Helper constructs that enable rapid regeneration in cotton and methods of using the same

Abstract

This invention relates to polynucleotides comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation and expression cassettes, vectors, and compositions comprising the same for the purpose of enabling rapid somatic regeneration in a nonembryonic cell, as well as methods of making and methods of using the same.

Description

[0001] Attorney Docket No. 9662.81.WO

[0002] HELPER CONSTRUCTS THAT ENABLE RAPID REGENERATION IN COTTON AND METHODS OF USING THE SAME

[0003] STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0004] A Sequence Listing in XML format, entitled 9662-81WO ST26. xml, 29,672 bytes in size, generated on October 28, 2025 and filed herewith, is hereby incorporated by reference into the specification for its disclosures.

[0005] STATEMENT OF PRIORITY

[0006] This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 63 / 715,898 filed on November 4, 2024, the entire contents of which are incorporated by reference herein.

[0007] FIELD OF THE INVENTION

[0008] This invention relates to polynucleotides comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation and expression cassettes, vectors, and compositions comprising the same for the purpose of enabling rapid somatic regeneration in a nonembryonic cell, as well as methods of making and methods of using the same.

[0009] BACKGROUND OF THE INVENTION

[0010] Constrains in plant genetic transformation is a primary bottleneck in deploying precious trait genetics broadly for the improvement of cropping systems. Current techniques for somatic regeneration are slow, laborious, tedious, and often have a low frequency of results.

[0011] The present invention overcomes shortcomings in the art by providing polynucleotides, expression cassettes, vectors, compositions and transformed plants and plant parts for expedited somatic regeneration in crop plants such as cotton.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is based, in part, on the development of polynucleotides comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation and expression cassettes, vectors, and compositions comprising the same for the purpose of enabling rapid somatic regeneration in a nonembryonic cell.

[0014] Thus, one aspect of the invention relates to a polynucleotide comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation. Attorney Docket No. 9662.81.WO

[0015] A further aspect of the invention relates to an expression cassette comprising a polynucleotide comprising a LEC1 A open reading frame in antisense orientation.

[0016] A further aspect of the invention relates to a vector comprising a polynucleotide and / or an expression cassette of the present invention.

[0017] A further aspect of the invention relates to a transformed plant cell comprising a polynucleotide, expression cassette, and / or vector of the present invention.

[0018] A further aspect of the invention relates to a transgenic plant produced by (e.g., grown from) a transformed plant cell of the present invention.

[0019] A further aspect of the invention relates to a transformed plant or plant part comprising a polynucleotide, expression cassette, vector, and / or transformed plant cell of the invention.

[0020] A further aspect of the invention relates to a plant seed comprising a polynucleotide, expression cassette, vector, and / or transformed plant cell of the invention.

[0021] A further aspect of the invention relates to a transgenic plant produced by (e.g., grown from) a seed (e.g., a transformed plant seed) of the present invention.

[0022] A further aspect of the invention relates to a composition comprising a polynucleotide, expression cassette, vector, transformed plant cell, or transformed plant or plant part (e.g., transformed seed) of the present invention, in an agronomically acceptable carrier.

[0023] A further aspect of the invention relates to a method of expressing an antisense LEC1 A open reading frame in a plant cell, comprising: (a) contacting the plant cell with a polynucleotide, expression cassette, vector, and / or composition of the present invention, wherein the polynucleotide incorporates into the plant cell, and (b) contacting the plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame; thereby expressing the antisense LEC1 A open reading frame in the plant cell.

[0024] A further aspect of the invention relates to a method of reprogramming a nonembryonic plant cell for somatic regeneration, comprising: (a) contacting the nonembryonic plant cell with t a polynucleotide, expression cassette, vector, and / or composition of the present invention, wherein the polynucleotide incorporates into the nonembryonic plant cell, and (b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; thereby reprogramming the nonembryonic plant cell for somatic regeneration.

[0025] A further aspect of the invention relates to a method of inducing somatic embryogenesis in a nonembryonic plant cell, comprising: (a) contacting the nonembryonic plant cell with a Attorney Docket No. 9662.81.WO polynucleotide, expression cassette, vector, and / or composition of the present invention, wherein the polynucleotide incorporates into the nonembryonic plant cell, and (b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; thereby inducing somatic embryogenesis in the nonembryonic plant cell.

[0026] A further aspect of the invention relates to a method of producing a plant from a nonembryonic plant cell, comprising: (a) contacting the nonembryonic plant cell with a polynucleotide, expression cassette, vector, and / or composition of the present invention, (b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; and (c) growing the somatically regenerating plant cell of (b) to produce an adult plant; thereby producing a plant from the nonembryonic plant cell.

[0027] A further aspect of the invention relates to a method of producing a plant capable of somatic regeneration from a nonembryonic plant cell thereof, comprising: (a) culturing a seed comprising a polynucleotide of the present invention stably expressed in the seed plant genome (e.g., a transformed plant seed of the present invention) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell(s) of the seed gain functional capability for somatic regeneration; and (b) growing the somatically regenerating plant cell(s) to produce an adult plant; thereby producing a plant capable of somatic regeneration from a nonembryonic plant cell thereof.

[0028] It is noted that aspects of the invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination. Applicant reserves the right to change any originally filed claim and / or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and / or incorporate any feature of any other claim or claims although not originally claimed in that manner. These and other objects and / or aspects of the present invention are explained in detail in the specification set forth below. Further features, advantages and details of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments that follow, such description being merely illustrative of the present invention. Attorney Docket No. 9662.81.WO

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows a linear representation of the LEC1 A 'helper-gene' in anti-sense orientation driven by the LEXA (P-estradiol-inducible promoter) with the 5 ’untranslated region (UTR) and the NOS terminator.

[0031] FIG. 2 shows a circular representation of the 'helper T-DNA' with selectable markers hygromycin (HYG) and BAR.

[0032] FIG. 3 shows representative images of developmental stages from explant preparation to differentiated whole-plants with the 'helper system'.

[0033] DETAILED DESCRIPTION

[0034] The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0036] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or Attorney Docket No. 9662.81.WO combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0037] As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0038] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0039] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0040] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0041] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

[0042] The term "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0043] As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed Attorney Docket No. 9662.81.WO invention. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

[0044] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0045] The term "consists essentially of (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and / or 3’ or N-terminal and / or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together. The term "materially altered," as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term "materially altered," as applied to polypeptides of the invention, refers to an increase or decrease in biological activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0046] The term "open reading frame" or "ORF" as used herein, refers to the portion of a polynucleotide, e.g., a gene, that encodes a polypeptide.

[0047] The term "codon-optimized," as used herein, refers to a gene coding sequence that has been optimized to increase expression by substituting one or more codons normally present in a coding sequence (for example, in a wild-type sequence, including, e.g., a coding sequence for FIG4) with a codon for the same (synonymous) amino acid. In this manner, the protein encoded by the gene is identical, but the underlying nucleobase sequence of the gene or corresponding mRNA is different. In some embodiments, the optimization substitutes one or more rare codons (that is, codons for tRNA that occur relatively infrequently in cells from a particular species) with synonymous codons that occur more frequently to improve the efficiency of translation. For example, in human codon-optimization one or more codons in a coding sequence are replaced by codons that occur more frequently in human cells for the same amino acid. Codon optimization can also increase gene expression through other mechanisms Attorney Docket No. 9662.81.WO that can improve efficiency of transcription and / or translation. Strategies include, without limitation, increasing total GC content (that is, the percent of guanines and cytosines in the entire coding sequence), decreasing CpG content (that is, the number of CG or GC dinucleotides in the coding sequence), removing cryptic splice donor or acceptor sites, and / or adding or removing ribosomal entry sites, such as Kozak sequences. Desirably, a codon- optimized gene exhibits improved protein expression, for example, the protein encoded thereby is expressed at a detectably greater level in a cell compared with the level of expression of the protein provided by the wild-type gene in an otherwise similar cell.

[0048] The term "sequence identity," as used herein, has its standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 45:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 55:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12 :387 (1984), preferably using the default settings, or by inspection.

[0049] A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-BLAST-2 to maximize the alignment score are ignored).

[0050] In a similar manner, percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.

[0051] The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences that contain either more or fewer nucleotides than the polynucleotides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of Attorney Docket No. 9662.81.WO nucleotides in the shorter sequence, in one embodiment. In percent identity calculations, relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[0052] In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.

[0053] As used herein, the term "nucleic acid" encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid may be double-stranded or single-stranded. The nucleic acid may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

[0054] The terms "nucleic acid segment," "nucleotide sequence," "nucleic acid molecule," or more generally "segment" will be understood by those in the art as a functional term that includes both genomic DNA sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, small regulatory RNAs, operon sequences and smaller engineered nucleotide sequences that express or may be adapted to express, proteins, polypeptides or peptides. The term "nucleic acid" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The "nucleic acid" may also optionally contain non-naturally occurring or modified nucleotide bases. The term "nucleotide sequence" or "nucleic acid sequence" refers to both the sense and antisense strands of a nucleic acid, either as individual single strands or in the duplex. The term "ribonucleic acid" (RNA) is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), shRNA (short / small hairpin RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), long non-coding RNA (IncRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and cRNA (complementary RNA), and the term "deoxyribonucleic acid" (DNA) is inclusive of cDNA and genomic DNA and DNA-RNA hybrids. Nucleic acids of the present disclosure may also be synthesized, either completely or in part, by methods known in the art. Thus, all or a portion of the nucleic acids of the present codons may be synthesized using codons preferred by a selected Attorney Docket No. 9662.81.WO host. Species-preferred codons may be determined, for example, from the codons used most frequently in the proteins expressed in a particular host species. Other modifications of the nucleotide sequences may result in mutants having slightly altered activity.

