WO2014071266A2 - Alteration of sex determination in flowering plants - Google Patents

Abstract

The invention provides methods for the production of unisexual plants. According to the invention, the plants can be either unisexual male or unisexual female plants. In the methods of the invention, the expression of the modified B Class floral identity gene results in the suppression of the development of the fourth floral whorl, which results in male-only floral development. In other methods of the invention, suppression of B Class gene expression by an altered DELLA protein results in female-only floral development in the plant.

Description

ALTERATION OF SEX DETERMINATION IN FLOWERING PLANTS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of US provisional application no. 61/721,665, filed on 2 November 2012, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 1, 2013, is named 5006.012PCTl_SL.txt and is 52,303 bytes in size.

BACKGROUND OF THE INVENTION

New cultivated varieties of seeds are developed by crossing existing isogenic or near isogenic lines. Frequently, the end result is a highly heterozygous (hybrid) seed line, which is sold directly to farmers and growers. Due to genetic recombination, the produced seed is not identical to the seed that is planted. Therefore, the farmer must repurchase seed annually. To produce large quantities of hybrid seed, companies must emasculate plants to control the crossing process. This typically requires a large amount of field space and the time consuming individualized manipulation of plants, which result in increases in time and cost to produce the desired seed products.

An alternative method of creating new seed varieties is genetic modification. However, public resistance to the development and use of genetically modified organisms (GMOs) remains strong. Public resistance to GMOs originates from the concern that genes may "escape" from cultivated fields, combine with wild relative species in natural settings, and cause detrimental effects to the wild species and the environment in general. The genetic "escape" is generally expected to occur through pollen transfer.

Improved methods for creating varieties of seeds are needed that are affordable, reliable and acceptable to the public.

SUMMARY OF THE INVENTION

Applicants have discovered methods for the production of unisexual plants. The invention provides methods of producing a unisexual male plant, where a genetically modified B Class floral identity gene is introduced into the genome of the plant, and where the expression of the modified B Class floral identity gene results in the suppression of the development of the fourth floral whorl, and the plant exhibits male-only floral development.

Further, provided are methods of producing unisexual male plants where the plants do not develop carpels, or where the carpals are completely absent or non-functioning. The methods also provide plants that exhibit functional, pollen producing stamen. Certain methods provide plants where the flowers of said plants contain artifacts of female flower organ development and do not produce seed. Also provided are methods that provide plants where artifacts of female flower organ development are absent in said plant, and where the plant is non-seed producing. Alternatively, the methods provide plants where artifacts of female flower organ development are present in the plant, and where the plant is non-seed producing.

Also provided is a method of producing a unisexual female plant, comprising inserting into the genome of a plant a modified nucleic acid sequence for a DELLA protein (SEQ ID NO: 9 or 10) in the SpGAI gene, where the modified sequence for the DELLA protein produces a modified DELLA protein (SEQ ID NO: 11), which suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development.

Also provided are methods of producing a unisexual female plant, comprising inserting into the genome of a plant a modified DELLA protein (SEQ ID NO: 11), which contains an amino acid encoding a deletion in the SpGAI gene, where the DELLA protein suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development.

Further, provided are methods of producing a unisexual female plant, comprising inserting into the genome of a plant an isolated, purified, modified DELLA protein sequence, wherein the modified DELLA protein sequence contains an amino acid deletion in the SpGAI gene (SEQ ID NO: 9 or 10), and wherein the modified DELLA protein sequence, when expressed, produces a modified DELLA protein (SEQ ID NO: 11), and wherein said modified DELLA protein suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development.

In certain embodiments, the modified DELLA protein (SEQ ID NO: 11) is not ubiquitinated and not degraded by the 28S proteasome. In other methods, the deletion is a deletion of the amino acid sequence DELLA VLGYKVRS S DM A (SEQ ID NO: 12) of the SpGAI gene. In other methods, the modified DELLA protein acts as an irreversible repressor protein. Also in the methods of the invention, the suppression of B Class gene expression results in the lack of development of stamen.

In other embodiments, provided is a method of producing a unisexual female plant, comprising inserting into the genome of a plant a genetically modified DELLA protein sequence, where said genetically modified DELLA protein sequence contains an amino acid deletion in the SpGAI gene, and where said genetically modified DELLA protein sequence produces a modified DELLA protein, and where said modified DELLA protein contains an amino acid deletion in the SPGAI gene, and where said modified DELLA protein suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development. In any of the methods, the plant is an angiosperm. In another method, the plant is a

Spinach plant. In an embodiment, the plant can be any plant.

In an embodiment, the methods of the claims are used for the production of targeted hybrid plants and seeds. In a further embodiment, a plant is provided that does not produce ornamental fruits. In another embodiment, the release of pollen from the plants produced by the methods is controlled or prevented. The invention also provides for the plants produced by any of the methods of the invention.

In certain embodiments, methods are provided for preventing the unauthorized use of seeds, comprising producing a unisexual male plant, comprising introducing into the genome of a plant a genetically modified B Class floral identity gene, wherein the expression of the modified B Class floral identity gene results in the suppression of the development of the fourth floral whorl, and the plant exhibits male-only floral development.

In other embodiments, methods are provided for preventing the unauthorized use of seeds, comprising producing a unisexual female plant, comprising inserting into the genome of a plant a DELLA protein containing an amino acid deletion in the SpGAI gene, wherein the expression of said DELLA protein containing an amino acid deletion in the SpGAI gene suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development. Provided is an isolated, purified recombinant polypeptide, comprising an amino acid sequence defined by SEQ ID NO: 8, 11, 12, 13, or 14, or an amino acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% identical to SEQ ID NO: 8, 11, 12, 13, or 14. The polypeptides provides herein can be DELLA proteins, or isolated, purified DELLA proteins, or genetically modified DELLA proteins, or portions thereof, or a combination thereof.

Provided is an isolated, purified recombinant polypeptide, comprising an amino acid sequence defined by SEQ ID NO: 11, or an amino acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO. 11.

Provided is an isolated, purified recombinant polypeptide, comprising an amino acid sequence defined by SEQ ID NO: 8, or an amino acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO. 8.

