ ID NO: 7. Alternatively, the method may comprise expressing in a dicot explant a first heterologous BBM nucleotide sequence encoding any one of SEQ ID NOs: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80 and a LEC2 nucleotide sequence encoding any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, or 27. In another example, the method can include expressing a first heterologous BBM nucleotide sequence encoding any one of SEQ ID NO:29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78 or 80 and a LEC1 nucleotide sequence encoding SEQ ID NO:11 in a dicot explant.

[0085] In some aspects, the method of producing dicot somatic embryos comprises expressing a morphogenetic cassette comprising a first heterologous nucleotide sequence encoding a RKD polypeptide (e.g., SEQ ID NO: 9) and a second heterologous nucleotide sequence encoding a LEC polypeptide. The LEC polypeptide can be a LEC2 polypeptide of SEQ ID NO: 7 or a LEC2 polypeptide of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, or 27. In various examples, the first heterologous nucleotide sequence encodes RKD4 (e.g., SEQ ID NO: 9), and the second heterologous nucleotide sequence encodes LEC1 (e.g., SEQ ID NO: 11).

[0086] The method for introducing the morphogenic gene pair (i.e., the first heterologous nucleotide sequence and the second heterologous nucleotide sequence) into the explant can vary depending on the dicotyledonous plant or explant source being targeted. Suitable methods for introducing nucleotide sequences into explants include microinjection, electroporation, direct gene transfer, biolistic-mediated gene transfer, and bacterial-mediated transformation (e.g., Agrobacterium-mediated transformation and Ochrobactrum-mediated transformation).

[0087] In some aspects, a first heterologous nucleotide sequence encoding a first morphogenic gene and a second heterologous nucleotide sequence encoding a second morphogenic gene are introduced into the explant via transferred DNA (T-DNA). As used herein, "transferred DNA" or "T-DNA" refers to a portion of a Ti plasmid inserted into the genome of a host plant cell. The morphogenic genes of the present disclosure can be integrated into a T-DNA transfer cassette, i.e., a T-DNA comprising a morphogenic gene expression cassette flanked by a right border and a left border. Agrobacterium tumefaciens and Agrobacterium rhizogenes are examples of plant pathogens that can transfer bacterial genes encoded by plasmids located on T-DNA into plant cells in a manner that depends on bacterial virulence (Vir) protein translocation. In some aspects, the method uses a disarmed Agrobacterium strain (such as, but not limited to, AGL-1, EHA105, GV3101, LBA4404, LBA4404 THY-, and LBA4404 THY-TD) to transform the explant with a first heterologous nucleotide sequence and a second heterologous nucleotide sequence.

[0088] Methods described herein, expression cassettes and vectors can be used to produce or derive somatic embryos in dicot explants, including those in the following orders as exemplified herein: Ranunculales, Beans, Cucurbitales, Malvaceae, Cruciferae, Solanales, Chrysanthemeales and Umbelliferae. Methods described herein, expression cassettes and vectors can be used to produce or derive somatic embryos in true dicot explants in Platycodon branches, Labiales branches, Malvaceae branches or Bean branches and in Papaveraceae, Fabaceae, Cucurbitaceae, Malvaceae, Cruciferae, Solanaceae, Compositae or Umbelliferae.

[0089] In some aspects, the explant is from cotton, Arabidopsis, poppy, carrot, alfalfa, soybean, flax, cassava, bean, cowpea, tomato, potato, sugar beet, grape, eucalyptus, poplar, citrus, papaya, cocoa, cucumber, apple, Capsicum, melon, or Brassica.

[0090] Suitable explant sources for somatic embryo induction, which may vary depending on the dicot, include cotyledons, hypocotyls, epicotyls, leaves, stems, and roots.

[0091] In some respects, dicotyledon explant can be derived from transgenic plant.As used herein, " transgenic plant " and " transformed plant " refer to the plant that comprises heterologous polynucleotide (for example, transgenic) in the genome, and this heterologous polynucleotide is usually stably integrated in the genome of transgenic or transformed plant, so that polynucleotide is passed to continuous generation.Heterologous polynucleotide or transgenic can be integrated into the genome individually or as the part of recombinant DNA construct.In this respect, the disclosure contains that transgenic dicotyledon explant is transformed again to produce somatic embryo.

[0092] In some aspects, the dicot explants can be derived from non-transgenic plants.

[0093] In some aspects, the dicot explant is from a resistant variety.

[0094] Somatic Embryogenesis in Cotton and Regeneration of Transgenic Cotton Plants

[0095] The disclosure provides the method for inducing somatic embryo formation in cotton explant, and the method comprises transforming cotton explant with the construct comprising the morphogenesis gene box disclosed herein.Suitable cotton explant source comprises cotyledon, hypocotyl, epicotyl, leaf, stem and root.As shown in this paper example, disclosed method not only improves the efficiency of somatic embryo formation, and surprisingly finds that inducing somatic embryo formation in a plurality of cotton lines (comprising elite cotton varieties), these cotton lines are originally resistant to somatic embryo induction.Therefore, in an example of each kind of method for inducing somatic embryo formation disclosed herein for cotton, the cotton variety converted can be the variety that somatic embryo induction has resistance before the application discloses.

[0096] In one aspect, a method of inducing somatic embryo formation comprises transforming a cotton (e.g., Pima cotton or other varieties) explant with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming a cotton explant with a first nucleotide sequence encoding a BBM1 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78 or 80 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming cotton (e.g., Pima or other varieties) explants with a first nucleotide sequence encoding a BBM1 polypeptide (e.g., SEQ ID NO:5) and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0097] In a second aspect, a method of inducing somatic embryo formation comprises transforming a cotton (e.g., Pima cotton or other varieties) explant with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming a cotton (e.g., Pima cotton or other varieties) explant with a first nucleotide sequence encoding a RKD4 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 9 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming cotton (e.g., Pima or other varieties) explants with a first nucleotide sequence encoding an RKD4 polypeptide and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0098] In a third aspect, a method of inducing somatic embryogenesis comprises transforming cotton (e.g., Pima or other varieties) explants with a first nucleotide sequence encoding a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding CBF1A disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:70, or it may encode a CBF1A polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:71.

[0099] In a fourth aspect, a method of inducing somatic embryo formation comprises transforming cotton (e.g., Pima or other varieties) explants with a first nucleotide sequence encoding BBM and a second nucleotide sequence encoding CBF1A coupled to a LEC polypeptide (e.g., LEC1 or LEC2). In another variation, both the first nucleotide sequence encoding BBM and the second nucleotide sequence encoding a LEC polypeptide are each coupled to a sequence encoding CBF1A.

[0100] In a fifth aspect, a method of inducing somatic embryogenesis comprises transforming cotton (e.g., Pima or other varieties) explants with a nucleotide sequence encoding CBF1A coupled to a truncated form of a BBM polypeptide, wherein the truncated BBM comprises a BBMAP2 domain and a C-terminus, but lacks a BBM N-terminus.

[0101] In a sixth aspect, a method of inducing somatic embryo formation comprises transforming a cotton (e.g., Pima cotton or other varieties) explant with a first nucleotide sequence encoding a glucocorticoid receptor (GR) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a seventh aspect, a method of inducing somatic embryo formation comprises transforming a cotton (e.g., Pima cotton or other varieties) explant with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a GR coupled to a LEC polypeptide (e.g., LEC1 or LEC2). In another variation, both the first nucleotide sequence encoding the BBM and the second nucleotide sequence encoding the LEC polypeptide are each coupled to a sequence encoding the GR. The nucleotide sequence encoding the GR domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:73, or it may encode a GR polypeptide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:74.

[0102] In an eighth aspect, a method of inducing somatic embryo formation comprises transforming cotton (e.g., Pima or other varieties) explants with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a FUSCA3 (FUS3) polypeptide. The FUS3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:57.

