Apomictic plants and production thereof

Field of the invention

The present invention relates to novel transgenic apomictic plants, seeds and/or methods of the production of such transgenic apomictic plants. The instant means and methods are useful for plant breeding, particularly in maintaining heterozygosity in hybrid plants and/or in the production of hybrid seeds.

Background of the invention

Apomixis refers to asexual reproduction leading to the production of seeds without meiosis and fertilization, that is to offspring which is genetically identical to the mother plant (Koltunow, et al., (1995) Plant Physiol. 108: 1345-1352; Koltunow and Grossniklaus (2003) Ann. Rev. Plant Devi. 54: 547-74; Ravi, et al, (2008) Nature 451 : 1121-4). Apomixis is thus a reproductive process that bypasses female meiosis and syngamy to produce viable seeds containing embryos identical to the maternal parent. Apomixis increases the opportunity for developing superior gene combinations and facilitates the rapid incorporation of desirable traits. Apomixis not only provides reproductive assurance, but also avoids a loss of heterozygosity in the offspring because the offspring maintains the parental genotype. Apomixis therefore avoids the effects of loss of vigor due to inbreeding and may additionally confer some advantages because of the heterosis effects. Apomictic hybrids are true-breeding hybrids because seed-derived progeny of an apomictic plant are genetically identical to the maternal parent. In other words, apomixis allows the perpetual self-reproduction of hybrids because the progeny is clonal in origin.

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants. At the species level, apomixis occurs in less than 1 % of the species. Apomixis occurs in many wild species and in a few agronomically important species such as citrus and mango, but not in any of the major cereal crops (Eckhardt, (2003) Plant Cell 15: 1449- 1501). One form of apomixis is adventitious embryony (also known as sporophytic apomixis), where embryos are formed directly out of somatic tissues within the ovules outside the embryo sac. Adventitious embryony usually occurs in parallel to normal sexual reproduction. A second form of apomixis is diplospory, which displaces sexual reproduction because it takes place in the cell that usually would undergo meiosis. In diplospory, however, this cell aborts or omits meiosis and forms a non-reduced, nonrecombined egg cell which then goes through a process called parthenogenesis (embryogenesis without fertilization) to form a clonal embryo. A third form of apomixis is apopsory, which like adventitious embryony initiates in tissues outside the sexual embryo sac. Apospory involves the formation of an asexual, non-reduced and nonrecombined embryo sac whose egg cell - as in diplospory - goes through parthenogenesis to form a clonal embryo. Diplospory and apospory are collectively referred to as gametophytic apomixis. All three forms of apomixis rely on the production of an embryo without fertilization. Because apomixis offers the promise of the fixation and indefinite propagation of a desired genotype, including that of F1 hybrids, there is a great deal of interest in engineering this ability to produce clonal seeds into crops, especially cereals (Spillane, et al, (2001) Sex. Plant Reprod. 14: 179-87; Spillane, et al, (2004) Nat. Biotechnol. 22:687-91).

A molecular approach to engineer apomixis in commercial plant lines is highly desirable as a self-reproducing hybrid (SRH) system can result in a significant acceleration of the breeding process and a strong reduction in hybrid seed production costs. In other crops, such as soybean or wheat, it can result in the production increase and sale of hybrid seeds, something which has not been done. There are three key components to apomixis: (1) avoidance of meiosis (apomeiosis); (2) parthenogenic development of the embryo; and (3) achieving functional endosperm formation (Grossniklaus et al., (1998), in The Flowering of Apomixis: from Mechanisms to Genetic Engineering, eds. Savidan, Carman, Dresselhaus (CIMMYT, IRD, European Commission DG VI (FAIR), Mexico, DF), pp. 168-211 ; Grossniklaus, (2001), in Advances in Hybrid Rice Technology. Proceedings of the 3rd International Symposium on Hybrid Rice 1996, eds. Virmani, Siddiq, Muralidharan (International Rice Research Institute, Manila), pp. 187-211 ).

WO 2016/179522 discloses certain methods of producing viable non-reduced or viable non-reduced and non-recombined gametes.

WO 2015/061355 discloses certain methods of achieving propagation from one or more gametophytic or sporophytic cells in an ovule of a flowering plant in the absence of egg cell fertilization.

Khanday et al. (Nature, 2019, 565, 91) discloses certain rice embryogenic trigger redirected for asexual propagation through seeds.

Summary of the invention

It is an object of the present invention to provide a self-reproducing hybrid plant as well as methods and means of the production of such plants. In particular, the self reproducing hybrid plant should be configured to provide clonal seeds, genetically identical with the hybrid mother plant. It is a further object to provide a self reproducing plant wherein propagation of the plant is possible for several consecutive generations.

The technical problem is solved by the embodiments provided herein and as characterized in the claims.

The present invention concerns a transgenic apomictic plant, wherein an endogenous Nrf4 (Non-reduction in female4) gene is disrupted and a BBML (BABY BOOM-LIKE) transgene is expressed in the embryo sac (female gametophyte) or egg cell. It is noted that disrupting an endogenous Nrf4 and expressing a BBML transgene cannot currently be obtained exclusively by means of an essentially biological process. Systems for producing clonal seeds are known in the prior art; however, typically plants resulting from such seeds cannot be propagated. The present inventors have surprisingly found that propagation of a plant assuring phenotypic uniformity over apomictic generations (as it was demonstrated in natural apomicts) is possible over several consecutive generations if an endogenous Nrf4 gene is disrupted, leading to diplospory, and a BBML transgene is expressed in the egg cell or embryo sac, leading to parthenogenesis.

The present invention can be summarized in the following aspects. In a first aspect, the present invention relates to a transgenic apomictic plant, the plant characterized in that (a) an endogenous Nrf4 gene is disrupted; and (b) a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 is expressed preferably in the egg cell or embryo sac, preferably with the proviso that the plant is not exclusively obtained by means of an essentially biological process, preferably wherein the polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 has essentially retained the functionality of the polypeptide of SEQ ID NO.: 1.

In a particular aspect, the present invention relates to a transgenic apomictic plant further characterized in that the endogenous Nrf4 gene is disrupted by inhibiting expression and/or activity of a protein encoded by the endogenous Nrf4 gene.

In a further particular aspect, the present invention relates to a transgenic apomictic plant further characterized in that in addition to the endogenous Nrf4 gene, at least one other gene involved in meiosis is disrupted.

In a particular aspect, the present invention relates to a transgenic apomictic plant further characterized in that in addition to the endogenous Nrf4 gene, at least one other gene involved in recombination is disrupted.

In a further particular aspect, the present invention relates to a transgenic apomictic plant, wherein (a) is achieved by introducing a recombinant nucleic acid construct comprising a nucleotide sequence encoding an element that silences Nrf4 activity.

In yet further particular aspect, the present invention relates to a transgenic apomictic plant, wherein (b) is achieved by transforming the plant with a recombinant nucleic acid construct encoding a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 .

In a further particular aspect, the present invention relates to a transgenic apomictic plant, wherein the recombinant nucleic acid construct encoding a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 further comprises one or more untranslated region(s) (UTR) and optionally a promoter.

In yet further particular aspect, the present invention relates to a transgenic apomictic plant, wherein the plant is selected from maize, wheat, barley, sorghum, rye, oat, millet, turf grass, switchgrass, sugar cane, banana and rice.

In another aspect, the present invention relates to a seed of the transgenic apomictic plant of the present invention.

In another aspect, the present invention relates to a method for the production of an apomictic plant, comprising (a) providing a plant or a plant cell; (b) disrupting an endogenous Nrf4 gene in the plant or the plant cell; (c) inducing the expression of a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell; and (d) if the plant cell was used, regenerating the plant from the plant cell.

In a particular aspect, the present invention relates to a method for production of an apomictic plant, wherein the plant in (a) is selected from maize, wheat, barley, sorghum, rye, oat, millet, turf grass, switchgrass, sugar cane, banana and rice.

In a further particular aspect, the present invention relates to a method for production of an apomictic plant, wherein in (b) the endogenous Nrf4 gene is disrupted by inhibiting expression and/or activity of a protein encoded by the endogenous Nrf4 gene.

In again a further particular aspect, the present invention relates to a method for production of an apomictic plant, further comprising the step of disrupting at least one other gene involved in meiosis.

In again a further particular aspect, the present invention relates to a method for production of an apomictic plant, further comprising the step of disrupting at least one other gene involved in recombination. In a further particular aspect, the present invention relates to a method for production of an apomictic plant, wherein (b) is achieved by introducing a recombinant nucleic acid construct comprising a nucleotide sequence encoding an element that silences Nrf4 activity.

In again a further particular aspect, the present invention relates to a method for production of an apomictic plant, wherein (c) is achieved by transforming the plant with a nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1.

In again a further particular aspect, the present invention relates to a method for production of an apomictic plant, wherein the recombinant nucleic acid construct encoding a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 further comprises one or more untranslated region and optionally a promoter.

In another aspect, the present invention relates to an apomictic plant obtained according to the methods for production of an apomictic plant of the present invention.

As used herein, the term "plant" can include inclusively, as context indicates, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and/or plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, flowers, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and/or the like.

The terms "control", "control plant" or "control plant cell" are used interchangeable herein. It provides a reference point for measuring changes in phenotype of the subject plant provided herein and may be any suitable plant. A control plant may comprise, for example: (a) a wild-type or native plant, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the plant of the invention; (b) a plant of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant which is a non-transformed segregant among progeny of a plant of the invention; (d) a plant which is genetically identical to the plant of the invention but which is not exposed to the same treatment (e.g., herbicide treatment) as the plant of the invention; (e) the plant of the invention itself, under conditions in which the gene of interest is not expressed, in particular wherein a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 is not expressed and/or (f) a plant wherein the endogenous Nrf4 gene is not disrupted, in particular by inhibiting expression and/or activity of a protein encoded by the endogenous Nrf4 gene, for example which is not transformed with a construct containing a silencing element.

As used herein, an “apomictic plant” is defined as a flowering plant capable of asexually forming seeds that upon germination would give raise to a plant that genetically is a copy of a maternal plant. Such seeds are also referred to as clonal seeds. Therefore, in other words, an apomictic plant is a plant capable of producing clonal seeds. In nature, apomictic plants typically form seeds from either sporophytic or gametophytic tissues of the ovule, avoiding the processes of meiosis and fertilization that typically lead to embryo development. As used herein, the term “apomictic plant” also relates to a plant wherein apomixis was engineered, preferably by means of genetic engineering or genetic manipulations. Such a plant may also be referred to as a transgenic apomictic plant. As it will be known to the skilled person, in certain apomictic plants while an embryo can form from maternal tissue, fertilization may still be required for seeds to develop, in particular in order for the endosperm to maintain the correct genetic material content and develop correctly.

As used herein, the term "transgenic" describes a non-naturally occurring plant that contains a genome modified by man, wherein the plant includes in its genome a nucleic acid molecule, which can be derived from the same or a different species, including non-plant species. The nucleic acid molecule can be a gene regulatory element such as a promoter, enhancer, or other regulatory element, or can contain a coding sequence, which can be linked to a native or heterologous gene regulatory element. T ransgenic plants that arise from sexual crossing or by selfing are descendants of such a plant.