Provided is an isolated, purified recombinant polypeptide, comprising a nucleic acid sequence defined by SEQ ID NO: l, 2, 3, 4, 5, 6, 7, 9, or 10, or a nucleic acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, or 10.

Provided in an embodiment is an isolated, purified recombinant polypeptide, comprising an nucleic acid sequence defined by SEQ ID NO: 9 or 10, or a nucleic acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO: 9 or 10.

Also provided is an isolated, purified recombinant polypeptide, comprising an nucleic acid sequence defined by SEQ ID NO: 5, 6, or 7, or a nucleic acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO: 5, 6 or 7. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and

accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

Figure 1 depicts the floral development of spinach.

Figure 2 depicts pWSRi:SpGAI treated plants.

Figure 3 depicts a map of pWSRi.

Figure 4 illustrates the non-nucleotide substitution sequence evolution of AP3. Figure 4 discloses SEQ ID NOS 15 and 15-23, respectively, in order of appearance.

Figure 5 illustrates the non-nucleotide substitution sequence evolution of UFO. Figure 5 discloses SEQ ID NOS 24-34, respectively, in order of appearance.

Figure 6 illustrates floral phenotype on male inflorescences treated with MG132.

Figure 7 illustrates the results of in situ hybridization using SpAP3 (a-b) and SpAG (d-g) probes. Fig. 7a illustrates a stage 1 male. Fig.7b illustrates a stage 3 male. Fig. 7c illustrates a stage 3 female. Fig. 7d illustrates a stage 1 male. Fig. 7e illustrates a cross section of anther. Fig. 7f illustrates a stage 1 female. Fig. 7g illustrates a stage 4 female. OW: ovary wall; Mi:

microsporangia. Figure 8 illustrates results of functional testing of spinach floral homeotic genes. Fig. 8 a-c illustrates male flowers on male plants treated to silence SpPI. Fig. 8 d-e illustrate female and male flowers on plants treated to silence SpAG. Fig. 8a illustrates a hermaphroditic flower with 4 sepals, 4 stamens, and a central carpel. Fig. 8b illustrates a flower with 2 whorl three stamens, 2 whorl three carpels, and undifferentiated whorl four tissue. Fig. 8c illustrates a female flower among male flowers. Fig. 8d illustrates a female indeterminate flower with bractlike organs. Fig. 8 e illustrates a male flower with sterile whorl three organs on left and transformation to inflorescence on right.

Figure 9 depicts a schematic of hormone action through derepression: repressor degradation SCF complexes.

Figure 10 provides a diagram of single locus sex regulation.

Figure 11 illustrates a spinach female treated with pWSRi:SpGAI.

Figure 12 illustrates a spinach female treated with pWSRi:SpGAI.

Figure 13 illustrates the effect of SpGAI silencing in female spinach plants.

Figure 14 illustrates a spinach male treated with MG132.

Figure 15 illustrates a spinach male treated with MG132.

Figure 16 depicts the results of Y2H interactions. The "deltaDELLA" or "ADELLA" sequences referenced in this figure are disclosed as SEQ ID NOS 9-11.

Figure 17 depicts the BiFC interaction results.

Figure 18 provides a summary of in vivo protein-protein interactions. The "dDELLA" sequences referenced in this figure are disclosed as SEQ ID NOS 9-11.

Figure 19 depicts the results of in situ hybridization of pWSRi:SpPI treated plants. Figure 20 depicts a schematic of hormone action through derepression: JA and GA crosstalk defense/growth.

Figure 21 depicts a schematic of hormone action through derepression: TPL (auxin pathway) crosstalk.

Figure 22 provides a spinach sex determination model.

Figure 23 provides a spinach sex determination model.

Figure 24 provides a proposed sex determination model.

Figure 25 shows the internal and external cues that regulate floral development and sex expression. Figure 25D discloses SEQ ID NOS 15-23, respectively, in order of appearance.

Figure 26 provides photographs of the results of functional testing of spinach

feminization and masculinization pathways, a) Wildtype female spinach flower; b) Female plant silenced for DELLA protein (SpGAI) displaying stamens of the female flower; c) pair of flowers on a female plant with one wildtype female flower and one flower with a central stamen; d) male plant treated with proteasome inhibitor with female stigma visible within flowers.

Figure 27. Spinach B Class gene function and evolution, a). Photograph of Wild type male flower, b) Photograph of SpPI silenced male with hermaphroditic flower, c) Photograph of SpAP3 silenced male with multiple hermaphroditic flowers, d) Carboxyl end sequences of AP3 from multiple Chenopods.

Figure 28. Photographs of Arabidopsis thaliana transformed with constitutively expressed SpAP3. a. Flower showing stamen and reduced gynoecium. b. Inflorescences showing no developing siliques. c. Inflorescence showing no developing siliques.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a plant" includes a plurality of such plants. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. The term "about" can refer to a variation of + 5%, + 10%, + 20%, or + 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values and ranges proximate to the recited range that are equivalent in terms of the functionality of the composition, or the embodiment.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of reagents or ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into subranges as discussed above. In the same manner, all ratios recited herein also include all sub- ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, as used in an explicit negative limitation.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, in vitro, or in vivo.

An "effective amount" refers to an amount effective to bring about a recited effect.

The phrases "genetic information" and "genetic material", as used herein, refer to materials found in the nucleus, mitochondria and /or cytoplasm of a cell, which play a fundamental role in determining the structure and nature of cell substances, and capable of self- propagating and variation. The phrase "genetic material" of the present invention may be a gene, a part of a gene, a group of genes, DNA, RNA, nucleic acid, a nucleic acid fragment, a nucleotide sequence, a polynucleotide, a DNA sequence, a group of DNA molecules, double- stranded RNA (dsRNA), small interfering RNA or small inhibitory RNA (siRNA), or microRNA (miRNA)or the entire genome of an organism. The genetic material of the present invention may be naturally occurring.

As used herein, the term "nucleic acid" and "polynucleotide" refers deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 (1994).

Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. A "nucleic acid fragment" is a fraction or a portion of a given nucleic acid molecule. The terms "nucleic acid", "nucleic acid molecule", "nucleic acid fragment", "nucleic acid sequence or segment", or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.

