Parthenocarpic plants comprising a loss of function mutation in MBP22 and methods of producing same

Abstract

A Solanaceous plant exhibiting a facultative parthenocarpy and at least 80 % of fruit yield being devoid of homeotic aberrations and comprising a loss-of-function mutation in &MBP22 gene is provided. Also provided are methods of producing and using same.

Description

[0001] PARTHENOCARPIC PLANTS COMPRISING A LOSS OF FUNCTION MUTATION IN

[0002] MBP22 AND METHODS OF PRODUCING SAME

[0003] RELATED APPLICATIONS

[0004] This application claims priority from U.S. Provisional Patent Application No, 63 / 615,332 filed 28 December 2023, which is hereby incorporated by reference in its entirety.

[0005] SEQUENCE LISTING STATEMENT

[0006] The XML file, entitled 101869SequenceListing.xml, created on 30 October 2024, comprising 86,016 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0007] FIELD AND BACKGROUND OF THE INVENTION

[0008] The present invention, in some embodiments thereof, relates to parthenocarpic plants comprising a loss of function mutation in MBP22 and methods of producing same.

[0009] In flowering plants, fruit development is critical for the completion of their life cycle because the biological function of fruiting is the production and dissemination of seeds. Fruits develop from various floral tissue(s) that expand, and "true" fruits, such as tomato (Solarium ly coper sicum), originate from the flower ovary that contains one or more ovules (seed precursors) (Gillaspy et al., 1993). The ovules are the site for sexual plant reproduction where megagametogenesis, fertilization and embryogenesis take place. At anthesis, the mature tomato ovule consists of a female gametophyte (embryo sac) that is enclosed by a single maternal integument containing 6-7 cell layers of which, the innermost one is differentiated into a typical endothelium. The funiculus, a stalk-like maternal structure, anchors the ovule to the placenta (Cooper, 1931). In tomato, the unpollinated ovary, which develops in concert with the rest of the flower whorls, enters a temporary growth-arrest phase 1-2 days before anthesis (Gillaspy et al., 1993). It is well established that the developmental switch that turns a quiescent ovary into a rapidly growing fruit (fruit set) depends on successful flower pollination and ovule fertilization (Seymour et al., 2013). In contrast, in the absence of fertilization, ethylene is produced, promoting ovary senescence and flower abscission (Llop-Tous et al., 2000; Carbonell-Bejerano et al., 2010). This scenario implies that a fertilized ovule emits a signal that induces the quiescent ovary to set a fruit. Among the phytohormones identified as playing a role in the transition from arrested ovary to a growing fruit, several lines of evidence singled out auxin and gibberellins (GAs) as prominent positive regulators of fruit set, with auxin operating both independent and upstream to GA (Serrani et al., 2008; Vriezen et al., 2008; de Jong et al., 2009; Molesini et al., 2020; Fenn and Giovannoni, 2021). In tomato, following double fertilization, auxin levels increase in the ovules and in the ovary inducing active GA accumulation in the ovary. The increased auxin and GA levels in the ovary activate corresponding signaling pathways that in turn trigger ovary growth ( / '.<?., fruit set) by regulating cell division and cell expansion (Fenn and Giovannoni, 2021). Based on the demonstrated ability of Auxin Response Factors (ARFs), Auxin / Indole-3 -Acetic Acid (Aux / IAA) and DELLA proteins to physically interact, a cross-talk between the auxin and GA pathways has been suggested (Hu et al., 2018). This crosstalk was proposed to involve the gaseous hormone ethylene acting to prevent GA perception and tomato fruit set (Shinozaki et al., 2018a). However, the exact regulatory mechanisms translating these hormonal signals to tomato ovary arrest before anthesis, and its release in response to fertilization or auxin application remain unclear (Joldersma and Liu, 2018; Fenn and Giovannoni, 2021).

[0010] However, fruit set sometimes occurs in an alternative, fertilization-independent pathway referred to as parthenocarpy, which means 'virgin fruiting’ in Greek (Joldersma and Liu, 2018). Previously it was shown that the tomato AGAMOUS-Like6 (SIAGL6) MADS-box transcription factor (TF) loss-of-function mutant produces seeded as well as parthenocarpic fruits with wildtype characteristics (Klap et al., 2017). And a similar phenotype was later reported for the agl6 / Pat-k mutant (Takisawa et al., 2018). The expression of S1AGL6 peaks in the anthesis flower ovary and declines in the set fruit (Klap et al., 2017), as well as, in unfertilized ovaries triggered to set fruit either by auxin and GA application (Tang et al., 2015; Hu et al., 2018) or by silencing their signaling components AUX / IAA9 (Wang et al., 2009) and S1DELLA (Shinozaki et al., 2020), respectively. Within the developing ovary, S1AGL6 is predominantly expressed in the immature ovule integument, and upon ovule maturation, its expression shifts to the endothelium. Consistent with this, unfertilized slagl6CR'sglmutant ovules are enlarged due to integument over-proliferation and lack a characteristic endothelium, the integument’s innermost layer. RNA-Seq analysis of unfertilized slagl6CR'sglovules transcriptome indicated that it profoundly differs from that of wildtype ovules and underwent vast reprogramming that mimics the transcriptional changes occurring in wild-type ovules following fertilization. In particular, genes that were found to preferentially express in the mature unfertilized ovule were downregulated and those that were preferentially express in 4 DPA fertilized ovule integument and funiculus were upregulated (Gupta et al., 2021). These studies suggest that S1AGL6 acts from within the ovule integument as a switch that, unless turned off, prevents fruit set by suppressing fertilization-associated transcriptional reprogramming.

[0011] The SIAGL6 gene encodes a MIKCctype II MADS-box TF, and as such it is formed by the MADS (M), intervening (I), keratin-like (K) and C-terminal domains (Kaufmann et al., 2005a). Seed plants MIKCcgroup proteins function as master developmental regulators by repressing or activating target genes (Smaczniak et al., 2012a; Schilling et al., 2018). This is accomplished by binding through the highly conserved MADS domain, as homo- or heterodimers, to the DNA- binding motif CArG-box (CC(A / T)6GG) (Aerts et al., 2018), or as tetramers formed by complexing of two dimers attached to two spaced CArG-box motifs (TheiBen et al., 2016). The K domain acts as a determinant of oligomerization strength and specificity, playing roles in both dimerization and tetramerization (Kaufmann et al., 2005a). It was suggested that the petunia AGL6, which is highly similar to S1AGL6, has a SEPALLATA (SEP)-like function in floral patterning (Rijpkema et al., 2009). Namely it functions like an E-class protein which, according to the ABCDE and the floral quartet models, participates and serves as central protein- protein interaction hub in the various tetrameric complexes determining floral organ identity (Kaufmann et al., 2005b; Smaczniak et al., 2012b; TheiBen et al., 2016). Indeed, AGL6 homologs from petunia and other plants showed a conserved and broad spectrum of interactions with other MIKCcMADS-box proteins (Immink et al., 2003; Rijpkema et al., 2009; Dreni and Zhang, 2016).

[0012] Additional related background art:

[0013] Li et al. 10.21203 / rs.3.rs-519249 / vl;

[0014] Li et al. (2020) Plant Science 301: 110672;

[0015] WO2017 / 125931

[0016] SUMMARY OF THE INVENTION

[0017] According to an aspect of some embodiments of the present invention there is provided a Solanaceous plant exhibiting a facultative parthenocarpy and at least 80 % of fruit yield being devoid of homeotic aberrations and comprising a loss-of-function mutation in a MBP22 gene.

[0018] According to some embodiments of the invention, the plant of claim is a tomato.

[0019] According to some embodiments of the invention, the plant is a tomato, eggplant or pepper. According to some embodiments of the invention, the plant further exhibits at least one of:

[0020] (i) a fruit yield / plant at least the same as that of a non-parthenocarpic plant of the same genetic background under fertilization permissive conditions;

[0021] (ii) an average fruit weight / plant at least the same as that of a non-parthenocarpic plant of the same genetic background under fertilization permissive conditions; (iii) when the plant is tomato the fruit comprises a jelly fill;

[0022] (iv) enlarged ovules within the seedless fruits developed from non-fertilized ovaries;

[0023] (v) petals color and sepals length of a flower having wild type AGL6 alleles;

[0024] According to some embodiments of the invention, the fruit yield is mature fruit yield.

[0025] According to some embodiments of the invention, the plant is a processing tomato.

[0026] According to some embodiments of the invention, the plant is a determinate tomato.

[0027] According to some embodiments of the invention, the plant is an indeterminate tomato.

[0028] According to some embodiments of the invention, the plant is a semi-determinate tomato.

[0029] According to some embodiments of the invention, the plant is an elite line.

[0030] According to some embodiments of the invention, the plant is transgenic.

[0031] According to some embodiments of the invention, the tomato is of a species selected from the group consisting of Lycopersicon esculentum, Lycopersicon cerasiforme, Lycopersicon pimpinellifolium, Lycopersicon cheesmanii, Lycopersicon parviflorum, Lycopersicon chmielewskii, Lycopersicon hirsutum, Lycopersicon penellii, Lycopersicon peruvianum, Lycopersicon chilense and Solanum lycopersicoides.

[0032] According to some embodiments of the invention, the tomato is selected from the group consisting of a single fruit per truss, branched tomato and cherry tomato.

[0033] According to some embodiments of the invention, the facultative parthenocarpy is manifested under heat or cold stress.

[0034] According to some embodiments of the invention, the plant is an inbred.

[0035] According to some embodiments of the invention, the loss-of-function mutation is in a homozygous form.

[0036] According to some embodiments of the invention, the plant further comprises a loss-of- function mutation in an AGL6 gene.

[0037] According to some embodiments of the invention, the plant comprises a silencing agent for suppressing expression of an MBP22 gene and optionally an AGL6 gene.

[0038] According to some embodiments of the invention, the plant exogenously expresses a nuclease selected from the group consisting of a meganuclease, an RNA-guided DNA endonuclease, a zinc-finger nuclease and a TALEN.

[0039] According to an aspect of some embodiments of the present invention there is provided a fruit of the plant as described herein.

[0040] According to an aspect of some embodiments of the present invention there is provided a seed of the plant as described herein. According to an aspect of some embodiments of the present invention there is provided a hybrid seed produced of the plant as described herein.

[0041] According to an aspect of some embodiments of the present invention there is provided an edible processed product of the plant or fruit as described herein.

[0042] According to some embodiments of the invention, the processed product is selected from the group consisting of a tomato paste, a ketchup, a tomato sauce a tomato soup, a tomato juice, a tomato powder, a tomato dice, a crushed tomato, a chopped tomato and a tomato concentrate.