[0103] In cotton, each of the aforementioned aspects of inducing somatic embryogenesis can further include regenerating transgenic cotton plants from somatic embryos.Usually, regenerating plants from the somatic embryo tissues of conversion can include embryo transfer to embryo germination medium and / or rooting and extension medium to regenerate transgenic cotton plants.In a preferred embodiment, the first nucleotide sequence and the second nucleotide sequence of the morphogenesis gene box used to induce somatic embryogenesis are inactivated before or during the regeneration of transgenic cotton plants.This inactivation can be completed by any suitable means, for example, by making the expression inactivation of two kinds of encoded heterologous morphogenesis proteins or suppressing their expression or by excising the heterologous nucleotide sequence of the encoding morphogenesis protein.Therefore, for example, after somatic embryo induction, the first heterologous nucleotide sequence and the second heterologous nucleotide sequence of the morphogenesis gene box can be excised before completing the process of regenerating transgenic plants.

[0104] Techniques and media that can be used for cotton transformation are described in U.S. Pat. Nos. 5,846,797, 5,159,135, 5,004,863, and 6,624,344, and also in protocols published electronically by IP.com using permanent public identifiers IPCOM000033402D, IPCOM000033403D, IPCOM000033404D (these identifiers can be retrieved using an online database search portal hosted by IP.com), and in the Examples provided herein.

[0105] Somatic Embryo Formation in Alfalfa and Regeneration of Transgenic Alfalfa Plants

[0106] The present disclosure provides a method for inducing somatic embryo formation in alfalfa explants, the method comprising transforming the alfalfa explants with a construct comprising the morphogenetic gene cassette disclosed herein. Suitable sources of alfalfa explants include leaves, stems and roots.

[0107] In one aspect, the method of inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM1 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78 or 80 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM1 polypeptide (e.g., SEQ ID NO:5) and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0108] In a second aspect, a method of inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the RKD4 disclosed herein may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8, or encodes a RKD4 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 9. In one example, the method comprises transforming alfalfa explants with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming alfalfa explants with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0109] In a third aspect, a method of inducing somatic embryogenesis comprises transforming alfalfa explants with a first nucleotide sequence encoding a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding CBF1A disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:70, or it may encode a CBF1A polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71.

[0110] In a fourth aspect, a method of inducing somatic embryogenesis comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a CBF1A coupled to a LEC polypeptide (eg, LEC1 or LEC2).

[0111] In a fifth aspect, a method for inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM AP2 domain and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, the first nucleotide sequence can encode a BBM AP2 domain coupled to CBF1A, and the second nucleotide sequence encodes a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the BBM AP2 domain disclosed herein can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 68, or it can encode a BBM AP2 domain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 69.

[0112] In a sixth aspect, a method of inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a glucocorticoid receptor (GR) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, a method of inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a GR coupled to a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the GR domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 73, or it may encode a GR polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 74.

[0113] In a seventh aspect, a method of inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a FUS3 polypeptide. The FUS3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:57.

[0114] In an eighth aspect, a method of inducing somatic embryo formation comprises transforming alfalfa explants with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding an ABI3 polypeptide. The ABI3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 64.

[0115] In alfalfa, inducing somatic embryogenesis in the aforementioned aspect of formation can further comprise from somatic embryo regeneration transgenic alfalfa plant.Usually, regeneration plant from the somatic embryo tissue of conversion can comprise embryo transfer to embryo germination medium and / or rooting and extension medium with regeneration transgenic alfalfa plant.In a preferred embodiment, the first nucleotide sequence and the second nucleotide sequence of the morphogenesis gene box that are used to induce somatic embryo formation are inactivated before or during regeneration of transgenic alfalfa plant.This inactivation can be completed by any suitable means, for example, by making the expression inactivation of two kinds of heterologous morphogenesis proteins of coding or suppressing their expression or by excision of the heterologous nucleotide sequence of coding morphogenesis protein.Therefore, for example, after somatic embryo induction, can excise the first heterologous nucleotide sequence and the second heterologous nucleotide sequence of the morphogenesis gene box before completing the process of regeneration transgenic plant.

[0116] Techniques and media that can be used for alfalfa transformation are described in U.S. Pat. Nos. 4,801,545, 6,566,137; 8,124,848; and 9,771,597; Samac et al. (Samac DA, Austin-Phillips S. (2006) Alfalfa (Medicago sativa L.): Wang K. (ed.) Agrobacterium Protocols. Methods in Molecular Biology, Vol. 343), and in the Examples provided herein.

[0117] Somatic embryogenesis in canola and regeneration of transgenic canola plants

[0118] The present disclosure provides a method for inducing somatic embryo formation in Brassica napus or canola rape explants, the method comprising transforming canola rape explants with a construct comprising a morphogenetic gene cassette disclosed herein. Suitable canola rape explant sources include cotyledons, hypocotyls, epicotyls, leaves, stems and roots. As used herein, the term "canola rape" refers to any Brassica napus, including spring or winter (vernalized) varieties.

[0119] In one aspect, a method of inducing somatic embryo formation comprises transforming a canola explant with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming a canola explant with a first nucleotide sequence encoding a BBM1 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78 or 80 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming canola explants with a first nucleotide sequence encoding a BBM1 polypeptide (e.g., SEQ ID NO:5) and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0120] In a second aspect, a method of inducing somatic embryo formation comprises transforming a canola explant with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming a canola explant with a first nucleotide sequence encoding a RKD4 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 9 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming canola explants with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0121] In a third aspect, a method of inducing somatic embryogenesis comprises transforming canola explants with a first nucleotide sequence encoding a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding CBF1A disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:70, or it may encode a CBF1A polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:71.

[0122] In a fourth aspect, a method of inducing somatic embryogenesis comprises transforming canola explants with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a CBF1A coupled to a LEC polypeptide (eg, LEC1 or LEC2).

[0123] In a fifth aspect, a method of inducing somatic embryo formation comprises transforming canola rape explants with a first nucleotide sequence encoding a BBM AP2 domain and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, the first nucleotide sequence may encode a BBM AP2 domain coupled to CBF1A, and the second nucleotide sequence encodes a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the BBM AP2 domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 68, or it may encode a BBM AP2 domain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 69.

[0124] In a sixth aspect, a method of inducing somatic embryo formation comprises transforming a canola explant with a first nucleotide sequence encoding a glucocorticoid receptor (GR) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, a method of inducing somatic embryo formation comprises transforming a canola explant with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a GR coupled to a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the GR domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 73, or it may encode a GR polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 74.

[0125] In a seventh aspect, a method of inducing somatic embryo formation comprises transforming canola rape explants with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a FUS3 polypeptide. The FUS3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:57.

[0126] In an eighth aspect, a method of inducing somatic embryogenesis comprises transforming canola rape explants with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding an ABI3 polypeptide. The ABI3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 64.

[0127] Each of the aforementioned aspects of inducing somatic embryogenesis in canola canola can further include regenerating transgenic canola canola plants from somatic embryos.Usually, regenerating plants from the somatic embryo tissue of conversion can include transferring embryos to embryo germination medium and / or rooting and extension medium to regenerate transgenic canola canola plants.In a preferred embodiment, the first nucleotide sequence and the second nucleotide sequence of the morphogenic gene box used to induce somatic embryogenesis are inactivated before or during the regeneration of transgenic canola canola plants.This inactivation can be completed by any suitable means, for example, by inactivating the expression of two kinds of heterologous morphogenic proteins encoded or suppressing their expression or by excising the heterologous nucleotide sequence of the morphogenic protein encoded.Therefore, for example, after somatic embryo induction, the first heterologous nucleotide sequence and the second heterologous nucleotide sequence of the morphogenic gene box can be excised before completing the process of regenerating transgenic plants.

[0128] Techniques and culture media that can be used for canola rape transformation are described in U.S. Patent No. 5,750,871 and Chu et al. (2020) Genotype-independent transformation and genome editing of brassicanapus using a novel explant material. [Genotype-independent transformation and genome editing of brassicanapus using a novel explant material]. Frontiers in Plant Sciences 11(579524): 1-11 and in the examples provided herein.

[0129] Somatic embryogenesis in California poppy, cucumber, carrot or tomato and regeneration of transgenic California poppy, cucumber, carrot or tomato plants

[0130] The present disclosure provides a method for inducing somatic embryo formation in California poppy, cucumber, carrot or tomato explants, the method comprising transforming California poppy explants, cucumber explants, carrot explants or tomato explants with a construct comprising a morphogenetic gene cassette disclosed herein. Suitable sources of California poppy, cucumber, carrot or tomato explants include cotyledons, hypocotyls, epicotyls, leaves, stems and roots.