The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

"Naturally occurring" is used to describe an object that can be found in nature as distinct from being artificially produced. For example, nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.

The invention encompasses isolated or substantially purified nucleic acid compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

The term "modified" as used herein refers to the alteration of genetic material, such genetic material includes but is not limited to DNA, RNA, and polymers thereof. The alteration or modification of the genetic material may be one or more deletions of genetic material, one or more additions or insertions of genetic material, one or more mutations of genetic material, and the like.

The term "chimeric" refers to any gene or DNA that contains 1) DNA sequences, including regulatory and coding sequences that are not found together in nature or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.

A "transgene" refers to a gene that has been introduced into the genome by

transformation and is stably maintained. Transgenes may include, for example, DNA that is either heterologous or homologous to the DNA of a particular cell to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.

A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions.

"Recombinant DNA molecule" is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook and Russell, Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (3.sup.rd edition, 2001).

The terms "heterologous DNA sequence," "exogenous DNA segment" or "heterologous nucleic acid," each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.

A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

"Wild- type" refers to the normal gene, or organism found in nature without any known mutation.

"Genome" refers to the complete genetic material of an organism.

A "vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

"Cloning vectors" typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.

"Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

"Coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein. An intron can also refer to the original sequence in the genomic DNA.

The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms

"initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides ( codon ) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

A "functional RNA" refers to an antisense RNA, ribozyme, or other RNA that is not translated. The term "RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect

complimentary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA" (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a single- or a double- stranded DNA that is complementary to and derived from mRNA.

"Regulatory sequences" and "suitable regulatory sequences" each refer to 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 enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences. As is noted above, the term "suitable regulatory sequences" is not limited to promoters. However, some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive promoters, tissue- specific promoters, development- specific promoters, inducible promoters and viral promoters.

"5' non-coding sequence" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner et al., Mol. Biotech., 3:225 (1995).

"3' non-coding sequence" refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.

"Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA -box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

"Constitutive expression" refers to expression using a constitutive or regulated promoter. "Conditional" and "regulated expression" refer to expression controlled by a regulated promoter.

"Operably-linked" refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

"Expression" refers to the transcription and/or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

"Transcription stop fragment" refers to nucleotide sequences that contain one or more regulatory signals, such as polyadenylation signal sequences, capable of terminating

transcription. Examples of transcription stop fragments are known to the art.

"Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not "chromosomally integrated" they may be "transiently expressed." Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus.

Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.

The terms "transfection" and "transformation", as used herein, refer to the introduction of foreign DNA into eukaryotic or prokaryotic cells, or the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

"Transformed," "transgenic," and "recombinant" refer to a host cell or organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) (1989). See also Innis et al., PCR Protocols, Academic Press (1995); and Gelfand, PCR Strategies, Academic Press (1995); and Innis and Gelfand, PCR Methods Manual, Academic Press (1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector- specific primers, partially mismatched primers, and the like. For example, "transformed," "transformant," and "transgenic" cells have been through the transformation process and contain a foreign gene integrated into their chromosome.

The term "untransformed" refers to normal cells that have not been through the transformation process. The genetic material of the present invention may be of eukaryotic (including plant kingdom members), prokaryotic, fungal, archaeal or viral origin.

As used herein, the term abortion refers to the termination of androecium or gynoecium development, resulting in nonfunctional organs.

As used herein, the term "unisexual plant" refers to a plant that is male or female, or having only one type of functional sexual organ. That is, the plant has the functional

reproductive organs of only one sex.

As used herein, the term "hermaphroditic plant" refers to a plant that is functionally male and female, or having both functional male and female sexual organs.

As used herein, the term "female flower" refers to a flower in which the gynoecium is functional and the androecium is absent or nonfunctional; alternate terms for the female flower include but are not limited to pistillate, carpellate or malesterile flower.

As used herein, the term "Male flower" refers to a flower in which the androecium is functional and the gynoecium is absent or nonfunctional; alternate terms for male flower include, but are not limited to staminate or female-sterile flower.

As used herein, the term Pistillode refers to an aborted gynoecium. This term is often used to describe structures that are visible but nonfunctional in mature flowers.

As used herein, the phrase "Sex-differentiation genes" refers to those genes that determine sexual function and lead to the production of differentiated gametes. In plants, these can be differentially expressed in organs, meristems and/or individuals.

Also used herein, the phrase "Sex-determination genes" refers to genes that determine exclusive male or female function in individuals and segregate with gender. As used herein, the phrase "Sexual system" refers to the distribution and function of gamete-producing morphological structures (the androecium and gynoecium) within and among individuals. 'Sexual system' is often confused with mating system, a term used to describe realized mating patterns (e.g. self-fertilization or out-crossing).

As used herein, the term Monoecy refers to male and female flowers borne on the same plant s used herein, the term Andromonoecy refers to male and hermaphroditic flowers borne on the same plant. As used herein, the term Gynomonoecy refers to female and hermaphroditic flowers borne on the same plant. As used herein, the term Dioecy refers to male and female flowers borne on separate plants. As used herein, the term Androdioecy refers to male and hermaphroditic flowers borne on different plants. As used herein, the term Gynodioecy refers to female and hermaphroditic flowers borne on different plants.

As used herein, the term "protogynous" refers to a hermaphroditic plant having the female reproductive organs come to maturity before the male reproductive organs.

As used herein, the term staminode refers to an aborted stamen. This term or similar terms are often used to describe structures that are visible but nonfunctional in mature flowers.

As used herein, the phrase "Type I flower" refers to a flower that is unisexual by abortion of organs. As used herein, the phrase "Type II flower" refers to a flower that is unisexual from inception.

As used herein, the phrase "DELLA protein" refers to a member of a family of proteins called DELLA proteins that negatively regulate plant responses to gibberillic acid. DELLA proteins represent a class of negative transcription factors that are degraded through a pathway that is regulated by the plant hormone gibberellic acid (GA). Early work (Chailakhyan and Khryanin, 1978) demonstrated that GA has a masculinizing effect on spinach female inflorescences.