[0043] According to an aspect of some embodiments of the present invention there is provided a method of producing a processed product, the method comprising processing the fruit as described herein to produce an edible processed product.

[0044] According to an aspect of some embodiments of the present invention there is provided a method of producing the plant as described herein, the method comprising down-regulating expression or activity of MBP22 gene in the plant and detecting a mutation in MBP22 gene in the plant.

[0045] According to some embodiments of the invention, the down-regulating is effected by treating the plant with an RNA silencing agent.

[0046] According to some embodiments of the invention, the down-regulating is effected by treating the plant with an DNA editing agent.

[0047] According to some embodiments of the invention, the method further comprises downregulating expression or activity of AGL6 gene in the plant and detecting a mutation in AGL6 gene in the plant.

[0048] According to an aspect of some embodiments of the present invention there is provided a method of breeding comprising selfing or crossing the plant as described herein.

[0049] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0050] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0051] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0052] In the drawings:

[0053] Figs. 1A-F show the characterization of S1AGL6 interactors. (A) Schemes of S1AGL6 and interactor proteins identified through Y2H screen. The MADS-box domain (yellow), Intermediate domain (blue), K domain (pink) and C-terminal domain (black line) are indicated. Protein length (aa) is indicated on the right, and the parts identified in the Y2H clones are indicated by white dotted lines. For each interactor, the interaction category and number of identified clones are indicated in parenthesis. (B-C) Expression profiles of S1AGL6 and SIMBP22 in laser dissected anthesis ovary (0 DPA) and set fruit (4-5 DPA) tissues of S.pimpinellifolium (B) or S.lycopersicum cv M82 (C). The expression intensity is in reads per million (RPM). Expression data was retrieved from the Tomato Expression Atlas (http: / / tea(dot)solgenomics(dot)net / ) (Shinozaki et al., 2018b). ov, ovule; pl, placenta; sp, septum; p, pericarp; em, embryo; en, endosperm; sc, seed coat; f, funiculus; s, seeds; c, columela; It, locular tissue; tp, total pericarp. (D) Expression levels of SIAGL6 and SIMBP22 in Micro-Tom tomato 2 days ahead of anthesis (2DAA) ovaries following flower emasculation, and 4 days post their treatment at anthesis by artificial pollination (4DPAP), exogenous application of 2,4-D (4DPAT) or GA3 (4DPGT), or 6 days post flower emasculation (6DPE). The expression intensity is in number of transcripts per million clean tags (TPM). Expression data was retrieved from (Tang et al., 2015). (E) RT-qPCR quantitation of S1AGL6 and SIMBP22 in developing ovaries (developmental stages according to Gupta et al., 2021) and 4 DPA set fruits. Relative expression levels were normalized to SITIP41 (Solycl0g049850') as the reference gene and calculated by the comparative delta delta Ct (AACt) method. Error bars indicate +SD over at least two biological replicates. The indicated P-values were determined by Student’s t test. (F) Pull-down experiments confirming the interaction between S1AGL6 and S1MBP22. The proteins used as "bait" and "prey" in each experiment and the antibody (ctHalo and aH A) used for their detection are indicated. The pull-down was performed with the HaloLink™ resin. Molecular weights (MW) are indicated to the left of each panel.

[0054] Figs. 2A-D show CRISPR mutants of the SIMBP22 gene and their leaf and flower phenotypes. (A) Schematic representation of the SIMBP22 gene structure. Black boxes and lines indicate exons and introns, respectively. White asterisk indicates the location of the start codon in the 1stexon and half arrows indicate the locations of primers used for mutant genotyping. The gRNA targeted sequences and protospacer adjacent motifs (red) are shown in the expanded region. (B) Nucleotide sequences of the slmbp22CR'20(a non-contiguous portion of SEQ ID NO: 6) and slmbp22CR'10(a non-contiguous portion of SEQ ID NO: 6) mutant alleles aligned to the SIMBP22 wild type sequence (SEQ ID NO: 6). The gRNAs target sequences are highlighted in red, ~ indicates omitted nucleotides and the number after A indicates deletion size in bp. (C) Pictures of indicated genotypes full expanded leaf. Scale bars = 1 cm. (D) Representative pictures of intact 2 DPA flowers and their isolated organs of the indicated genotypes. Left to right, whole flower, sepals, petals, anther cone, stamens, and pistil. Scale bars = 2 mm.

[0055] Figs. 3A-I show fruit and seed phenotypes of the slmbp22CRmutants. (A) Pictures of representative red fruits and their respective cross-sections of indicated genotypes (below). Only seedless fruits are shown in the case of slmbp22CR'10, slmbp22CR'20and slagl6CR'sgl. Scale bars = 1 cm. (B) Pictures of representative seeds and soft seed-like structures (right) extracted from seeded and seedless (when applicable) red ripe fruits, respectively, of indicated genotypes. Scale bar = 1 mm. (E) Binocular images of manual longitudinal sections of representative seeds of indicated genotypes. Scale bar = 500 pM. SC, seed coat; en, endosperm; em, embryo. (D) Mean rate (%) of seedless, under seeded and seeded fruits in slmbp22CR'20and its parental line MP-1 (wild type). Numbers of analyzed fruits are indicated. (C, F-G) Measurements of MP-1 (wild-type) and slmbp22CR'20fruit weight (C), seed weight (F) and area (G). In E-G, box center line, median; box limits, upper and lower quartiles; whiskers, min and max values; points, individual values; n = number of fruits (E) or seeds (F-G). The indicated P-value was determined by Student’s t test. (H- I) Mean rate (%) of seed germination of indicated genotypes. Error bars indicate +SD over four biological replicates (I, n = 50 each) or two biological replicates (H, n = 50 each).

[0056] Figs. 4A-D show the characterization of slmbp22CR'20mutant ovules. (A) Pictures of representative plastic -embedded longitudinal sections of indicated genotypes stage 18 ovaries. The middle and rightmost panels show close up views of left panel ovules, which were sectioned at the equatorial position. Scale bars = 50 pm. Numbers indicate integument layer position, pe, pericarp; pl, placenta; ov, ovule; f, funiculus; et, endothelium; es, embryo sac; it, integumentary tapetum; pa, parenchyma cell layers; ep, epidermis. (B-C) Measurements of wild type and slmbp22CR'20stage 18 ovules embryo sac area (B) and lengths (C). Box center line, median; box limits, upper and lower quartiles; whiskers, min and max values; points, individual values; average, plus sign; n = number of ovules; P-values were determined by Student’s t test. (D) Number of different integument cell layers in stage 18 ovules of wild type and slmbp22CR'20ovules.

[0057] Figs. 5A-I show global gene expression profiles in slmbp22CR'20ovules. (A) Principal component analysis of all expressed genes showing distinct wild-type and slmbp22CR'20groups. (B) Total number of DEGs between wild-type and slmbp22CR'20ovules. (C) Number of unique and shared DEGs between the slmbp22CR'20and slagl6CR'sglovules. (D, G) Heat maps showing the differential expression (Log2FC) of parthenocarpy (D) and hormone (G) associated DEGs in slmbp22CR'20and slagl6CR'sglovules, and their expression in 0 DPA and 4 DPA ovary tissues according to Pattison et al., (2015). Ovary tissues are: Ov, ovule; Pl, placenta; S, septum, Pe, pericarp; Em, embryo; En, endosperm; Sc, seed coat; F, seed funiculus. Scaled log2 (left) or relative (right) expression values are shown from blue (lowest) to red (highest) expression levels. Gray indicates expression below detection level. (E-F,H-I) Quantitation by RT-qPCR of SIKLUH (E), SIARF5 (F), SIAGL6 (H) and SIMBP22 (I) in stage 18 ovules of wild-type and indicated mutant genotype. Relative expression levels were normalized by SITIP41 as a reference gene and calculated by the comparative delta Ct (AACt) method. Error bars indicate +SD over three biological replicates. The indicated P-values were determined by Student’s t-test.

[0058] Fig. 5J shows functional classification of slmbp22CR'20ovule DEGs according to Pattison et al. (2015).

[0059] Figs. 6A-D show profiling of cluster-related slmbp22CR'20ovule DEGs. (A) Number of slmbp22CR'20ovule DEGs belonging to indicated clusters as defined by Pattison et al. (2015). (B- D) Heat maps showing the differential expression (Log2FC) of cluster 23 (unfertilized ovules) (B), cluster 14 (4 DPA seed coat ) (C) and cluster 21 (4 DPA seed funiculus) (D) DEGs in slmbp22CR'20and slagl6CR'sglovules, and their relative expression in 0 DPA and 4 DPA ovary tissues according to Pattison et al., (2015). Ovary tissues are: Ov, ovule; Pl, placenta; S, septum, Pe, pericarp; Em, embryo; En, endosperm; Sc, seed coat; F, seed funiculus. Scaled log2 (left) or relative (right) expression values are shown from blue (lowest) to red (highest) expression levels. Gray indicates expression below detection level.

[0060] Figs. 7A-F show phenotypic characterization of the slagl6CR'sglslmbp22CR'20double mutant (DM). (A) Representative pictures of whole anthesis flowers and their isolated organs from wild type and DM. Left to right, whole flower, sepals, petals, anther cone, anthers, and pistil. Scale bars = 5 mm. (B) Pictures of representative wild-type and DM red-ripe fruits (left, scale bars = 1 cm), their manual cross-sections (middle, scale bars = 1 cm), and representative seeds extracted from seeded fruits (wild type, DM) and tiny soft seed-like structures extracted from seedless DM fruits (right), scale bars = 1 mm. (C) Mean rate (%) of seedless, under-seeded and seeded fruits in DM and wild type. Numbers of analyzed fruits are indicated above columns. (D) Binocular images of manual longitudinal sections of representative DM seeds. Scale bar = 500 m. (E) Average seed germination percentage of indicated genotypes in soil. Error bars indicate +SD over three biological replicates (n=36 / replicate). (F) Pictures of representative plastic-embedded longitudinal sections of stage 18 ovaries of wild type and DM stained with toluidine blue. Scale bar = 100 pm. Numbers indicate integument layer position, pe, pericarp; pl, placenta; ov, ovule; f, funiculus; et, endothelium; es, embryo sac; it, integumentary tapetum; pa, parenchyma cell layers; ep, epidermis.

[0061] Fig. 8 shows multiple alignment of the tomato protein sequence of MPB22 to the pepper and eggplant sequences (SEQ ID Nos: 27-32).