The term polynucleotide can include the terms "nucleic acid", "nucleic acid sequence", and "oligonucleotide", as those terms are generally understood in the art. What is included in a specific instance will be appreciated by the person of skill in the art as indicated by the context, but where no particular context limits the scope, then the term will be understood to be broadly inclusive. Therefore, the term polynucleotide can also include DNAs and/or RNAs that contain one or more modified bases. Thus, DNAs and/or RNAs with backbones modified for stability or for other reasons are "polynucleotides", as that term is intended herein. Moreover, DNAs and/or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically and/or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

As used herein, an "isolated" or "purified" polynucleotide or polypeptide or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or polypeptide 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. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e. , sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A polypeptide that is substantially free of cellular material includes preparations of polypeptides having less than about 30%, 20%, 10%, 5% or 1 % (by dry weight) of contaminating protein. When the polypeptide of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1 % (by dry weight) of chemical precursors or non-protein-of-interest chemicals. As used herein, a polynucleotide or polypeptide is "recombinant" when it is at least partially artificial or engineered or derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A polypeptide expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example, a variant of a naturally occurring gene is recombinant.

As used herein, the term “recombinant nucleic acid” relates to a polynucleotide produced by recombinant DNA technology. In one embodiment a recombinant polynucleotide may be produced by separation from substantially all other molecules normally associated with it in its native state. A recombinant nucleic acid may be greater than 60% free, greater than 75% free, greater than 90% free, or greater than 95% free from the other molecules (exclusive of solvent) present in the natural mixture. In another embodiment, a recombinant nucleic acid may be separated from nucleic acids which normally flank the polynucleotide in nature. Thus, polynucleotides fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered recombinant nucleic acids herein. Such molecules are considered recombinant nucleic acid even when present, for example in the chromosome of a host cell, or in a nucleic acid solution. The term recombinant polynucleotide as used herein is not intended to encompass molecules present in their native state.

As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. In an aspect, the promoter is a heterologous promoter.

"Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having a deletion (i.e., truncations) at the 5' and/or 3' end and/or a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the Nrf4 polypeptides. Naturally occurring 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 as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis or gene synthesis but which still encode a polypeptide of interest.

"Variant" protein is intended to mean a protein derived from the protein by deletion (i.e., truncation at the 5' and/or 3' end) and/or a deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, have non-reduction activity. Such variants may result from, for example, genetic polymorphism or from human manipulation.

As used herein, a “fragment” of a gene, sequence, polynucleotide or polypeptide relates to a part of a gene, sequence, polypeptide or a polynucleotide that is smaller than the whole gene, sequence, polynucleotide or a polypeptide. As understood herein, the sequence of the fragment of gene, sequence, polynucleotide or polypeptide is contained within the sequence of said gene, sequence, polynucleotide or polypeptide. As used herein, a "construct" or "gene construct" can refer to a polynucleotide which codes for the particular gene of the gene construct. Such polynucleotides can be operably linked to one or more untranslated regions (UTRs), and/or one or more transcriptional initiation regulatory sequence/promoter regulatory region which is capable of directing the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant, thereby regulating expression of a given gene. Expression of a given gene can be determined in terms of the amount of gene product or protein expressed., and a variety of methods can be used for detecting protein expression levels, including, for example, enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations, and immunofluorescence, and the like.

As used herein, a "vector" can refer to any nucleic acid construct which is able to enter a plant cell, including circular or linear nucleic acids, and/or bacterial, viral, fungal, plant and synthesized nucleic acids, as well as homologous or heterologous nucleic acid constructs.

As used herein, the terms "transform", "transformed", and "transforming" can refer to the introduction of a foreign gene into a plant. Numerous methods for introducing foreign genes into plants are known and can be used to insert nucleic acid sequences into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al. (1993) "Procedure for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, ed. Glick and Thompson (CRC Press, Inc., Boca Raton), pages 67-88. The methods chosen can vary with the host plant, and many such methods are known to those in the art; these include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium tumefaciens or other bacteria, electroporation, microinjection, and biolistic bombardment. Expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are known and available. See, for example, Gruber et al. (1993) "Vectors for Plant Transformation," in Methods in Plant Molecular Biology and Biotechnology, ed. Glick and Thompson (CRC Press, Inc., Boca Raton), pages 89-119.

As used herein, the terms "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties {e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are often said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif).

As used herein, a "target sequence" or "target polynucleotide" comprises any sequence that one desires to decrease the level of expression.

The term "silencing element" is intended to relate to a polynucleotide that is capable of reducing or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby. The silencing element employed can decrease or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript or, alternatively, by influencing translation and thereby affecting the level of the encoded polypeptide. Methods to assay for functional silencing elements that are capable of decreasing or eliminating the level of a sequence of interest are known to the skilled person. A single polynucleotide employed in the methods can comprise one or more silencing elements to the same or different target polynucleotides. The silencing element can be produced in vivo (i.e. , in a host cell such as a plant or microorganism) or in vitro. As used herein, the term "dsRNA" is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, for example, short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), post- transcriptional gene silencing RNA (ptgsRNA), and others.

As used herein, Cas gene relates to a gene that is generally coupled, associated or close to, or in the vicinity of flanking CRISPR loci. The terms "Cas gene", "CRISPR- associated (Cas) gene" are used interchangeably herein. The number of Cas genes at a given CRISPR locus can vary between species.

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

As used herein, the term "guide polynucleotide" relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence). Optionally, the guide polynucleotide can comprise at least one nucleotide, phosphodiester bond or linkage modification such as, but not limited to, Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine, 2'-Fluoro A, 2'-Fluoro U, 2'-0-Methyl RNA, phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer (hexaethylene glycol chain) molecule, or 5' to 3' covalent linkage resulting in circularization. A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a "guide RNA".

The term "Cas endonuclease recognition domain" or "CER domain" of a guide polynucleotide is used interchangeably herein and includes a nucleotide sequence (such as a second nucleotide sequence domain of a guide polynucleotide) that interacts with a Cas endonuclease polypeptide. The CER domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see for example modifications described herein), or any combination thereof.

The preferred definitions given in the "Definition'-section apply to all of the embodiments described below unless stated otherwise.

Description of the Figures

Figure 1. Engineering diplosporous apomixis with pseudogamous development of the endosperm. (A) - crossing scheme to generate apomictically reproducing individuals in maize and their propagation for two generations. A1 - apomictic generation 1 , A2 - apomictic generation2, S1 - siblings of A1 , S2 - siblings of A2. (B) - a family of F2 nrf4/nrf4' PsASGR:BBML M3A-H1-1 crossed to 4n W23 R1-nj male. (C) - results of SSR analysis of putative clonal individuals. Each column represents one individual and each row one SSR marker. The chromosomal bin of each SSR marker is shown next to the row. The color of the cells reflects the state of the SSR marker in each individual: light red - heterozygous, red - homozygous. M stands for maternal individual, progeny individuals are indicated by numbers. Individuals with the same number and differing only by letter are twins.

Figure 2. Boxplots of ten phenotypes in A1 - apomictic plants generation 1 , S1 — siblings of the apomictic plant generation 1 , A2 -apomictic plant generation 2, S2 - siblings of the apomictic plant generation 2. Significance codes: *** 0.001 ; ** 0.01 ; * 0.05; ‘ 0.1.

Detailed description of the invention

The embodiments of the present invention will be described in the following. It is to be understood that all possible combinations of the following definitions are also envisaged. In one embodiment, the present invention relates to a transgenic apomictic plant. The transgenic apomictic plant is characterized in that an endogenous Nrf4 gene is disrupted; and a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 is expressed, preferably in the egg cell or embryo sac. Said polypeptide is also referred to as BBML transgene. Preferably, the transgenic apomictic plant of the present invention is not exclusively obtained by the means of an essentially biological process. Further preferably, the polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 is herein understood as having essentially retained the functionality of the polypeptide of SEQ ID NO. 1 .

The amino acid sequence referenced above, i.e. the sequence according to SEQ ID NO. 1 , is included in a separate sequence listing and is also shown in the following. In the case of conflict between the sequence specified in the sequence listing and the corresponding sequence shown further below, the present invention relates to both sequences.

The amino acid sequence according to SEQ ID NO. 1 is as follows:

Met Gly Ser Thr Asn Asn Trp Leu Arg Phe Ala Ser Phe Ser Gly Gly 1 5 10 15

Gly Gly Ala Lys Asp Ala Ala Ala Leu Leu Pro Leu Pro Pro Ser Pro

20 25 30

Arg Gly Asp Vai Asp Glu Ala Gly Ala Glu Pro Lys Leu Glu Asp Phe

35 40 45

Leu Gly Leu Gin Glu Pro Ser Ala Ala Ala Vai Gly Ala Gly Arg Pro

50 55 60

Phe Ala Vai Gly Gly Gly Ala Ser Ser He Gly Leu Ser Met He Arg 65 70 75 80

Asn Trp Leu Arg Ser Gin Pro Ala Pro Ala Gly Pro Ala Ala Gly Vai 85 90 95

Asp Ser Met Vai Leu Ala Ala Ala Ala Ala Ser Thr Glu Vai Ala Gly

100 105 110

Asp Gly Ala Glu Gly Gly Gly Ala Vai Ala Asp Ala Vai Gin Gin Arg 115 120 125

Lys Ala Ala Ala Vai Asp Thr Phe Gly Gin Arg Thr Ser l ie Tyr Arg

130 135 140

Gly Vai Thr Lys His Arg Trp Thr Gly Arg Tyr Glu Ala His Leu Trp 145 150 155 160 Asp Asn Ser Cys Arg Arg Glu Gly Gin Thr Arg Lys Gly Arg Gin Vai 165 170 175

Tyr Leu Gly Gly Tyr Asp Lys Glu Glu Lys Ala Ala Arg Ala Tyr Asp

180 185 190

Leu Ala Ala Leu Lys Tyr Arg Gly Thr Thr Thr Thr Thr Asn Phe Pro

195 200 205

Met Ser Asn Tyr Glu Lys Glu Leu Glu Glu Met Lys His Met Ser Arg 210 215 220

Gin Glu Tyr Vai Ala Ser Leu Arg Arg Lys Ser Ser Gly Phe Ser Arg 225 230 235 240 Gly Ala Ser He Tyr Arg Gly Vai Thr Arg His His Gin His Gly Arg