[0055] MicroRNAs are a class of noncoding small RNAs that originate from primary miRNA (pri-miRNA) transcripts that are encoded by miRNA genes. The pri-miRNA transcripts are processed into smaller 19-24 nucleotide RNAs, which can regulate gene expression, for example, through silencing reactions mediated by translational inhibition or cleavage.

[0056] As used herein, the term "polypeptide" encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise. The terms "polypeptide," "peptide" and "protein" may be used interchangeably to refer to polymers of amino acids of any length. The terms "nucleic acid," "nucleic acid sequence," and "polynucleotide" may be used interchangeably to refer to polymers of nucleotides of any length. As used herein, the terms "nucleotide sequence," "polynucleotide," "nucleic acid sequence," "nucleic acid molecule" and "nucleic acid fragment" refer to a polymer of RNA, DNA, or RNA and DNA that is single- or double-stranded, optionally containing synthetic, non-natural and / or altered nucleotide bases.

[0057] As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and 5’ and 3’ untranslated regions). A gene may be "isolated" by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in chemically synthesizing the nucleic acid.

[0058] As used herein, the terms "gene of interest," "nucleic acid of interest" and / or "protein of interest" refer to that gene / nucleic acid / protein desired under specific contextual conditions.

[0059] As used herein, "complementary" polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. For example, the sequence " A-G-T" binds to the complementary sequence "T-C-A." It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other. Attorney Docket No. 9662.81.WO

[0060] The terms "complementary" or "complementarity," as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be "partial," in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0061] As used herein, the terms "substantially complementary" or "partially complementary" mean that two nucleic acid sequences are complementary at least about 50%, 60%, 70%, 80% or 90% of their nucleotides. In some embodiments, the two nucleic acid sequences can be complementary at least at 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides. The terms "substantially complementary" and "partially complementary" can also mean that two nucleic acid sequences can hybridize under high stringency conditions and such conditions are well known in the art.

[0062] The term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. The region in a nucleic acid sequence or polynucleotide in which one or more regulatory elements are found is referred to as a "regulatory region."

[0063] The term coding region as used herein, refers to the portion of a polynucleotide, e.g., a gene, that encodes a polypeptide.

[0064] As used herein with respect to nucleic acids, the term "operably linked" refers to a functional linkage between two or more nucleic acids. For example, a promoter sequence may be described as being "operably linked" to a heterologous nucleic acid sequence because the promoter sequence initiates and / or mediates transcription of the heterologous nucleic acid sequence. In some embodiments, the operably linked nucleic acid sequences are contiguous and / or are in the same reading frame.

[0065] As used herein, the term "binding site" refers to any general structural feature that acts as a location for binding between components. As applied to nucleic acids or polynucleotides, the term "binding site" can refer to, though is not limited to, a nucleotide sequence in a specific motif of primary, secondary, or tertiary structure wherein that motif provides a binding location for an interacting molecule, which may comprise other nucleic acids or proteins. As applied to peptides, polypeptides, or proteins, the term "binding site" can refer to, though is not limited to, a sequence of amino acids in a specific motif of primary, secondary, tertiary or quaternary Attorney Docket No. 9662.81.WO structure wherein that motif provides a binding location for an interacting molecule, which may comprise other nucleic acids or proteins.

[0066] A "recombinant" nucleic acid, polynucleotide or nucleotide sequence is one produced by genetic engineering techniques.

[0067] A "recombinant" polypeptide is produced from a recombinant nucleic acid, polypeptide or nucleotide sequence.

[0068] As used herein, an "isolated" polynucleotide (e.g., an "isolated nucleic acid" or an "isolated nucleotide sequence") means a polynucleotide at least partially separated from at least some of the other components of a source material from which the polynucleotide is isolated, including but not limited to an in vitro mixture, or a naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide. Optionally, but not necessarily, the "isolated" polynucleotide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twentyfold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

[0069] An "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of a source material from which the polypeptide is isolated, including but not limited to an in vitro mixture, or the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide. Optionally, but not necessarily, the "isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

[0070] Furthermore, an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature. For example, an isolated cell can be a cell in culture medium and / or a cell in a pharmaceutically acceptable carrier.

[0071] The term "endogenous" refers to a component naturally found in an environment, i.e., a gene, nucleic acid, miRNA, protein, cell, or other natural component expressed in the subject (e.g., a plant cell, plant, or plant part), as distinguished from an introduced component, i.e., an "exogenous" component. Attorney Docket No. 9662.81.WO

[0072] As used herein, the term "heterologous" refers to a nucleotide / polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0073] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein and refer to a nucleic acid molecule and / or nucleotide sequence that is not naturally occurring in the virus. Generally, the heterologous nucleic acid may comprise an open reading frame that encodes a protein, protein fragment, peptide or nontranslated RNA of interest (e.g., for delivery to a plant, plant part and / or plant cell). In some embodiments, a heterologous nucleic acid of the invention may comprise and / or encode a synthetic product of interest such as but not limited to a gene or fragment thereof, a protein or fragment thereof, a DNA and / or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), or any combination thereof).

[0074] As used herein with respect to nucleic acids, the term "fragment" refers to a nucleic acid that is reduced in length relative to a reference nucleic acid and that comprises, consists essentially of and / or consists of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference nucleic acid. Such a nucleic acid fragment may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, the nucleic acid fragment comprises, consists essentially of or consists of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive nucleotides. In some embodiments, the nucleic acid fragment comprises, consists essentially of or consists of less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive nucleotides.

[0075] As used herein with respect to polypeptides, the term "fragment" refers to a polypeptide that is reduced in length relative to a reference polypeptide and that comprises, consists essentially of and / or consists of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference polypeptide. Such a polypeptide fragment may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, the polypeptide fragment comprises, consists essentially of or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive Attorney Docket No. 9662.81.WO amino acids. In some embodiments, the polypeptide fragment comprises, consists essentially of or consists of less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive amino acids.

[0076] As used herein with respect to nucleic acids, the term "functional fragment" or "active fragment" refers to nucleic acid that encodes a functional fragment of a polypeptide.

[0077] As used herein with respect to polypeptides, the term "functional fragment" or "active fragment" refers to polypeptide fragment that retains at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of at least one biological activity of the full-length polypeptide (e.g., the ability to up- or down-regulate gene expression). In some embodiments, the functional fragment actually has a higher level of at least one biological activity of the full-length polypeptide.

[0078] As used herein, the term "modified," as applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from a source sequence due to one or more deletions, additions, substitutions, or any combination thereof. Modified sequences may also be referred to as "modified variant(s)."

[0079] A "vector" or "construct" as used herein refers to a compound used as a vehicle to carry foreign genetic material into another cell, where it can be replicated and / or expressed. A vector or construct containing foreign nucleic acid may be termed a recombinant vector or recombinant construct (e.g., expression construct). Examples of nucleic acid vectors or nucleic acid constructs are plasmids, viral vectors, cosmids, expression cassettes, and artificial chromosomes. In the current state of the art, recombinant vectors typically contain an origin of replication, a multicloning site, and a selectable marker. The nucleic acid sequence typically consists of an insert (recombinant nucleic acid or transgene) and a larger sequence that serves as the "backbone" of the construct. The purpose of a construct which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Expression vectors (expression constructs or expression cassettes) are for the expression of the exogenous gene and / or gene product in the target cell, and generally have a promoter sequence that drives expression of the exogenous gene and / or gene product. Insertion of a vector or construct into the target cell is referred to transformation or transfection for bacterial and eukaryotic cells, although insertion of a viral vector is often called transduction. The term "vector" or "construct" may also be used in general to describe items to that serve to carry foreign genetic material into another cell, such as, but not limited to, a transformed cell or a nanoparticle. Attorney Docket No. 9662.81.WO

[0080] As used herein, by "isolate" or "purify" (or grammatical equivalents) a vector or construct, it is meant that the vector or construct is at least partially separated from at least some of the other components in the starting material.

[0081] The terms "enhance" and "increase" refer to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold.

[0082] The terms "inhibit" and "reduce" or grammatical variations thereof as used herein refer to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0083] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts may be referred to as "transcription products" and encoded polypeptides may be referred to as "translation products." Transcripts and encoded polypeptides may be collectively referred to as "gene products. " If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression product itself, e.g., the resulting nucleic acid or protein, may also be said to be "expressed." An expression product can be characterized as intracellular, extracellular or secreted. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside a cell. A substance is "secreted" by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.