[0062] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0063] The present invention, in some embodiments thereof, relates to parthenocarpic plants comprising a loss of function mutation in MBP22 and methods of producing same.

[0064] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0065] Fruit set, the initiation of the fruit developmental program, is a key developmental landmark that normally occurs following ovule fertilization inside the quiescent ovary. Previously a loss-of- function mutant in slagl6 (slagl6CR~'gl) gene was shown to be capable of fertilization-independent setting of normal, yet seedless (parthenocarpic) fruits, suggesting that the S1AGL6 MADS-box TF is a suppressor of fertilization-independent fruit set. However, its mode of action remained obscure.

[0066] Whilst conceiving embodiments of the invention and reducing them to practice, the present inventors aimed at identifying S1AGL6 interacting proteins that play a role in fertilizationdependent fruit set. A yeast two-hybrid library of ovaries before and during fruit set was screened with the S1AG16 full-length protein, resulting in the identification of eight putative MADS-box partners. Surprisingly of these eight genes only MADS-BOX PROTEIN 22 (S1MBP22) was found to be a true S1AGL6 interacting protein that plays a role in fertilization-dependent fruit set. In vitro pull-down assay validated the interaction between S1AGL6 and S1MBP22. Similar to S1AGL6, SIMBP22 is predominantly expressed in unfertilized ovules and downregulated following fertilization. slmbp22 CRISPR knockout mutants (slmbp22CR) exhibited a parthenocarpy that closely resembles that of slagl6CR'sgl. Accordingly, slmbp22CRplants set normal-looking parthenocarpic fruits along with seeded fruits, but had no other obvious phenotypes, i.e., devoid of homeotic aberrations. Like slagl6CR'sglovules, the integument of slmbp22CRovules is enlarged, but lacks a characteristic endothelium. Consistent with this, RNA-seq analysis revealed a considerable overlap between the transcriptomes of unfertilized slmbp22CR~2(iand slagl6CR'sglovules, suggesting that a similar set of ovule- specific genes is regulated by both MADS-box transcription factors. In contrast to slagl6 mutant plants, slmbp22 mutant plants exhibit normal sepals lenth and petal color.

[0067] Taken together, the present results show that a protein complex containing S1MBP22 and S1AGL6 regulates ovule endothelium differentiation and suppress fertilization-independent fruit set.

[0068] These findings can be harnessed towards the development of parthenocarpic tomato plants.

[0069] Importantly, whilst further conceiving the embodiments of the invention, the present inventors have realized that the same teachings can be applied toward other Solanaceous plants having fleshy fruit i.e., pepper and eggplant, that share a high level of identity with the tomato gene and protein product (see Figure 8). Hence, according to an embodiment of the invention the present disclosure is directed to this subclass of Solaneceous plants though each plant species is considered an independent embodiment.

[0070] Thus, according to an aspect there is provided a Solanaceous plant exhibiting a facultative parthenocarpy and comprising a loss-of-function mutation in an MBP22 gene.

[0071] Alternatively or additionally, there is provided a tomato plant exhibiting a facultative parthenocarpy and comprising a loss-of-function mutation in an MBP22 gene.

[0072] Alternatively or additionally, there is provided a Solanaceous plant exhibiting a facultative parthenocarpy and at least 80 % of fruit yield being devoid of homeotic aberrations and comprising a loss-of-function mutation in a MBP22 gene.

[0073] Alternatively or additionally, there is provided a tomato plant exhibiting a facultative parthenocarpy and at least 80 % of fruit yield being devoid of homeotic aberrations and comprising a loss-of-function mutation in a MBP22 gene.

[0074] The term '"plant" as used herein encompasses whole plants, a grafted plant, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots, rootstock, scion, cuttings and plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.

[0075] According to a specific embodiment, the plant is in a form of a cutting.

[0076] According to another specific embodiment, the plant part is a seed (e.g., a hybrid seed).

[0077] According to another specific embodiment, the plant part is a fruit.

[0078] According to another specific embodiment, the plant is a plantlet.

[0079] The tomato plant can be of a cultivated genetic background or a wild (e.g., tomato) genetic background. As used herein a “solanaceous plant” refers to a crop plant (domesticated) of this family, including tomatoes, potatoes, eggplant, bell, and peppers.

[0080] As used herein, the term "tomato" refers to a plant, line or population within the species Solanum lycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersicon esculentum) or formerly known under the genus name of Lycopersicon including but not limited to L. cerasiforme, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newly proposed scientific name for L. esculentum is S. pennellii. Similarly, the names of the wild species may be altered. L. pennellii has become S. pennellii, L. hirsutum may become S. habrochaites, L. peruvianum may be split into S. 'N peruvianum' and S. 'Callejon de Hueyles', S. peruvianum, and S. comeliomuelleri, L. parviflorum may become S. neorickii, L. chmielewskii may become S. chmielewskii, L. chilense may become S. chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense, and L. pimpinellifolium may become S. pimpinellifolium.

[0081] Generally a cultivated tomato refers to tomato which is suitable for consumption and meets the requirements for commercial cultivation, e.g. typically classified as Solanum lycopersicum. In addition to the tomato plants themselves, and the parts thereof suitable for consumption, such as the fruit, the invention comprises parts or derivatives of the plant suitable for propagation. Examples of parts suitable for propagation are organ tissues, such as leaves, stems, roots, shoots and the like, protoplasts, somatic embryos, anthers, petioles, cells in culture and the like. Derivatives suitable for propagation are for instance seeds. The plants according to the invention can be cultivated or propagated in the conventional manner but also by means of tissue culture techniques from plant parts.

[0082] The present invention is aimed at using any cultivars, such as of domestic use or industrial use.

[0083] Specifically, the present invention is aimed at using any tomato cultivars, such as of domestic use, fresh market tomatoes and processing tomatoes as well as heirloom varieties.

[0084] As used herein “open-field tomato” refers to tomato grown by commercial growers in direct seeded, large fields - sprawled for machine harvesting and at times artificial ripening off the vine.

[0085] The choice of the variety depends on market demand, regional adaptability, disease resistance and the end use of the product. Exemplary segments for fresh market tomatoes include, but are not limited to, Beef (fruit weight of about 220-400 gr), Standard (fruit weight of about 160- 220 gr) and Cluster (uniform fruit weight of about 120-180 gr). Such varieties are available from major seed companies e.g., Grodena, Macarena, Estatio, Zouk, Climbo and Climstar, all available from Syngenta. Other varieties can be proprietary or available from other vendors, including but not limited to, Cherry-micro (up to 5 gr) round cherry, mini round cherry (7.5-15 gr), mini plum elongated cherry (10-25 gr). Examples for these varieties are: Creative (Clause), Batico (Nirit seeds), Shiren (Hazera Genetics). Cocktail round and elongated (25-40 gr): Romanita, Cherry and Cocktail with red, yellow, orange, pink, zebra, chocolate background. Examples include, but are not limited to, Summer sun (Hazera Genetics), Black pearl (Burpee) Tyty (Tomodori). Roma determinate and indeterminate. 120-200gr. Examples for the intermediate marker include, but are not limited to, lancelot (Vilomorin) and Parsifal (Vilomorin). Pink tomato divided to beef (220- 400), standard (160-220) and cluster (120-180). Example: Momotaro type, Cor di bue tomato, (150-350 gr), Pinton (250-300 gr), open field tomato- determinate or semi-determinate (180- 400gr).

[0086] Exemplary cultivars of processing tomatoes include, but are not limited to, Roma, SUN 6366, AB 2, Heinz 9780, Heinz 9557, Halley 3155 and Hypeel 303.

[0087] There are two major types of tomato growth determinate and indeterminate. Determinate growth produces "bush" tomatoes and which are bred for compactness. The entire plant stops growing once the terminal fruit ripens, the remainder of the fruit all ripen nearly simultaneously, and then the plant dies. Indeterminate growth produces tomatoes that can grow up to 10 feet in height (so-called "vining" tomatoes) and will only stop growing when killed (e.g. by frost). Their fruits ripen sequentially. In a typical plant, all growth arises from the reiteration of modular sympodial units that each produce three leaves and a multiflowered inflorescence. Most field- grown varieties of tomato, including MP-1, are determinate plants whose shoots produce an average of six sympodial units, each harboring a single inflorescence, within which leaf number gradually decreases before a precocious termination of growth. In general, determinate tomatoes are suitable for open field production. Semi-determinate and indeterminate "cultivated" varieties are suitable for staked cultivation in the open field or protected nets and for glasshouse cultivation.

[0088] According to an embodiment of the invention the tomato plant is a determinate tomato.

[0089] According to an embodiment of the invention the tomato plant is an indeterminate tomato.

[0090] According to an embodiment of the invention the tomato plant is a semi-determinate tomato.

[0091] According to an embodiment, the tomato is selected from the group consisting of a single fruit per truss, branched tomato and cherry tomato.

[0092] As used herein "pepper" refers to the cultivated species "Capsicum (hereinafter, referred to as "C") annuum", or wild species "C. pubescens", "C. baccatum", "C. chinense", and "C. frutescens”. Moreover, "pepper" is a concept that encompasses plants called by names other than "pepper", e.g., horticultural crops called "piment", "paprika", and "sweet pepper". As used herein “eggplant" refers to the cultivated species "Solanum (hereinafter, referred to as "S") melongena" or wild species "S. incanum", "S. torvum", "S. nigrum", "S. aethiopicum", "S. macrocarpon", and "S. quitoense”.

[0093] It will be appreciated that the terms "parthenocarpy", "parthenocarpic fruit formation" "seedlessness" and "fertilization-independent fruit formation" are used interchangeably herein.

[0094] As used herein “parthenocarpy” refers to fruit production in the absence of fertilization. Hence, the parthenocarpic fruits according to some embodiments are characterized by no or less than 5 seeds per fruit.

[0095] Maintenance of sexual reproduction capability is evident upon the production of seed bearing fruit.

[0096] For example, in tomato seed-bearing fruits are considered as at least 1 seed per fruit.

[0097] Facultative parthenocarpy refers to seedless fruit formation under fertilization restrictive conditions such as abiotic stress conditions e.g., temperature stress i.e., heat or cold stress, humidity, light intensity that can be acute or chronic.

[0098] In general, deviation of 5-15 °C from the optimal temperature hamper fertilization dependent fruit set.