[0131] In one aspect, a method of inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming an explant with a first nucleotide sequence encoding a BBM1 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78, or 80 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming the explant with a first nucleotide sequence encoding a BBM1 polypeptide (e.g., SEQ ID NO: 5) and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0132] In a second aspect, a method of inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming an explant with a first nucleotide sequence encoding a RKD4 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 9 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming the explant with a first nucleotide sequence encoding an RKD4 polypeptide and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0133] In a third aspect, a method of inducing somatic embryogenesis comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding CBF1A disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:70, or it may encode a CBF1A polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:71.

[0134] In a fourth aspect, a method of inducing somatic embryogenesis comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a CBF1A coupled to a LEC polypeptide (e.g., LEC1 or LEC2).

[0135] In a fifth aspect, a method of inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a BBM AP2 domain and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one variation of this aspect, the first nucleotide sequence may encode a BBM AP2 domain coupled to CBF1A, and the second nucleotide sequence may encode a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the BBM AP2 domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 68, or it may encode a BBMAP2 domain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 69.

[0136] In a sixth aspect, a method for inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a glucocorticoid receptor (GR) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, a method for inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a GR coupled to a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the GR domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:73, or it may encode a GR polypeptide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:74.

[0137] In a seventh aspect, a method of inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a FUS3 polypeptide. The FUS3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 57.

[0138] In an eighth aspect, a method of inducing somatic embryo formation comprises transforming an explant (i.e., a California poppy explant, a cucumber explant, a carrot explant, or a tomato explant) with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding an ABI3 polypeptide. The ABI3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 64.

[0139] Each of the aforementioned aspects of inducing somatic embryogenesis in California poppy, cucumber, carrot or tomato can further include regenerating transgenic plants from somatic embryos. Usually, regenerating plants from the somatic embryo tissue of conversion can include transferring embryos to embryo germination medium and / or rooting and extension medium to regenerate transgenic California poppy, cucumber, carrot or tomato plants. In a preferred embodiment, the first nucleotide sequence and the second nucleotide sequence of the morphogenic gene box used for inducing somatic embryogenesis are inactivated before or during the regeneration of transgenic plants. This inactivation can be completed by any suitable means, for example, by inactivating the expression of two kinds of encoded heterologous morphogenic proteins or suppressing their expression or by excising the heterologous nucleotide sequence of the encoding morphogenic protein. Therefore, for example, after somatic embryo induction, the first heterologous nucleotide sequence and the second heterologous nucleotide sequence of the morphogenic gene box can be excised before completing the process of regenerating transgenic plants.

[0140] Techniques and media that can be used to transform California poppy, cucumber, carrot, and tomato are described in Park et al. (Park, S.U., Facchini, P. Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis. Plant Cell Reports 19, 1006-1012 (2000)); techniques and media that can be used for transformation are described in Burza et al. (Burza W., Zuzga S., Yin Z., Malepszy S. (2006) Cucumber (Cucumissativus L.). Wang K. (ed.) Agrobacterium Protocols. Methods in Molecular Biology. Biology [Molecular Biology Methods], Vol. 343), Hardegger et al. (Hardegger, M., Sturm, A. Transformation and regeneration of carrot (Daucus carota L.). [Carrot (carrot) (Daucus carota L.)) transformation and regeneration] Molecular Breeding [Molecular Breeding] 4, 119-127 (1998)), Sharma et al. (Sharma, MK, Solanke, AU, Jani, D. et al. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato. [Simple and efficient Agrobacterium-mediated procedure for transformation of tomato] J Biosci [Journal of Biological Sciences] 34, 423-433 (2009)) and in the examples provided herein.

[0141] Somatic embryogenesis in sunflower and regeneration of transgenic sunflower. The present disclosure provides a method for inducing somatic embryogenesis in sunflower (Helianthus annuus) explants, the method comprising transforming the sunflower explants with a construct comprising a morphogenetic gene cassette disclosed herein. Suitable sources of sunflower explants include cotyledons, hypocotyls, epicotyls, leaves, stems, and roots.

[0142] In one aspect, the method of inducing somatic embryo formation comprises transforming a sunflower explant with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming a sunflower explant with a first nucleotide sequence encoding a BBM1 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO: 5, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 76, 78 or 80 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming sunflower explants with a first nucleotide sequence encoding a BBM1 polypeptide (e.g., SEQ ID NO:5) and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0143] In a second aspect, a method of inducing somatic embryo formation comprises transforming a sunflower explant with a first nucleotide sequence encoding a RKD4 polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In one example, the method comprises transforming a sunflower explant with a first nucleotide sequence encoding a RKD4 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 9 and a second nucleotide sequence encoding a LEC2 polypeptide (e.g., SEQ ID NO: 7). In another example, the method comprises transforming sunflower explants with a first nucleotide sequence encoding an RKD4 polypeptide and a second nucleotide sequence encoding a LEC2 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any one of SEQ ID NO:7, 13, 15, 17, 19, 21, 23, 25 or 27.

[0144] In a third aspect, a method of inducing somatic embryo formation comprises transforming sunflower explants with a first nucleotide sequence encoding a CCAAT binding factor 1A activation domain (CBF1A) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding CBF1A disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:70, or it may encode a CBF1A polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71.

[0145] In a fourth aspect, a method of inducing somatic embryogenesis comprises transforming a sunflower explant with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a CBF1A coupled to a LEC polypeptide (eg, LEC1 or LEC2).

[0146] In a fifth aspect, a method for inducing somatic embryo formation comprises transforming sunflower explants with a first nucleotide sequence encoding a BBM AP2 domain and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, the first nucleotide sequence may encode a BBM AP2 domain coupled to CBF1A, and the second nucleotide sequence may encode a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the BBM AP2 domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 68, or it may encode a BBM AP2 domain polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 69.

[0147] In a sixth aspect, a method of inducing somatic embryo formation comprises transforming a sunflower explant with a first nucleotide sequence encoding a glucocorticoid receptor (GR) coupled to a BBM polypeptide and a second nucleotide sequence encoding a LEC polypeptide (e.g., LEC1 or LEC2). In a variation of this aspect, a method of inducing somatic embryo formation comprises transforming a sunflower explant with a first nucleotide sequence encoding a BBM and a second nucleotide sequence encoding a GR coupled to a LEC polypeptide (e.g., LEC1 or LEC2). The nucleotide sequence encoding the GR domain disclosed herein may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 73, or it may encode a GR polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 74.

[0148] In a seventh aspect, a method of inducing somatic embryo formation comprises transforming a sunflower explant with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding a FUS3 polypeptide. The FUS3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO:57.

[0149] In an eighth aspect, a method of inducing somatic embryo formation comprises transforming a sunflower explant with a first nucleotide sequence encoding a BBM polypeptide (e.g., BBM1 or BBM2) and a second nucleotide sequence encoding an ABI3 polypeptide. The ABI3 polypeptide may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 64.

[0150] Each of the aforementioned aspects of inducing somatic embryogenesis in sunflower can further include regenerating transgenic sunflower plants from somatic embryos.Usually, regenerating plants from the somatic embryo tissue of conversion can include transferring embryos to embryo germination medium and / or rooting and extension medium to regenerate transgenic sunflower plants.In a preferred embodiment, the first nucleotide sequence and the second nucleotide sequence of the morphogenic gene box used to induce somatic embryogenesis are inactivated before or during the regeneration of transgenic canola rape plants.This inactivation can be completed by any suitable means, for example, by inactivating the expression of two kinds of encoded heterologous morphogenic proteins or suppressing their expression or by excising the heterologous nucleotide sequence of the morphogenic protein encoding.Therefore, for example, after somatic embryo induction, the first heterologous nucleotide sequence and the second heterologous nucleotide sequence of the morphogenic gene box can be excised before completing the process of regenerating transgenic plants.