As used herein, "DELLA protein SpGAIADELLA" (SEQ ID NO: 11) or "DELLA protein SpGAIdeltaDELLA" (SEQ ID NO: 11) refers to a DELLA protein which has been modified by Applicants. As used herein, SpGAI refers to a naturally occurring DELLA protein (SEQ ID NO: 8) found in spinach. In this instance, DELLA protein SpGAIADELLA or SpGAIdeltaDELLA (SEQ ID NO: 11) was prepared by inserting into the genome of a plant a modified nucleic acid sequence for a DELLA protein (SEQ ID NO: 9 or 10) containing an amino acid deletion in the SpGAI gene (SEQ ID NO: 12), where the modified DELLA protein (SEQ ID NO: 11), when expressed, suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development. DELLA protein SpGAIADELLA (SEQ ID NO: 11) is not ubiquitinated and not degraded by the 28S proteasome. The amino acid deletion can be a deletion of the amino acid sequence DELLA VLGYKVRS S DM A (SEQ ID NO: 12) of the SpGAI gene (SEQ ID NO: 5). DELLA protein SpGAIADELLA (SEQ ID NO: 11) can act as an irreversible repressor protein. The suppression of B Class gene expression results in the lack of development of stamen.

As used herein, the term "stamen" refers to the pollen-producing reproductive organ of a flower, usually consisting of a filament and an anther.

As used herein, the term "pistil" refers to the female, ovule-bearing organ of a flower, including the stigma, style, and ovary.

As used herein, the term "filament" refers to the stalk that bears the anther in a stamen.

As used herein, the term "anther" refers to the pollen-bearing part of the stamen. As used herein, the term "carpel" is one of the structural units of a pistil, representing a modified, ovule-bearing leaf.

As used herein, the term "non-seed producing" refers to plants that do not produce or bear seed. As used herein, the term "flower" refers to the reproductive structure of some seed- bearing plants, characteristically having either specialized male or female organs or both male and female organs, such as stamens and a pistil, enclosed in an outer envelope of petals and sepals.

The phrase "repressor protein" refers to a protein in which, in its binding to the operator, inhibits the transcription of one or more genes. A repressor protein can be reversible or irreversible.

The term hybrid, as used herein, refers to a plant or seed derived from the sexual reproduction involving two genetically distinct parents.

The phrase "ornamental fruits" refers to fruit that are borne on non-crop trees and that are not cultivated specifically to be harvested as its main function.

The term pollen, as used herein, refers to the microspores of seed plants; the powdery mass of microspores shed from anthers.

As used herein, the term "UFO" refers to the UNUSUAL FLORAL ORGANS gene in plants, including Arabidopsis.

As used herein, the "LFY" protein refers to the plant- specific LEAFY (LFY) protein. The LFY protein is necessary and sufficient for the vital switch from vegetative to reproductive development in angiosperm plant species. LFY controls the production of the flowers, which are formed in lieu of secondary inflorescences from the flanks of the shoot apical meristem. Because LFY is required for all of the major features that differentiate flowers from inflorescence branches, it is referred to as a meristem identity gene.

After initiating the meristem identity switch, LFY has a second role in the activation of the floral homeotic genes that specify the identity of organs in the flower. The two roles of LFY are separable genetically and molecularly. LFY exerts its developmental effects by means of transcriptional regulation; LFY has been shown to be a transcription factor in vivo.

Despite the critical importance of this regulator, only three direct targets of LFY have been identified. Two of the targets, AGAMOUS and APETALA3, are floral homeotic genes that act directly downstream of LFY in flower morphogenesis. Only one known direct LFY target gene product, APETALA1 (API), acts in the meristem identity pathway.

Bimolecular fluorescence complementation (BiFC) is a type of analysis that enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments.

Y2H, as used herein, refers to the yeast two-hybrid screening method.

MG132 refers to a proteasome inhibitor.

As used herein, the term "angiosperm" refers to any member of the more than 250,000 species of flowering plants (division magnoliophyta) having roots, stems, leaves, and well- developed conductive tissues (xylem and phloem). Angiosperms are the largest group of plants whose reproductive organs are in their flowers (flowering plants). Angiosperms are a superclass in the seed plants (Spermatophyta) division belonging to the vascular plants (Tracheophyta) phylum of the plant kingdom. The Angiosperms are divided into four groups: Basal

angiosperms (a mixed group including Amborella, water lilies, Austrobailey), Magnollids (including magnolia, laurel, wintergreen, spicebush), Monocots (including lily, iris, orchid, grasses), and Eudicots (including dandelion, roses, violet, tobacco) . Angiosperms are often differentiated from gymnosperms by their production of seeds within a closed chamber (the ovary). In angiosperms, the ovary wall is actually the fruit. Their ovules are enclosed in the carpel and pollen travels through the pollen tube to reach it.

One subclass of Core Eudicots is Caryophyllidae, which contains 3 orders

(Caryophyllales, Plumbaginales, and Polygonales) and at least 2600 accepted taxa overall. The Order Caryophyllales contains 11 families and over 1800 accepted taxa overall. At present, the families of Caryophyllales include Achatocarpaceae, Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Caryophyllaceae, Chenopodiaceae, Molluginaceae, Nyctaginaceae, Phytolaccaceae and Portulacaceae. The family Chenopodiaceae is commonly known as the goosefoot family, and includes such species as sugar beets, quinoa, tumbleweed, and spinach. At present, Chenopodiaceae contains 34 genera and 304 accepted taxa overall. One genus of

Chenopodiaceae is Spinacia, which includes Spinacia tetranda, Spinacia turkistanica, and Spinacia oleracea.

Development of unisexual plants

Applicants' invention provides methods for imparting desired traits in plants via genetic modification with enhanced safety to the consumer and environment. Applicants' methods utilize the advances made in genetically modifying plants and crops to the benefit of the consumers and growers, yet do so without the possibility of pollen escaping. Using Applicants methods, the desired GMO plants could be planted as female only, thereby not producing any pollen to escape. Similarly, female only lines can be planted with the possibility of increased yield per plant. By the use of Applicants' methods to produce unisexual varieties of selected parental lines, time, personnel, and space can be saved in the hybrid seed production process.