[0084] As used herein, the terms "transgenic" and / or "transgene" refer to a nucleic acid sequence containing a functional coding region for a gene that comprises one or more exogenous nucleic acids. The exogenous nucleic acid can be stably integrated within the genome such that the polynucleotide is passed on in successive cell divisions. The exogenous nucleic acid can be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" may be used to designate any substrate the genotype of which has been altered by the presence of an exogenous nucleic acid.

[0085] By "pharmaceutically acceptable" it is meant a material that is not toxic or otherwise undesirable, / .< ., the material may be administered to a subject without causing any undesirable biological effects. Attorney Docket No. 9662.81.WO

[0086] By "agronomically acceptable" it is meant a material that is not toxic or otherwise undesirable in an agricultural setting, z.e., the material may be administered to a subject (e.g., a plant or plant part, e.g., a crop plant or plant part) without causing any undesirable biological effects in the plant or plant part.

[0087] The terms "administering" and "administration" of a synthetic gene, expression cassette, vector, plasmid, transformed cell, or composition to a plant, plant part or plant cell include any route of introducing or delivering to a plant, plant part or plant cell a compound to perform its intended function. Administration can be carried out by any suitable route such as known in the art.

[0088] As used herein, the terms "contacting," "introducing" and "administering" are used interchangeably, and refer to a process by which a nucleotide and / or polypeptide of the present invention or a nucleic acid molecule encoding a polypeptide of this invention is delivered to a cell, in order to inhibit or alter or modify expression of a target gene. The nucleotide and / or polypeptide may be administered in a number of ways, including, but not limited to, direct introduction into a cell (z.e., intracellularly) and / or extracellular introduction into a cavity, interstitial space, or into the circulation of the plant, plant part thereof or plant cell.

[0089] "Introducing" in the context of a plant, plant part thereof or plant cell means presenting the nucleic acid molecule to the plant, plant part thereof or plant cell in such a manner that the nucleic acid molecule gains access to the interior of a cell. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into cells in a single transformation event or in separate transformation events. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient.

[0090] "Transient transformation" or "transient transfection" in the context of a polynucleotide means that a polynucleotide is introduced into a cell and does not integrate into the genome of the cell.

[0091] "Stable transformation" or "stable transfection" as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. "Genome" as used herein includes the nuclear and mitochondrial genome, and therefore includes integration of the nucleic acid into, for example, the mitochondrial genome. Stable transformation or stable Attorney Docket No. 9662.81.WO transfection as used herein can also refer to a transgene that is maintained extra- chromasomally, for example, as a minichromosome.

[0092] Transient transformation or transient transfection may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation or stable transfection of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation or stable transfection of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0093] Embodiments of the invention are directed to expression cassettes designed to express the nucleic acids of the present invention. As used herein, "expression cassette" means a nucleic acid molecule having at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable interaction with the nucleotide sequences for the nucleotide and / or polypeptide of the invention are provided in expression cassettes for expression in a plant, plant part thereof or plant cell.

[0094] As used herein, the term "promoter" refers to a region of a nucleotide sequence that incorporates the necessary signals for the efficient expression of a coding sequence. This may include sequences to which an RNA polymerase binds, but is not limited to such sequences and can include regions to which other regulatory proteins bind together with regions involved in the control of protein translation and can also include coding sequences.

[0095] Furthermore, a "promoter" of this invention is a promoter capable of initiating transcription in a plant, plant part thereof or plant cell. Such promoters include those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner, as these various types of promoters are known in the art.

[0096] For purposes of the invention, the regulatory regions ( / .< ., promoters, transcriptional regulatory regions, and translational termination regions) can be native / analogous to the plant Attorney Docket No. 9662.81.WO or cell and / or the regulatory regions can be native / analogous to the other regulatory regions. Alternatively, the regulatory regions may be heterologous to the plant or cell and / or to each other (z.e., the regulatory regions). Thus, for example, a promoter can be heterologous when it is operably linked to a polynucleotide from a species different from the species from which the polynucleotide was derived. Alternatively, a promoter can also be heterologous to a selected nucleotide sequence if the promoter is from the same / analogous species from which the polynucleotide is derived, but one or both (z.e., promoter and polynucleotide) are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0097] In addition to the promoters described above, the expression cassette also can include other regulatory sequences. As used herein, "regulatory sequences" means nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences and polyadenylation signal sequences.

[0098] The expression cassette also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part thereof or plant cell. As used herein, "selectable marker" means a nucleic acid that when expressed imparts a distinct phenotype to the plant, plant part thereof or plant cell expressing the marker and thus allows such a transformed plant, plant part thereof or plant cell to be distinguished from those that do not have the marker. Such a nucleic acid may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0099] As used herein, "antisense orientation" or "antisense configuration" refers to the position wherein the open reading frame (ORF) is inverted (e.g., 3' to 5') for transcription and translation as compared to one or more other components of a construct (e.g., 5' to 3'). While not wishing to be bound to theory, the inverted configuration of the ORF may lead to the transcription of an antisense transcript of the ORF, otherwise referred to as "antisense RNA" or "antisense transcript", a single stranded RNA that is complementary to a protein coding messenger RNA (mRNA) with which it can hybridize. The term "antisense oligonucleotide" Attorney Docket No. 9662.81.WO

[0100] (including "antisense RNA") as used herein, refers to a nucleic acid that is complementary to and specifically hybridizes to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids that encode the same can be made in accordance with conventional techniques. See, e.g., U.S. Patent No. 5,023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al.

[0101] Those skilled in the art will appreciate that it may not be necessary that the antisense oligonucleotide be fully complementary to a target sequence as long as the degree of sequence similarity is sufficient for the antisense nucleotide sequence to specifically hybridize to its target and reduce production of a protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0102] To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be carried out under conditions of reduced stringency, medium stringency or even stringent conditions. Suitable conditions for achieving reduced, medium and stringent hybridization conditions are as described herein.

[0103] Alternatively stated, in particular embodiments, antisense oligonucleotides of the invention may have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity with the complement of a target sequence and reduce production of a protein product. In some embodiments, an antisense sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with a target sequence. Methods of determining percent identity of nucleic acid sequences are described in more detail elsewhere herein.

[0104] The length of an antisense oligonucleotide may not be critical as long as it specifically hybridizes to the intended target and reduces production of the protein product and can be determined in accordance with routine procedures. In general, an antisense oligonucleotide is at least about eight, ten or twelve or fifteen nucleotides in length and / or less than about 20, 30, 40, 50, 60, 70, 80, 100 or 150 nucleotides in length.

[0105] "Plant tissue" as used herein means a group of plant cells organized into a structural and / or functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, explants, tissue culture and any group of plant cells assembled together to provide a structural and / or functional unit. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0106] An "explant" as used herein refers to a plant tissue that is used as starting material for a tissue culture. In some embodiments, an explant comprises a wounded plant tissue. In some Attorney Docket No. 9662.81.WO embodiments, an explant comprises a plant shoot or portion thereof, a plant root or portion thereof, a plant leaf or a portion thereof, a plant meristem tissue or a portion thereof, etc.

[0107] As used herein, "modified" in reference to a plant, plant part, shoot, root, tissue, or cell (e.g., a modified plant, plant part, shoot, root, tissue, or cell) refers to a plant, plant part, shoot, root, tissue, or cell, respectively, that has undergone a physical (e.g., genetic) change compared to the plant, plant part, shoot, root, tissue, or cell, respectively, at a prior time point (e.g., prior to contact with a polynucleotide, expression cassette, vector, and / or composition of the present invention and / or prior to a method of the present invention). In some embodiments, "modified" in reference to a plant, plant part, shoot, root, tissue, or cell (e.g., a modified plant, plant part, shoot, root, tissue, or cell) refers to a plant, plant part, shoot, root, tissue, or cell, respectively, that has been subjected to genome editing, genetic transformation (e.g., introduction of a transgene), or a combination thereof. Accordingly, in some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transgenic cell and / or an edited cell (i.e., a target nucleic acid of the cells has been modified). In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transgenic, non-edited cell and / or a transgenic, edited cell. In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a non-transgenic, edited cell. In some embodiments, a modified plant or plant part, shoot, root, tissue, or cell comprises a transformed, edited cell.

[0108] An "edited cell," "edited plant," "edited plant part," "edited root," "edited tissue," "edited plantlet," and / or the like as used herein refer to a cell, plant, plant part, root, tissue, plantlet, and / or the like, respectively, that comprises a modified nucleic acid in that a target nucleic acid has been modified using an editing system as described herein to provide the modified nucleic acid. Thus, an "edited cell," "edited plant," "edited plant part," "edited root," "edited tissue," "edited plantlet," and / or the like comprise a nucleic acid that has been modified and / or changed compared to its unmodified or native sequence and / or structure (i.e., a modified nucleic acid). The term "non-edited" refers to a condition in which a nucleic acid in the genome of a host cell or tissue or organism of interest has not been modified using an editing system as described herein.