245 250 255

Trp Gin Ala Arg He Gly Ser Vai Ala Gly Asn Lys Asp Leu Tyr Leu

260 265 270

Gly Thr Phe Ser Thr Gin Glu Glu Ala Ala Glu Ala Tyr Asp l ie Ala

275 280 285

Ala He Lys Phe Arg Gly Leu Asn Ala Vai Thr Asn Phe Asp Met Ser

290 295 300

Arg Tyr Asp Vai Lys Ser He He Glu Ser Ser Ser Leu Pro Vai Gly 305 310 315 320

Gly Thr Pro Lys Arg Leu Lys Glu Vai Pro Asp Gin Ser Asp Met Gly 325 330 335

He Asn He Asn Gly Asp Ser Ala Gly His Met Thr Ala He Asn Leu

340 345 350

Leu Thr Asp Gly Asn Asp Ser Tyr Gly Ala Glu Ser Tyr Gly Tyr Ser

355 360 365

Gly Trp Cys Pro Thr Ala Met Thr Pro He Pro Phe Gin Phe Ser Asn 370 375 380

Gly His Asp His Ser Arg Leu Trp Cys Lys Pro Glu Gin Asp Asn Ala 385 390 395 400

Vai Vai Ala Ala Leu His Asn Leu His His Leu Gin His Leu Pro Ala

405 410 415

Pro Vai Gly Thr His Asn Phe Phe Gin Pro Ser Pro Vai Gin Asp Met

420 425 430

Thr Gly Vai Ala Asp Ala Ser Ser Pro Pro Vai Glu Ser Asn Ser Phe

435 440 445

Leu Tyr Asn Gly Asp Vai Gly Tyr His Gly Ala Met Gly Gly Ser Tyr

450 455 460

Ala Met Pro Vai Ala Thr Leu Vai Glu Gly Asn Ser Ala Gly Ser Gly

465 470 475 480

Tyr Gly Vai Glu Glu Gly Thr Gly Ser Glu l ie Phe Gly Gly Arg Asn

485 490 495

Leu Tyr Ser Leu Ser Gin Gly Ser Ser Gly Ala Asn Thr Gly Lys Ala

500 505 510

Asp Ala Tyr Glu Ser Trp Asp Pro Ser Met Leu Vai He Ser Gin Lys

515 520 525

Ser Ala Asn Vai Thr Vai Cys His Gly Ala Pro Vai Phe Ser Vai Trp

530 535 540

Lys

545

In a further embodiment, the present invention relates to a method for the production of an apomictic plant. The said method comprises the steps of providing a plant or a plant cell; disrupting an endogenous Nrf4 gene in the plant or the plant cell; and inducing the expression of a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell. If the plant cell was used, the method for the production of an apomictic plant further comprises a step of regenerating the plant from the plant cell. It is noted that the steps of disrupting an endogenous Nrf4 gene in the plant or the plant cell; and inducing the expression of a polypeptide having at least 70% sequence identity, preferably of at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell can be performed in any order, separately or at the same time. In again a further embodiment, the present invention relates to an apomictic plant obtained according to the method for the production of an apomictic plant of the present invention.

Apomixis differs from normal sexual reproduction in (1 ) the avoidance or omission of meiosis (apomeiosis) resulting in unreduced, unrecombined gametes, (2) the development of the unreduced egg cell into an embryo in the absence of fertilization (parthenogenesis), and (3) the functional development of endosperm, an embryonourishing tissue of the seed.

Because not only the egg cell is unreduced but also the cell giving rise to the endosperm (central cell), the sperm have to be diploid (preferably come from a tetrapioid plant) to yield a genetically balance endosperm (for example in maize it requires a ratio of maternal to paternal genomes of 2:1). In some natural apomicts, this ratio does not matter and in some the endosperm develops without fertilization (autonomous endosperm) but this could not yet be engineered, so a special pollen donor is required to ensure functional endosperm formation. As the endosperm does not contribute to the next generation, the seed is clonal nonetheless.

According to the present invention, the avoidance or omission of meiosis (apomeiosis) resulting in unreduced, unrecombined gametes is accomplished disrupting an endogenous Nrf4 gene in the plant or the plant cell.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of an apomictic plant of the present invention, the endogenous Nrf4 gene is disrupted by inhibiting expression and/or activity of a protein encoded by the endogenous Nrf4 gene. Manipulating Nrf4 expression and/or activity provides an opportunity to alter chromosome reduction during meiosis. Decreasing Nrf4 expression and/or activity in a plant may be used to create a plant with a non-reduced gamete, i.e. where the gamete retains a full chromosome set and does not have its genetic material reduced in half. Moreover, decreasing Nrf4 expression and/or activity in a plant may be used prevent recombination during sporogenesis. Provided herein are methods and compositions for producing plants having viable non-reduced, or non-reduced and non- recombined, gametes as well methods and compositions for maintaining heterozygosity in a progeny plant.

The term endogenous Nrf4 gene is herein understood as selected from: a) a polynucleotide having at least 70% sequence identity, as determined by the GAP algorithm under default parameters, to the full length sequence of a polynucleotide selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11 , 12, 14, 15, 16, 19, 20, 22, 26, and 27, and wherein the polynucleotide encodes a polypeptide that has the function of reducing, or reducing and recombining, female sporocytes, female gametophytes, or female gametes during meiosis; b) a polynucleotide having at least 80% sequence identity, as determined by the GAP algorithm under default parameters, to the full length sequence of a polynucleotide selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11 , 12, 14, 15, 16, 19, 20, 22, 26, and 27, and wherein the polynucleotide encodes a polypeptide that has the function of reducing, or reducing and recombining, female sporocytes, female gametophytes, or female gametes during meiosis; c) a polynucleotide having at least 90% sequence identity, as determined by the GAP algorithm under default parameters, to the full length sequence of a polynucleotide selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11 , 12, 14, 15, 16, 19, 20, 22, 26, and 27, and wherein the polynucleotide encodes a polypeptide that has the function of reducing, or reducing and recombining, female sporocytes, female gametophytes, or female gametes during meiosis; d) a polynucleotide selected from the group consisting of SEQ I D NOs: 7, 8, 9, 10, 11 , 12, 14, 15, 16, 19, 20, 22, 26, and 27; e) a polynucleotide that is a variant of the polynucleotide of (a), (b), (c) or

(d); f) a polynucleotide that is a fragment of the polynucleotide of (a), (b), (c), (d) or (e); and g) a polynucleotide that is complementary to the polynucleotide of (a), (b), (c), (d), (e) or (f); and h) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (a), (b), (c), (d), (e), (f) or (g).

As understood herein, the endogenous Nrf4 gene as disclosed herein encodes for a Nrf4 polypeptide selected from: a) a polypeptide comprising an amino acid sequence being identical to or having at least 70% identity with SEQ ID NOs: 5, 6, 13, 17, 18, 21 , 23, 24 or 25, or an ortholog thereof; b) a polypeptide comprising an amino acid sequence being identical to or having at least 80% identity with SEQ ID NOs: 5, 6, 13, 17, 18, 21 , 23, 24 or 25, or an ortholog thereof; c) a polypeptide comprising an amino acid sequence being identical to or having at least 90% identity with SEQ ID NOs: 5, 6, 13, 17, 18, 21 , 23, 24 or 25, or an ortholog thereof; d) a polypeptide comprising an amino acid sequence being identical to or having at least 100% identity with SEQ ID NOs: 5, 6, 13, 17, 18, 21 , 23, 24 or 25, or an ortholog thereof; e) a polypeptide comprising an amino acid sequence, which is a variant of the amino acid sequence of (a), (b), (c) or (d); and f) a polypeptide comprising an amino acid sequence which is a fragment of the amino acid sequence of (a), (b), (c), (d), or (e).

Means and methods for decreasing meiotic reduction in a female gametophyte or female gamete by modulating Nrf4 expression or activity are described herein. As used herein, "Nrf4 activity" refers to one or more of the following activities: (i) causing the reduction in meiosis during plant reproduction, for example, in an ovule, female sporocyte, female gametophyte, or female gamete, (ii) the recombination during the reduction in meiosis in a plant ovule, for example, in a female sporocyte. Nrf4 activity may occur in vitro or in vivo. In some embodiments, decreasing Nrf4 activity in a plant includes decreasing the expression of one or more Nrf4 genes in a plant cell, for example, in an ovule primordia, ovule, female gametophyte, or female gamete. In some embodiments, the methods include disrupting an endogenous Nrf4 gene in a plant.

Means and methods of the present invention for decreasing Nrf4 activity in a female sporocyte, female gametophyte or female gamete in a plant can include decreasing the expression level of one or more Nrf4 sequences, for example, gene, cDNA, polypeptide, in the female plant germline, for instance a female sporocyte, female gametophyte or female gamete. In some embodiments, methods of the present invention employ a silencing element that decreases the level of expression and/or activity of Nrf4. In other words, in certain embodiments of the present invention, an endogenous Nrf4 gene is disrupted by introducing a recombinant nucleic acid construct comprising a nucleotide sequence encoding an element that silences Nrf4 activity. Targets sequences for Nrf4 polynucleotides include wild-type Nrf4 polynucleotide or Nrf4 polypeptide sequences, Nrf4 variant polynucleotides, Nrf4 variant polypeptides, cognate promoter sequences, ortholog sequences, variants or fragments thereof. Silencing elements that decrease the expression of one or more of the Nrf4 target sequences and thereby decrease or inhibit the reduction and recombination in meiosis, e.g. Nrf4 activity, can be designed in view of these target polynucleotides.

In specific embodiments, decreasing the level of the Nrf4 target sequence in the female sporocyte, the female gametophyte orfemale gamete prevents or decreases reduction and/or recombination in meiosis. Non-limiting examples of target sequences include those set forth in the sequence listing, any wild-type Nrf4 polynucleotide or Nrf4 polypeptide sequences, Nrf4 variant polynucleotides, Nrf4 variant polypeptides, cognate promoter sequences, ortholog sequences, variants or fragments thereof. Decreasing the level of expression of one or more of these target sequences results in the non-reduction and non-recombination during meiosis in the plant female sporocyte, female gametophyte orfemale gamete. In certain embodiments of the present invention, the Nrf4 target sequence is endogenous to the plant. In other embodiments, while the silencing element regulates non-reduction and non-recombination of meiosis, preferably the silencing element has no effect on the parts of the plant that do not constitute the female germline (female sporocyte, female gametophyte, and female gamete).

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by introducing a recombinant nucleic acid construct comprising a nucleotide sequence encoding an element that silences Nrf4 activity. Such a recombinant nucleic acid construct may also be referred to as silencing element.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, silencing elements can include, but are not limited to, a sense suppression element, an antisense suppression element, a double stranded RNA, a siRNA, an amiRNA, a miRNA, ora hairpin suppression element. Non-limiting examples of silencing elements that can be employed to decrease expression of these target sequences or additionally sequences targeting genes involved in recombination comprise fragments and variants of the sense or antisense sequence or consists of the sense or antisense sequence of any of the sequences of Nrf4 set forth in the sequence listing, wild-type Nrf4 polynucleotide or Nrf4 polypeptide sequences, Nrf4 variant polynucleotides, Nrf4 variant polypeptides, cognate promoter sequences, ortholog sequences, variants or fragments thereof. The silencing element can further comprise additional sequences that advantageously affect transcription and/or the stability of a resulting transcript. For example, the silencing elements can comprise at least one thymine residue at the 3' end. This can aid in stabilization. Thus, the silencing elements can have at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more thymine residues at the 3' end.

The silencing element can be introduced in a variety of ways. In certain embodiment of the present invention, the silencing element can be expressed in a specific manner, for example, using inducible or tissue-preferred or developmentally regulated promoters that are discussed elsewhere herein. In one embodiment, the silencing element is operably linked to an Nrf4 promoter, variant or fragment thereof. In specific embodiments, the silencing element is expressed in a plant ovule primordium, plant ovule, plant female sporocyte, plant female gametophyte, or plant female gamete.