[0151] Techniques and culture media that can be used for transformation of sunflower are described, for example, in International Application Publication No. WO 2021 / 195058, Sankara et al., "Agrobacterium-Mediated Transformation of Sunflower (Helianthus Annuus L.) A Simple Protocol," Annals of Botany, Academic Press (London, GB), Vol. 83(4): 347-354 (1999), and Schrammeijer et al., Plant Cell Reports, 9: 55-60 (1990), as well as in the Examples herein.

[0152] The following are examples of specific embodiments of some aspects of the present invention. These examples are provided for illustrative purposes only, and they are not intended to limit the scope of the present invention in any way. With regard to the numbers used (e.g., amounts, temperatures, etc.), efforts have been made to ensure their accuracy, but some experimental errors and deviations should still be allowed.

[0153] Examples

[0154] The plasmids used for transformation in the examples described herein are described in Table 1, which shows the T-DNA elements and the corresponding sequence listing identifiers. The composition of the culture medium is described in Table 2. Unless otherwise stated, seeds of different species were surface sterilized with 70% ethanol for 2 min and 20% bleach solution containing 2-4 drops of Tween 20 for 10 min. In some cases, seeds were surface sterilized for 30 minutes using a 3% hydrogen peroxide solution containing 2-4 drops of Tween 20. The seeds were rinsed thoroughly with sterile water and germinated on semi-solid culture medium. Unless otherwise stated, an Agrobacterium suspension in 20A culture medium (Table 2) (adjusted to OD 1.0 at 600 nm) was used for infection; and explants were incubated in 100x 25 mm culture dishes with an Agrobacterium suspension supplemented with 100 μM acetosyringone for 10 min. The Agrobacterium suspension was poured off, and the explants were placed in low light (12 μmol m -2 s -1) in the growth chamber and cultivate 2-4 days together.Then explant is transferred to the selection medium and continues 3 weeks.If necessary, the other 3 weeks of explant subculture are used for somatic embryo induction.Through under 45 ℃, 70% RH, somatic embryo is separated by 24 hours heat shock twice and continues 2h to excise the morphogenesis gene.The embryo of heat shock is transferred in the regeneration medium and per 3 weeks subculture is until the plant that obtains rooting is obtained.

[0155] Table 2

[0156]

[0157]

[0158] Example 1: Generation of somatic embryos in cotton explants and regeneration of transgenic cotton plants.

[0159] As described above, the seeds of various cotton (upland cotton) genotypes are surface sterilized. After sterilization, the seeds are washed in sterile water, then germinated and transferred to liquid extension medium to continue for 10-14 days at 28°C; and the expanded cotyledon explants are cut into pieces of 1x 1mm to 5x 5mm. In an alternative procedure, after sterilization and washing, the seeds (including some Pima cotton varieties) are then germinated on a semi-solid medium 14580C in the dark at 28°C for 1-5 days, and the cotyledons that emerge are cut into pieces of 1x 1mm or 2x 2mm.

[0160] Agrobacterium strain LBA4404 THY-TD containing a T-DNA expression construct was used for transformation. The expression construct comprises one or more of the following coding sequences: (a) a single morphogenetic gene (i.e., LEC2, BBM, or RKD4); (b) two morphogenetic genes: BBM1 and LEC2 (SEQ ID NO: 1); (c) two morphogenetic genes: RKD4 and LEC2 (SEQ ID NO: 2); or (d) no morphogenetic gene (control). Each of the aforementioned expression constructs with morphogenetic genes also comprises a heat-inducible CRE recombinase coding sequence and loxp excision sites flanking the BBM1 / LEC2 or RKD4 / LEC2 expression cassette; and all three expression constructs comprise spectinomycin resistance and a visual DS-RED marker conferred by an SPCN sequence. Actively growing Agrobacterium was suspended in 14580AJ liquid medium and adjusted to an OD of 0.3 at 600 nm. The explants were incubated with Agrobacterium suspension in 100 x 25 mm culture dishes for 1 hour. The suspension medium was subsequently removed and the explants were co-cultured for T-DNA transfer. The explants were incubated with the aforementioned 14580AJ (thymidine + AS) medium in a growth chamber under low light (12 μmol m -2s -1 ) and co-cultivated on wet filter paper for 3 days. In some cases, the explants were then transferred to 14580K selection medium containing spectinomycin (spectinomycin resistance is conferred by the SPCN sequence on each expression construct). In other cases, explants of varieties including the Pima cotton variety were transferred to (ii) 14580AM selection medium containing spectinomycin or (iii) 14580P medium (for constructs without SPCN).

[0161] After induction of somatic embryos, explants were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks to induce CRE expression and excision of loxp-flanked morphogenetic genes. Heat shocked somatic embryos were transferred to drying medium 17862 for 7 days and then to germination medium 199M. Germinated somatic embryos were transferred to rooting and extension medium 14582F for regeneration of transgenic plants.

[0162] PCR analysis of transgenic shoots confirmed the excision of the morphogenetic genes and the presence of a single copy of the SPCN transgene and DS-RED (which were located outside of the flanking sites in the initial construct).

[0163] The ability of BBM1 and LEC2 ("BBM1 / LEC2") or RKD4 and LEC2 ("RKD4 / LEC2") to produce somatic embryos was tested in 11 different genotypes: Coker 312 and ten normally resistant elite cotton varieties. The results are shown in Table 3.

[0164] Table 3.

[0165]

[0166] The above results demonstrate the unexpected ability of BBM1 / LEC2 or RKD4 / LEC2 to produce somatic embryos in cotton genotypes that were previously shown to be resistant to somatic embryo induction. The above results also show that BBM1 / LEC2 unexpectedly increases somatic embryo formation in the non-resistant variety Coker 312, improving somatic embryo formation by more than 100%.

[0167] Table 4 shows the percentage of cotton explants that regenerated TO plants in the nine different genotypes tested.

[0168] Table 4.

[0169]

[0170] In addition, cotyledon explants of the Pima 800 cotton (Gossypium barbadense) genotype were transformed as described above. Several hundred T0 events were recovered from somatic embryos formed after heat shock-mediated excision of the morphogenetic genes BBM1 and LEC2. 180 T0 plants transformed with the Agrobacterium strain LBA4404 THY-TD containing the BBM1 / LEC2 expression cassette were randomly selected and qPCR analyzed. 178 of the 180 T0 events were qPCR positive for the transgene, and 60 T0s showed successful removal of the Cre and BBM1 / LEC2 expression cassettes and contained only the gene of interest (GOI) cassette after heat shock treatment. The use of a heat shock induction treatment resulted in an excision frequency of 33%. In the Pima 800 genotype, single copy events of the GOI cassette were generated at a frequency of 25%.

[0171] The foregoing results demonstrate what is believed to be the first reported successful regeneration of transgenic cotton plants from cotton varieties that do not have BBM1 / LEC2 or RKD4 / LEC2 and are resistant to somatic embryo induction and transformation, including the Pima cotton variety. The foregoing also shows consistent and greatly improved transformation efficiency in explants undergoing BBM1 / LEC2-supported embryogenesis.

[0172] Example 2: Fertility of transgenic events in cotton transformed with BBM / LEC2. Transgenic shoots obtained from the transformation of different cotton genotypes in Example 1 were transferred to soil and grown in a greenhouse. After 4-5 months, cotton bolls were produced and seeds were collected. Almost all genotypes that produced events had fertility of 50% or more. The number of events that produced seeds and the percentage of fertile events are shown in Table 5.

[0173] Table 5

[0174] genotype event Events that generate seeds Fertile events% Excellent series 1 43 26 60.5％ Excellent series 3 2 1 50％ Excellent series 4 1 0 0％ Excellent series 5 8 6 75％ Excellent series 6 2 1 50％ Excellent series 7 4 2 50％ Excellent series 8 4 2 50％

[0175] The foregoing results demonstrate what is believed to be the first reported successful regeneration of fertile transgenic cotton plants from most of these cotton varieties. The foregoing also demonstrates the robust efficiency of the disclosed methods in generating fertile transgenic cotton plants.