[0109] As used herein, the term "agonist" refers to a compound that in combination with a binding site can produce a cellular response. An agonist may be a ligand that directly binds to the binding site on a target (e.g., on / in a promoter, e.g., on / in a receptor). Alternatively an agonist may combine with a target comprising a binding site on indirectly by for example (a) forming a complex with another molecule that directly binds to the target, or (b) otherwise Attorney Docket No. 9662.81.WO resulting in the modification of another compound so that the other compound directly binds to the target.

[0110] As used herein, the terms "somatic regeneration" or "somatic embryogenesis (SE)" refers to a mode of stimulated plant cell totipotency wherein embryos form without fertilization either through somatic or vegetative origins. SE has two main phases: induction and a developmental phase. The induction phase comprises cell proliferation and dedifferentiation, whereas the developmental phase involves the differentiation of somatic embryos under appropriate stimuli. As such, SE is categorized into two main types: direct (without an intervening callus) and indirect (includes a callus phase). Further description of SE can be found in Sivanesan et al., 2022, Genetic and epigenetic modes of the regulation of somatic embryogenesis: a review, Biologia Futura 73:259-277, the disclosures of which are incorporated herein in their entirety.

[0111] This invention relates to polynucleotides comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation and expression cassettes, vectors, and compositions comprising the same for the purpose of enabling rapid somatic regeneration in a nonembryonic cell, as well as methods of making and methods of using the same.

[0112] One aspect of the invention relates to a polynucleotide comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation.

[0113] In some embodiments, a LEC1A open reading frame of the invention comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least about 70% sequence identity (e.g., at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, or any value or range therein) thereto. In some embodiments, a LEC1A open reading frame of the invention comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides have been substituted, added, and / or deleted compared to the wild-type nucleotide sequence.

[0114] Another aspect of the invention relates to an expression cassette comprising a polynucleotide comprising a LEC1A open reading frame in antisense orientation. In some embodiments, an expression cassette of the invention comprises a polynucleotide of the present invention, e.g., a polynucleotide comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation; e.g., a polynucleotide comprising a LEC1A open reading frame in antisense orientation of the invention, wherein the LEC 1 A open reading frame comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least about 70% sequence identity thereto. Attorney Docket No. 9662.81.WO

[0115] The LEC1A open reading frame in the expression cassette may be operably linked to one or more expression elements that may enhance expression of one or more component of the expression cassette, e.g., the antisense transcript of the LEC1 A ORF. For example, in some embodiments, the LEC1A open reading frame is operably linked to a promoter. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a non-plant promoter (e.g., a promoter not naturally occurring and / or source from a plant or plant cell, e.g., mammalian promoter, e.g., a human promoter). In some embodiments, the promoter is a beta-estradiol inducible promoter (e.g., a LEXA promoter). In some embodiments, the promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence having at least about 70% sequence identity (e.g., at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, or any value or range therein) thereto.

[0116] Those skilled in the art will further appreciate that a variety of promoter / enhancer elements may be used depending on the level and tissue-specific expression desired. The promoter / enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0117] Promoter / enhancer elements can be native to the target cell or plant to be treated and / or native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter / enhancer element is a non-plant promoter / enhancer element. The promoter / enhance element may be constitutive or inducible.

[0118] Inducible expression control elements are generally used in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence(s). Inducible promoters / enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, and include hormone-inducible and metalinducible elements. Exemplary inducible promoters / enhancer elements include, but are not limited to, a beta-estradiol inducible promoter.

[0119] In embodiments wherein the LEC1A open reading frame is transcribed and then translated in a target cell, specific initiation signals may be generally employed for efficient translation of inserted protein coding sequences. These exogenous translational control Attorney Docket No. 9662.81.WO sequences, which may include the ATG initiation codon (i.e., translation start site) and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0120] In some embodiments, the expression cassette further comprises at least one selection marker. As used herein, "selection marker" means a polynucleotide sequence that when expressed imparts a distinct phenotype or characteristic to the host cell expressing the marker and thus allows such transformed and / or transgenic cells to be distinguished from those that do not have the marker. The selection marker may be a sequence itself or a polynucleotide encoding a transcribable selection marker (also referred to as a "selectable marker"). Such a polynucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, a herbicide, and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., fluorescence). Many examples of suitable selection markers are known in the art and can be used in the expression cassettes described herein.

[0121] In some embodiments, an expression cassette of the present invention may further comprise an antibiotic selection marker. Non-limiting examples of an antibiotic selection marker of use in the present invention include selection markers selective for (e.g., which allow for screening by application to a culture of) hygromycin (HYG) or kanamycin, e.g., wherein transformed and / or transgenic cells comprising the selection marker survive co-culture with the antibiotic selective for the selection marker, whereas cells not comprising the selection marker are selectively killed by presence of the antibiotic.

[0122] In some embodiments, an expression cassette of the present invention may further comprise a herbicide selection marker. Non-limiting examples of a herbicide selection marker of use in the present invention include selection markers selective for (e.g., which allow for screening by application to a culture of) glufosinate (BAR; also known as phosphinothricin), e.g., wherein transformed and / or transgenic cells comprising the selection marker survive coculture with the herbicide selective for the selection marker, whereas cells not comprising the selection marker are selectively killed by presence of the herbicide.

[0123] In some embodiments, an expression cassette of the present invention comprises a nonplant promoter, the LEC1 A open reading frame in antisense orientation, an antibiotic selection marker, and a herbicide selection marker, optionally in the recited order.

[0124] In some embodiments, an expression cassette of the present invention comprises an inducible non-plant promoter, the LEC1A open reading frame in antisense orientation, an antibiotic selection marker, and a herbicide selection marker, optionally in the recited order. Attorney Docket No. 9662.81.WO

[0125] In some embodiments, an expression cassette of the present invention comprises a betaestradiol inducible promoter, the LEC1A open reading frame in antisense orientation, an antibiotic selection marker, and a glufosinate (BAR) herbicide selection marker, optionally in the recited order.

[0126] In some embodiments, an expression cassette of the present invention comprises a betaestradiol inducible promoter, the LEC1A open reading frame in antisense orientation, a hygromycin (HYG) antibiotic selection marker, and a glufosinate (BAR) herbicide selection marker, optionally in the recited order.

[0127] In some embodiments, an expression cassette of the present invention comprises a betaestradiol inducible promoter, the LEC1A open reading frame in antisense orientation, a kanamycin antibiotic selection marker, and a glufosinate (BAR) herbicide selection marker, optionally in the recited order.

[0128] In some embodiments, an expression cassette of the present invention comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:2 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

[0129] In some embodiments, an expression cassette of the present invention comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:3 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

[0130] Another aspect of the invention relates to a vector comprising a polynucleotide and / or expression cassette of the present invention. Suitable vectors include, but are not limited to, a plasmid, phage, viral vector (e.g., an AAV vector, a lentiviral vector, an adenovirus vector, a herpesvirus vector, an alphavirus vector, or a baculovirus vector), bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC). In some embodiments, the vector is a delivery vehicle such as a particle (e.g., a microparticle or nanoparticle) or a liposome to which the expression cassette is attached or in which the expression cassette is embedded. The vector may be any delivery vehicle suitable to carry the expression cassette into a cell.

[0131] In some embodiments, the vector is a plasmid. In some embodiments, the plasmid is a T-DNA plasmid. In some embodiments, the plasmid is a pCAMBIA T-DNA plasmid.

[0132] An additional aspect of the invention relates to a transformed and / or transgenic cell comprising the polynucleotide, expression cassette, and / or vector of the invention. In some embodiments, the polynucleotide, expression cassette, and / or vector is stably incorporated into the cell genome (e.g., the plant cell genome). Attorney Docket No. 9662.81.WO

[0133] The transformed and / or transgenic plant cell may be an in vitro, ex vivo, or in vivo cell. In some embodiments, the transformed and / or transgenic plant cell is a nonembryonic cell. In some embodiments, the transformed and / or transgenic plant cell is a parenchymal plant cell. In some embodiments, the transformed and / or transgenic plant cell is a callus plant cell. As used herein, a "callus plant cell," "callus," "calluses" or "calli" refer to one or more undifferentiated plant cell in a growing mass of unorganized plant parenchyma cells. Without wishing to be bound to theory, in living plants, callus cells are those cells that cover a plant wound. Callus growths are also used in biological research and biotechnology, where callus formation may be induced from plant tissue samples ("explants") after surface sterilization and plating onto tissue culture medium in vitro. The culture medium may be supplemented with plant growth regulators, such as but not limited to auxin, cytokinin, and gibberellin, to initiate callus formation, also referred to "somatic embryogenesis" or "somatic regeneration". Callus initiation has been described for all major groups of land plants. Further description of callus cells and current methods in the art for plant somatic regeneration or somatic embryogenesis can be found as described in Long et al., 2022 "New Insights Into Tissue Culture Plant- Regeneration Mechanisms," Front Plant Sci. 13:926752, the disclosures of which are incorporated herein by reference in their entirety.

[0134] In some embodiments, the plant cell is a cotton (Gossypium,' e.g., Gossypium hirsutum, Gossypium barbadense, or Gossypium arboreum) plant cell. In some embodiments, the plant cell is an upland cotton (Gossypium hirsutum) plant cell. In some embodiments, the plant cell is a Coker 312 genotype G. hirsutum upland cotton plant cell.