Although Applicants' technology was developed using Spinach species, the methods provided by the invention are applicable to a wide selection of plants.

B Class Floral Organ Identity genes (referred to as B Class genes from hereon) are identified as encoding transcription factors that are necessary for the development of petals (sterile organs) and stamen (male organs) in the flowers of flowering plants. Applicants have demonstrated that in the flowering crop plant, spinach, B Class genes have the original function for differentiating organ primordia (undifferentiated initial tissue growth) into stamen. Loss of activity of B Class Floral Organ Identity genes results in the formation of female carpels. However, the flowering crop plant spinach is uncommon in that it produces plants with flowers that either lack male organs (female plants) or female organs (male plants).

At present, unisex crops and plants are created using manual procedures which are costly, time consuming, and are burdensome to the environment as they generally require large areas of land. Applicants' invention provides alternative methods for the generation of strictly unisexual (male or female) plants for a variety of uses, including but not limited to agricultural and horticultural uses. Applicants' methods are applicable to and may be used in a broad range of plant species, both dicotyledonous (including major fruit and vegetable producing species) and monocotyledonous plants (including major grain producing species). In an embodiment of the invention, the unisexual plants produced by Applicants' methods can be used, for example, by seed companies for rapid production of hybrid seed lines, by crop scientists for research purposes, or by crop producers to increase yield and reduce gene transfer. Applicants' technology uses two independent genetic controls. The first control utilizes genes for suppression of carpel (female organ) development and which are also are required for stamen (male development). The second control uses a gene that suppresses the activation of the required male genes, and makes its activation controlled. In one embodiment, Applicants' two genetic controls can be used singly to produce male or female only lines. In an alternative embodiment, Applicants' genetic controls can be used in combination, to produce stable populations with males and females.

A. Suppression of Female Organ Development in Plants

Applicants have discovered that in addition to normal organ identity function, the spinach B Class genes have a unique property of suppressing the development of the female organs in their normal positions in the flower. Partial loss of spinach B Class function results in the generation of the female organs where they are normally absent in normal male spinach flowers. Mutations in the spinach B Class genes have resulted in specific

modifications to the encoded transcription factor proteins. These are used in the invention to generate carpel- suppressed (male) flowers in other species.

Applicants have discovered that in spinach, the male determining genes are expressed early in floral development only in males. Applicants' have unexpectedly discovered that the spinach genes differ from homologous genes in other organisms in that they are involved in suppression of the initiation of the female organs. The reduction of the expression of either of the two male genes, as discovered by Applicants, results in development of the female organ in spinach. Applicants have demonstrated, by sequence analysis that the carboxyl termini of both male determining genes in spinach differ from conserved domains in homologous proteins in other species. Applicants' invention uses a new complex of gene products from the B Class floral organ identity genes from spinach for directed regulation of flower development, leading to flowers with only stamens (male organs). As it targets nearly universally utilized genes in flower development, Applicants' invention has broad target potential.

In one embodiment, the invention provides for the production of unisexual varieties in multiple crop species for the efficient production of hybrid crop seed for sales. In a further embodiment, the invention provides for unisexual male horticultural plants that have the potential for developing ornamental varieties that do not produce nuisance fruits.

B. Suppression of Male Organ Development in Plants

Applicants have further demonstrated that spinach B Class genes are present in the female genome but are not expressed. Applicants have also shown that the plant hormone, gibberellic acid (GA), can cause female plants to develop male flowers. Applicants have subsequently demonstrated that the repressor protein SpGAI, which is a target for degradation in the normal GA regulatory pathway, contributes to suppression of B Class expression and hence stamen development. Applicants' invention utilizes controlled expression of a SpGAI protein to suppress stamen development in other plant species and hence generate unisexual female flowers. The SpGAI protein is not ubiquitinated and not degraded by the 28S proteasome

The invention uses a new complex of gene products from the GA regulatory pathway for directed regulation of flower development. As the invention targets nearly universally utilized genes in flower development, it has broad target potential.

It is known that the plant hormone gibberellic acid (GA) can masculinize female plants. Applicants have demonstrated that applications of a GA inhibitor feminize male plants.

Applicants have further isolated an inhibitor protein that is involved in the GA response pathway and have demonstrated that reduction of the expression of the gene encoding the inhibitory protein results in the production of male organs in females. This result implies that the inhibitor protein blocks the expression of the male genes. Applicants have tested the effects of a protein degradation inhibitor and found that female flowers can develop on male plants. Applicants' results support the hypothesis that sex determination can be controlled by alternative repression of the male determining genes in females or degradation of the repressor and thus de-repression of the male genes in males.

Applicants' invention provides the ability to generate unisexual flowers in a variety of plant species. In one embodiment, the invention provides for the production of unisexual varieties in multiple crop species for the efficient production of hybrid crop seeds. In another embodiment, the invention provides unisexual female plants that have the potential for increased yields in many plant species. In a further embodiment, the invention provides female only plants that can be used to prevent gene escape through pollen release.

Applicants have cloned the male determining genes into vectors, which are used to transform plants in a stable manner. Applicants have produced stable, transgenic plants with the male spinach genes.

Applicants have cloned the female determining gene encoding the repressor protein. Applicants have also generated a mutant variant that lacks the degradation signal and therefore is that is not ubiquitinated and not degraded by the 28S proteasome. Applicants have created inducible vectors for the purpose of plant transformation.

Applicants have determined that feminization must be related to suppression or inactivation of B Class genes. Feminization can be modulated by applications of GA.

Feminization does not involve sex chromosomes. Feminization must entail upstream regulatory pathway of B Class genes (LFY plus UFO). Additionally, Applicants have determined that DELLA protein transcription repressors are GA insensitive.

Applicants have also determined that B Class genes are expressed in males only (stamen identity function, fourth whorl suppression function). Also, Applicants have determined that C class genes are expressed in both sexes (stamen and ovule function, meristem determination).