Decreasing the expression level of a polynucleotide or a polypeptide encoded thereby is intended to mean, the polynucleotide or polypeptide level of the target sequence is lower than the polynucleotide level or polypeptide level of the same target sequence in an appropriate control (e.g. control plant), in which the silencing element is not introduced. In certain embodiments, decreasing the polynucleotide level and/or the polypeptide level of the target sequence results in less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the polynucleotide level, or the level of the polypeptide encoded thereby, of the same target sequence in an appropriate control. Methods to assay for the level of the RNA transcript, the level of the encoded polypeptide, or the activity of the polynucleotide or polypeptide are known to the skilled person.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene through decreasing expression of an Nrf4 polypeptide may be achieved by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a polypeptide in the "sense" orientation. Overexpression of the RNA molecule may result in decreased expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show the desired degree of inhibition of polypeptide expression.

Typically, a sense suppression element has substantial sequence identity to the target polynucleotide, typically greater than about 65% sequence identity, greater than about 85% sequence identity, about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The sense suppression element can be any length so long as it allows for the suppression of the targeted sequence. The sense suppression element can be, for example, 15, 16, 17, 18 19, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 900, 1000, 1100, 1200, 1300 nucleotides or longer of the target polynucleotides set forth in any of SEQ ID NO:7-12, 14, 15, 16, 19, 20, 22, , 26 or 27. In other embodiments, the sense suppression element can be, for example, about 15-25, 25-100, 100-150, 150- 200, 200-250, 250-300, 300-350, 350-400, 450- 500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900- 950, 950-1000, 1000-1050, 1050-1100, 1100- 1200, 1200-1300, 1300-1400, 1400- 1500, 1500-1600, 1600-1700, 1700-1800 nucleotides or longer of the target polynucleotides set forth in any of SEQ ID NO: 7-12, 14, 15, 16, 19, 20, 22, 26 or 27.

The polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the polypeptide, all or part of the 5' and/or 3' untranslated region of a polypeptide transcript or all or part of both the coding sequence and the untranslated regions of a transcript encoding a polypeptide. In some embodiments where the polynucleotide comprises all or part of the coding region for the polypeptide, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated.

Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the sense sequence and 5' of the polyadenylation signal. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity

In some embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the polypeptide. Over expression of the antisense RNA molecule may result in decreased expression of the target gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the desired degree of inhibition of polypeptide expression.

The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the target sequence encoding the polypeptide, all or part of the complement of the 5' and/or 3' untranslated region of the target transcript or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the polypeptide. In addition, the antisense polynucleotide may be fully complementary (i.e. , 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. In addition, the antisense suppression element may be fully complementary (i.e. , 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target polynucleotide. In specific embodiments, the antisense suppression element comprises at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the target polynucleotide. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. Furthermore, the antisense suppression element can be complementary to a portion of the target polynucleotide. Generally, sequences of at least 15, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater of the sequence set forth in any of SEQ ID NO: 7-12, 14, 15, 16, 19, 20, 22, 26, or27 may be used. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the antisense sequence and 5' of the polyadenylation signal.

In some embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by double-stranded RNA (dsRNA) interference. A "double stranded RNA silencing element" or "dsRNA" comprises at least one transcript that is capable of forming a dsRNA. Thus, a "dsRNA silencing element" includes a dsRNA, a transcript or polyribonucleotide capable of forming a dsRNA or more than one transcript or polynucleotide (herein RNA polynucleotide, or polyribonucleotide) capable of forming a dsRNA. "Double stranded RNA" or "dsRNA" refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. The dsRNA molecule(s) employed in the methods and compositions mediate the decrease of expression of a target sequence, for example, by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. The dsRNA is capable of decreasing or eliminating the level or expression of a target polynucleotide or the polypeptide, herein, Nrf4.

The dsRNA can decrease or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript, by influencing translation and thereby affecting the level of the encoded polypeptide, or by influencing expression at the pre- transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). Methods to assay for functional dsRNA that are capable of decreasing or eliminating the level of a sequence of interest are known to the skilled person.

For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA. Expression of the sense and antisense molecules may be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the desired degree of inhibition of polypeptide expression.

In some embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a singlestranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Alternatively, the basepaired stem region may correspond to a portion of a promoter sequence controlling expression of the gene whose expression is to be inhibited. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes and the RNA interference they induce is inherited by subsequent generations of plants.

For ihpRNA, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference.

Any region of the target polynucleotide (target sequence) can be used to design the domain of the silencing element that shares sufficient sequence identity to allow expression of the hairpin transcript to decrease the level of the target polynucleotide. For instance, the domain can be designed to share sequence identity to the 5' untranslated region of the target polynucleotide(s), the 3' untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof. In specific embodiments, a domain of the silencing element shares sufficient homology to at least about 15, 16, 17, 18, 19, 20, 22, 25 or 30 consecutive nucleotides from about nucleotides 1-50, 25- 75, 75-125, 50-100, 125-175, 175-225, 100-150, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375, 375-425, 300- 350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575, 575-625, 550-600, 625-675, 675-725, 600-650, 625-675, 675-725, 650-700, 725-825, 825-875, 750-800, 875-925, 925- 975, 850-900, 925-975, 975-1025, 950- 1000, 1000-1050, 1025-1075, 1075-1 125, 1050- 1 100, 1125-1 175, 1 100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425, 1300-1400, 1425-1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625, 1625-1675, 1675- 1725, 1725-1775, 1775-1825, 1825-1875, 1875-1925, 1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175-2225, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900- 2000 of the target sequence. In some instances, to optimize the siRNA sequences employed in the hairpin, the synthetic oligodeoxyribonucleotide/RNAse H method can be used to determine sites on the target mRNA that are in a conformation that is susceptible to RNA silencing.

The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transcriptional gene silencing (TGS), which may be accomplished through use of a hairpin suppression element where the inverted repeat of the hairpin shares sequence identity with the promoter region of a target polynucleotide to be silenced.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by using amplicon expression cassettes. Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e. , the messenger RNA for the polypeptide). In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by RNA interference by expression of a gene encoding a micro RNA (miRNA) or short-interfering RNA (siRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. The miRNA can be an "artificial miRNA" or "amiRNA" which comprises a miRNA sequence that is synthetically designed to silence a target sequence. For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. For example, the miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). In some embodiments, the 22-nucleotide sequence is selected from a transcript sequence from a Nrf4 gene and contains 22 nucleotides of the Nrf4 gene in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. In some embodiments, in addition to targeting Nrf4, genes involved in recombination may also be targeted. Accordingly, in some embodiments, the 22- nucleotide sequence is selected from a transcript sequence from a gene involved in recombination and contains 22 nucleotides of the gene involved in recombination in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.

The heterologous polynucleotide being expressed need not form the dsRNA by itself, but can interact with other sequences in the plant cell to allow the formation of the dsRNA. For example, a chimeric polynucleotide that can selectively silence the target polynucleotide can be generated by expressing a chimeric construct comprising the target sequence for a miRNA or siRNA to a sequence corresponding to all or part of the gene or genes to be silenced. In this embodiment, the dsRNA is "formed" when the target for the miRNA or siRNA interacts with the miRNA present in the cell. The resulting dsRNA can then decrease the level of expression of the gene or genes to be silenced. The construct can be designed to have a target for an endogenous miRNA or, alternatively, a target for a heterologous and/or synthetic miRNA can be employed in the construct. If a heterologous and/or synthetic miRNA is employed, it can be introduced into the cell on the same nucleotide construct as the chimeric polynucleotide or on a separate construct. Any method known to the skilled person can be used to introduce the construct comprising the heterologous miRNA.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the recombinant nucleic acid construct encodes a zinc finger protein that binds to a gene encoding a polypeptide, resulting in decreased expression of the gene. In certain embodiments, the zinc finger protein binds to a messenger RNA encoding a polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins are known to the skilled person.

In some embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the recombinant nucleic acid construct encodes an antibody that binds to at least one Nrf4 polypeptide and decreases the activity of Nrf4. In certain embodiments, the binding of the antibody results in increased turnover of the antibody- Nrf4 complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the activity of a Nrf4 polypeptide is decreased or eliminated by disrupting the gene encoding the polypeptide, in other words by disrupting an endogenous Nrf4 gene in the plant or the plant cell. The gene encoding the Nrf4 polypeptide may be disrupted by any method known in the art, for example, by genome editing, transposon tagging or mutagenizing plants using random or targeted mutagenesis and selecting for plants that have decreased activity.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the target sequence, herein Nrf4, can be modified using gene editing technology, including without limitation double-strand-break-inducing agent, such as but not limited to a CRISPR-Cas guideRNA or other polynucleotide-guided double strand break reagent, a Zinc Finger endonuclease, a meganuclease, or a TALEN endonuclease.

CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) (also known as SPIDRs-SPacer Interspersed Direct Repeats) constitute a family of recently described DNA loci. CRISPR loci consist of short and highly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to 140 times, also referred to as CRISPR- repeats) which are partially palindromic. The repeated sequences (usually specific to a species) are interspaced by variable sequences of constant length (typically 20 to 58 bp depending on the CRISPR locus. The repeats are short elements that occur in clusters, which are always regularly spaced by variable sequences of constant length.

Cas endonuclease relates to a Cas protein encoded by a Cas gene, wherein said Cas protein is capable of introducing a double strand break into a DNA target sequence. The Cas endonuclease is guided by a guide polynucleotide to recognize and optionally introduce a double strand break at a specific target site into the genome of a cell. The guide polynucleotide/Cas endonuclease system includes a complex of a Cas endonuclease and a guide polynucleotide that is capable of introducing a double strand break into a DNA target sequence. The Cas endonuclease unwinds the DNA duplex in close proximity of the genomic target site and cleaves both DNA strands upon recognition of a target sequence by a guide RNA if a correct protospacer-adjacent motif (PAM) is approximately oriented at the 3' end of the target sequence.

The Cas endonuclease gene can encode Cas9 endonuclease, or a functional fragment thereof, such as but not limited to the Cas9 genes listed in W02007/025097, published March 1 , 2007. The Cas endonuclease gene can be a plant, maize or soybean optimized Cas9 endonuclease, such as but not limited to a plant codon optimized Streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the form N(12-30)NGG. The Cas endonuclease can be introduced directly into a cell by any method known in the art, for example, but not limited to, transient introduction methods, transfection, and/or topical application. The guide polynucleotide can be a double molecule (also referred to as duplex guide polynucleotide), comprising a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that is complementary to a nucleotide sequence in a target DNA and a second nucleotide sequence domain (referred to as Cas endonuclease recognition domain or CER domain) that interacts with a Cas endonuclease polypeptide. The CER domain of the double molecule guide polynucleotide comprises two separate molecules that are hybridized along a region of complementarity. The two separate molecules can be RNA, DNA, and/or RNA-DNA- combination sequences. In some embodiments, the first molecule of the duplex guide polynucleotide comprising a VT domain linked to a CER domain is referred to as "crDNA" (when composed of a contiguous stretch of DNA nucleotides) or "crRNA" (when composed of a contiguous stretch of RNA nucleotides), or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The crNucleotide can comprise a fragment of the cRNA naturally occurring in Bacteria and Archaea. In one embodiment, the size of the fragment of the cRNA naturally occurring in Bacteria and Archaea that is present in a crNucleotide disclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the second molecule of the duplex guide polynucleotide comprising a CER domain is referred to as "tracrRNA" (when composed of a contiguous stretch of RNA nucleotides) or "tracrDNA" (when composed of a contiguous stretch of DNA nucleotides) or "tracrDNA- RNA" (when composed of a combination of DNA and RNA nucleotides. In one embodiment, the RNA that guides the RNA/Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA- tracrRNA.