[0135] Another aspect of the invention relates to a transgenic plant or plant part produced by (e.g., grown from) a transformed and / or transgenic plant cell of the present invention, e.g., grown from a seed comprising the polynucleotide, expression cassette, vector, and / or the transformed and / or transgenic cell of the invention, optionally wherein the polynucleotide is stably incorporated into the seed plant cell genome.

[0136] Another aspect of the invention relates to a transformed and / or transgenic plant or plant part comprising the polynucleotide, expression cassette, vector, and / or the transformed cell of the invention, optionally transiently expressing the polynucleotide of the invention.

[0137] In some embodiments, a transformed and / or transgenic plant or plant part of the present invention is a seed. In some embodiments, the seed comprises the polynucleotide, expression cassette, or vector stably incorporated into seed cell genome.

[0138] In some embodiments, the transformed and / or transgenic plant or plant part is a cotton Gossypium,' e.g., Gossypium hirsutum, Gossypium barbadense, or Gossypium arboreum) Attorney Docket No. 9662.81.WO plant or plant part. In some embodiments, the plant or plant part is an upland cotton (Gossypium hirsutum) plant or plant part. In some embodiments, the plant or plant part is a Coker 312 genotype G. hirsutum upland cotton plant or plant part.

[0139] Another aspect of the invention relates to a composition comprising a polynucleotide, expression cassette, vector, transformed and / or transgenic plant cell, or transformed and / or transgenic plant or plant part of the present invention in an agronomically acceptable carrier.

[0140] In some embodiments, a composition of the present invention further comprises Agrobacterium (including but not limited to Agrobacterium tumefaciens . e.g., for use in transformation co-culturing techniques, as would be known to one of ordinary skill in the art. For example, in some embodiments, a composition of the present invention further comprises Agrobacterium comprising (e.g., transformed with) the polynucleotide, expression cassette, and / or vector of the present invention, e.g., for use in delivering or otherwise introducing the polynucleotide into a plant or plant cell or plant part thereof. In some embodiments, the Agrobacterium is Agrobacterium tumefaciens. In some embodiments, the Agrobacterium tumefaciens is a A. tumefaciens cell line, e.g., including but not limited to, the EHA105 Agrobacterium tumefaciens cell line, the EHA101 cell line, the LBA4404 cell line, and the Auxo-Agro™ EHA105 cell line.

[0141] In some embodiments, the polynucleotide, expression cassette, vector, composition, transformed and / or transgenic cell and / or transformed and / or transgenic plant or plant part of the invention is isolated.

[0142] In some embodiments, the polynucleotide, expression cassette, vector, composition, transformed and / or transgenic cell and / or transformed and / or transgenic plant or plant part of the invention is purified.

[0143] In some embodiments, the polynucleotide, expression cassette, vector, composition, transformed and / or transgenic cell and / or transformed and / or transgenic plant or plant part of the invention may be for use in somatic embryogenesis of a cotton plant or plant part.

[0144] In some embodiments, the polynucleotide, expression cassette, vector, composition, transformed and / or transgenic cell and / or transformed and / or transgenic plant or plant part of the invention may be for use in cellular reprogramming of a cotton plant or plant part.

[0145] In some embodiments, the polynucleotide, expression cassette, vector, composition, transformed and / or transgenic cell and / or transformed and / or transgenic plant or plant part of the invention may be for use in cellular reprogramming of a cotton plant or plant part.

[0146] The present invention also relates to methods for delivering a LEC1A open reading frame in antisense orientation to a plant or plant cell or plant part thereof, e.g., to induce or Attorney Docket No. 9662.81.WO enhance somatic embryogenesis, e.g., to induce or enhance cellular reprogramming, e.g., for agricultural or research purposes in vitro, ex vivo, or in vivo.

[0147] Another aspect of the invention relates to a method of expressing an antisense LEC1A open reading frame in a plant cell, comprising: (a) contacting the plant cell with a polynucleotide, expression cassette, vector, and / or composition of the present invention, wherein the polynucleotide incorporates into the plant cell, and (b) contacting the plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame; thereby expressing the antisense LEC1 A open reading frame in the plant cell.

[0148] Another aspect of the invention relates to a method of reprogramming a nonembryonic plant cell for somatic regeneration, comprising: (a) contacting the nonembryonic plant cell with a polynucleotide, expression cassette, vector, and / or composition of the present invention, wherein the polynucleotide incorporates into the nonembryonic plant cell, and (b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; thereby reprogramming the nonembryonic plant cell for somatic regeneration.

[0149] Another aspect of the present invention relates to a method of inducing somatic embryogenesis in a nonembryonic plant cell, comprising: (a) contacting the nonembryonic plant cell with a polynucleotide, expression cassette, vector, and / or composition of the present invention, wherein the polynucleotide incorporates into the nonembryonic plant cell, and (b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; thereby inducing somatic embryogenesis in the nonembryonic plant cell.

[0150] Another aspect of the present invention relates to a method of producing a plant from a nonembryonic plant cell, comprising: (a) contacting the nonembryonic plant cell with a polynucleotide, expression cassette, vector, and / or composition of the present invention, (b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; and (c) growing the somatically regenerating plant cell of (b) to produce an adult plant; thereby producing a plant from the nonembryonic plant cell. Attorney Docket No. 9662.81.WO

[0151] In some embodiments, contacting the plant cell with the polynucleotide, expression cassette, vector, and / or composition comprises culturing the plant cell in a medium in combination (e.g., co-culturing) with an Agrobacterium (e.g., Agrobacterium tumefaciens) comprising the polynucleotide, expression cassette, vector and / or composition. In some embodiments, the Agrobacterium is Agrobacterium tumefaciens. In some embodiments, the Agrobacterium tumefaciens is a A. tumefaciens cell line, e.g., including but not limited to, the EHA105 Agrobacterium tumefaciens cell line, the EHA101 cell line, the LBA4404 cell line, and the Auxo-Agro™ EHA105 cell line.

[0152] In some embodiments, the plant cell is an explant derived from a plant (e.g., is comprised in an explant derived from a plant).

[0153] The plant cell may be an in vitro, ex vivo, or in vivo cell. In some embodiments, the plant cell is a nonembryonic cell. In some embodiments, the plant cell is a parenchymal plant cell. In some embodiments, the plant cell is a callus plant cell. In some embodiments, the plant cell is a cotton Gossypium,' e.g., Gossypium hirsutum, Gossypium barbadense, or Gossypium arboreum) plant cell. In some embodiments, the plant cell is an upland cotton (Gossypium hirsutum) plant cell. In some embodiments, the plant cell is a Coker 312 genotype G. hirsutum upland cotton plant cell.

[0154] In some embodiments, contacting the plant cell with the polynucleotide, expression cassette, vector, and / or composition stably incorporates into the polynucleotide into the plant cell genome.

[0155] In some embodiments, contacting the plant cell with the polynucleotide, expression cassette, vector, and / or composition transiently expresses the polynucleotide in the plant cell.

[0156] In some embodiments, a method of the present invention may further comprise contacting the plant cell with a second polynucleotide, expression cassette, vector and / or composition comprising a gene editing tool of interest.

[0157] Another aspect of the present invention relates to a method of producing a plant capable of somatic regeneration from a nonembryonic plant cell thereof, comprising: (a) culturing a seed comprising a polynucleotide of the present invention stably expressed in the seed plant genome (e.g., a transformed and / or transgenic plant seed of the present invention) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell(s) of the seed gain functional capability for somatic regeneration; and (b) growing the somatically regenerating plant cell(s) to produce an adult plant; thereby producing a plant capable of somatic regeneration from a nonembryonic plant cell thereof. Attorney Docket No. 9662.81.WO

[0158] In some embodiments, a method of the present invention may further comprise contacting the plant cell with a polynucleotide, expression cassette, vector and / or composition comprising a gene editing tool of interest to generate a modified transformed plant.

[0159] In some embodiments, frequency of embryo formation from the nonembryonic plant cell is increased at least about 5% to about 100% (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% or more or any value or range therein) as compared to the frequency of embryo formation from a control plant cell, optionally wherein the control plant cell is a plant cell not contacted with a polynucleotide, expression cassette, vector, and / or composition of the present invention, or optionally wherein the control plant cell is a plant cell contacted with a polynucleotide, expression cassette, vector and / or composition devoid of a LEC1 A open reading frame in antisense orientation.

[0160] In some embodiments, the nonembryonic plant cell gains functional capability for somatic regeneration at a rate of less than about 1 in 500 events (e.g., at a rate of less than about 1 in 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 events or less or any value or range therein) as compared to the rate of events in which a control plant cell spontaneously gains functional capability for somatic regeneration, optionally wherein the control plant cell is a plant cell not contacted with a polynucleotide, expression cassette, vector, and / or composition of the present invention, or optionally wherein the control plant cell is a plant cell contacted with a polynucleotide, expression cassette, vector and / or composition devoid of a LEC1A open reading frame in antisense orientation. "Events" is used herein to refer to the number of individuals cells contacted with and / or transformed by a polynucleotide, expression cassette, vector, and / or composition of the present invention (e.g., "contact events"; "transformation events").