[0176] Example 3: somatic embryos are produced in alfalfa explants. As mentioned above, the pruning surfaces of greenhouse-grown alfalfa (Medicago sativa) of various genotypes were sterilized. After sterilization, the leaves of the three leaves were cut into at least four. The alfalfa explants cut were incubated with the Agrobacterium strain LBA4404 THY-TD containing the T-DNA expression construct of BBM1 and LEC2 (SEQ ID NO:1), RKD4 and LEC2 (SEQ ID NO:2) or RKD4 and LEC1 (SEQ IDNO:3). Explants were incubated in a culture dish with Agrobacterium suspension for 5 min. The explants were then removed and co-cultivated in 17202B culture medium for 3 days. The explants were transferred to the selection medium 17202I, and the percentage of alfalfa explants that formed somatic embryos from the explants of three different genotypes is shown in Table 6.

[0177] Table 6

[0178]

[0179] The foregoing results demonstrate the unexpected ability of RKD4 and LEC2 ("RKD4 / LEC2") or BBM1 and LEC2 ("BBM1 / LEC2") to produce somatic embryos in alfalfa genotypes that were found to be resistant to somatic embryo induction.

[0180] Example 4: Regeneration of transgenic alfalfa plants from somatic embryos. As described in Example 3, somatic embryos from alfalfa (Medicago sativa) explants were generated by transformation with a T-DNA BBM1 / LEC2 expression construct (SEQ ID NO: 1), a T-DNA with an RKD4 / LEC2 expression construct (SEQ ID NO: 2), or an RKD4 / LEC1 expression construct (SEQ ID NO: 3). In addition to the morphogenetic genes, each expression construct also contained a heat-inducible CRE recombinase coding sequence and loxp excision sites flanking the BBM1 / LEC2, RKD4 / LEC1, or RKD4 / LEC2 expression cassette. After somatic embryo induction, the somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks, to induce CRE expression and excise the morphogenetic gene expression cassette. The heat-shocked somatic embryos were dried for 2 days and then moved to germination medium 17203H for regeneration of transgenic plants. PCR analysis of transgenic shoots confirmed the excision of morphogenetic genes and the presence of single copies of SPCN and DS-RED transgenes.

[0181] Table 7 shows the percentage of alfalfa explants that regenerated TO plants in the three different genotypes.

[0182] Table 7

[0183]

[0184] The foregoing results demonstrate the ability of RKD4 and LEC2 ("RKD4 / LEC2") or BBM1 and LEC2 ("BBM1 / LEC2") to regenerate transgenic plants from somatic embryos in alfalfa genotypes that were found to be resistant to such transformation methods. For example, the variety alfalfa line 1 was previously found to be resistant to transformation. Thus, this represents the first reported successful transformation of such a resistant variety.

[0185] Example 5: Production of somatic embryos in canola explants. Seeds of canola (Brassica napus) genotype 4PYZE50B were surface sterilized as described above. After sterilization, the seeds were washed in sterile water and germinated in 90 medium for 7-10 days. The hypocotyls were cut into 3-4 mm pieces, and the expanded cotyledon explants were cut into 5 x 5 mm pieces. The cut explants were incubated with Agrobacterium strain LBA4404 THY-TD containing T-DNA expression constructs of BBM1 and LEC2 (SEQ ID NO: 1), RKD4 and LEC2 (SEQ ID NO: 2) or no morphogenetic genes (control). In addition to the morphogenetic genes, each expression construct also contained a heat-inducible CRE recombinase coding sequence and loxp excision sites flanking the BBM1 / LEC2 or RKD4 / LEC2 genes. The explants were infected with an Agrobacterium suspension in a culture dish for 10 min. The Agrobacterium suspension was then removed and the explants were grown in 20A medium under low light (12 μmol m -2 s -1 ) and co-cultivated in a growth chamber for 3 days.

[0186] The explants were then transferred to somatic embryo induction medium 70AY. The percentages of canola cotyledon and hypocotyl explants that formed somatic embryos are shown in Table 8.

[0187] Table 8

[0188]

[0189] The foregoing demonstrates the unexpected ability of RKD4 and LEC2 ("RKD4 / LEC2") or BBM1 and LEC2 ("BBM1 / LEC2") to produce somatic embryos in Brassica genotypes and tissue types that were found to be resistant to other methods of somatic embryo induction.

[0190] Example 6: Regeneration of transgenic Brassica plants from somatic embryos. As described in Example 5, somatic embryos from canola (Brassica napus) explants were generated by transformation with Agrobacterium strain LBA4404 THY-TD containing T-DNA expression constructs of BBM1 / LEC2 (SEQ ID NO: 1), RKD4 / LEC2 (SEQ ID NO: 2) or no morphogenetic genes (control). After somatic embryo induction, the somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a day, for 2 days to induce CRE expression and excise morphogenetic genes. The heat-shocked somatic embryos were transferred to 90A medium for regeneration of transgenic plants. The number of somatic embryos and canola explants of regenerated transgenic T0 plants was counted 40-56 days after infection. The percentage of somatic embryos derived from cotyledons or hypocotyls of regenerated plants is shown in Table 9.

[0191] Table 9

[0192]

[0193] The foregoing demonstrates the unexpected ability of BBM1 and LEC2 ("BBM1 / LEC2") to regenerate transgenic plants from hypocotyl-derived somatic embryos in Brassica lines that are otherwise resistant to such transformation methods.

[0194] Example 7: Producing somatic embryos in hypocotyls from different Brassica genotypes. As described in Example 5, Brassica napus somatic embryos were produced from hypocotyls of various genotypes. Explants were transformed with Agrobacterium strain LBA4404THY-TD containing BBM1 / LEC2 expression constructs, heat-inducible CRE genes, DS-RED genes, and SPCN selective marker genes (SEQ ID NO: 1). Somatic embryos were counted 41 days after infection, and the percentages of Brassica explants exhibiting somatic embryos in 3 different genotypes (including Brassica napus elite breeding lines) are shown in Table 10.

[0195] Table 10

[0196]

[0197] The foregoing demonstrates the ability of BBM1 and LEC2 ("BBM1 / LEC2") to efficiently generate somatic embryos in hypocotyl tissue from elite Brassica lines.

[0198] Example 8: Production of somatic embryos in California poppy explants and transgenic plants thereof. Seeds of California poppy (Eschscholzia californica) were surface sterilized as described above. After sterilization, the seeds were washed in sterile water and germinated in medium 90 for 7-10 days.

[0199] The hypocotyls were cut into 3-4 mm pieces, and the expanded cotyledon explants were cut into 5x5 mm pieces. The cut explants were incubated with Agrobacterium strain LBA4404 THY-TD containing T-DNA with an expression construct (SEQ ID NO: 1), which contained BBM1 / LEC2, a heat-inducible CRE gene, a DS-RED gene, and a SPCN selective marker gene. The explants were incubated in a culture dish with an Agrobacterium suspension for 10 minutes. The Agrobacterium suspension was subsequently removed and the explants were grown in 20A medium under low light (12 μmol m -2 s -1 ) in a growth chamber for 2-4 days. The infected explants were transferred to 70AY medium to induce somatic embryos.

[0200] After somatic embryo induction, somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks to induce CRE expression and excise the morphogenetic gene expression cassette. Heat shocked somatic embryos were transferred to regeneration medium and subcultured every 3 weeks until rooted transgenic plants were obtained.

[0201] Somatic embryos were counted 31 days after Agrobacterium infection, and the percentage of poppy explants exhibiting somatic embryos is shown in Table 11. The percentage of explants from which TO transgenic plants were recovered is also shown in Table 11.

[0202] Table 11

[0203]

[0204] The foregoing demonstrates the unexpected efficiency of BBM1 and LEC2 ("BBM1 / LEC2") in generating somatic embryos in opium poppy plant explants; and the ability of the foregoing protocol to generate transgenic opium poppy plants from somatic embryos.

[0205] Example 9: Producing somatic embryos in tomato explants and transgenic plants thereof. As described above, seeds of tomato (Solanum lycopersicum) cultivar Roma tomatoes were surface sterilized. After sterilization, the seeds were washed in sterile water and germinated in half-strength MS medium with 90 basis. Seedlings with 6-10 cm long hypocotyls and expanded cotyledons were divided. The hypocotyls were further cut into 3-4 mm pieces, and the expanded cotyledon explants were cut into 5 x 5 mm pieces. The cut explants were incubated with Agrobacterium strain LBA4404 THY-TD containing a T-DNA expression construct (SEQ ID NO: 1) of BBM1 / LEC2, heat-inducible CRE gene, DS-RED gene, and SPCN selective marker gene. The explants were infected with an Agrobacterium suspension in a culture dish for 10 minutes. The Agrobacterium suspension was subsequently removed and the explants were grown in 20A medium under low light (12 μmol m -2 s -1 ) in a growth chamber for 2-4 days. The explants were then transferred to 70AY medium for somatic embryo induction.