[0099] According to some exemplary embodiments, the mean daily temperature range for stable fruit set is provided as follows: 13-25 °C for tomato, 16-25 °C for eggplant, and 18-25 °C for sweet pepper (as reviewed by: Karapanos et al 2008, Kawasaki 2015). Consequently, cold stress relate to such low temperatures that prevent viable pollen production. For most tomato cultivars it means temperatures below 10 °C for over 3-4 h during the night, but reduced viability is encountered already below 12 °C, especially during the post meiosis stage of pollen development, i.e. -5 to +2 days post anthesis. Temperatures below 10 °C also damage pollen adherence to stigma and its germination and pollen tube elongation (Picken 1984). Typically, heat stress occurs when temperatures rise 5-15 °C above the optimum for plant growth and development (Sato et al 2006, Mesihovic et al 2016, and references therein). In tomato, pending on the variety, day temperature of above 36 °C-38 °C for 2-4 h and night temperatures of 18-20 °C and above damage the process of microsporogenesis, which is especially sensitive to heat stress with temperatures > 35 °C for 2- 4h between -9 to -5 days post anthesis and hence lead to severe reduction in fruit set.

[0100] In pepper: the process of pollen production is sensitive to heat stress. In particular, exposure to temperatures of 33 °C during early flower development, corresponding to microspore mother cell meiosis (14-17 days before anthesis), and during late flower development, corresponding to microspore maturation, anthesis, and pollination (-2 to 0 days before anthesis) lead to most severe adverse effect on fruit set (Erickson and Markhart 2002). Pepper male fertility is severely damaged if the flower buds are exposed to 10 °C or lower. Yet fruit set is already damaged when nigh temperatures are lower than 15.5° C or higher than 24°C. In eggplant, the crop is sensitive to low temperatures and fruit set is damaged at night temperatures of 10 °C or lower. Pollen production and fertilization are also damaged at temperatures above 35 °C during microsporogenesis (Karapanos et al 2008, Kawasaki 2015).

[0101] Thus, according to some embodiments of the invention, fruit is produced under heat and cold stress conditions as well as high humidity (> 90%) or low light intensity that hamper fertilization-dependent fruit set.

[0102] Facultative parthenocarpy is of high commercial value in this case since eggplant, tomatoes and pepper are propagated from seeds. Alternatively or additionally fruits are generated even under abiotic stress that preferentially hamper pollen production and / or fertilization (e.g., moderately extreme temperatures, extreme high or low humidity) or any other conditions which hamper fertilization e.g., genetic male sterility.

[0103] According to a specific embodiment, parthenocarpy is considered facultative when the parthenocarpy rate is less than 100 %.

[0104] As used herein “parthenocarpy rate” refers to the proportion or percentage of fruits that develop without fertilization in a given crop.

[0105] According to a specific embodiment, the parthenocarpy rate is at least 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more

[0106] According to a specific embodiment, the parthenocarpy rate is at least 70 %. For example, the present inventors have shown that the double mutant slagl6 slmbp22 / + resulted in 72.3 % parthenocarpy rate, where + indicates a wild type allele (i.e., heterozygous).

[0107] As used herein “MADS-BOX PROTEIN 22 (MBP22y refers to the genomic sequence, or encoded RNA or protein of the MBP22 gene.

[0108] When the Solanaceous plant is tomato then the MBP22 gene is SXMBP22 gene i.e., Solycl lg005120, SEQ ID NO: 6, XP_019066630.1.

[0109] When the Solanaceous plant is eggplant then the MBP22 gene is SMEL4.1_04g011260.1 SEQ ID NO: 2, 3.

[0110] When the Solanaceous plant is pepper then the MBP22 gene is for example, CAOOgl881O + CA00gl8820 + CAOOgl883O, Capsicum annuum zunla Capana04g001527

[0111] Capsicum annuum glabriusculum Capang04g001434

[0112] As used herein “AGL6” refers to a transcription factor which is a key regulator gene of the transition between the state of ‘ovary arrest’ imposed towards anthesis and the fertilization- triggered fruit set.

[0113] When the Solanaceous plant is tomato then the AGL6 gene is S1AG6 gene i.e., Solyc01g093960 coding sequence, SEQ ID NO: 33-35.

[0114] When the Solanaceous plant is eggplant then the AGL6 gene is Sme2.5_06058.1 SEQ ID NO: 39-41.

[0115] When the Solanaceous plant is pepper then the AGL6 gene is Capana01g001334 (ChrOl- 44476983-44483323) SEQ ID NO: 36-38.

[0116] The skilled artisan would know how to uncover sequence information for different cultivars within the cultivated species.

[0117] As used herein, the phrase “loss-of-function alterations” refers to any mutation in the DNA sequence of a gene (in this case MBP22 and optionally AGL6), which results in downregulation of the expression level and / or activity of the expressed product, i.e., the mRNA transcript and / or the translated protein. Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the regulatory activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the regulatory activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the regulatory activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the regulatory activity of the non-mutated polypeptide; a readthrough mutation due to a frame- shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with an abolished regulatory activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in down-regulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame- shift mutation or an inframe mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, z.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame- shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, z.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation z.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, z.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.

[0118] According to a specific embodiment, the loss of function mutation is in a coding sequence of the gene and / or in a regulatory sequence which is upstream or downstream of the coding region.

[0119] According to a specific embodiment, the loss of function mutation is in exon 1 of the MBP22 gene (e.g., SIMBP22'). According to a specific embodiment, the loss of function mutation is a deletion which deletes at least a portion or all of exon 1 or exon 1 and sequences upstream and / or downstream thereto. For example, as shown in the Examples section which follows, the deletion encompasses the entire 1stexon, including the start codon and 141 bp upstream to it. Alternatively, the slMBP22CR'20mutant allele harbors a 391 bp deletion that eliminates 46 bp upstream of the 1stexon, the entire 1stexon and 61 bp of the 1stintron (Figure 2A-B). Such mutants are expected to produce truncated, non-functional mutant proteins, which would lack the MADS- box domain encoded by their 1stexon, and probably other domains, due to introduction of in-frame premature stop codons. This structure of the two alleles strongly suggests that they represent null alleles.

[0120] According to specific embodiments loss-of-function alteration of a gene may comprise at least one allele of the gene.

[0121] The term "allele" as used herein, refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0122] According to other specific embodiments, a loss-of-function alteration of a gene comprises both alleles of the gene. In such instances the e.g. MBP22 and optionally AGL6 (as in a double mutant) may be in a homozygous form or in a heterozygous form. According to this embodiment, homozygosity is a condition where both alleles at the e.g. MBP22 locus are characterized by the same nucleotide sequence. Heterozygosity refers to different conditions of the gene at the e.g. MBP22 locus.

[0123] According to a specific embodiment the loss of function mutation is in a homozygous or heterozygous form yet both encode for dis-functioning products. According to a specific embodiment parthenocarpic phenotype as described herewith is evident when both alleles of AGL6 comprise a loss of function mutation and the MBP22 is heterozygous or homozygous for the loss-of-function mutation.

[0124] According to a specific embodiment, the loss of function mutation is a deletion e.g., exon 1 of e.g., S1MBP22.

[0125] According to a specific embodiment, the loss of function mutation causes a premature stop codon.

[0126] According to some embodiments, the facultative parthenocarpic plant (as used herein “the plant”) of the invention exhibits at least one of:

[0127] (i) a fruit yield / plant at least about the same (e.g., 80 %, 90 %, 100 %, 110 %) as that of a non- parthenocarpic plant e.g., tomato, of the same genetic background under fertilization permissive conditions;

[0128] (ii) an average fruit weight / plant at least about the same as that of a non-parthenocarpic plant e.g., tomato of the same genetic background under fertilization permissive conditions of the non- parthenocarpic tomato;

[0129] (iii) comprising jelly fill when the plant is a tomato plant;

[0130] (iv) at least 80 % of the fruit yield being devoid of homeotic aberrations; and

[0131] (v) when the plant is tomato it comprises enlarged ovules in the red seedless parthenocarpic fruit.

[0132] As used herein “control” refers to a plant of the same genetic background and growth stage, subjected to the same growth conditions as the plant of the present invention, however it carries both wild type alleles of MBP3 and optionally AGL6.

[0133] According to some embodiments, the characteristic of the plant can be attained from studies on a plurality of plants (open field or greenhouse) and the values provided herein relate to an average.

[0134] As used herein “about the same” refers to ± 10 % or 20 %, at the same developmental stage and under the same conditions.

[0135] As used herein “fruit yield” refers to the total weight of the marketable harvested fruit, which is the product of the number of fruits per plant multiplied by the average weight of the harvested fruits.

[0136] The “same genetic background” refers to at least 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % of the genome is shared between the plant and the non-parthenocarpic plant.

[0137] As used herein “jelly fill” refers to the fluid to semi-fluid filling in the locular cavity of the fruit. As used herein “homeotic aberrations” refers to developmental aberrations in the anatomic structure of the plant, e.g., floral or fruit structures, which deviate from the normal flower shape with regards to whorls number or shape of the organs comprising the wild type (WT) flower, or fruit shape, size and internal structure clearly different from that of seeded fruit, besides the lack of normal seeds, which is inherent to parthenocarpy.

[0138] As used herein “enlarged ovules” refers to the small pseudo-seeds observed in mature seedless fruits.

[0139] The enlarged ovules are accompanied by the absence of a characteristic endothelium layer which is present in the wild type ovary (e.g. as in Figure 4A).

[0140] However, with respect to eggplant it should be noted that the fruits are harvestable at different sizes.

[0141] According to a specific embodiment, when the plant is a determinate tomato plant the fruit yield is a yield concentration where fruits along the 4-6 stages ripe almost simultaneously.

[0142] According to a specific embodiment, the plant is of an elite line. Examples of tomato elite lines are known in the art and some of which are listed herein.

[0143] Examples of Elite pepper cultivars: include, but are not limited to, Bastille, Rampart, Bayonet, Cutlass, Lafayette, Crusader, Pageant, Rising Sun, Trifecta (Syngenta); Atir, Gilad, Serenada, Vilmorin: E5661 Fl, RIFLESSI, Lussac, Vivaldi, Tyson (Hazera Genetics), Razer, E20B 10015 (Enza Zaden), Alma Paprika Peppe, and others.

[0144] Examples of Eggplant elite cultivars include, but are not limited to, Hybrid cultivars: Classic, Dancer, Dusky, Fairy Tale, Ghostbuster, Nadia, Purple Rain,

[0145] According to a specific embodiment, the plant is a transgenic plant (e.g., for a genome editing agent or for an RNA silencing agent, as described herein below).