[0161] In some embodiments, the time to produce an adult plant from the nonembryonic plant cell upon gaining functional capability for somatic regeneration is reduced (e.g., reduced to no more than 16 months, e.g., 16, 15, 14, 13, 12, 11, or 10 months or any value or range therein) as compared to the time to produce an adult plant from nonembryonic plant cell of a control plant cell upon spontaneously gaining functional capability for somatic regeneration, optionally wherein the control plant cell is a plant cell not contacted with a polynucleotide, expression cassette, vector, and / or composition of the present invention, or optionally wherein the control plant cell is a plant cell contacted with a polynucleotide, expression cassette, vector and / or composition devoid of a LEC1 A open reading frame in antisense orientation. Attorney Docket No. 9662.81.WO

[0162] In some embodiments, the time to produce an adult plant from the nonembryonic plant cell upon gaining functional capability for somatic regeneration is about 16 months or less (e.g., about 16, 15, 14, 13, 12, 11, or 10 months or less).

[0163] In some embodiments, a method of the present invention may comprise or further comprise the steps of: (a) incubating a culture of Agrobacterium tumefaciens (e.g., inoculating a suspension of A. tumefaciens comprising a polynucleotide, expression cassette, vector, and / or composition of the present invention, in a medium (e.g., comprising rifampicin at a concentration of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / L or any value or range therein and / or kanamycin at a concentration of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / L or any value or range therein and / or acetosyringone at a concentration of about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 pM or any value or range therein) at a temperature of about 28 °C (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 °C or any value or range therein), (b) introducing one or more explants in the Agrobacterium suspension culture (e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min), thereby introducing the antisense LEC1A into the one or more explants, (c) removing the one or more explants from the suspension culture, drying the one or more explants (e.g., via blotting with sterile filter paper) and transferring the dried one or more explants to a solid medium surface, (d) incubating the one or more explants on the solid medium surface under room temperature (e.g., about 23 °C; e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 °C or any value or range therein) conditions devoid of light for about 72 hours (e.g., about 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hrs or any value or range therein), (e) placing the one or more explants under selective pressure (e.g., transferring the one or more explants onto a new solid medium surface comprising a selective agent (e.g., an antibiotic, e.g., hygromycin, e.g., kanamycin, e.g., a herbicide) for about 4 weeks (e.g., about 1, 2, 3, 4, 5, 6, 7, or 8 weeks or any value or range therein) such that the explant forms callus tissue comprising the antisense LEC1A under selective pressure, (f) contacting the selectively pressured one or more explants with an agonist with binding specificity for the promoter (e.g., "pulsing" the transformed callus tissue formed under selective pressure on the explant; e.g., transferring the selectively pressured one or more explant comprising callus tissue formed under selective pressure to a new medium comprising an agonist with binding specificity to the promoter) and incubating for at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days or any value or range therein, (g) subculturing the selectively pressured explant comprising callus tissue (e.g., transferring the selectively pressured explant comprising callus tissue to fresh culture medium) about every 30 Attorney Docket No. 9662.81.WO days (e.g., about every 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days or any value or range therein) until somatic embryo tissue develops, (h) differentiating the somatic embryo tissue (e.g., transferring the developed somatic embryo tissue to a differentiation medium) until the embryo tissue germinates to form one or more roots and / or shoots, (i) incubating the germinated embryo tissue of (h) under conditions such that plantlets (e.g., 3-4 leaves stage plants) with roots and shoots develop (e.g., incubating by transferring the germinated embryo tissue of (h) to a rooting medium), and (j) transferring the plantlets to soil and incubating under conditions for mature plant development.

[0164] The compositions can be presented in unit / dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0165] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile composition of this invention in a unit dosage form in a sealed container can be provided. The composition can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject (e.g., a plant or plant part, e.g., a crop plant or plant part). The unit dosage form can be from about 1 pg to about 10 grams of the composition of this invention, or any value or range therein, e.g., from about 1 pg to about 5 grams, about 2 pg to about 10 pg, about 1.5 pg to about 7.5 grams, or about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 20 pg, about 50 pg, about 100 pg, about 250 pg, about 500 pg, about 750 pg, about 1 gram, about 1.25 grams, about 1.5 grams, about 1.75 grams, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, or about 10 grams. When the composition is substantially water-insoluble, a sufficient amount of emulsifying agent, which is physiologically acceptable, can be included in sufficient quantity to emulsify the composition in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0166] The present invention further comprises a kit or kits to carry out the methods of this invention. A kit of this invention can comprise reagents, buffers, and apparatus for mixing, measuring, sorting, labeling, etc., as well as instructions and the like as would be appropriate for using a polynucleotide, expression cassette, vector, composition, plant, plant part, plant cell, and / or plant seed of the present invention. Attorney Docket No. 9662.81.WO

[0167] In some embodiments, the invention provides a kit for comprising one or more polypeptide(s) of the present invention and / or nucleic acid construct(s) of the present invention, and / or expression cassette(s), vectors, composition9s), plant(s), plant part(s), plant seed(s) and / or plant cell(s) comprising the same as described herein, with optional instructions for the use thereof.

[0168] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.

[0169] EXAMPLES

[0170] Example 1: A helper construct that enables rapid regeneration in Upland cotton.

[0171] The study relates to the precise genetic modification of Gossypium hirsutum (upland cotton) genotype Coker 312, specifically targeting the LEAFY COTYLEDON 1 (LEC1A) gene. The LEC1 A gene was cloned in an antisense configuration and placed under the control of a P-estradiol-inducible promoter to drive targeted genetic and cellular reprogramming in non-embryogenic callus tissue. This reprogramming initiated somatic embryogenesis.

[0172] Following transformation via Agrobacterium tumefaciens-mediated co-cultivation, inducible LEC1 A expression resulted in the formation of spherical pre-embryogenic structures within approximately three months. These structures further developed into plantlets by six months, with fully mature plants emerging between seven and eight months, and producing T1 generation seeds within 11 to 12 months post-initiation.

[0173] In contrast, non-transformed wild-type Coker 312 typically requires a minimum of 18 months for embryo formation and up to 24 months for fully differentiated plant development. Furthermore, the frequency of embryo formation is significantly enhanced, exceeding 50% in LEClA-induced lines, compared to the 5-15% observed in the wild type.

[0174] Binary plasmid system. The modified pCAMBIA T-DNA plasmid contains a helper gene and a multiple cloning site (MCS). This system includes the LEC1A helper gene in an antisense orientation, regulated by an inducible LEXA promoter (see FIG. 1). It was designed for transient expression, allowing a controlled expression pulse during somatic regeneration. This results in a significantly increased frequency of embryo formation and a reduced time to embryo development compared to the wild type. The binary plasmid is available both as purified DNA and as a recombinant Agrobacterium tumefaciens strain (EH105A) stored as a glycerol stock. True-to-type Coker 312 seeds are also provided. Selectable markers on the T- Attorney Docket No. 9662.81.WO

[0175] DNA include hygromycin (HYG) for antibiotic selection and glufosinate (BAR) gene for herbicide selection, FIG. 2.

[0176] Recombinant Coker312 system. This system features a transgenic Coker 312 line with the LEC1A gene and the LEXA inducible promoter stably integrated into the genome in a homozygous state. Since this line also contains the HYG resistance gene, selection during regeneration was performed using an alternative antibiotic, such as kanamycin.

[0177] Methods for Transformation and Regeneration. This protocol presents a highly efficient, standardized approach for Agrobacterium-mediated transformation in Coker 312. It details the various media compositions necessary throughout the transformation process. Furthermore, it provides a comprehensive procedure for transforming hypocotyls employed as explants, selecting transformed calli, and regenerating plants.

[0178] Stock Solutions:

[0179] B5 vitamin stock

[0180] Antibiotics Hormones and solution A

[0181] Media (Autoclave the media at 121°C for 20 min at 15 lbs pressure. When the media cools to

[0182] <50°C, add hormones and antibiotics according to different media requirements. Attorney Docket No. 9662.81.WO a) Germination Media for cotton seeds (pH 6.1-6.2) b) Suspension media for Agrobacterium c) Co-cultivation media, selection media and induction media (pH 5.85-5.95) Co-cultivation media = Hormones (100 pl / L each from 2,4-D and Kinetin stocks)

[0183] Selection media = Hormones (100 l / L each from 2,4-D and Kinetin stocks), antibiotics (1 ml each from stock solution of hygromycin-B 20mg / L, cefotaxime 250mg / L and timentin lOOmg / L)

[0184] Induction media = Hormones (100 pl / L each from 2,4-D and Kinetin stocks), antibiotics (1 ml each from stock solution of hygromycin-B 20mg / L, cefotaxime 250mg / L and timentin lOOmg / L) and 1 ml of solution A from (10 mM / L) stock d) Differentiation media (pH 6.1-6.2) Attorney Docket No. 9662.81.WO

[0185] Hormones = (Kinetin 150 pl / L and IBA 500 pl / L from stocks) e) Rooting Media (pH 5.90-5.95)

[0186] Hormones = (IBA 500 pl / L from stock) Procedures for Cotton Seed Germination Delinting of cotton seeds (carry out this step in the acid / organics fume hood)

[0187] Place about 20 fuzzy cotton seeds in a 50 ml Falcon tube. Add concentrated sulfuric acid (H2SO4) to cover the seeds. Shake the falcon tube for 1-2 minutes for the uniform and effective treatment. Drain off sulfuric acid. Wash the seeds 4-5 times with cold water. For the complete removal of acid, seeds need to be soaked in TrisCi for 15. Washing needs to be repeated 3-4 times. Surface sterilization of cotton seeds.