[0206] After somatic embryo induction, somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks to induce CRE expression and excise the morphogenetic gene expression cassette. Heat shocked somatic embryos were transferred to regeneration medium and subcultured every 3 weeks until rooted transgenic plants were obtained.

[0207] The number of explants producing somatic embryos was counted 49 days after Agrobacterium infection, and the percentage of tomato explants exhibiting somatic embryos is shown in Table 12. The percentage of explants from which TO transgenic plants were recovered is also shown in Table 12.

[0208] Table 12

[0209]

[0210] The foregoing demonstrates the ability of BBM1 and LEC2 ("BBM1 / LEC2") to produce somatic embryos in tomato plants and to regenerate transgenic tomato plants from hypocotyl-derived somatic embryos.

[0211] Example 10: Somatic embryos are produced in cucumber explants and transgenic plants thereof. As described above, seeds and leaf surfaces of cucumber (Cucumber) (Cucumis sativus) cultivar Summer Dance were sterilized. After sterilization, the seeds were washed in sterile water and germinated in medium 90. Seedlings with 6-10 cm long hypocotyls and expanded cotyledons were divided. The hypocotyls were further cut into 3-4 mm pieces, and the expanded cotyledon explants and leaves were each cut into 5 x 5 mm pieces. The cut explants were incubated with Agrobacterium strain LBA4404 THY-TD containing T-DNA with BBM1 / LEC2 expression constructs, heat-inducible CRE recombinase coding sequences, and loxp excision sites (SEQ ID NO: 1) flanking the BBM1 / LEC2 gene. The explants were incubated with the Agrobacterium suspension in a petri dish for 10 min, after which the Agrobacterium suspension was removed and the explants were grown in 20A medium under low light (12 μmol m -2 s -1 ) in a growth chamber for 2-4 days. The explants were then transferred to 70AY medium for somatic embryo induction.

[0212] After somatic embryo induction, somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks to induce CRE expression and excise the morphogenetic gene expression cassette. Heat shocked somatic embryos were transferred to regeneration medium and subcultured every 3 weeks until rooted transgenic plants were obtained.

[0213] Somatic embryos were counted 35 days after infection, and the percentage of cucumber explants exhibiting somatic embryos is shown in Table 13. The percentage of explants from which TO transgenic plants were recovered is also shown in Table 13.

[0214] Table 13

[0215]

[0216] The foregoing demonstrates the ability of BBM1 and LEC2 ("BBM1 / LEC2") to generate somatic embryos from cotyledon, hypocotyl, and leaf-derived cucumber tissues with very high efficiency. The foregoing results also demonstrate the ability of the disclosed methods to efficiently regenerate transgenic cucumber plants from these somatic embryos.

[0217] Example 11: Production of somatic embryos in sunflower. Seeds of sunflower (Helianthus annuus) were surface sterilized and germinated on medium 90. Seedlings with 6-10 cm long hypocotyls, expanded cotyledons and leaves were divided into 3-4 mm pieces (hypocotyls) and 5 x 5 mm squares (cotyledons and leaves). The explants were incubated with Agrobacterium strain LBA4404 THY-TD containing T-DNA with BBM1 / LEC2 or RKD4 / LEC2 expression cassettes, heat-inducible CRE recombinase coding sequences and loxp excision sites flanking the BBM1 / LEC2 gene (SEQ ID NO: 1) or RKD4 / LEC2 gene (SEQ ID NO: 2). The explants were incubated with the Agrobacterium suspension in a culture dish for 10 minutes, then the Agrobacterium suspension was removed and the explants were grown in 20A medium under low light (12 μmol m -2 s -1 ) in a growth chamber for 2-4 days. The explants were then transferred to 70AY medium for somatic embryo induction.

[0218] After somatic embryo induction, somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks to induce CRE expression and excise the morphogenetic gene expression cassette. Heat shocked somatic embryos were transferred to regeneration medium and subcultured every 3 weeks until rooted transgenic plants were obtained.

[0219] The foregoing provides a method for producing somatic embryos from cotyledon, hypocotyl or leaf derived sunflower tissues using BBM1 / LEC2 or RKD4 / LEC2. The foregoing also provides regeneration of transgenic sunflower plants from these somatic embryos.

[0220] Example 12: Production of somatic embryos in carrot. Seeds of carrot (Daucus carota) were surface sterilized and germinated on medium 90. Seedlings with 6-10 cm long hypocotyls, expanded cotyledons and leaves were cut into 3-4 mm pieces (hypocotyls) and 5 x 5 mm squares (cotyledons and leaves). The excised explants were incubated with Agrobacterium strain LBA4404THY-TD containing T-DNA with BBM1 / LEC2 or RKD4 / LEC2 expression cassettes, heat-inducible CRE recombinase coding sequences and loxp excision sites flanking the BBM1 / LEC2 gene (SEQ ID NO: 1) or RKD4 / LEC2 gene (SEQ ID NO: 2). The explants were incubated with the Agrobacterium suspension in a petri dish for 10 minutes, then the Agrobacterium suspension was removed and the explants were grown in 20A medium under low light (12 μmol m -2s -1 ) in a growth chamber for 2-4 days. The explants were then transferred to 70AY medium for somatic embryo induction.

[0221] After somatic embryo induction, somatic embryos were subjected to heat treatment at 45°C for 2 hours, once a week for 4 weeks to induce CRE expression and excise the morphogenetic gene expression cassette. Heat shocked somatic embryos were transferred to regeneration medium and subcultured every 3 weeks until rooted transgenic plants were obtained.

[0222] The foregoing provides a method for producing somatic embryos from carrot tissue derived from cotyledons, hypocotyls or leaves using BBM1 / LEC2 or RKD4 / LEC2. The foregoing also provides regeneration of transgenic carrot plants from these somatic embryos.

[0223] Example 13: Production of somatic embryos in cotton using different sources of LEC2 genes. Cotton cotyledon explants were prepared and transformed as described in Example 1 above, with the following changes. Sterilized seeds were germinated on culture medium for 5 days and then cut to expose the cotyledons. The cotyledons were cut into 1-2 mm pieces and infected with an Agrobacterium suspension as described above. The Agrobacterium strain contained a T-DNA having a BBM1 / LEC2 expression cassette, a heat-inducible CRE recombinase coding sequence, and loxp excision sites flanking the BBM1 / LEC2 gene (SEQ ID NO:1), except that the LEC2 gene sequence in the BBM1 / LEC2 gene cassette in SEQ ID NO:1 was replaced with the coding sequence of the LEC2 gene from each source indicated in Table 14. A phylogeny showing the relative positions of different dicot species used as sources of LEC2 sequences is shown in Figure 2 The number of explants producing somatic embryos after 5 weeks was recorded, and the frequency of somatic embryogenesis is given in Table 14.

[0224] Table 14

[0225]

[0226]

[0227] The foregoing demonstrates that LEC2 sequences from a variety of sources can be used in the methods disclosed herein for inducing somatic embryos using BBM1 / LEC2, which methods include using BBM1 / LEC2 to produce somatic embryos with high (70%-90%) to very high (90%-100%) efficiency.

[0228] Example 14: Production of somatic embryos in cotton using different sources of BBM genes. Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that the BBM1 gene in SEQ ID NO: 1 was replaced with the coding sequence of the BBM gene from each source indicated in Table 15. A phylogeny showing the relative positions of different dicot species used as sources of BBM1 sequences is shown in Figure 3 The number of somatic embryos and T0 plants was assessed 4-5 weeks after infection.