[0146] According to a specific embodiment, the plant may be a transgenic plant but the transgene may not be associated with (i.e., not the cause for) facultative parthenocarpy, as described herein. For example, the transgene may function to improve biotic stress resistance, pesticide resistance or abiotic stress resistance.

[0147] According to a specific embodiment, the plant comprises a diploid genome.

[0148] According to a specific embodiment, the plant is an inbred.

[0149] According to a specific embodiment, the plant is a hybrid plant or the seed is a hybrid seed, where e.g., each of the parental lines is homozygous for a loss-of-function mutation in MBP22 and optionally AGL6 as described herein. Methods of producing the plant as described herein may rely on the use of mutagens e.g., EMS or genetic engineering which is naturally a more directed method and therefore involves less breeding steps.

[0150] Thus, according to an aspect of the invention there is provided a method of producing the plant as described herein, the method comprising down-regulating expression or activity of MBP22 and optionally AGL6 gene in the plant.

[0151] Following is a non-limiting description of methods of inducing loss-of-function mutation(s) in the MBP22 gene and optionally A GL6 gene which can be used to produce the plant.

[0152] Thus, according to some embodiments of the invention, down-regulating MBP22 and optionally A GL6 is effected by treating the plant or a regenerative portion thereof with a mutagen. In such a case the plant is non-genetically modified with an agent for inducing down-regulation of MBP22 and optionally A GL6.

[0153] Alternatively or additionally, occurrence of the genetic event responsible for the facultative parthenocarpic trait may be achieved by exposing a plant (i.e., tomato, pepper, eggplant) or part thereof to a chemical or physical mutagen (as described in the Examples section). Examples of chemical mutagens include, but are not limited to nitrous acid, alkylating agents such as ethyl methanesulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES), and base analogs such as 5-bromo-deoxyuridine (5BU). Physical mutagens include radiation (e.g. fast neutron, gamma radiation).

[0154] Initial exposure is typically followed by additional steps of selfing, selection, crossing and selfing or combinations thereof, where any step can be repeated more than once, as long as the loss-of-function in the MBP22 gene and optionally A GL6 gene is in a homozygous form. Selection can be phenotypic or using marker-assisted breeding as further described hereinbelow.

[0155] According to another specific embodiment, the non-genetically modified plant of the invention results from a spontaneous genetic event incurred by multiple crossings / selfings.

[0156] Below is a description of platform technologies for effecting knock-out (also referred to as “genome editing”) and transcriptional silencing in plants.

[0157] Methods of introducing nucleic acid alterations to a gene of interest (in this case MBP22 and optionally AGL(5)are well known in the art [see for example Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent Nos. 8771945, 8586526, 6774279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases, PB transposases and genome editing by engineered nucleases. Agents for introducing nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.

[0158] Following is a description of various exemplary methods used to introduce nucleic acid alterations to a gene of interest and agents for implementing same that can be used according to specific embodiments of the present invention.

[0159] Any of the below methods can be directed to any part of the MBP22 gene and optionally AGL6 gene as long as a loss-of-function is achieved. When needed further steps of selfing are effected in order to achieve a homozygous form of the mutation.

[0160] As used herein ’’target sequence” refers to the MBP22 DNA coding or RNA transcript or regulatory sequences of the gene. It will be appreciated that MBP22 can also be down-regulated at the protein level using an MBP22 antibody or chemical inhibitor. Although this option is not discussed here at length, it is still considered an embodiment for producing the plant.

[0161] Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double- stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDR) and non-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends in a double-stranded break, while HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location. To overcome this challenge and create site-specific single- or doublestranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR / Cas system.

[0162] Meganucleases - Meganucleases are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location. This can be exploited to make site-specific double- stranded breaks in genome editing. One of skill in the art can use these naturally occurring meganucleases, however the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize a new sequence. Alternatively, DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., US Patent 8,021,867). Meganucleases can be designed using the methods described in e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent Nos. 8,304,222; 8,021,867; 8, 119,381; 8, 124,369; 8, 129,134; 8,133,697; 8,143,015; 8,143,016; 8, 148,098; or 8, 163,514, the contents of each are incorporated herein by reference in their entirety. Alternatively, meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0163] ZFNs and TALENs - Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator- like effector nucleases (TALENs), have both proven to be effective at producing targeted double- stranded breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

[0164] Basically, ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the doublestranded break.

[0165] Thus, for example to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. Upon transient expression in cells, the nucleases bind to their target sites and the FokI domains heterodimerize to create a double-stranded break. Repair of these double- stranded breaks through the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site. The deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have successfully been generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). In addition, when a fragment of DNA with homology to the targeted region is introduced in conjunction with the nuclease pair, the doublestranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011; Miller et al., 2010; Umov et al., 2005).

[0166] Although the nuclease portions of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Approaches for making site-specific zinc finger endonucleases include, e.g., modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low- stringency selection of peptide domains vs. triplet nucleotides followed by high- stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

[0167] Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53. A recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org). TALEN can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA). Another agent capable of downregulating MBP22 and optionally AGL6 is a RNA-guided endonuclease technology e.g. CRISPR system (that is exemplified in great details in the Examples section which follows).

[0168] As used herein, the term "CRISPR system" also known as Clustered Regularly Interspaced Short Palindromic Repeats refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated genes, including sequences encoding a Cas9 gene (e.g. CRISPR-associated endonuclease 9), a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat) or a guide sequence (also referred to as a "spacer") including but not limited to a crRNA sequence (i.e. an endogenous bacterial RNA that confers target specificity yet requires tracrRNA to bind to Cas) or a sgRNA sequence (i.e. single guide RNA, see Table 1 of the Examples section which follows, that is deemed to be part of the specification). It will be appreciated that due to the high sequence homology the same gRNA can be used for the indicated Solaneceous plants e.g., gRNAl (SEQ ID NO: 9).

[0169] In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system (e.g. Cas) is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Neisseria meningitides, Streptococcus thermophilus or Treponema denticola.

[0170] In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0171] In the context of formation of a CRISPR complex, "target sequence" in this case MBP22 and optionally AGL6 refers to a sequence to which a guide sequence (i.e. guide RNA e.g. sgRNA or crRNA) is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Thus, according to some embodiments, global homology to the target sequence may be of 50 %, 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or 99 %. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0172] Thus, the CRISPR system comprises two distinct components, a guide RNA (gRNA) that hybridizes with the target sequence, and a nuclease (e.g. Type-II Cas9 protein), wherein the gRNA targets the target sequence and the nuclease (e.g. Cas9 protein) cleaves the target sequence. The guide RNA may comprise a combination of an endogenous bacterial crRNA and tracrRNA, i.e. the gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA (required for Cas9 binding). Alternatively, the guide RNA may be a single guide RNA capable of directly binding Cas.

[0173] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.

[0174] In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, a complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or 99 % of sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0175] Introducing CRISPR / Cas into a cell may be effected using one or more vectors driving expression of one or more elements of a CRISPR system such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. A single promoter may drive expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron).

[0176] “Hit and run” or “in-out” - involves a two-step recombination procedure. In the first step, an insertion-type vector containing a dual positive / negative selectable marker cassette is used to introduce the desired sequence alteration. The insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, transformed into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.

[0177] The “double-replacement” or “tag and exchange” strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive / negative selectable cassette near the location where the mutation is to be introduced. After transformation and positive selection, homologously targeted clones are identified. Next, a second targeting vector that contains a region of homology with the desired mutation is transformed into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

[0178] Site-Specific Recombinases - The Cre recombinase derived from the Pl bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.

[0179] Basically, the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue-specific manner. Of note, the Cre and Flp recombinases leave behind a Lox or FRT “scar” of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3' UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function.

[0180] Thus, Cre / Lox and Flp / FRT recombination involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.

[0181] Silencing at the MBP22 and optionally AGL6 transcript (RNA) level can be effected using the below exemplary platforms.

[0182] As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0183] As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene (MBP22 and optionally AGL6). In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and / or expression) of an mRNA molecule through a post- transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.

[0184] In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

[0185] According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA and does not cross inhibit or silence other targets or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene; as determined by PCR, Western blot, Immunohistochemistry and / or flow cytometry.

[0186] RNA interference refers to the process of sequence- specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).

[0187] Following is a detailed description on RNA silencing agents that can be used according to specific embodiments of the present invention.

[0188] DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.

[0189] Accordingly, some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.

[0190] According to one embodiment dsRNA longer than 30 bp are used. Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects - see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004;13:115- 125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P.J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004;573:127-134],

[0191] According to some embodiments of the invention, dsRNA is provided in cells where the interferon pathway is not activated, see for example Billy et al., PNAS 2001, Vol 98, pages 14428- 14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089 / 154545703322617069.

[0192] According to an embodiment of the invention, the long dsRNA are specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.

[0193] Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.

[0194] The term "siRNA" refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is suggested to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.

[0195] It has been found that position of the 3 '-overhang influences potency of a siRNA and asymmetric duplexes having a 3 '-overhang on the antisense strand are generally more potent than those with the 3'-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.

[0196] The strands of a double- stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned, the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).

[0197] The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'-CAAGAGA-3' and 5’-UUACAA-3’ (International Patent Application Nos. WO2013126963 and WO2014107763). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem- loop or hairpin structure comprising a doublestranded region capable of interacting with the RNAi machinery.

[0198] Synthesis of RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the MBP22 mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3’ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and / or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5’ UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www(dot)ambion(dot)com / techlib / tn / 91 / 912(dot)html).

[0199] Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov / BLAST / ). Putative target sites which exhibit significant homology to other coding sequences are filtered out.

[0200] Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G / C content as these have proven to be more effective in mediating gene silencing as compared to those with G / C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

[0201] Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The coding sequence constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The genetic construct can be an expression vector wherein the nucleic acid sequence is operably linked to one or more regulatory sequences allowing expression in the plant cells.

[0202] Plant cells may be transformed stably or transiently with the nucleic acid constructs of the present invention. In stable transformation, the nucleic acid molecule of the present invention is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.

[0203] There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205- 225; Shimamoto et al., Nature (1989) 338:274-276).

[0204] The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:

[0205] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Amtzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

[0206] (ii) direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6:559-563; McCabe et al. Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217;

[0207] Glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

[0208] The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

[0209] There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

[0210] Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.

[0211] However other methods of production are also contemplated including sexual reproduction (and selection for the phenotype whether morphologically or using molecular markers as described herein), tissue culture and more.

[0212] Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generated plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

[0213] Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet gradually increased so that it can be grown in the natural environment. Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV, TRV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87 / 06261.