[0188] From here on, the entire procedure has to be done under plant tissue culture hood to avoid fungal / microbial contamination. Transfer the seeds into a clean, sterile falcon tube and immerse them in 30-40 ml of 70% ethanol for 2 min with continuous shaking. Discard the ethanol and wash the seeds with sterile water. Immerse seed again in 20 % bleach containing 0.1% Tween 20 (v / v) for 15-20 minutes. Wash the seeds with sterile MilliQ ultrapure H2O (3-4 times). Change the falcon tubes with each wash. Place surface sterilized seeds in germination media Attorney Docket No. 9662.81.WO bottles and keep them in the dark for 7-10 days. Seedlings will germinate after one week, and clean, etiolated seedlings will be used as an explant for transformation.

[0189] Procedures for Agrobacterium-mediated Cotton Transformation

[0190] Grow Agrobacterium on LB + Rifampicin 50mg / L + Kanamycin 50mg / L plate, 28 °C for two days. Restreak Agrobacterium again on LB plates to make it highly active before starting the transformation.

[0191] Day 1:

[0192] • Inoculate 30 ml of suspension medium in 50 ml falcon tube containing rifampicin 50mg / L + kanamycin

[0193] • Grow this culture at 28°C on an incubator shaker at 220 rpm until OD600 reads 0.30- 0.35.

[0194] • Cut the hypocotyls into 5-7 mm size explants.

[0195] • Immerse 80-100 explants in 30 mL of an Agrobacterium suspension culture for 5 min, shaking occasionally by hand.

[0196] • Once the explants have been immersed for 5 minutes, remove them by carefully pouring the suspension media from the tube into a collection dish. Blot-dry the explants with sterile filter paper.

[0197] • After blot drying, explants will be transferred to sterilized filter paper on the surface of co-culture media to prevent Agrobacterium overgrowth. Place the culture in the dark for ~72 hrs at room temperature (23 °C). Do not stack the explants; just place them separately.

[0198] Day 3 : Transfer the explants onto the selection medium.

[0199] Day 25: Subculture the explants onto the selection medium.

[0200] Day 40: After four weeks on selection media, hygromycin-resistant callus (transformed) will form on the cut edges of the explants. Transfer the explants to new induction media to facilitate an expression pulse of the TFs. Keep on this media for 25-30 days.

[0201] Day 70: Transformed calli will be moved to new media every ~ 30 days and monitored for polar structure formation with high-resolution imaging.

[0202] Day 100: Subculture all the explants onto the fresh selection media plate until the somatic embryos form. Transfer the embryos to the differentiation media for shoot and root development.

[0203] Day 130: Continue subculturing embryogenic tissue to differentiation media until embryos start to germinate and form roots and shoots. Attorney Docket No. 9662.81.WO

[0204] Day 160: Rooted embryos will be transferred to magenta boxes containing rooting media.

[0205] Day 190: Regenerated plantlets (3-4 leaves stage) with satisfactory roots will be transferred to soil in small pots, kept in a mist chamber for hardening, and then transferred to the greenhouse in 3-4 gallons pots. Representative example images of resultant plant growth is shown in FIG. 3.

[0206] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

Attorney Docket No. 9662.81.WOWHAT IS CLAIMED IS:

1. A polynucleotide comprising a LEAFY COTYLEDON 1 (LEC1A) open reading frame in antisense orientation.

2. The polynucleotide of claim 1, wherein said LEC1 A open reading frame comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least about 90% identity thereto.

3. An expression cassette comprising a polynucleotide comprising a LEC1 A open reading frame in antisense orientation.

4. The expression cassette of claim 3, wherein the polynucleotide is the polynucleotide of claim 1 or 2.

5. The expression cassette of claim 3 or 4, wherein the LEC1 A open reading frame is operably linked to a promoter, optionally a heterologous promoter.

6. The expression cassette of claim 5, wherein the promoter is an inducible promoter.

7. The expression cassette of claim 5 or 6, wherein the promoter is a non-plant promoter (e.g., a mammalian promoter, e.g., a human promoter).

8. The expression cassette of any one of claims 5 to 7, wherein the promoter is a betaestradiol inducible promoter (e.g., a LEXA promoter).

9. The expression cassette of any one of claims 3-8, further comprising an antibiotic selection marker.

10. The expression cassette of claim 9, wherein the antibiotic selection marker is selective for an antibiotic selected from the group consisting of hygromycin and kanamycin.

11. The expression cassette of any one of claims 3-10, further comprising a herbicide selection marker.Attorney Docket No. 9662.81.WO12. The expression cassette of claiml 1, wherein the herbicide selection marker is selective for a herbicide selected is glufosinate (BAR).

13. The expression cassette of any one of claims 3-12, wherein the expression cassette comprises a non-plant promoter, the LEC1 A open reading frame in antisense orientation, an antibiotic selection marker, and a herbicide selection marker.

14. The expression cassette of any one of claims 3-13, wherein the expression cassette comprises an inducible non-plant promoter, the LEC1A open reading frame in antisense orientation, an antibiotic selection marker, and a herbicide selection marker.

15. The expression cassette of any one of claims 3-14, wherein the expression cassette comprises a beta-estradiol inducible promoter, the LEC1 A open reading frame in antisense orientation, an antibiotic selection marker, and a glufosinate (BAR) herbicide selection marker.

16. The expression cassette of any one of claims 3-15, wherein the expression cassette comprises a beta-estradiol inducible promoter, the LEC1 A open reading frame in antisense orientation, a hygromycin (HYG) antibiotic selection marker, and a glufosinate (BAR) herbicide selection marker.

17. The expression cassette of any one of claims 3-15, wherein the expression cassette comprises a beta-estradiol inducible promoter, the LEC1 A open reading frame in antisense orientation, a kanamycin antibiotic selection marker, and a glufosinate (BAR) herbicide selection marker.

18. The expression cassette of claim 16, comprising the nucleotide sequence of SEQ ID NO:2 or a sequence at least about 90% identical thereto.

19. The expression cassette of claim 17, comprising the nucleotide sequence of SEQ ID NO: 3 or a sequence at least about 90% identical thereto.

20. A vector comprising the polynucleotide of claim 1 or 2 or the expression cassette of any one of claims 3-19.Attorney Docket No. 9662.81.WO21. The vector of claim 20, wherein the vector is a plasmid.

22. The vector of claim 21, wherein the plasmid is a T-DNA plasmid.

23. The vector of claim 22, wherein the plasmid is a pCAMBIA T-DNA plasmid.

24. A transformed plant cell comprising the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, and / or the vector of any one of claims 20-23.

25. The transformed plant cell of claim 24, wherein the polynucleotide, expression cassette, and / or vector is stably incorporated into the plant cell genome.

26. The transformed plant cell of claim 24 or 25, wherein the plant cell is a nonembryonic cell.

27. The transformed plant cell of any one of claims 24-26, wherein the plant cell is a parenchymal plant cell.

28. The transformed plant cell of any one of claims 24-27, wherein the plant cell is a callus plant cell.

29. The transformed plant cell of any one of claims 24-28, wherein the plant cell is a cotton (Gossypium e.g., Gossypium hirsutum, Gossypium bctrbadense. or Gossypium arboreum) plant cell.

30. The transformed plant cell of any one of claims 24-29, wherein the plant cell is an upland cotton (Gossypium hirsutum) plant cell.

31. The transformed plant cell of any one of claims 24-30, wherein the plant cell is a Coker 312 genotype G. hirsutum upland cotton plant cell.

32. A transgenic plant produced by (e.g., grown from) the transformed plant cell of any one of claims 24-31.Attorney Docket No. 9662.81.WO33. A transformed plant or plant part comprising the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the transformed plant cell of any one of claims 24-31.

34. The transformed plant or plant part of claim 33, wherein the plant part is a seed.

35. The transformed plant seed of claim 34, wherein the seed comprises the polynucleotide, expression cassette, or vector stably incorporated into seed cell genome.

36. A transgenic plant produced by (e.g., grown from) the transformed plant seed of claim 34 or 35.

37. The transformed plant or plant part of any one of claims 32 to 35 or transgenic plant of claim 36, wherein the plant or plant part is a cotton (Gossypium e.g., Gossypium hirsutum, Gossypium bar hade use. or Gossypium arboreum) plant or plant part.

38. The transformed plant or plant part of any one of claims 32 to 35 or 37, or transgenic plant of claim 36, wherein the plant or plant part is an upland cotton (Gossypium hirsutum) plant or plant part.