[0229] Table 15

[0230]

[0231]

[0232] The foregoing demonstrates the use of BBM1 sequences from a variety of dicotyledonous and monocotyledonous (maize) sources in the methods disclosed herein, which use BBM1 / LEC2 to induce somatic embryos with a range of efficiencies, including relatively low efficiency (0-19%), moderate efficiency (20%-69%), high efficiency (70%-89%), and very high efficiency (90%-100%).

[0233] Example 15: Production of somatic embryos and TO transgenic plants in cotton using different promoters regulating BBM1 or LEC2. Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that LEC2 was regulated by the Arabidopsis thaliana ubiquitin 3 (UBQ3) promoter. Cotton explants were transformed with Agrobacterium strain LBA4404 THY-TD containing T-DNA containing an expression construct, which further contained a UBQ10::BBM-UBQ3::LEC2 expression cassette (SEQ ID NO:65) or a UBQ10::BBM-UBQ14::LEC2 expression cassette (SEQ ID NO:1) in addition to a heat-inducible CRE gene, a DS-RED gene, and a SPCN gene. The frequency of explants producing somatic embryos, mature somatic embryos, mature somatic embryos transferred to root, and regenerated TO plants is given in Table 16. The quality of transgenic plants was assessed by PCR, and T0 produced with the UBQ3 promoter regulating LEC2 generally showed higher quality than T0 produced with the UBQ14 promoter regulating LEC2. The frequency of high quality events with a single copy of the gene and complete excision of the morphogenetic gene is given in Table 17.

[0234] Table 16

[0235]

[0236] Table 17

[0237]

[0238] In other examples, LEC2 is regulated using the soybean (Glycine max) elongation factor TU 2 (GM-EFTU2; SEQ ID NO: 66) or Rubisco activase (GM-RUBACT; SEQ ID NO: 67) promoter. When the GM-EFTU2 promoter regulated LEC2, 96.9% of the explants produced somatic embryos, which was comparable to the UBQ3 promoter (see Table 16). The GM-RUBACT promoter did not produce any explants with somatic embryos. In other examples, one or more of BBM1, LEC2, and CRE are regulated by the auxin-inducible DR5 promoter (SEQ ID NO: 72), the AT-UBI11 promoter (SEQ ID NO: 81), or the CSVMV promoter (SEQ ID NO: 82).

[0239] The foregoing demonstrates that different promoters operably linked to BBM1, LEC2 and CRE can be used in the methods disclosed herein to induce somatic embryos and regenerate transgenic plants very efficiently.

[0240] Example 16: Somatic embryo generation in cotton using CCAAT binding factor 1A activation domain (CBF1A) coupled to BBM or LEC2. Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that each morphogenic gene (BBM1 or LEC2) was coupled to a CCAAT binding factor 1A activation domain (CBF1A) to prepare BBM-CBF1A or LEC2-CBF1A. Cotton explants were transformed with Agrobacterium strain LBA4404 THY-TD containing T-DNA containing an expression construct, which, in addition to the heat-inducible CRE gene, DS-RED gene, and SPCN gene, further contained a BBM-CBF1A / LEC2 expression cassette (SEQ ID NO: 58), a BBM / LEC2-CBF1A (SEQ ID NO: 59) expression cassette, or a BBM-CBF1A / LEC2-CBF1A (SEQ ID NO: 60) expression cassette. The number of explants that produced somatic embryos and subsequently regenerated TO transgenic plants was evaluated. The results are shown in Table 18.

[0241] Table 18

[0242]

[0243] The foregoing examples demonstrate the use of CBF1A-coupled morphogenic genes in the disclosed methods for producing somatic embryos in dicotyledonous plants and / or regenerating transgenic plants from such somatic embryos.

[0244] Example 17: Production of somatic embryos in cotton using an N-terminally truncated BBM with or without coupled CBF1A and using the BBM AP2 domain with or without coupled CBF1A.

[0245] Cotton cotyledon explants were prepared and transformed as described in Example 16 above, except that the BBM1 gene sequence of the expression construct was replaced with a truncated form comprising the BBM AP2 domain and the C-terminus but lacking the N-terminal portion, such that the expression construct comprised either the truncated BBM1 coupled to CBF1A or the truncated BBM1 alone. Somatic embryos were generated from the truncated BBM1 alone or the truncated BBM1 coupled to CBF1A. The number of explants that produced somatic embryos and subsequently regenerated T0 transgenic plants was assessed.

[0246] In a separate experiment, cotton cotyledon explants were prepared and transformed as described in Example 16 above, except that the BBM1 gene sequence of the expression construct was replaced with a truncated form containing the BBM AP2 domain, such that the expression construct contained either the BBM AP2 domain coupled to CBF1A (SEQ ID: 61 containing the BBM1 AP2-CBF1A / LEC2 sequence) or BBM1 AP2 alone (SEQ ID: 61 containing the BBM1 AP / LEC2 gene). Neither BBM1 AP2 alone nor the BBM1 AP2 domain coupled to CBF1A produced somatic embryos.

[0247] The foregoing examples demonstrate the use of CBF1A-coupled truncated morphogenic genes in the disclosed methods for producing somatic embryos in dicotyledonous plants and / or regenerating transgenic plants from such somatic embryos.

[0248] Example 18: Somatic embryos are produced in cotton using a glucocorticoid receptor (GR) coupled to BBM and / or LEC2. Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that each morphogenic gene (BBM1 or LEC2) was coupled to a glucocorticoid receptor (GR; SEQ ID NO: 73) to prepare BBM-GR or LEC2-GR. Cotton explants were transformed with Agrobacterium strain LBA4404 THY-TD containing a T-DNA containing an expression construct, which further contained a BBM-GR / LEC2 expression cassette, a BBM / LEC2-GR expression cassette, or a BBM-GR / LEC2-GR expression cassette in addition to a heat-inducible CRE gene, a DS-RED gene, and a SPCN gene. Morphogenic genes were induced by adding 0.5 mg / L triamcinolone acetonide (TA) to the culture medium. For all of the above constructs, most explants produced somatic embryos after induction with TA. In the absence of TA, somatic embryos were not produced for GR-linked morphogenic genes. For explants transformed with BBM1 / LEC2, somatic embryos were generated regardless of the presence or absence of TA. Somatic embryos were visualized by bright field microscopy and also by DS RED fluorescence as shown in Table 19 (+ and - indicate the presence or absence of red fluorescent somatic embryos, respectively).

[0249] Table 19

[0250]

[0251] The foregoing examples demonstrate the use of GR-coupled morphogenic genes in the disclosed methods for producing somatic embryos in dicotyledonous plants and / or regenerating transgenic plants from such somatic embryos.

[0252] Example 19: Retransformation of transgenic cotton germplasm containing dexamethasone-inducible BBM-GR and LEC2-GR to generate transgenic events with trait genes. Seeds from the TO plants produced in Example 18 were transformed with Agrobacterium strain LBA4404 THY-TD containing T-DNA containing an expression construct with the trait gene as described in Example 13. Explants were placed on medium supplemented with 5 mg / l of dexamethasone to induce somatic embryos and transgenic TO plants with the trait gene. The morphogenetic gene cassette was removed by segregation in subsequent generations, leaving transformed plants with the trait gene.

[0253] The foregoing examples provide a method for retransformation using transgenic germplasm by producing somatic embryos in dicotyledonous plants and / or regenerating transgenic plants from such somatic embryos.

[0254] Example 20: Excision of BBM and LEC2 genes using auxin-inducible CRE to produce somatic embryos in cotton. Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that CRE was regulated by the auxin-inducible promoter DR5 (SEQ ID NO:72). The Agrobacterium strain contained T-DNA with a BBM1 / LEC2 expression cassette, an auxin-inducible DR5-CRE recombinase coding sequence, and loxp excision sites flanking the BBM1 / LEC2 gene. After inducing somatic embryos, auxin was added to the culture medium to induce expression of CRE for excision of the BBM1 and LEC2 gene cassettes. The number of explants that produced somatic embryos and subsequently regenerated T0 transgenic plants was assessed.

[0255] The foregoing examples provide a method for using an auxin-inducible DR5 promoter that regulates CRE expression to excise morphogenic genes in the disclosed methods for producing somatic embryos in dicotyledonous plants and / or regenerating transgenic plants from such somatic embryos.