The guide polynucleotide can also be a single molecule comprising a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that is complementary to a nucleotide sequence in a target DNA and a second nucleotide domain (referred to as Cas endonuclease recognition domain or CER domain) that interacts with a Cas endonuclease polypeptide. By "domain" it is meant a contiguous stretch of nucleotides that can be RNA, DNA, and/or RNA-DNA-combination sequence. The VT domain and/or the CER domain of a single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or an RNA-DNA-combination sequence. In some embodiments, the single guide polynucleotide comprises a crNucleotide (comprising a VT domain linked to a CER domain) linked to a tracrNucleotide (comprising a CER domain), wherein the linkage is a nucleotide sequence comprising an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. The single guide polynucleotide being comprised of sequences from the crNucleotide and tracrNucleotide may be referred to as "single guide RNA" (when composed of a contiguous stretch of RNA nucleotides) or "single guide DNA" (when composed of a contiguous stretch of DNA nucleotides) or "single guide RNA- DNA" (when composed of a combination of RNA and DNA nucleotides). In one embodiment of the disclosure, the single guide RNA comprises a cRNA or cRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a plant genomic target site, enabling the Cas endonuclease to introduce a double strand break into the genomic target site. One aspect of using a single guide polynucleotide versus a duplex guide polynucleotide is that only one expression cassette needs to be made to express the single guide polynucleotide.

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

The nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or an RNA- DNA combination sequence. In one embodiment, the nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27,

28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50,

51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73,

74, 75, 76, 77, 78, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95,

96, 97, 98, 99 or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the crNucleotide and the tracrNucleotide of a single guide polynucleotide can comprise a tetraloop sequence, such as, but not limiting to, a GAAA tetraloop sequence.

Nucleotide sequence modification of the guide polynucleotide, VT domain and/or CER domain can be selected from, but not limited to, the group consisting of a 5' cap, a 3' polyadenylated tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides for tracking, a modification or sequence that provides a binding site for proteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2'- Fluoro A nucleotide, a 2'-Fluoro U nucleotide, a 2'-0-M ethyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5 ' to 3 ' covalent linkage, or any combination thereof. These modifications can result in at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group of a modified or regulated stability, a subcellular targeting, tracking, a fluorescent label, a binding site for a protein or protein complex, modified binding affinity to complementary target sequence, modified resistance to cellular degradation, and increased cellular permeability.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transposon tagging. Transposon tagging is used to decrease or eliminate the activity of one or more polypeptides. Transposon tagging comprises inserting a transposon within an endogenous gene in the pathway to decrease or eliminate expression of the polypeptide. In this embodiment, the expression of one or more polypeptides is decreased or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the polypeptide. A transposon that is within an exon, intron, 5' or 3' untranslated sequence, a promoter or any other regulatory sequence of a gene may be used to decrease or eliminate the expression and/or activity of the encoded polypeptide. Methods for the transposon tagging of specific genes in plants are well known in the art.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may also be achieved by using additional methods for decreasing or eliminating the expression of endogenous genes in plants that are also known in the art. Thus, disrupting an endogenous Nrf4 gene in the plant or the plant cell may also be achieved by using forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines, in which the endogenous gene has been mutated or deleted. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. Mutations may impact gene expression or interfere with the activity of an encoded Nrf4 protein. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the activity of the encoded protein. Conserved residues of plant polypeptides suitable for mutagenesis with the goal to eliminate activity have been described. Such mutants may be isolated according to well-known procedures and mutations in different Nrf4 loci may be stacked by genetic crossing. In another embodiment of the present invention, dominant mutants may be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, Kusaba et al, (2003) Plant Cell 15: 1455-1467.

The present invention further encompasses additional methods for decreasing or eliminating the activity of one or more target polypeptides. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the level of expression or activity of a Nrf4 polypeptide is modified in a plant cell, for example, in an ovule primordia, ovule, female sporocyte, female gametophyte, or female gamete. In some embodiments, a plant cell is transformed with a DNA construct or expression cassette for expression of at least one silencing element. In certain embodiments, modulation of Nrf4 expression level and/or activity of the Nrf4 polypeptide promotes non-reduction, or non-reduction and nonrecombination, during meiosis resulting in the production of non-reduced, or nonreduced and non-recombined, female gametes.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by an expression construct comprising an element that when expressed decreases Nrf4 polynucleotide and/or Nrf4 polypeptide expression level or activity and is operably linked to a promoter functional in a plant cell. In certain embodiments, the promoter is a female sporogenesis-related promoter, in particular a promoter expressing in ovule primordia, ovule tissue, or sporocytes, including but not limited to an Nrf4 promoter. The expression cassette can include 5' and 3' regulatory sequences operably linked to a polynucleotide of interest, e.g. a silencing element, or an active variant or fragment thereof. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest, for example, a silencing element. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional polynucleotides can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the element to be under the transcriptional regulation of the promoter. The expression cassette may additionally contain selectable marker genes. In certain embodiments of the present invention, the expression cassette will include in the 5'-3' direction of transcription an ovule-specific promoters, ovule-preferred promoters, sporocyte-specific, sporocyte-preferred, female-gametophyte specific promoters, female-gametophyte preferred promoters, female-gamete-specific promoters, female-gamete-preferred promoters, a female sporogenesis-related promoter or an active variant or fragment thereof, a silencing element and a transcriptional and translational termination region (i.e., termination region) functional in the host cell (i.e., the plant). The regulatory regions and/or the silencing elements may be heterologous to the host cell, e.g. plant cell, or to each other.

The termination region may be native with the transcriptional initiation region, may be native with the operably linked silencing element or with the ovule tissue-preferred promoter sequences, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the silencing element, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase gene termination regions.

Where appropriate, the recombinant nucleic acid constructs, or polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats and other such well- characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. The constructs or expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis Virus 5' noncoding region) potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), MDMV leader (Maize Dwarf Mosaic Virus), and human immunoglobulin heavy-chain binding protein (BiP), untranslated leader from the coat protein mRNA of Alfalfa Mosaic Virus (AMV RNA 4), tobacco mosaic virus (TMV) leader, and Maize Chlorotic Mottle Virus (MCMV) leader. Other methods known to enhance translation can also be utilized, for example, introns.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, substitutions, e.g., transitions and transversions, may be involved.

The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers, such as p-galactosidase and fluorescent proteins, for example green fluorescent protein (GFP), cyan florescent protein (CYP), yellow florescent protein (PhiYFP™ from Evrogen) and red fluorescent protein (DsRED). The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the methods and compositions include a plant cell that has the modified endogenous Nrf4 gene or silencing element targeting Nrf4. Additionally, the modified Nrf4 gene or Nrf4 silencing element can be combined with other genes that are modified and/or other silencing elements that target other genes of interest.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, in addition to the endogenous Nrf4 gene, at least one other gene involved in meiosis is disrupted. Accordingly, the plant or the plant cell having the modified endogenous Nrf4 gene or silencing element targeting Nrf4 may be combined or stacked with a silencing element that targets genes that play a role in recombination in order to create transgenic apomictic plants of the present invention. For example, the recombination target genes include but are not limited to SPO11, PRD1, OSD1, TAM, REC8, Ago104, AMI, AM2, PAM1, PAM2, AS1, DSY1, DY1, ST1, ELI, DV1, VA1, VA2, POI, and the like and combinations thereof.

In certain embodiments of the present invention, the at least one other gene involved in meiosis may be an /Vrf4-like gene. Such a gene is present, for example, in maize, and the present inventors have demonstrated that disrupting said gene in addition to the endogenous Nrf4 gene is advantageous in the production of the apomictic plant of the present invention. In a preferred embodiment, the Nrf4-like gene as defined herein encodes a polypeptide according to SEQ ID NO.: 28.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, in addition to the endogenous Nrf4 gene, at least one other gene involved in recombination is disrupted. Accordingly, the plant cell having the modified endogenous Nrf4 gene or silencing element targeting Nrf4 may be combined with one or more genes involved in recombination that have been modified, for example, by gene-editing technologies, to have decreased recombination activity in order to create transgenic apomictic plants of the present invention.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by RNA interference.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by using amplicon expression cassettes.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transcriptional gene silencing (TGS).

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by double-stranded RNA (dsRNA) interference.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by antisense suppression.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene through decreasing expression of a Nrf4 polypeptide may be achieved by sense suppression or cosuppression. In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transposon tagging.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may also be achieved by using forms of mutagenesis.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell is done on a plant or a plant cell that already expresses, preferably under the control of an ovule-specific, ovule-preferred, germline-specific, germline-preferred, embryo sac-specific, embryo sac-preferred, egg cell-specific, or egg cell-preferred promoter, the BBML gene, a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 which has essentially retained the functionality of the polypeptide of SEQ ID NO. 1 .

As understood herein, the polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 which has essentially retained the functionality of the polypeptide of SEQ ID NO. 1 is preferably a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide corresponding to the residues 135 to 335 of SEQ ID NO. 1 , which has essentially retained the functionality of the polypeptide of SEQ ID NO. 1 .

According to the present invention, the development of the unreduced egg cell into an embryo in the absence of fertilization, also referred to as parthenogenesis, is achieved by expressing a BBML transgene in the plant or the plant cell. As understood herein, a BBML transgene refers to a gene expressing a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 which has essentially retained the functionality of the polypeptide of SEQ ID NO. 1 . As preferably understood herein, the functionality of the polypeptide of SEQ ID NO.: 1 encompasses enabling the development of the unreduced egg cell into an embryo in the absence of fertilization, also referred to as parthenogenesis.

Due to the protein similarity of the ASGR-BBM-LIKE {ASGR-BBML) gene to BBM (Baby Boom), and its characterized role in parthenogenesis as a key gene in the induction of parthenogenesis in the apomictic pathway, the expression of the ASGR- BBML gene can be used to induce parthenogenesis in plants. Herein the BBML transgene, or ASGR-BBML gene, is understood preferably as gene sequence as encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 which has essentially retained the functionality of the polypeptide of SEQ ID NO. 1 .