39. The transformed plant or plant part of any one of claims 32 to 35, 37 or 38, or transgenic plant of claim 36, wherein the plant or plant part is a Coker 312 genotype G. hirsutum upland cotton plant or plant part.

40. A composition comprising the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, the transformed plant cell of any one of claims 24-31, and / or the transformed plant or plant part of any one of claims 32- 35, 37 or 38 in an agronomically acceptable carrier.

41. The composition of claim 40, for use in somatic embryogenesis of a cotton plant or plant part.

42. The composition of claim 41, for use in cellular reprogramming of a cotton plant or plant part.Attorney Docket No. 9662.81.WO43. The composition of any one of claims 40-42, further comprising Agrobacterium comprising (e.g., transformed with) the polynucleotide, expression cassette, or vector.

44. The composition of claim 43, wherein the Agrobacterium is a EHA105 Agrobacterium tumefaciens cell line.

45. A method of expressing an antisense LEC1 A open reading frame in a plant cell, comprising:(a) contacting the plant cell with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44, wherein the polynucleotide incorporates into the plant cell, and(b) contacting the plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1 A open reading frame; thereby expressing the antisense LEC1A open reading frame in the plant cell.

46. A method of reprogramming a nonembryonic plant cell for somatic regeneration, comprising:(a) contacting the nonembryonic plant cell with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44, wherein the polynucleotide incorporates into the nonembryonic plant cell, and(b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; thereby reprogramming the nonembryonic plant cell for somatic regeneration.

47. A method of inducing somatic embryogenesis in a nonembryonic plant cell, comprising:(a) contacting the nonembryonic plant cell with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / orAttorney Docket No. 9662.81.WO the composition of any one of claims 40-44, wherein the polynucleotide incorporates into the nonembryonic plant cell, and(b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; thereby inducing somatic embryogenesis in the nonembryonic plant cell.

48. A method of producing a plant from a nonembryonic plant cell, comprising:(a) contacting the nonembryonic plant cell with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44,(b) contacting the nonembryonic plant cell of (a) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell gains functional capability for somatic regeneration; and(c) growing the somatically regenerating plant cell of (b) to produce an adult plant; thereby producing a plant from the nonembryonic plant cell.

49. The method of any one of claims 45-48, wherein contacting the plant cell with the polynucleotide, expression cassette, vector, and / or composition comprises culturing the plant cell in a medium in combination (e.g., co-culturing) with an Agrobacterium comprising the polynucleotide, expression cassette, vector and / or composition.

50. The method of claim 49, wherein the Agrobacterium is a EHA105 Agrobacterium tumefaciens cell line.

51. The method of any one of claims 45-50, wherein the plant cell is an explant derived from a plant (e.g., is comprised in an explant derived from a plant).

52. The method of any one of claims 45-51, wherein the plant cell is a cotton (Gossypium,' e.g., Gossypium hirsutum, Gossypium bar hade use. or Gossypium arboreum) plant cell.Attorney Docket No. 9662.81.WO53. The method of any one of claims 45-52, wherein the plant cell is an upland cotton (Gossypium hirsutum) plant cell.

54. The method of any one of claims 45-52, wherein the plant cell is a Coker 312 genotype G. hirsutum upland cotton plant cell.

55. The method of any one of claims 45-54, wherein the plant cell is a nonembryonic cell.

56. The method of any one of claims 45-55, wherein the plant cell is a parenchymal plant cell.

57. The method of any one of claims 45-56, wherein the plant cell is a callus plant cell.

58. The method of any one of claims 45-56, wherein contacting the plant cell with the polynucleotide, expression cassette, vector, and / or composition stably incorporates into the polynucleotide into the plant cell genome.

59. The method of any one of claims 45-56, wherein contacting the plant cell with the polynucleotide, expression cassette, vector, and / or composition transiently expresses the polynucleotide in the plant cell.

60. The method of any one of claims 45-59, further comprising contacting the plant cell with a second polynucleotide, expression cassette, vector and / or composition comprising a gene editing tool of interest.

61. A method of producing a plant capable of somatic regeneration from a nonembryonic plant cell thereof, comprising:(a) culturing a seed comprising the polynucleotide of claim 1 or 2 stably expressed in the seed plant genome (e.g., the transformed plant seed of claim 34 or 35) with an agonist, wherein the agonist binds the promoter of the polynucleotide thereby inducing expression of the antisense LEC1A open reading frame, whereby the nonembryonic plant cell(s) of the seed gain functional capability for somatic regeneration; and(b) growing the somatically regenerating plant cell(s) to produce an adult plant;Attorney Docket No. 9662.81.WO thereby producing a plant capable of somatic regeneration from a nonembryonic plant cell thereof.

62. The method of claim 61, further comprising contacting the plant cell with a polynucleotide, expression cassette, vector and / or composition comprising a gene editing tool of interest to generate a modified transformed plant.

63. The method of any one of claims 45-62, wherein frequency of embryo formation from the nonembryonic plant cell is increased at least about 5% to about 100% (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% or more or any value or range therein) as compared to the frequency of embryo formation from a control plant cell, optionally wherein the control plant cell is a plant cell not contacted with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44, or optionally wherein the control plant cell is a plant cell contacted with a polynucleotide, expression cassette, vector or composition devoid of a LEC1 A open reading frame in antisense orientation.

64. The method of any one of claims 45-63, wherein the nonembryonic plant cell gains functional capability for somatic regeneration at a rate of less than about 1 in 500 events (e.g., at a rate of less than about 1 in 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 events or less or any value or range therein) as compared to the rate of events in which a control plant cell spontaneously gains functional capability for somatic regeneration, optionally wherein the control plant cell is a plant cell not contacted with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44, or optionally wherein the control plant cell is a plant cell contacted with a polynucleotide, expression cassette, vector or composition devoid of a LEC1 A open reading frame in antisense orientation.

65. The method of any one of claims 45-64, wherein the time to produce an adult plant from the nonembryonic plant cell upon gaining functional capability for somatic regeneration is reduced (e.g., reduced to no more than 16 months, e.g., 16, 15, 14, 13, 12, 11, or 10 months or any value or range therein) as compared to the time to produce an adult plant from nonembryonic plant cell of a control plant cell upon spontaneously gaining functionalAttorney Docket No. 9662.81.WO capability for somatic regeneration, optionally wherein the control plant cell is a plant cell not contacted with the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44, or optionally wherein the control plant cell is a plant cell contacted with a polynucleotide, expression cassette, vector or composition devoid of a LEC1 A open reading frame in antisense orientation.

66. The method of any one of claims 45-65, wherein the time to produce an adult plant from the nonembryonic plant cell upon gaining functional capability for somatic regeneration is about 16 months or less (e.g., about 16, 15, 14, 13, 12, 11, or 10 months or less).

67. The method of any one of claims 45-65, comprising:(a) incubating a culture of Agrobacterium (e.g., inoculating a suspension of A. tumefaciens comprising the polynucleotide of claim 1 or 2, the expression cassette of any one of claims 3-19, the vector of any one of claims 20-23, and / or the composition of any one of claims 40-44, in a medium (e.g., comprising rifampicin 50mg / L and / or kanamycin 50mg / L and / or acetosyringone 200pM) at a temperature of about 28 °C,(b) introducing one or more explants in the. Agrobacterium suspension culture (e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min), thereby introducing the antisense LEC1A into the one or more explants,(c) removing the one or more explants from the suspension culture, drying the one or more explants (e.g., via blotting with sterile filter paper) and transferring the dried one or more explants to a solid medium surface,(d) incubating the one or more explants on the solid medium surface under room temperature (e.g., about 23 °C) conditions devoid of light for about 72 hours,(e) placing the one or more explants under selective pressure (e.g., transferring the one or more explants onto a new solid medium surface comprising a selective agent (e.g., an antibiotic, e.g., hygromycin, e.g., kanamycin, e.g., a herbicide) for about 4 weeks such that the explant forms callus tissue comprising the antisense LEC1 A under selective pressure,(f) contacting the selectively pressured one or more explants with an agonist with binding specificity for the promoter (e.g., "pulsing" the transformed callus tissue formed under selective pressure on the explant; e.g., transferring the selectively pressured one or more explant comprising callus tissue formed under selective pressure to a new mediumAttorney Docket No. 9662.81.WO comprising an agonist with binding specificity to the promoter) and incubating for at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 5 days,(g) subculturing the selectively pressured explant comprising callus tissue (e.g., transferring the selectively pressured explant comprising callus tissue to fresh culture medium) about every 30 days until somatic embryo tissue develops,(h) differentiating the somatic embryo tissue (e.g., transferring the developed somatic embryo tissue to a differentiation medium) until the embryo tissue germinates to form one or more roots and / or shoots,(i) incubating the germinated embryo tissue of (h) under conditions such that plantlets (e.g., 3-4 leaves stage plants) with roots and shoots develop (e.g., incubating by transferring the germinated embryo tissue of (h) to a rooting medium), and(j) transferring the plantlets to soil and incubating under conditions for mature plant development.

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