[0256] Example 21: Somatic embryo production in cotton using FUSCA3 (FUS3). Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that the LEC2 gene sequence of the expression construct was replaced with the Arabidopsis thaliana FUSCA3 gene (DNA sequence SEQ ID NO:56 encoding the protein of SEQ ID NO:57), the Arabidopsis thaliana ABI3 gene (SEQ ID NO:63; encoding the protein of SEQ ID NO:64), or the Arabidopsis thaliana LEC1 gene (SEQ ID NO:10; encoding the protein of SEQ ID NO:11). The number of explants that produced somatic embryos and subsequently regenerated TO transgenic plants was evaluated. The results are shown in Table 20.

[0257] Table 20

[0258] Construct % of explants with somatic embryos BBM1+AT-FUS3 7.4％ BBM1+AT-ABI3 0％ BBM1+AT-LEC1 0％

[0259] The foregoing examples provide a method for using the FUSCA3 morphogenetic gene in the disclosed method for producing somatic embryos in dicotyledonous plants to produce somatic embryos at a lower frequency relative to LEC2. When LEC2 was replaced with AT-ABI3 or AT-LEC1, no explants formed somatic embryos.

[0260] Example 22: Production of transgenic plants in cotton using a glucocorticoid receptor (GR) coupled to CRE for excision of morphogenetic genes. Cotton cotyledon explants were prepared and transformed as described in Example 13 above, except that the CRE gene sequence of the expression construct was coupled to the glucocorticoid receptor (GR; SEQ ID NO: 73) to prepare a BBM1+LEC2+constitutive promoter::CRE-GR construct, wherein BBM / LEC2 is flanked by loxP sites. Transformed cotton explants on which somatic embryos are formed are transferred to a medium containing TA (0.5 mg / L) to induce CRE-GR and promote excision of BBM1 and LEC2. The number of regenerated T0 plants is evaluated.

[0261] The foregoing examples provide a method for using a glucocorticoid-inducible constitutive promoter that regulates CRE expression to excise morphogenic genes in the disclosed methods for producing somatic embryos in dicotyledonous plants and / or regenerating transgenic plants from such somatic embryos.

[0262] Example 23: Automated explant preparation. Cotyledons from 3-15 day old cotton seeds were cut and transferred to an Agrobacterium suspension in the bowl of a Cuisinart Mini Blender (Model DLC-1SS) food processor. In other examples, hypocotyl tissue from canola or tomato or California poppy or cucumber, or leaf tissue from alfalfa or cucumber, or cotyledon tissue from canola, California poppy, tomato or cucumber was used instead of cotton cotyledons as explants.

[0263] The Cuisinart mini blender food processor is then pulse driven until the average explant length is approximately 2-3mm. The explant is then taken out and contacted with the Agrobacterium suspension for 10 minutes to infect, or the Agrobacterium suspension is added to a bowl to infect for 10 minutes. The Agrobacterium suspension and explant are then poured through a 0.4 μm nylon net to collect the explant at the top. The nylon net is then inverted and repeatedly patted to move the tissue to the filter paper placed on the solid co-cultivation medium, for 3 days. The explant is then moved to a suitable culture medium to induce somatic embryos. Explants are expected to produce somatic embryos after 4-5 weeks, and produce T0 plants.

[0264] In a variation of the aforementioned method, after infection with the Agrobacterium suspension, the explants are removed from a Cuisinart mini-blender food processor and evenly distributed on filter paper, which is then placed on the co-cultivation medium. Subsequently, in each subculture step, the filter paper supporting the explants is picked up with tweezers and moved to fresh culture medium. The batch transfer of the explants on filter paper causes the rapid formation of somatic embryos.

[0265] In additional variations, explants are prepared for transformation using various food processors and automated or semi-automated grinding devices, including the use of automated or semi-automated chopping and / or grinding devices.

Claims

1. A method for producing dicotyledonous somatic embryos, the method comprising: expressing a first heterologous nucleotide sequence encoding a BBM or RWP-RK domain (RKD) polypeptide in a dicot explant; expressing a second heterologous nucleotide sequence encoding a Leafy Cotyledon (LEC) polypeptide or a FUSCA3 (FUS3) polypeptide in the dicot explant; and Somatic embryogenesis is induced in the explants.

2. The method of claim 1, wherein the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide.

3. The method of claim 1, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide.

4. The method of claim 1, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide.

5. The method of claim 1, wherein the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a FUS3 polypeptide.

6. The method of claim 1, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a FUS3 polypeptide.

7. The method of any one of claims 1 to 6, wherein the explant is from a eudicot.

8. The method of any one of claims 1-6, wherein the explant is from the orders Ranunculales, Fabales, Cucurbitales, Malvales, Brassicales, Solanales, Asterales, or Apiales.

9. The method of any one of claims 1 to 6, wherein the dicot explant is from a resistant dicot variety.

10. The method of any one of claims 1-9, wherein the explant tissue is a hypocotyl, epicotyl, cotyledon, leaf, stem or root.

11. A method for producing a transgenic dicotyledonous plant, the method comprising: transforming a dicot explant with a first heterologous nucleotide sequence encoding a BBM polypeptide or a RWP-RK domain (RKD) polypeptide and a second heterologous nucleotide sequence encoding a LEC polypeptide or a FUSCA3 (FUS3) polypeptide; transforming the explant with a transgene; inducing somatic embryogenesis in the explants to produce transgenic somatic embryos; as well as The transgenic somatic embryonic tissue is cultured under germination, rooting and sprouting conditions to form a transgenic dicotyledonous plant comprising the transgene.

12. The method of claim 11, wherein the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide encodes a LEC2 polypeptide.

13. The method of claim 11, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide.

14. The method of claim 11, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide.

15. The method of claim 11, wherein the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a FUS3 polypeptide.

16. The method of claim 11, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a FUS3 polypeptide.

17. The method of any one of claims 11 to 16, wherein the first heterologous nucleotide sequence and the second heterologous nucleotide sequence are flanked by recognition sites for a site-specific recombinase.

18. The method of claim 17, further comprising providing the site-specific recombinase to the explant, and excising the first heterologous nucleotide sequence and the second heterologous nucleotide sequence after somatic embryo induction.

19. The method of any one of claims 11 to 18, wherein the dicot explant is from a resistant variety.

20. The method of any one of claims 11-18, wherein the explant is a hypocotyl, epicotyl, cotyledon, leaf, stem or root.

21. The method of any one of claims 11-18, wherein the transformation is an Agrobacterium-mediated transformation of the explant.

22. The method of any one of claims 11-18, wherein the dicot explant is an explant from cotton, alfalfa, Brassica napus, poppy, tomato, cucumber, sunflower or carrot.

23. An expression construct comprising: (a) a first promoter operably linked to a first heterologous nucleotide sequence encoding a BBM or RWP-RK domain (RKD) polypeptide; and (b) a second promoter operably linked to a second heterologous nucleotide sequence encoding a Leafy Leaf (LEC) polypeptide or a FUSCA3 (FUS3) polypeptide.

24. The expression construct of claim 23, wherein the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide.

25. The expression construct of claim 23, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC1 polypeptide.

26. The expression construct of claim 23, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a LEC2 polypeptide.

27. The expression construct of claim 23, wherein the first heterologous nucleotide sequence encodes a BBM polypeptide and the second heterologous nucleotide sequence encodes a FUS3 polypeptide.

28. The expression construct of claim 23, wherein the first heterologous nucleotide sequence encodes a RKD4 polypeptide and the second heterologous nucleotide sequence encodes a FUS3 polypeptide.

29. An expression construct as described in any one of claims 23-28, wherein the first promoter and / or the second promoter is an inducible promoter, a developmentally regulated promoter or a constitutive promoter.

30. The expression construct of any one of claims 23-29, wherein the first heterologous nucleotide sequence and the second heterologous nucleotide sequence are flanked by recognition sites for a site-specific recombinase.

31. The expression construct of claim 30, further comprising a nucleotide sequence encoding the site-specific recombinase.

32. An expression construct as claimed in claim 31, wherein the nucleotide sequence encoding the site-specific recombinase is operably linked to a third promoter selected from an inducible promoter, a developmentally regulated promoter or a constitutive promoter.

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