A BBM-LIKE gene from Zea mays called ODP2 has previously been described. However, the apomixis-specific BBM differs substantially from the maize BBM genes outside of the AP2-domain. The BBM, BBM-LIKE, and ASGR-BBM-LIKE proteins share a conserved bbm-1 domain that has not been identified in other members of the euANT lineage. Deletion of the bbm-1 domain has eliminated the ability of transgenic plants to induce somatic embryogenesis on cotyledons. A distinct clade of proteins from Oryza sativa, including the ASGRBBMs, BBM1 (Osl lgl9060), and proteins from Setaria italica and Panicum virgatum were found to be formed in the majority of phylogenic trees constructed. No functional studies on the genes within this clade, other than PsASGR-BBML, have been reported, although the UniGene database at NCBI and the Rice Oligonucleotide Array Database contains limited expression data for BBM1.

While BBM genes have been expressed from sexual species in ovules prior to fertilization, neither embryo development nor an apomixis phenotype has been observed to date. The expression of BnBB/W has been observed in microspores 3-4 days post-induction (at the time they were destined to become embryogenic), persisting throughout the time frame tested (28 days post-induction). BnBB/W expression, as determined by RT-PCR, has also been observed in 3-day-old seeds/globular embryos, and expression was found to persist throughout embryo development. BnBB/W and BnLECI (LEAFY COTYLEDON1), whose expression primarily occurs during microspore and zygotic embryogenesis, are considered to be markers for embryogenesis. BBM was also found to be detectable in Arabidopsis ovules in free-nuclear endosperm, as established by in situ hybridization. While expression was found to decline in endosperm once cellularization was initiated, to date there has been no published evidence for expression of BBM in egg or zygote cells within ovules. BnBB/W was originally considered to provide a route to induction of adventitious embryony in seeds, hence maternally derived embryos as a form of apomixis. In adventitious embryony, no alteration in embryo sac development occurs; rather, somatic cells of the ovule, usually nucellus, directly divide to form embryos. Adventitious embryony is therefore sporophytic apomixis.

While amino acid similarity between the AP2 regions of ASGR-BBML and BnBBM is high (96%), the similarity declines significantly outside of this region (35% similarity upstream and 27% similarity downstream). Three highly conserved genomic duplications of PsASGR-BBML have been identified from ASGR-linked BACs p203, p207, and p208 to date. The p208 PsASGR-BBML sequence is identical to the p207 PsASGR-BBML2 sequence (EU559277.1), with the exception of the number of AT repeats (11 vs. 17) found in intron 1 . The conservation of gene sequence means that it is not clear which of the PsASGR-BBML genomic regions are transcribed. The PsASGR-BBML transcript encodes a 545 amino acid protein derived from the splicing of 8 exons, a 73 bp 5' UTR, and multiple 3' UTRs, with lengths ranging from 30 to 258 bp. The PsASGR-BBML gene contains two AP2 DNA-binding domains and is therefore predicted to function as a transcription factor. Two ASGR-linked copies of ASGR- BBML also have been found to be present in apomictic Cenchrus ciliaris both of which are transcribed. CcASGR-BBM-LIKE1 contains a full open reading frame nearly identical to PsASGR-BBML whereas CcASGR-BBM-LIKE2 contains two nonsense mutations, the first of which is located within the first AP2 domain. Two related, but ASGR-unlinked, BBML (non-ASGR-BBML) genes also have been isolated from C. ciliaris, and orthologs have been found to be present in P. squamulatum. A previous comparative study with rice showed that ASGR-BBML was most closely related to a BBM gene at rice locus Osl lgl9060, for which no function has been identified to date; expression has been documented in seed (5 days after pollination) and embryo (25 days after pollination), but not pistil. Furthermore, ASGR-BBML is conserved among eight apomictic, but absent from seven sexual, Pennisetum species tested.

In some embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the ASGR-BBML gene construct further includes one or more untranslated region (UTR). In some embodiments, the ASGR-BBML gene construct further including one or more UTR can have at least 70% sequence identity to SEQ ID NO: 2 or a fully complementary strand thereof. In some embodiments, the ASGR- BBML gene construct further includes a promoter. In some embodiments, the ASGR- BBML gene construct can have at least 70% sequence identity to SEQ ID NO: 3 or a fully complementary strand thereof.

In the transgenic apomictic plant of the present invention, as well as in the method for the production of the apomictic plant of the present invention, the embryo is preferably to be formed from an unreduced egg or another unreduced cell of the embryo sac or ovule, more preferably the embryo is to be formed from an unreduced egg.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is expressed, preferably in the egg cell or embryo sac, upon introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1. In particular embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention the recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 further comprises one or more untranslated region(s) (UTR) and optionally a promoter.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation. Transformation as understood herein may include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium tumefaciens or other bacteria, electroporation, microinjection, and biolistic bombardment. In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the present invention, an endogenous Nrf4 gene is disrupted and/or a BBML transgene is expressed by introduction of (a) polypeptide(s) or polynucleotide(s) into a plant. "Introduction" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the nucleic acid or protein gains access to the interior of a cell of the plant. The methods disclosed herein do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, transfection, electroporation, biolistic bombardement, microinjection, and delivery by macromolecular systems or other deliverty reagents. "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and ballistic particle acceleration.

In certain embodiments, the various sequences employed in the methods disclosed herein (e.g., the silencing elements or constructs, Nrf4 promoters or variants and fragments thereof, ASGR-BBML gene) can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the various sequences employed in the methods disclosed herein (e.g., the silencing elements or constructs, Nrf4 promoters or variants and fragments thereof, ASGR-BBML gene) directly into the plant or the introduction of the transcript into the plant. Such methods include, for example, microinjection or particle bombardment.

Alternatively, in certain embodiments of the present invention the various sequences employed in the methods and compositions disclosed herein (e.g., the silencing elements or constructs, Nrf4 promoters or variants and fragments thereof, ASGR- BBML gene) can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use particles coated with polyethyleneimine (PEI; Sigma #P3143).

In certain embodiments of the present invention, the recombinant nucleic acid construct(s), or the polynucleotide(s) according to the present invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the disclosure within a viral DNA or RNA molecule. It is recognized that the various sequences employed in the methods and compositions disclosed herein (e.g., the silencing elements or constructs, Nrf4 promoters or variants and fragments thereof, ASGR-BBML gene) may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters disclosed herein also encompass promoters utilized for transcription by viral RNA polymerases.

Methods are known in the art for the targeted insertion of a polynucleotide, preferably a recombinant nucleic acid construct, at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system, known to the skilled person. Briefly, the polynucleotide of the disclosure can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

Additional methods for targeted mutagenesis in vivo are known. For example, a DNA sequence having the desired sequence alteration can be flanked by sequences homologous to the genomic target. One can then select or screen for a successful homologous recombination event. Generally, such a vector construct is designed having two regions of homology to the genomic target which flank a polynucleotide having the desired sequence. Introduction of the vector into a plant cell will allow homologous recombination to occur and to produce an exchange of sequences between the homologous regions at the target site.

Such methods of homologous recombination can further be combined with agents that induce site-specific genomic double-stranded breaks in plant cells. Such double strand break agents can be engineered to produce the break at a targeted site and thereby enhance the homologous recombination events. The cells that have been transformed may be grown into plants in accordance with conventional ways, known to the skilled person. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by RNA interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by using amplicon expression cassettes, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transcriptional gene silencing (TGS), and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by double-stranded RNA (dsRNA) interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by antisense suppression, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene through decreasing expression of an Nrf4 polypeptide may be achieved by sense suppression or cosuppression, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transposon tagging, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may also be achieved by using forms of mutagenesis, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of transformation.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell is done on a plant or a plant cell that already expresses BBML, a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 provided that said polypeptide essentially retains the functionality of the polypeptide of SEQ ID NO.: 1 .

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by RNA interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by using amplicon expression cassettes, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transcriptional gene silencing (TGS), and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, more preferably at least 75% sequence identity, even more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by double-stranded RNA (dsRNA) interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70 % sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by antisense suppression, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene through decreasing expression of an Nrf4 polypeptide may be achieved by sense suppression or cosuppression, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid. In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transposon tagging, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may also be achieved by using forms of mutagenesis, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of contacting a plant or a plant cell with a virus or a viral nucleic acid.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by RNA interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by using amplicon expression cassettes, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transcriptional gene silencing (TGS), and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by double-stranded RNA (dsRNA) interference, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by antisense suppression, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene through decreasing expression of an Nrf4 polypeptide may be achieved by sense suppression or cosuppression, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may be achieved by transposon tagging, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, disrupting an endogenous Nrf4 gene in the plant or the plant cell may also be achieved by using forms of mutagenesis, and introducing a recombinant nucleic acid construct encoding a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 is accomplished by the means of targeted insertion of a recombinant nucleic acid construct at a specific location in the plant genome. In certain embodiments of the present invention, once a single transformed plant has been obtained, e.g., a plant transformed with a desired gene, conventional plant breeding methods can be used to transfer the structural gene and associated regulatory sequences via crossing and backcrossing. In general, such plant breeding techniques are used to transfer a desired gene into a specific plant, e.g., a crop plant or another type of plant used for commercial purposes. Accordingly, the methods of the claimed invention can be used in, for example, plant breeding, plant improvement, propagation of unstable and/or recessive genotypes, seed production, and trait propagation, as well as other purposes involving the reproduction of plants.

According to the method of the present invention, any plant species may be so modified that an endogenous Nrf4 gene is disrupted and a BBML transgene is expressed, including, but not limited to, monocots. Examples of plant species of interest include, but are not limited to, corn also known as maize (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), wheat (Triticum aestivum), coconut (Cocos nucifera), pineapple (Ananas comosus), banana (Musa spp.), sugarcane (Saccharum spp.), oats (Avena sativa), barley (Hordeum vulgare), switchgrass (Panicum virgatum) and monocotyledonous vegetables and ornamentals.

Monocotyledonous vegetables include onion, garlic, and leek (all Allium spp.), asparagus (Asparagus officinalis), ginger (Zingiber spp.), and turmeric (Curcuma spp.) Monocotyledonous ornamentals include tulips (Tulipa spp.), daffodils (Narcissus spp.), orchids (Orchideacea), ornamental bananas (Musa spp.), and decorative plants of the ginger family (Zingiberales).

In certain embodiments, transgenic apomictic plants of the present invention are crop plants (for example, corn, sorghum, wheat, millet, sorghum, barley, oats, etc.). In preferred embodiments, corn, sorghum and barley plants are transgenic apomictic plants of the present invention. In most preferred embodiments corn is a transgenic apomictic plant of the present invention. Other plants suitable for practicing the present invention include grain plants that provide seeds of interest, monocotyledonous oil-seed plants, monocotyledonous biomass/forage/bioenergy species, other important crops such as banana, plantains, and sugarcane, and spice plants. Seeds of interest include grain seeds, such as com, wheat, barley, rice, sorghum, rye, oats etc. Monocotyledonous oil-seed plants include maize, palm, coconut, etc., and spice plants include ginger, cardamom, curcuma, etc.

In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the plant is selected from maize, wheat, barley, sorghum, rye, oat, millet, turf grass, switchgrass, sugar cane, banana, and rice. In certain embodiments of the transgenic apomictic plant of the present invention, as well as in certain embodiments of the method for the production of the apomictic plant of the present invention, the plant is selected from maize, wheat, rice, sorghum, barley, oat, lawn grass, rye, millet, sugarcane,. Preferably, the plant is selected from maize, wheat, barley, rice, and sorghum. More preferably, the plant is maize.