The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

EXAMPLES

Example 1. The Feminization Pathway: Identification and Role of DELLA Protein

SpGAI in Spinach Feminization

Applicants hypothesized that female suppression of male development is achieved by suppression of male functions through the activity of the DELLA proteins. The masculinizing effect of GA in spinach then would be manifested by the degradation of the repressive DELLA proteins post-translation.

To determine the mechanism of this effect, and hence to uncover the feminization pathway in spinach, Applicants isolated and purified a 2229 bp fragment that includes the complete coding region of a GAI homologue (a DELLA gene)(SEQ ID NO: 5) from spinach.

The translated protein (SEQ ID NO: 8) is 616 amino acids long and has the conserved GID1 binding motifs DELLA (SEQ ID NO: 13) and VHYNP (SEQ ID NO: 14). BLAST searches indicate low E values (0 to e-160) with other GAI or DELLA proteins, and simple phylogenetic analysis (PhylML) shows the spinach sequence as a sister to the Glycine max GAIL RT-PCR experiments using total RNA from male and female inflorescences resulted in positive amplification from tissue of both genders, indicating that the gene is expressed in both genders.

As a preliminary test, Applicants suppressed the expression of the targeted DELLA protein in female plants through the use of a gene silencing vector previously developed in our laboratory (Golenberg et al., 2009). A 400bp fragment of SpGAI from the variable region 3' of the DELLA sequence (but including the VHYNP (SEQ ID NO: 14) encoding sequence) was subcloned into Applicants' silencing vector, pWSRi (Fig. 3). (See Golenberg, E.M. et al, "Development of a Gene Silencing DNA Vector Derived from a Broad Host Range

Geminivirus," Plant Methods, 5:9 (2009), which is incorporated by reference in its entirety herein). Plants were biolistically treated with the engineered vector at the two to four leaf stage of development.

Many of the plants displayed stunted growth and some necrosis starting at the margins of the leaves. From these experiments, plants that have flowered and had exclusively male flowers are wild-type. On plants that appear to be female, the predominant majority of the flowers are phenotypically wild-type (Figures 2a, 2b, 26a, and 26b). However, a few flowers develop anthers.

In figure 2a and 26b, a dehisced anther on a long filament can be seen among a small cluster of female flowers. In figure 2b and 26c, two adjacent flowers are seen in a cluster. The leftmost flower is a wild- type female with two sepals and a single central gynoecium. The rightmost flower has two sepals but a single, central stamen (male organ). The remainder of the flowers in the background and foreground are normal female flowers. On additional plants, female, male, and hermaphroditic flowers have been found (Figure 2c, Fig. 26d). Thus, male flowers or male floral organs are able to be detected on predominantly female plants, using the methods developed by Applicants.

Applicants have caused homeotic changes among female flowers as opposed to homeotic changes in male flowers as Applicants have shown previously. Causing homeotic changes among female flowers is a significant advance, as have silenced a non-floral organ identity gene and obtained floral organ identity transformation. These results demonstrate the functional relationship between the masculinization effect of GA on females and the suppression of male floral organs in females is due to a role of DELLA proteins in regulating male development expression.

Example 2: The Role of Proteasome Mediated Protein Degradation on Spinach Floral

Development

As an additional, independent test of Applicants' model in which DELLA protein SpGAI (SEQ ID NO: 8) acts as a repressor of B Class expression, solutions of the proteasome inhibitor MG132 (MG132 is a chemical inhibitor of the expected protein degradation system) were applied directly to emerging inflorescences. Because B Class expression in males results from the GA activated proteosomal degradation of SpGAI, it was expected that inhibition of the proteasome would have a feminizing effect.

As in previous experiments, treated plants displayed a variety of phenotypes. No morphological transformations were detected in female plants treated with MG132. In male plants, there were hermaphroditic flowers with four stamen and a central carpel (Fig. 6a) and female flowers (Fig. 6b). From the sample, homeotic transformations from stamens to carpels were not detected The results of MG132 applications are comparable with results gained through the direct silencing of the male organ identity genes SpPI and SpAP3 (Sather et al., 2010). Therefore, the results of proteasome inhibition in the male flowers recapitulate the suppression of B Class expression as predicted in the model.

Example 3: Development of Commercialization of Feminization of Crop Plants

The invention of the floral feminization process is based on the controlled expression of a

DELLA protein that is non-degradable by the proteasome pathway in the floral initials. The DELLA protein, DELLA Protein SpGAI (SEQ ID NO: 8), acts as an irreversible repressor protein that suppresses B Class gene expression in the flowers. The absence of expressed B Class protein results in the lack of development of stamens, the male floral organs, and hence feminizes the flower.

Applicants have engineered a modified SpGAI DELLA protein (DELLA protein

SpGAIdeltaDELLA) (SEQ ID NO: 11) that encodes a DELLA protein that is not ubiquitinated and not degraded by the 28S proteasome. Applicants cloned DELLA protein

SpGAIdeltaDELLA into the binary vector pMDC7. Specifically, a full length SpGAI coding gene and a SpGAI gene with a 17 amino acid deletion spanning the sequence

DELLA VLGYKVRSSDMA (SEQ ID NO: 12) were cloned into pDONR221. This sequence was recombined into the binary vector pMDC7. This vector has a kanamycin resistance gene for bacterial selection and a hygromycin resistance gene for selection in plants. The expression of the inserted DELLA gene in transformed plants is controlled by an estradiol promoter. The plasmid was transformed into ID 1428 Agrobacterium strain. Arabidopsis thaliana Col-0 plants were treated with the transformed Agrobacterium. Harvested seed can be screened for transformants. Transformants can be screened by PCR. Transcription can be verified by spinach sequence specific RT-PCR. Transformants can be treated with estradiol to induce expression of the modified spinach DELLA proteins (SEQ ID NO: 11) at the induction of flowering. Plants may be scrutinized for aberrant petal and/or stamen development.

Induction of the DELLA protein that is not ubiquitinated and not degraded by the 28S proteasome will suppress male floral organ development and hence produce female only flowers. Parallel studies can be executed in Nicotiana benthamiana. This technology is beneficial to agriculturally and commercially important crop species.