[0214] Regardless of the method used to produce the Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) of some embodiments of the invention, once plants or any reproductive material is at hand, it is selected for the facultative parthenocarpic trait.

[0215] Thus, according to an aspect of the invention there is provided a method of selecting a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant being facultative parthenocarpic, the method comprising detecting in a genome of a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant a loss of function mutation in the MBP22 gene and optionally AGL6 gene, wherein presence of the mutation is indicative of a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) having being facultative parthenocarpic.

[0216] Many methods are known in the art for analyzing for mutations including for example single base extension (SBE), allele- specific primer extension sequencing (ASPE), DNA sequencing, RNA sequencing, microarray-based analyses, universal PCR, Melting Curve SNP method, allele specific extension, hybridization, mass spectrometry, ligation, extension-ligation, Flap Endonuclease-mediated assays, restriction fragment length polymorphism (RFLP), electrophoresis, sequence alignment, allelic specific oligonucleotide hybridization (ASO) and random amplified polymorphic DNA (RAPD).

[0217] Thus, the present invention contemplates oligonucleotides (e.g. Primers) that can be used to distinguish between the mutated and non-mutated form of MBP22.

[0218] Thus, once a plant carrying the loss of function genetic alteration is identified it is considered as being facultative parthenocarpic. This plant material can be used as a breeding material in the development of Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) varieties having agriculturally desired traits.

[0219] According to one embodiment, the plants of the present invention are of a hybrid variety - i.e. are generated following the crossing (i.e. mating) of two non-isogenic plants both being homozygous for a loss of function mutation in the MBP22 gene. The hybrid may be an Fi Hybrid.

[0220] An "Fi Hybrid" as used herein, refers to first generation progeny of the cross of two non- isogenic plants. The development of Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) hybrids of the present invention requires the development of stable parental lines. In breeding programs desirable traits from two or more germplasm sources or gene pools are combined to develop superior breeding varieties. Desirable inbred or parent lines are developed by continuous self-pollinations and / or backcrosses and selection of the best breeding lines, sometimes utilizing molecular markers to speed up the selection process.

[0221] Once the parental lines that give the best hybrid performance have been identified e.g., both carrying the loss of function mutation as described above e.g., in the MBP22 gene, the hybrid seed can be produced indefinitely, as long as the homozygosity of the parents are maintained. According to one embodiment the Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plants of the present invention are stable parent plant lines (carrying the loss of function mutation e.g., in the MBP22 gene in a heterozygous form or a homozygous form).

[0222] As defined herein, the phrase "stable parental lines" refers to open pollinated, inbred lines, stable for the desired plants over cycles of self-pollination and planting. According to a specific embodiment, 95% of the genome is in a homozygous form in the parental lines of the present invention.

[0223] A common practice in plant breeding is using the method of backcrossing to develop new varieties by single trait conversion.

[0224] The phrase "single trait conversion" as used herein refers to the incorporation of new single gene into a parent line wherein essentially all of the desired morphological and physiological characteristics of the parent lines are recovered in addition to the single gene transferred.

[0225] The term "backcrossing" as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plants. The parental Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant which contributes the gene for the desired characteristic is termed the non-recurrent or donor parent. This terminology refers to the fact that the non-recurrent parent is used one time in the backcross protocol and therefore does not recur. The parental Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant to which the gene from the non-recurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.

[0226] In a typical backcross protocol, a plant from the original varieties of interest (recurrent parent) is crossed to a plant selected from second varieties (non-recurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the non-recurrent parent.

[0227] Thus, near-isogenic lines (NIL) may be created by many backcrosses to produce an array of individuals that are nearly identical in genetic composition except for the trait or genomic region under interrogation in this case loss of function genetic alteration e.g., in the MBP22 gene.

[0228] Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the parent lines. Marker assisted breeding (selection) as described above can be used in this method.

[0229] According to a specific embodiment, down-regulating MBP22 gene and AGL6 is in the same plant. The resultant plant can be subjected to further breeding to improve agriculturally desirable traits as known in the art.

[0230] Alternatively, down-regulating MBP22 gene and A GL6 is done in different plants that may be crossed to result in a plant where MBP22 gene and AGL6 are down-regulated. The resultant plant can be subjected to further breeding to improve agriculturally desirable traits as known in the art.

[0231] According to a specific embodiment, the plant or the plant seed is an inbred.

[0232] According to a specific embodiment, the plant is a hybrid plant or the seed is a hybrid seed.

[0233] The invention also relates to progeny of the tomato, eggplant, pepper plants of the invention. Such progeny can be produced by sexual or vegetative reproduction of a plant of the invention or a progeny plant thereof. The regenerated progeny plant grows fruits independent of fertilization in the same or a similar way as the facultative parthenocarpic parent. In addition to this, the progeny plant may be modified in one or more other characteristics. Such additional modifications are for example effected by mutagenesis or by transformation with a transgene.

[0234] As used herein the word "progeny" is intended to mean the offspring or the first and all further descendants from a cross with a plant of the invention that shows fertilization independent fruit formation. Progeny of the invention are descendants of any cross with a plant of the invention that carries the mutation (in a homozygous form) trait that leads to fertilization independent fruit formation.

[0235] "Progeny" also encompasses plants that carry the trait of the invention which are obtained from other plants of the invention by vegetative propagation or multiplication.

[0236] As mentioned, embodiments described herein, furthermore, relate to hybrid seed and to a method of producing hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed. In this case the trait is recessive, therefore both parent plants need to be homozygous for the fertilization independent fruit formation trait in order for all of the hybrid seed to carry the trait of the invention. They need not necessarily be uniform for other traits.

[0237] Embodiments described herein also relate to the germplasm of the plants. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the facultative fertilization independent fruit formation trait of the invention.

[0238] Embodiments described herein also relate to cells of the plants that show the facultative fertilization independent fruit formation trait. Each cell of such plants carries the genetic information (i.e., loss of function mutation in MBP22 and optionally AGL6) that leads to the facultative parthenocarpy. The cell may be an individual cell or be part of a plant or plant part, such as the fruit.

[0239] The present teachings further relate to consumed products which comprise the genomic (DNA) information (i.e., loss of function mutation in MBP22 and optionally AGL6) that leads to the facultative parthenocarpy.

[0240] Fruits of any of the plants described herein may be selected or qualified for fruit color, Brix, pH, sugars, organic acids and defect levels (insect damage, mold, etc.) at ripening or post harvest. For example, tomatoes are typically transported to a large processing facility, where they are collected and where they may subsequently be washed, typically using chlorinated water and rinsed using tap water and further selected to remove those that present defects (e.g., inadequate ripening, disease damage, molds etc.). Tomatoes may be stored (especially those exhibiting improved shelflife as described above) or immediately sent to the consumer (fresh-market tomatoes). Processing tomatoes may be processed into a wide variety of products.

[0241] For juice or pulp production, the tomatoes may be subject to oven dehydration and are comminuted and macerated (disintegrated and broken) to obtain a pumpable mass. As will be clear to the skilled person these operations per se are known and common in the field of tomato processing and any adjustments to the method can be made in this regard without departing from the scope.

[0242] Methods for processing tomatoes and / or producing tomato-based compositions are well known in the art, see generally U.S. Patent No. 6,924,420. Also reported are specific methods for preparing, for example, paste (U.S. Patent No. 7,074,451), sterile paste (U.S. Patent No. 4,206,239), puree (U.S. Patent No. 4,556,576), sauce (U.S. Patent No. 7,122,217), solidified sauce (U.S. Patent No. 4,038,424), barbecue sauce (U.S. Patent No. 6,869,634), salsa (U.S. Patent No. 5,914,146), ketchup (U.S. Patent No. 6,689,279), tomato fiber composition (U.S. Patent No. 7,166,315) and dehydrated tomato-product (U.S. Patent No. 5,035,909). Methods of modifying the texture and consistency of tomato paste, pulp, and puree has also been reported, see, for example, U.S. Patent No. 6,720,019.

[0243] Also provided is an edible processed tomato product comprising the tomato or an edible portion thereof (e.g., fruit or an edible part thereof).

[0244] Also provided is a tomato paste generated according to the present teachings.

[0245] Examples of such edible products include, but are not limited to, canned tomatoes (whole), a tomato paste, a ketchup, a tomato sauce a tomato soup, a dehydrated tomato, a tomato juice, a tomato powder, a tomato dice, a crushed tomato, a chopped tomato and a tomato concentrate.

[0246] Pepper products that can benefit from seedlessness include: varieties for fresh consumption, as well as for processed and preserved pepper. Cultivars grown for spices (paprika) production consist of dried, ground pods of Capsicum annuum L., sweet red pepper. Sweet paprika spice processing includes removal of the seeds before grinding of the pericarp, which is otherwise of reduced quality. Other products are made from paprika oleoresin, an oil-soluble extract from the fruits of Capsicum annuum which is primarily used as a coloring and / or flavouring in food products. It is also used to color cosmetics products including bath and beauty products and moisturizing lipstick.

[0247] Seedless eggplant are in great demand by the consumers as the seeds add bitterness and are associated with fruit flesh browning. Eggplant is consumed usually following cooking, backing, frying, or roasting. It is also consumed pickled or as dried, and dried baby eggplant skins serve for stuffing. It is also consumed as processed products like frozen entrees and specialty dips.

[0248] According to some embodiments, the products comprise the DNA (carrying a loss of function mutation in MBP22 and optionally AGL6 causing the facultative parthenocarpic phenotype) of the tomato, pepper or eggplant (e.g., paste, dried fruit, juice and the like)

[0249] As used herein the term “about” refers to ± 10 %.

[0250] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0251] The term “consisting of’ means “including and limited to”.

[0252] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0253] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0254] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0255] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0256] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0257] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0258] It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.

[0259] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0260] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0261] EXAMPLES

[0262] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0263] MATERIALS AND METHODS

[0264] Plant material and growth conditions

[0265] The tomato cultivar MP-1 (Barg et al., 1997) was used as the wild type. The slagl6CR'sgltomato MP-1 mutant was previously described in Klap et al., (2017) and Gupta et al., (2020). Tomato plants were grown in 4-liter pots under greenhouse conditions with temperatures ranging between 15 and 30 °C in a tuff-peat mix with nutrients. Germination and seedling growth took place in a growth chamber under a 16-h light, 8-h dark photoperiod (photosynthetic photon flux density: 50-70 pmol m’2s’1) at a constant temperature of 24 °C. At around one month post germination seedlings were transferred to the greenhouse.

[0266] Total RNA extraction

[0267] Total RNA was extracted from indicated tomato tissues using Bio-Tri RNA reagent (BioLab, Jerusalem, Israel) following the manufacturer’s protocol.