In certain embodiments of the method for the production of the apomictic plant of the present invention, if the plant cell was used, the method comprises the step of regenerating the plant from the plant cell. This step is known to the person skilled in the art.

In another embodiment, the present invention relates to a seed of the transgenic apomictic plant of the present invention. In a further aspect, the present invention relates to a seed of the apomictic plant obtained according to the method of the production of the apomictic plant of the present invention. Propagation of a plant through seeds wherein an endogenous Nrf4 gene is disrupted and a BBML transgene is expressed is possible over several consecutive generations, assuring phenotypic uniformity over apomictic generations as it was demonstrated in natural apomicts.

The seed of the present invention is a clonal seed that means it is genetically a copy of its maternal plant. In certain embodiments, in some apomicts, endosperm develops autonomously from the second female gamete, the central cell, without fertilization but in most apomicts, including all apomictic species in the grasses, endosperm develops after fertilization of the central cell (pseudogamy). Because in many species normal seed development requires a balanced endosperm with two maternal and one paternal genomes (2m: 1 p ratio), pseudogamy involves either endosperm that is insensitive to deviations from the 2m: 1 p ratio or adaptions in megagametogenesis, microgametogenesis, or double fertilization.

In another embodiment, the present invention relates to a method of increasing apomixis rate in a plant, comprising disrupting an endogenous Nrf4 gene in the plant or the plant cell; and inducing the expression of a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell. The means and methods for disrupting an endogenous Nrf4 gene in the plant or the plant cell according to the method for the production of the apomictic plant of the present invention as described herein, are applicable in the method for increasing apomixis rate in a plant of the present invention. The means and methods for inducing the expression of a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell according to the method for the production of the apomictic plant of the present invention as described herein, are applicable in the method for increasing apomixis rate in a plant of the present invention. In certain embodiments, wherein the plant cell was used, a plant is regenerated from the plant cells using the methods and means known to the skilled person.

As referred to herein, increasing apomixis rate is defined preferably as an increase of the frequency of clonal progeny in the engineered plants over controls, which can be selected from wild-type plant, a plant expressing BBML transgene, and a plant wherein Nrf4 expression is disrupted.

In certain embodiments of the method of increasing apomixis rate in a plant, the plant may be a non-apomictic plant, preferably selected from the plants suitable as transgenic apomictic plants of the present invention. In certain embodiments of the method of increasing apomixis rate in a plant, the plant is selected from maize, wheat, sorghum, barley, rye, oat, millet, turf grass, switchgrass, sugar cane, banana and rice. In certain embodiments of the method of increasing apomixis rate in a plant, the plant is selected from maize, wheat, rice, sorghum, barley, oat, lawn grass, rye, millet, sugarcane,. Preferably, the plant is selected from maize, wheat, barley, rice, and sorghum. More preferably, the plant is maize. In certain embodiments of the method of increasing apomixis rate in a plant, the plant may be a natural apomict.

In another embodiment, the present invention relates to use of one or more recombinant nucleic acid construct(s) for inducing apomixis in a plant. Herein, the one or more recombinant nucleic acid constructs are such that it/they are capable of disrupting an endogenous Nrf4 gene in the plant or the plant cell; and inducing the expression of a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell, provided that the said polypeptide retains functionality of the polylpetide of SEQ ID NO.: 1. The recombinant nucleic acid constructs suitable for disrupting an endogenous Nrf4 gene in the plant or the plant cell according to the method for the production of the apomictic plant of the present invention are described herein, and are applicable in the use for inducing apomixis in a plant of the present invention. The recombinant nucleic acid constructs suitable for inducing the expression of a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably 97% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell according to the method for the production of the apomictic plant of the present invention are described herein, and are applicable in the use for inducing apomixis in a plant of the present invention.

The present invention further relates to a vector for inducing apomixis in a plant, the vector comprising a recombinant nucleic acid construct suitable for disrupting an endogenous Nrf4 gene in the plant or the plant cell, and a recombinant nucleic acid constructs suitable for inducing the expression of a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 , as described herein, in the plant or in the plant cell. The recombinant nucleic acid constructs suitable for disrupting an endogenous Nrf4 gene in the plant or the plant cell according to the method for the production of the apomictic plant of the present invention are described herein and applicable in the vector for inducing apomixis in a plant of the present invention. The recombinant nucleic acid constructs suitable for inducing the expression of a polypeptide having at least 70% sequence identity, preferably at least 75% sequence identity to the polypeptide of SEQ ID NO. 1 in the plant or in the plant cell according to the method for the production of the apomictic plant of the present invention are described herein and are applicable in the vector for inducing apomixis in a plant of the present invention.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

The following examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention which is defined by the appended claims.

Examples

Herein, a successfully engineered diplosporous apomict with pseudogamous endosperm development in Zea mays (maize) by combining the non-reduction in female 4 (nrf4) mutant with a PsASGR-BBML transgene and crossing to a tetrapioid pollen donor is reported. Depending on the transgene insertion, the rate of clonal progeny was as high as 50%.

Plant material and growth conditions In this study the following Z. mays lines were used: the nrf4 mutant line in the W22 genetic background was generated previously at the University of Zurich (UZH) (US patent Application #20180142251 ); tetrapioid W23 line, carring the R1-nj allele of the r locus was generated at UZH by spontaneous doubling of the ploidy in the diploid W23 R1-nj line (Maize coop stock center M142V) with further selection for stability of the higher ploidy and a significant increase in seed set; several independent lines of Hi-Il Z. mays transformed with the PsASGR-BBML transgene that were generated at the University of Georgia (US patent Application #20160304901 A1). Plants were grown under controlled greenhouse conditions with 16 hours light at 25 °C and 8 hours dark with 22 °C and constant 60% humidity. Manual pollinations were done according to commonly used methods.

DNA extraction

DNA was extracted from the frozen leaf punches by ReliaPrep™ 96 gDNA Miniprep HT System 4 x 96 (A2671 , Promega) according to the manufacturer’s instructions.

Genotyping and SSR PCR

Primers used for genotyping the nrf4 mutation and BBML transgene are summarized in Table 1. PCR was perfomed using the Go-Taq PCR kit (Promega) according to the manufacturer’s instructions. 5-20 ng of DNA was used as template for PCR. The genotyping PCR amplicons were separated on a 1.5% agarose gel containing Midori dye and images were taken by a UVP Gel Imaging System. PCR products of SSR markers were analysed by a QIAxcel Advanced System (Qiagen) using the QIAxcel DNA High Resolution Kit (Qiagen).

Table 1. Primers and PCR conditions used in this study ddPCR

To quantify the copy number of the PsASGR-BBML transgene a digital droplet PCR was used. As the vector used for transformation contained the bialaphos resistance gene (bar), the copy number of bar in comparison to the maize Alcohol dehydrogenasel (ZmAdhl) single copy gene was quantified. ddPCR was performed using the Bio-Rad ddPCR platform according to the manufacturer’s recommendations. Primers and PCR conditions are listed in Table 1.

Ploidy analysis

Fresh leaf tissue was chopped in OTTO1 buffer (0.1 M citric acid, 0.5% Tween-20) using a sharp razor blade. The extract containing the nuclei was filtered through a 30 pm filter tip and 50 pl of the cleared extract was added to 400 pl of OTTO2 buffer (0.4 M Na2HPO4, 4 pg/ml DAPI). Ploidy measurements were done using a CytoFlex flowcytometer with the UV diode as light source and a 450 nm filter for emitted light.

Phenotypic measurements

Several different vegetative and reproductive traits were measured. Vegetative phenotypes included the following traits: Height of a plant to the flag leaf was measured as distance from soil to the collar of the flag leaf. Total number of leaves was counted when the flag leaf was visible. Number of internodes to the first ear was counted as the number of internodes below the lowest ear. Total height of plant was measured as the distance from the soil level to the top of the tassel when it was fully developed. The growth rate of the plants was also estimated. The height of the plant was measured as the distance from the soil level to the top of the highest leaf in 3-4 day intervals starting at one month after sowing and ending at two months after sowing. The growth rate was calculated as increase in the height of the plant from the previous measurement divided by the total height of the plant.

Reproductive traits included the number of ears per plant, number of branches on the tassel, days till silking, days till shedding pollen and the number of rows on an ear. Qualitative phenotypes included color of silks, color of anthers, and color of the cob.

Statistical analysis

ANOVA was performed using the Ime4 version 1.1-28 package for R. p values for ANOVA tables and significances were calculated using the ImerTest package for R. Graphics were generated by ggplot2 version 3.3.5 package for R.

Example 1 - Characterization of PsASGR-BBML transgenic lines

To test the efficiency of parthenogenesis five different transgenic lines of maize carrying either the genomic region of apomictic Pennisetum squamulatum that contained the promoter and gene sequence of PsASGR-BBML (lines M3A-H1-1 , 47A, and M24A) or only the gene sequence of PsASGR-BBML driven by the DD45 (also known as EC1.2; Sprunck et al, Science. 2012 Nov 23;338(6110): 1093-1097. doi: 10.1126/science.1223944) promoter (lines 45-17b-6 and 45-24a-9) (Conner et al, Plant Reprod. 2017 Mar;30(1):41-52. doi: 10.1007/s00497-017-0298-x) were analyzed. As none of the lines were fixed for the transgene, seedlings were genotyped for presence of the PsASGR-BBML transgene and plants carrying PsASGR-BBML (i.e., PsASGR-BBML positive plants) were crossed as females to nrf4 mutant plants (Figure 1A). If this cross was not possible due to asynchronous flowering time, nrf4 heterozygous plants were crossed as females to plants carrying the PsASGR-BBML transgene (i.e., PsASGR-BBML positive plants).

PsASGR-BBML transgene copy number analysis

F1 progeny of all successful crosses were genotyped for the presence of the PsASGR- BBML transgene and the nrf4 mutation. Individuals that carried a mutant allele of nrf4 and the PsASGR-BBML transgene (i.e., individuals carrying a mutant allele of nrf4 and positive for the PsASGR-BBML transgene) were subjected to transgene copy number analysis by ddPCR. As it is well documented that multiple transgene copies can lead to the silencing of the transgene through epigenetic mechanisms, work with lines carrying only one transgene copy was preferred. The bialaphos resistance gene (bar) was used to quantify the number of PsASGR-BBML transgenes, as barwas part of the vector sequence transformed into maize plants. ZmAdhl was used as a single copy gene control. As F1 plants from the cross of plants carrying the PsASGR-BBML transgene and nrf4 mutant plants were used, the transgene was expected to be hemizygous (one allele), whereas the internal control (ZmAd f) was expected to be homozygous (two alleles). The copy number of the PsASGR-BBML transgene varied in different lines (Table 2). Individuals with only one copy of the PsASGR-BBML transgene could be identified in lines M3A-H1-1 and M24A, while line 47A contained six copies and lines 45-24a-9 and 45-17b-6 three and two copies of the transgene, respectively. Further experiments were continued with individuals having the minimal number of insertions for each specific line.