Example 4: The Masculinization Process: Identification and Function of B Class Floral

Identity Protein SpAP3 and SpPI in Spinach Masculinization

B Class floral organ identity genes act to trigger the development of stamen, the male reproductive organs in flowering plants. Two B Class floral organ identity genes (referred to as B Class genes), SpAP3 and SpPI, are expressed in male flower primordia prior to initiation of floral organ primordia, but are not expressed in females at any developmental stage (Pfent, et al., 2005). Sequence analyses detected no allelic differentiation in coding and non-coding regions, including 5' upstream regions, between male and female plants in the cultivar America (Sather et al., 2010), indicating that alternative expression of B Class genes in spinach is regulated by transacting epistatic factors.

Spinach orthologues of the Arabidopsis B (AP3 and PI) and one C class (AG) genes from cDNA (Pfent et al., 2005; Sather et al., 2005) have been isolated and cloned. In situ

hybridization studies detected nearly identical patterns of expression of SpAP3 and SpPI. In males, both genes are expressed throughout the floral meristem with the exception of the LI cell layer before any organ primordia are formed (Figure 7a). As the flower develops, B Class genes are expressed in the early sepal primordia, then in the early stamen primordia, and then finally become restricted to the microsporangial and tapetal cells in the locules of the anthers (Figure 7b). No expression is found in the central area of the flower.

In contrast, B Class gene expression could not be detected at any stage of development in female flowers by in situ hybridization (Figure 7c). In comparison, SpAG is expressed at the earliest initiation of the floral meristem in both males and females (Figure 7d, f) and thus differs in this early pattern from both Arabidopsis and Antirrhinum homologues. As the flowers develop, SpAG is expressed in the microsporangia and in the nucellus in a sex-specific pattern (Figure 7e, g). Thus, in spinach, there is a novel region of expression that is sex specific and independent of the presence/absence of the organs involved.

Additionally, these results indicate that B Class expression in spinach precedes stamen development and is temporally correlated with sexual dimorphism.

To test the function of the floral homeotic genes, a VIGS (pWSRi) vector was developed that can silence the individual genes in spinach (Golenberg et al., 2009). Female plants bombarded with pWSRi:SpPI displayed no readily observable phenotypic abnormalities, as would be expected based on previous expression studies. Male plants developed with a range of flower morphologies, from normal males, hermaphrodites (having third whorl stamens and a single fourth whorl carpel, Figure 8a), mixed third whorl stamens and carpels (Figure 8b), flowers with four carpels in the third whorl, and completely normal appearing female flowers (two sepals, one carpel, Figure 8c). pWSRi:SpAP3 treated plants displayed similar ranges of phenotypes. SpAG silenced male plants have undeveloped stamen consistent with the microsporangial tissue expression patterns determined in the in situ studies, or fully sterile third whorl organs (flower to the left in Fig. 8e).

In female SpAG silenced plants, flowers have been detected with multiple whorls of bract-like organs sporting trichomes and no reproductive organs (Figure 8d). These flowers are phenocopies of Arabidopsis triple ag ap3 ap2 mutants (Bowman et al., 1989), consistent with the expectation that B Class genes are normally not expressed in females and the lack of expression of spinach APlhomologues in sepals (Sather and Golenberg, 2009). Additionally, both male and female pWSRi:SpAG treated plants were found with floral indeterminacies in which new inflorescences develop instead of flowers (Figure 8e).

Thus, RNAi knockdown experiments demonstrated that spinach B Class genes have acquired a masculinizing function in addition to the stamen identity function described in other species. Knockdown of SpAP3 and SpPI resulted in flowers with a developing fourth whorl organ indicating that SpAP3 and SpPI normally act to suppress fourth whorl and hence gynoecial development (Fig. 27A-C) (Sather et al., 2010). In summary, these results demonstrate that B Class genes affect organ identity, organ number, and, in extreme phenotypes, gender. In comparison, SpAG controls organ identity, pollen production, and floral determinacy in both males and females (Sather et al., 2010).

In Applicants' original reports on spinach B Class genes (Pfent et al., 2005), it was noted that both genes had deletion mutations in the 3' end of the gene that resulted in the truncation or complete loss of conserved angiosperm motifs. Applicants speculated that such mutations may be the masculinizing mutations and that they would not be present in closely related non- dioecious species. Recent results (Naeger and Golenberg, unpublished) confirm that the proposed deletion mutations are found only in Spinacia species, but not in closely related hermaphroditic or gynomonoecious species (Fig. 27D).

Example 5: Development of Commercialization of Masculinization of Crop Plants.

The invention of the masculinizing process is based on the novel effect of the B Class floral identity proteins to suppress development of the fourth floral whorl where the female organs develop. Mutations in the carboxyl end of the protein that have caused the loss of highly conserved motifs in these proteins likely contribute to the new specialized function.

Applicants have cloned spinach B Class genes and used these genes to genetically transform Agrobacterium. The full length spinach B Class floral organ identity genes SpAP3 and SpPI have been cloned into pDONR221 and pDONRZeo. Additionally, a chimeric gene with the spinach SpPI gene with its unique carboxyl end removed and replaced by the Arabidopsis PI carboxyl end has been constructed. These constructs have been recombined into the pEARLEYGATElOO binary vector. This vector has kanamycin bacterial selection and glufosate (BASTA) plant selection systems.

In one example, the cloned spinach B Class genes are under the control of a CaM35S constitutive promoter. The CaM35S promoter is one promoter out of many promoters that can be used in the present methods and systems of the invention.

The CaM35S promoter regulates transcription of the insertion genes, and activates expression of the genes throughout the growth of the plant in all tissues. (B Class floral organ identity genes are reported to have no effect in non-floral tissues.) The plasmids have been transformed into the GV3101 Agrobacterium strain. Arabidopsis thaliana Col-0 plants have been treated with the transformed Agrobacterium. Transformants are screened by PCR.