[0268] Yeast two-hybrid analysis

[0269] Yeast two-hybrid "prey" library construction and screening was performed by HYBRiGENiCS SERVICES (Paris, France; https: / / www(dot)hybrigenics-services(dot)com). For "prey" library construction, messenger RNA was purified from ~ 1 mg of total RNA extracted from tomato cv. MP-1 ovaries at stages 15 (-4 DPA) to stage 20 (0 DPA), and at 2-4 DPA, and used to produce a random-primed cDNA library. The cDNAs were ligated into the pP6 vector containing the LEU 3 selectable marker. For "bait" construction, the sequence corresponding to the full-length S1AGL6 ORF was codon optimized for yeast expression using the GeneUniversal service (https: / / www(dot)geneuniversal(dot)com / ) and cloned into the gateway pBD-GAL4 vector containing the TRP1 selectable marker. "Bait" and "prey" constructs were transformed into the yeast strain CG1945 (MATa Gal4-452 Gal80-538 ade2-101 his3-D200 leu2-3,112 trpl-901 ura3- 52 lys2-801 URA3::Gal4 Hiners (X3)-CyClTATA-LacZ lys2::GALlUAS-GALlTATA-HIS3 cyhR) and brought together by mating. Positive colonies were selected on dropout media lacking tryptophan, leucine, histidine and supplemented with 0.5 mM 3-Amino-l,2,4-triazole (3-AT), which was necessary to eliminate a weak self-activation background by S1AGL6. Around 66 million clones (six fold complexity of the library) were screened using bait pBD-GAL4-SlAGL6. The recovered preys were amplified by PCR and sequenced from their 5' and 3' ends and annotated following blast against the tomato genome cDNA database (ITAG release 2.4). A predicted biological score (PBS®) was then attributed to each positive clone according to technical parameters such as the number of independent "prey" fragments, their length and frame. The PBS® is computed as an e- value and thresholds are attributed to define categories from very high confidence (A) to lower confidence (D) interactions, with E and F as distinct categories flagging highly connected "prey" domains and technical false positives, respectively.

[0270] In-vitro pull-down assays

[0271] For pull-down experiments, the full length coding sequences of S1AGL6 and SIMBP22 were synthesized (GeneUniversal service) with art sites at both ends and then ER recombined into the pIX-Halo and pIX-3xHA vectors (ABRC, Columbus, OH ) to generate pIX-Halo-TF and pIX- 3xHA-TF. Then 1 pg of pIX-Halo-TF and pIX-3xHA-TF plasmid DNA were used for in vitro transcription and translation using the TNT coupled wheat germ extract system (Promega Corp, USA). Pulldown assays were performed with the HaloLink™ resin following the manufacturer instructions (Promega Corp, USA). Briefly, after washing and equilibration of the Halo resin with binding buffer (BB) (100 mM Tris pH 7.6, 150 mM NaCl, 0.5% NP-40), 40 pl of the "bait" Halo- TF fusion protein was incubated with the resin for 2 hours at room temperature on a rotator. Then samples were centrifuged at 800 g for 2 min and washed three times with 800 pl of washing buffer (WB) (100 mM Tris pH 7.6, 150 mM NaCl, 1 mg / ml of BSA and 0.5% triton) with centrifugation between each wash. After the third wash, the Halo resin was resuspended in 100 pl of BB and then 40 pl of the "prey" 3xHA-TF fusion protein was added to the mixture, incubated for 2 hours at room temperature on a rotator and washed again with WB three times. Elution of bound proteins from the resin was done by adding 20 pl of IX SDS protein loading buffer and incubating for 5 min at 90 °C. The "bait" Halo-TF, "prey" 3xHA-TF and eluted samples containing the pulled- down "prey" proteins were subjected to 12.5 % SDS-polyacrylamide gel electrophoresis and immunobloted with ctHalo antibody (Promega Corp, USA) and ctHA antibody (Invitrogen) to detect the baits and the preys, respectively.

[0272] Plasmid construction and tomato transformation

[0273] For the CRISPR / Cas9 mediated mutagenesis of SIMBP22, two gene-specific gRNAs targeting its coding sequence were designed (The gRNA sequences used are listed in Supplemental Table S5). Each gRNA was incorporated in silico into an sgRNA consisting of itself followed by a 76-bp generic scaffold and a 7xT Polymerase III terminator sequence. Then a construct delimited by 5’ -Mini and 3'-Hind\\\ and containing the SIMBP22 sgRNAs in tandem, each under the control of the synthetic Arabidopsis U6 promoter, was artificially synthesized and cloned into the pUC57 plasmid (GENEWIZ, USA). The pUC57-sgRNAs plasmid was digested with Mini and Hindlll, and the released U6::sgRNAl-U6::sgRNA2 fragment was ligated into the compatible sites of the pRCS binary vector alongside the plant codon-optimized version of Cas9 expressed under the constitutive CaMV 35S promoter, to generate pRCS-35S::Cas9-U6::SlMBP22-sgRNAs.

[0274] Tomato transformation

[0275] The binary plasmid pRCS-35S::Cas9-U6::SlMBP22-sgRNAs was transformed into tomato cultivar MP-1 by co-cultivation of cotyledons derived from 12-14-day-old seedlings with Agrobacterium tumefaciens strain EHA105 as described previously (Barg et al., 1997). Regenerated explants were selected on 100 mg / 1 kanamycin-containing media, where only transgenic seedlings developed a branched root system. The transgenic status of the kanamycin resistant seedlings was further validated by genomic DNA PCR with the primer pair Cas9-fwd and Cas9-rev to detect the presence of the Cas9 transgene. The primer sequences used are listed in Supplemental Table S5.

[0276] Isolation of CRISPR mutants

[0277] To isolate CRISPR mutants in the SIMBP22 gene, the primary (To) transformants were screened by genomic DNA PCR with specific primers flanking the gRNAs targeted sequences followed by agarose gel electrophoresis to detect amplicons, which length deviated from that of the wild-type one, indicating for the presence of relatively large indels. Identified To plants carrying large indels were backcrossed to wild-type, and the resulting Fi progeny were PCR genotyped as described above. Selected Fl CRISPR heterozygous mutants were selfed, and the resulting F2 progeny were PCR genotyped as described above to isolate homozygous SIMBP22 mutants followed by sequencing of the mutant loci amplicons. The verified homozygous mutants were further characterized. The primer sequences used are listed in Table 1 below. Table 1

[0278] Histology

[0279] Ovary tissues were fixed in PFA solution [4% paraformaldehyde; IX phosphate-buffered saline (PBS); 0.2% Tween-20 (v / v)] as described in Gupta et al., (2020). Microtome sliced sections (4-pm thick) of ovary tissue were spread on microscope slides and stained with 0.1% (w / v) Toluidine blue O for 30 seconds and then rinsed three times for 2 minutes with Distilled water. Slides were then dried at 37°C for several hours, mounted with Vectamount (Vector Laboratories, www(dot)vectorlabs(dot)com) and dried overnight at 70°C. After mounting, observations were performed under bright field using an Olympus DP73 microscope equipped with a digital camera. At least five ovaries were examined for each genotype.

[0280] Ovule isolation and measurements

[0281] Tomato ovules were isolated from ovaries as described in Gupta et al., (2020). Ovule dimensions were calculated from digitized images of Toluidine blue stained transverse sections at equatorial position of 30 ovules from at least 3 independent ovaries as described previously (Gupta et al., 2021).

[0282] Transcrip tome analysis of isolated ovules by RNA-seq

[0283] Tomato ovules were isolated from ovaries as described in Gupta et al., (2020). For RNA- seq, three true biological replicates of wild type and SIMBP22CR'20stage 18 isolated ovules (each replicate contained ca. 1,000 ovules collected from 30 ovaries from 3 plants) were used. RNA-seq libraries preparation and pair-end sequencing were performed at MACROGEN-EUROPE. Differential expression analysis was done at the ARO bioinformatic unit. Briefly, raw-reads were subjected to a filtering and cleaning procedure. The SortMeRNA tool was used to filter out rRNA. Next, the FASTX Toolkit (http: / / hannonlab(dot)cshl(dot)edu / fastx_toolkit / index(dot)html, version 0.0.13.2) was used to trim read-end nucleotides with quality scores <30, using the FASTQ Quality Trimmer, and to remove reads with less than 70% base pairs with a quality score <30 using the FASTQ Quality Filter. Clean reads were mapped to the reference genome of Tomato reference version 2.5 using Tophat2 software with an average mapping rate of 92.9%. Gene abundance estimation was performed using Cufflinks (v.2.2) from the Sol Genomics Network (https: / / solgenomics(dot)net / ; ITAG2.4 gene models). Principle Component Analysis (PCA) were performed using R Bioconductor. Gene expression values were computed as FPKM. Differential expression analysis was completed using the DESeq2 R package of no more than 0.01 and at least 2 fold change, were considered differentially expressed.

[0284] Reverse transcription quantitative PCR (RT-qPCR) assays

[0285] First-strand cDNA was prepared from 2 pg of total RNA with a Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Real-time quantification of gene expression was performed with at least three independent biological replicates for each sample, and quantification was performed in triplicate. QPCR was performed in StepOnePlus (Thermo Fisher Scientific) following the manufacturer’ s instructions and analyzed using StepOne software version 2.2.2. Relative expression levels were calculated by the comparative delta delta Ct (AACt) method and normalized to SITIP41 (Solycl0g049850') as a reference gene. The primers for RT-qPCR are listed in Supplemental Table S5.

[0286] Data availability

[0287] The RNA-seq data is available from the SRA database under the BioProject accession number PRJNA794989. The gene expression data of anthesis ovary and developing fruit tissues of Solarium pimpinellifolium is available in Supplemental data published by Pattison et al., (2015).