Table 2. Number of BBML transgene insertions in different BBML lines

Characterization of the parthenogenesis rate in PsASGR-BBML lines

In crosses where PsASGR-BBML transgenic plants were used as females and the nrf4 mutant as pollen donor, parthenogenesis rates were estimated by genotyping F1 individuals for presence of the nrf4 mutant allele and subsequent confirmation by ploidy analysis. It was reasoned that if embryos in PsASGR-BBML transgenic plants had developed parthenogenetically (if PsASGR-BBML transgenic plant develop parthenogenetic embryo), such embryos should not carry a nrf4 mutant allele and should be haploid due to the absence of fertilization. Although no haploids were observed in line M24A, the two other lines subjected to this analysis showed a high rate of parthenogenesis reaching up to 25% (Table 3). Interestingly, one case of twin seedlings in line 45-24a-9 where both of the twins were haploids was observed. On the other hand, there were 8 twin pairs from the M3A-H1-1 line and in all these cases one of the pair was diploid (developed upon fertilization) and the second one haploid (developed parthenogenetically), with one exception where both twins were haploid. Difference in parthenogenesis rate and observed phenotypes between different lines may arise due to position effects on the T-DNA insertions, (partial) epigenetic silencing of the transgene, and/or effects of the different promoters used. Each of these factors could change the level of expression of the PsASGR-BBML transgene in the maize embryo sac or egg cell.

It is noted that at least two out of three analyzed lines showed a high level of parthenogenesis, which is sufficient to perform a proof-of-principle experiment for the engineering of apomixis in maize.

Table 3. Estimation of the parthenogenesis rate in different PsASGR-BBML lines

Example 2 - Production of clonal seed in maize by diplosporous pseudogamous apomixis

To generate individuals that have a potential to reproduce apomictically, plants that are homozygous for the nrf4 mutation, which produce up to 35% of unreduced, unrecombined gametes (apomeiosis), and carry the PsASGR-BBML transgene, which induces the second component of apomixis, parthenogenesis, were generated. F1 individuals heterozygous for the nrf4 gene and carrying the PsASGR-BBML transgene were self-pollinated (Figure 1A). Not all self-pollinations were possible due to asynchrony in male and female flowering time in some individuals, but sufficient number of F2 kernels could be obtained for all combinations of nrf4 with the PsASGR- BBML lines, except for nrf4 PsASGR-BBML 47A; this line was thus not included in further analyses. 96 F2 kernels segregating nrf4 and PsASGR-BBML M3A-H1-1 or nrf4 and PsASGR-BBML 45-24a-9, and 192 F2 kernels segregating nrf4 and PsASGR- BBML M24A or nrf4 and PsASGR-BBML 45-17b-6 were germinated and genotyped to identify nrf4/nrf4', PsASGR-BBML-postive individuals. All such individuals were pollinated by a tetrapioid W23 line carrying the R1-nj allele. A tetrapioid pollen donor was used to ensure normal development of the endosperm with a balanced maternal to paternal genome ratio of 2m:1 p. Male plants carried the R1-nj allele to facilitate seed screening, as sexually derived embryos express the R1-nj allele that leads to a dark pigmentation of the embryo, whereas parthenogenetically derived embryos are unpigmented.

The majority of the nrf4/nrf4', PsASGR-BBML individuals had a reduced but sufficient (still satisfying) seed-set with almost no shrunken kernels, confirming the nearly full penetrance of the non-reduction phenotype in the nrf4 mutant (Figure 1 B). R1-nj screening of all kernels derived from all the crosses was performed to identify kernels with potentially parthenogenetically derived embryos (Table 4). The highest number of putative diploid/parthenogenetically derived kernels was observed in the PsASGR- BBML 45-17b-6 line, which was not analyzed previously. The lowest parthenogenesis rate was observed in the PsASGR-BBML 45-24a-9 line, which had quite a high parthenogenesis rate in the previous generation (see Table 2). This reduction may have been caused by silencing of the PsASGR-BBML transgene. Putative diploid kernels from some of the ears were germinated and a ploidy analysis of the seedlings was performed (Table 4). For three out of four independent lines, some diploid individuals were obtained. These individuals are putative clonal offspring because they are derived from unreduced female gametophytes upon parthenogenetic development of the egg cell, i.e., by apomixis. To confirm that these individuals are indeed clonal, that is genetically identical to their corresponding mother plant, SSR marker analysis of a subset of the individuals was performed. Progeny of plant NC2638-18 (5 individuals), plant NC2641-15 (16 individuals), and plant NC2642-12 (16 individuals) were used for this analysis. All five individuals in the progeny of plant NC2638-18 (nrf4/nrf4 PsASGR-BBML M3A-H1-1) lost heterozygosity to certain extent due to residual recombination, which was previously reported to occur in nrf4 mutants. On the other hand, six clonal individuals in the progeny of plant NC2641-15 and two clonal individuals in the progeny of plant NC2642-12 (Figure 1 C) were idenfied. Those individuals habe been named A1 for being the first apomictic generation. This result, i.e., the presence of clonal individuals in the progeny of nrf4/nrf4 PsASGR-BBML- positive plants, is a proof-of-principle demonstrating that it is possible to engineer diplosporous apomixis with pseudogamous endosperm development, as demonstrated herein in maize, the most widely grown crop plant world-wide. All the clonal individuals were grown to maturity and pollinated with the tetrapioid W23 R1-nj line to obtain the second apomictic seed generation (A2) and thereby assess whether propagation of maize for several consecutive generations by apomixis is possible and whether it will ensure phenotypic uniformity over apomictic generations as it was demonstrated in natural apomicts (Sailer et al., Curr Biol, 2016, 26(3):331-7, DOI: 10.1016/j.cub.2015.12.045).

Table 4. Screening for diploid progeny from nrf4/nrf4; PsASGR-BBML plants crossed to 4n W23 R1-n

PDK - putative diploid kernels identified by R1-nj screening PA - ploidy analysis

* - this ear was segregating PsASGR-BBML and only seedlings that carried the PsASGR-BBML transgene were screened by ploidy analysis

** - number of analyzed plants is greater than number of germinated kernels due to the presence of twin embryos in some kernels

^§ - a mother plant that gave rise to first and second apomictic generations used for subsequent phenotypic analyses

Phenotypic stability of apomicts maize

Apomictic reproduction leads to the propagation of identical genotypes that, in turn, are assumed to assure phenotypic stability of apomicts from generation to generation.

To test this assumption, clonal individuals from the two consequent apomictic generations A1 and A2 were grownt in a fully randomized fashion and various traits were measured in these plants. Together with clonal plants, sibling plants that arose from unreduced but recombined egg cells that developed parthenogenetically (S1 and S2 in Figure 1) were grown. These individuals lost heterozygosity to a certain extent and are supposed to show a higher phenotypic variability than apomicts. However, because of the absence of reduction and fertilization, they are genetically much closer to the apomicts than sexual progeny would be. Vegetative, reproductive, and growth- related phenotypes on six A1 plants and 13 corresponding sibling S1 plants with recombination, as well as on 12 A2 individuals and 20 corresponding sibling S2 plants, were measured (Figure 1).

We identified marginally significant difference between the two apomictic generations for total plant height (F=0.0771, P=0.1, Fig. 2A, Table 5), when analyzing only apomictically derived plants. The difference became insignificant when apomictic as well as sibling plants were analyzed, controlling for generation, parental plant, reproduction type, and interactions of those factors (Table 5). No differences between generations and also between modes of reproduction were found for plant height till the flag leaf (Fig. 2B, Table 5). Total number of leaves per plant did not vary between generations in apomicts but was significantly different between generations in siblings (F= 2.974, P=0.0949)', moreover, the number of leaves was significantly higher in apomicts than in siblings (F= 6.7727, P=0.01376, Fig. 2C, Table 5). Number of internodes below the first ear did not significantly vary between plants of the two apomictic generations but was significantly different between two generations of sibling plants (F= 4.619, P=0.0368, Fig. 2D, Table 5). We have not identified any significant difference in the growth rate between generations, either in apomicts or their siblings (Fig. 2E, Table 5). Interestingly, however, we identified significant differences in the expression of this trait in offspring of different mother plants, suggesting that this trait might be caused by a significant maternal effect (Table 5). Generation did not have significant influence for the days till silking phenotype, neither in apomicts nor in siblings, yet siblings were silking significantly later than apomicts (F= 4.1353, P=0.0161, Fig. 2F, Table 5). Days till shedding pollen did not show a significant difference either between generations in apomicts or between generations in siblings. Yet it was slightly but significantly higher in sibling plants (F= 2.6865, P=0.0873, Fig. 2G, Table 5). The present inventors did not identify a significant difference in number of kernel rows per ear neither between apomictic generations nor between sibling generations (Fig. 2I, Table 5). Yet. The present inventors found that parental plants have a significant influence on this trait, suggesting maternal effects (F= 5.721, P=0.0243, Table 5). Furthermore, apomicts have slightly but significantly more kernel rows per ear in comparison to siblings (F= 4.4915, P=0.0669, Fig. 2I, Table 5). Neither generation nor mode of the reproduction had a significant influence on the number of branches on the tassel (Fig. 2J, Table 5). Number of ears per plant was strikingly different from all other trats and showed quite a big and statistically significant difference between apomictic generations (F=15.4839, P=0.0013), whereas this trait was quite stable in two generations of the sibling plants (Fig. 2H, Table 5).

All the qualitative phenotypes, such as color of silks, color of anthers, color of a cob. were identical in all plants both of apomictic origin and in sibling plants.

Table 5. Mean values and standard deviations of the phenotypic traits

Conclusions

Taken together, the present inventors developed a method to engineer synthetic apomicts in maize. Furthermore, the present inventors tested two consequent apomictic generations as well as corresponding sibling plants for stability of the phenotypes, the present inventors identified that most of the phenotypes are stably inherited in apomictic plants from generation to generation. The only exception is the trait number of ears per plant, which was higher in first apomictic generation and went down to the level of the sibling plants in the second apomictic generation. This might be due to some environmental effect, which the present inventors did not account for, and/or partially due to a maternal effect. In five out of ten quantitative traits, the present inventors identified significant differences between apomictic and sibling plants. Apomicts were outperforming sibling plants in the total number of leaves per plant, number of ears per plant as well as number of kernel rows per ear. Furthermore, apomicts had slightly less days till shedding pollen and days till silking.

Thus, with the exception of plant height and number of ears per plant, all traits were stably inherited across apomictic generations. The two traits that differed are correlated and, as pointed out above, this difference is likely due to environmental or maternal effects and not the mode of reproduction. During summer, pollinations in the greenhouse are often unsuccessful, leading to low seed set on some ears resulting in larger seeds. These typically give rise to larger plants that have more ears. In contrast, half of the traits were significantly different between these apomictic generations and their siblings, indicating that even minor genetic differences can lead to phenotypic variation. As mentioned before, although recombination occurred, these siblings arose parthenogenetically from unreduced gametes and thus differed much less from the apomictic plants than normal sexual progeny would.

In summary, the results disclosed herein demonstrate that apomictic reproduction preserves not only genotypes but also phenotypic traits.