Transcription is verified by spinach sequence specific RT-PCR. Plants are scrutinized for flower development. Harvested seed from transformed Arabidopsis plants have been screened for transformants. Two transformed plants (with the native spinach B Class gene SpAP3) with distinctive phenotypes have been identified. Flowers develop with all four normal floral organs (sepals, petals, stamens, and carpels). The stamen show dishissed pollen (Fig. 28a). The carpels while present, appear reduced. When the plant is allowed to grow to maturity, multiple flowers develop on the inflorescences. However, there is no development of siliques (fruit) or seeds in these individuals (Figure 28b and 28c). These plants have been masculinized by the expression of the spinach B Class gene SpAP3. The lack of transformation of the carpel into male organs indicates that the suppression of fruit and seed set is not due to homeotic transformation of the carpels, but rather appears to be due to suppression of female reproductive function. Hence, these results establish proof-of-concept in novel transformed plants.

References:

1. Sather et al., "Duplication of API within the Spinacia oleracea L. AP1/FUL clade is followed by rapid amino acid and regulatory evolution," Planta (2009) 229: 507-521.

2. Golenberg et al., "Development of a gene silencing DNA vector derived from a broad host rang geminivirus," Plant Methods (2009) 5 (9): 1-14.

3. Diggle et al., "Multiple developmental processes underlie sex differentiation in angiosperms," Trends in Genetics (2011) 27 (9): 368-376.

4. Sather et al., "Sequence evolution and sex-specific expression patterns of the C class floral identity gene, SpAGAMOUS, in dioecious Spinacia oleracea L," Planta (2005) 222: 284- 292.

5. Pfent et al., "Characterization of SpAPETALA3 and SpPISTILLATA, B Class floral identity genes in Spinacia oleracea, and their relationship to sexual dimorphism," Dev Genes

Evol (2005) 215: 132-142.

6. Chailakhyan, M. K., and V. N. Khryanin. "Effect of growth regulators and role of roots in sex expression in spinach, " Planta (1978) 142: 207-210. 7. Sather, D. N., et al. "Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism," BMC Plant Biol (2010) 10: 46.

While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A method of producing a unisexual male plant, comprising introducing into the genome of a plant a genetically modified B Class floral identity gene, wherein the expression of the modified B Class floral identity gene results in the suppression of the development of the fourth floral whorl, and the plant exhibits male-only floral development.

2. The method of claim 1 , wherein the plant is an angiosperm.

3. The method of claim 1 , wherein the plant does not develop carpels.

4. The method of claim 1 , wherein the plant exhibits functional, pollen producing stamen.

5. The method of claim 1 , wherein the carpals of the plant are completely absent or are non-functioning.

6. The method of claim 1, wherein the flowers of said plant contain artifacts of female flower organ development and are non-seed producing.

7. The method of claim 1, wherein artifacts of female flower organ development are absent in said plant, and wherein the plant is a non-seed producing plant.

8. The method of claim 1, wherein artifacts of female flower organ development are present in the plant, and wherein the plant is a non-seed producing plant.

9. The method of claim 1, wherein the B Class floral identity gene is SpAP3 or SpPI.

10. A method of producing a unisexual female plant, comprising inserting into the genome of a plant a genetically modified DELLA protein sequence, wherein said genetically modified DELLA protein sequence contains an amino acid deletion in the SpGAI gene, and wherein said genetically modified DELLA protein sequence produces a modified DELLA protein, and wherein said modified DELLA protein contains an amino acid deletion in the SPGAI gene, and wherein said modified DELLA protein suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development.

11. The method of claim 10, wherein the plant is an angiosperm.

12. The method of claim 10, wherein the DELLA protein is not ubiquitinated and not degraded by the 28S proteasome.

13. The method of claim 10, wherein the amino acid deletion is a deletion of the amino acid sequence DELLA VLGYKVRS SDM A (SEQ ID NO: 12) of the SpGAI gene.

14. The method of claim 10, wherein the modified DELLA protein acts is DELLA protein SpGAIdeltaDELLA (SEQ ID NO: 11).

15. The method of claim 10, wherein the modified DELLA protein acts as an irreversible repressor protein.

16. The method of claims 1-16, wherein the suppression of B Class gene expression results in the lack of development of stamen.

17. The use of the methods of claims 1-16, for the production of targeted hybrid plants and seeds.

18. The use of the methods of claims 1-16, wherein the plant does not produce ornamental fruits.

19. The use of the methods of claims 10-15, wherein the release of pollen from the plant is controlled or prevented.

20. The plant produced by any of the methods of claims 1-16.

21. A method to prevent the unauthorized use of seeds, comprising producing a unisexual male plant, comprising introducing into the genome of a plant a genetically modified B Class floral identity gene, wherein the expression of the modified B Class floral identity gene results in the suppression of the development of the fourth floral whorl, and the plant exhibits male-only floral development.

22. A method to prevent the unauthorized use of seeds, comprising producing a unisexual female plant, comprising inserting into the genome of a plant a DELLA protein containing an amino acid deletion in the SpGAI gene, wherein the expression of said DELLA protein containing an amino acid deletion in the SpGAI gene suppresses B Class gene expression in the flower of the plant, and the plant exhibits female-only floral development.

23. A method of preventing the unauthorized use of seeds, comprising producing a unisexual male plant as in any of claims 1-9.

24. A method to prevent the unauthorized use of seeds, comprising producing a unisexual female plant as in any of claims 10-15.

25. An isolated, purified recombinant polypeptide, comprising an amino acid sequence defined by SEQ ID NOS. 8, 11, 12, 13 or 14, or an amino acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NOS. 8, 11, 12, 13 or 14.

26. An isolated, purified recombinant polypeptide, comprising an amino acid sequence defined by SEQ ID NO: 11, or an amino acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO. 11.

27. The polypeptide of claim 26, wherein the polypeptide is a modified DELLA protein.

28. An isolated, purified recombinant polypeptide, comprising an nucleic acid sequence defined by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, or 10, or a nucleic acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, or 10.

29. An isolated, purified recombinant polypeptide, comprising an nucleic acid sequence defined by SEQ ID NO: 9 or 10, or a nucleic acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO: 9 or 10.

30. An isolated, purified recombinant polypeptide, comprising an nucleic acid sequence defined by SEQ ID NO: 5, 6, or 7, or a nucleic acid sequence which is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to SEQ ID NO: 5, 6 or 7.