[0288] EXAMPLE 1

[0289] S1MBP22 is a putative S1AGL6 interactor

[0290] To identify putative S1AGL6 interactors, full length S1AGL6 protein was used as a "bait" to screen a Y2H "prey" library for interacting partner identification. Taking into account the accumulation of SIAGL6 in the maturing ovaries and its gradual decline post fertilization (Klap et al., 2017), its candidate interactors were screened among the genes expressed in developing and anthesis ovaries, when reaching its highest levels (Klap et al., 2017), and in early setting fruit. From a total of 66 million potential interactions tested, 373 positive clones were recovered, sequenced and annotated. Out of them, 282 were defined as category A (very high confidence in the interaction) and 16 as category B (high confidence in the interaction) and all encoded for MIKCcMADS-box proteins (Figure 1A). Among category A clones, 97 corresponded to MADS- BOX BINDING PROTEIN 3 (SIMBP3), 69 corresponded to MADS box gene no. 5 / LeSEP3 (TM5), 48 corresponded to MADS-BOX PROTEIN 22 (S1MBP22), 33 corresponded to SIAGAMOUS- LIKE 11 (SIAGLI I). 24 corresponded to MADS box gene no. 29 / EeSEPl (TM29) and 11 corresponded to S1AGAMOUS-LIKE 42-like (SlAGL42-like). Among category B clones, 11 corresponded to MACROCALYX (MC) and 5 corresponded to FOREVER YOUNG FLOWER like (S1FYFL) (Figure 1A). The remaining 75 positive clones were defined at the less confident interaction categories (C, D, and N / A) and corresponded to a number of different proteins, frequently represented by just one or only a few clones (not shown), and therefore not further characterized. From the eight identified MADS-box interactors, five were previously characterized. The tomato TM5 and TM29 protein were proposed to have SEP-like functions in floral meristem identity and floral organ development (Pnueli et al., 1994; Ampomah-Dwamena et al., 2002). The MC was shown to be required for pedicel abscission zone development (Nakano et al., 2012). The SIMBP3 was shown to regulate fruit locule gel formation and together with its close homolog SIAGL11, seed development (Zhang et al., 2019; Huang et al., 2021). However, as opposed to slagl6CR~sglfruits (Klap et al., 2017), silencing of MC, TM5 or TM29 (Nakano et al., 2012) and knockout of SIMBP3 or SIAGL11 (Huang et al., 2021) did not result in the set of normallooking seedless fruits, and thus their involvement in slagld-mediated parthenocarpy was less likely.

[0291] In silica analysis of the expression of the remaining previously uncharacterized Y2H interactors in ovary and set fruit tissues, revealed that the expression patterns of SIMBP22 closely resembled that of SIAGL6. In contrast to SIAGL42, which was barely detected in ovules, and SIFYFL, which was maximally expressed in anthesis ovary placenta (not shown), SIMBP22 expression was highest in the ovules compared to other anthesis ovary tissues, and it sharply declined in early developing seeds (Figure 1B-C). Moreover, similar to SI AG UY SIMBP22 was downregulated in the set fruit following artificial pollination or exogenous application of the fruit set promoting hormones auxin and gibberellin (GA) to unpollinated ovaries (Figure ID). mRNA quantitation in developing and fertilized ovaries indicated that SIMBP22 expression is relatively high and comparable to SIAGL6 in young ovaries (stage 15). Unlike SIAGL6, SIMBP22 levels slightly decline upon ovary maturation, but like SI AG UY its expression is strongly downregulated in the set fruit (Figure IE). Taken together, the above expression results indicate that, like S1AGL6, S1MBP22 is primarily expressed in ovules and its decline occurs upon fruit set. Thus, it was selected for further functional analysis.

[0292] SIMBP22 is predicted to encode a 238 aa MIKCctype II MADS -box protein (Figure 1A). Using BlastP to search against non-redundant protein sequences database (nr), S1MBP22 was found to have the strongest homology (68% identity, 79% similarity) to the Petunia FLORAL BINDING PROTEIN 24 (FBP24) protein. This homology extended over the M-LK-C domains (not shown). In Arabidopsis, S1MBP22 is most homologous (44% identity, 62% similarity) to the Bsister MADS-BOX protein TRANSPARENT TESTA16 (TT16), but unlike in FBP24, the homology is mostly contributed by the conserved MADS-box domain (not shown). The Y2H interaction between S1AGL6 and SMBP22 was verified by an in-vitro pull-down assay. The full length "bait" and "prey" proteins were expressed as Halo and 3xHA fusion proteins, respectively. As negative "bait" and "prey" controls served Halo, and 3xHA-GFP proteins respectively. As shown in Figure IF, the S1MBP22 "prey" protein was retained on the Halo Link™ resin only when S1AGL6 was used as "bait", thus confirming S1MBP22 as direct S1AGL6 interactor. In addition, neither S1AGL6 nor the S1MBP22 "prey" proteins were retained on the HaloLink™ resin when also served as "bait", suggesting that neither is able to homodimerize (Figure IF). EXAMPLE 2

[0293] No evident pleiotropic phenotypes are associated with slmbp22 loss-of-function

[0294] To support the involvement of S1MBP22 in SlAGL6-containing complex that inhibits the growth of the ovary at anthesis unless fertilized, loss-of-function mutants were generated and tested for their ability to uncouple fruit set from fertilization. Using CRISPR / Cas9 technology, the SIMBP22 gene was edited (Figure 2A), and two independent CRISPR mutant alleles (slmbp22CR') were isolated. The slmbp22CR'10mutant allele harbored a 317 bp deletion that eliminated almost the entire 1stexon, including the start codon and 141 bp upstream to it. The slmbp22CR'20mutant allele harbored a 391 bp deletion that eliminated 46 bp upstream of the 1stexon, the entire 1stexon and 61 bp of the 1stintron (Figure 2A-B). In the unlikely event that slmbp22CR'10and slmbp22CR'20mutant transcripts would be translated from an ectopic start codon, they are predicted to produce truncated, non-functional mutant proteins, which would lack the MADS-box domain encoded by their 1stexon, and probably other domains, due to introduction of in-frame premature stop codons. This structure of the two alleles strongly suggests that they represent null alleles.

[0295] Next, the present inventors analyzed the phenotypes of the slmbp22CRhomozygous mutant plants by growing them in the greenhouse along with the respective MP-1 parental line and slagl6CR'sglplants. The slmbp22CR'10and slmbp22CR'20homozygous mutants did not exhibit any obvious developmental phenotypes in plant architecture (data not shown), leaves and flowers (Figure 2C-D). Notably, the slmbp22CRmutants did not exhibit the faded petals hue, nor the longer sepals, characteristic of the slagl6CR'sglflower (Figure 2D), indicating that SIMBP22 does not complex with S1AGL6 to regulate perianth related features. In addition, at anthesis, the size and morphology of slmbp22CR'10and slmbp22CR'20ovaries were similar to those of wild type and slagl6CR'sgl, suggesting that fruit set was not initiated prior to anthesis (Figure 2D). In support of this assumption, comparative histological analysis of wild-type, slagl6CR'sgland the slmbp22CR~2(imutant stage 18 ovaries revealed no obvious differences between them (not shown).

[0296] EXAMPLE 3

[0297] The slmbp22CR'20loss-of-function mutation confers facultative parthenocarpy

[0298] The characterization of slmbp22CRred fruits showed that a fraction of them were seedless and contained tiny (< 2 mm) soft structures instead of seeds (as in the AGL6 mutant) but otherwise were normal looking with the characteristic jelly fill (Figure 3A-B). Further analysis was done on the slmbp22CR'20mutant. In line with its wild-type morphology, the average slmbp22CR'20fruit weight was not significantly different from wild type (Figure 3C). As for seed bearing, under pollination permissive conditions, -35% of the slmbp22CR'20fruits were seedless and -13% contained 10 or less seeds per fruit (Figure 3D). The seedless fruit phenotype raised the possibility that slmbp22 loss-of-function confers facultative parthenocarpy. To examine this, the present inventors emasculated wild-type MP-1, slagl6CR'sgland slmbp22CR'20stage 18 flowers (-2DPA) and tested for fruit set in the absence of fertilization. As expected, none of the emasculated wildtype flowers set fruit in the absence of pollination (0 / 32) and 62.5% (15 / 24) of the emasculated slagl6CR'sglflowers set parthenocarpic fruits. In line with its seedless phenotype, 15% (6 / 40) of the emasculated slmbp22CR'20flowers could set parthenocarpic fruits. These results indicate that the slmbp22CR'20mutation confer facultative parthenocarpy, albeit at lesser efficiency than the slagl6CR'sglloss-of-function mutant. To test whether the observed slmbp22CR'20parthenocarpic phenotype results from defects in the male or in the female reproductive organs, we performed reciprocal crossing between wild-type MP-1 and slmbp22CR'20. In both crosses, normal-looking seeded fruits were produced (data not shown), suggesting that slmbp22CR'20is maternally and paternally fertile. Manual sectioning of wild-type and mutant seeds revealed that slmbp22CR'20seeds exhibit wild-type morphology, whereas slagl6CR'sglseeds contain loosely-packed embryo and endosperm, compared with the tightly-packed embryo of wild type (Figure 3E). In addition, slmbp22CR'20seeds were heavier and larger than the wild-type and slagl6CR'sglseeds (Figure 3F- G). Germination assays indicated that slmbp22CR'20and slagl6CR'sglseeds have -76% and -54% reduction in the germination rate compared to wild-type seeds respectively (Figure 3H-I), suggesting that their mutant seed viability is compromised.

[0299] EXAMPLE 4

[0300] The slmbp22CR'20ovules have enlarged integument that fail to differentiate a typical endothelium

[0301] To address whether the slmbp22CR'20parthenocarpy is associated with ovule integument abnormalities, histological analysis was performed on stage 18 slmbp22CR'20ovaries and ovules compared to wild type and slagl6CR'sgl. The single integument of wild-type tomato ovules typically consists of 6-7 layers that surround a centrally-located female gametophyte (embryo sac) (Figure 4A). It is composed of single layer of outer epidermis, two-three layers of parenchyma cells, two layers of “integumentary tapetum” cells and a single layer of strongly stained endothelium cells that form the inner most integument layer (Figure 4 A, right panel). In contrast to wild-type and similar to slagl6CR'sglovules, the slmbp22CR'20ovules did not exhibit dark stained endothelium, and accordingly, their embryo sacs were enlarged (-2.8 fold) (Figures 4A-B). These observations suggested that endothelium differentiation was compromised. Moreover, the slmbp22CR'20ovules were 12% and 11% longer in vertical and horizontal lengths, respectively, than wild-type ovules (Figure 4C). Consistent with this, their integument contained more parenchyma cell layers than that of wild-type ovules (2.73+0.44 verses 4.41+0.57, P < 0.001) (Figure 4D). Comparable proliferation of the integument parenchyma was previously observed in slagl6CR~sglovules (Gupta et al., 2021).

[0302] EXAMPLE 5

[0303] Transcrip tomic analysis of slmbp22CR'20isolated ovules

[0304] The ability of S1AGL6 and S1MBP22 to interact (Figure 1A, F), together with the similar parthenocarpic phenotypes observed for their corresponding mutants (Figures 3A-I), suggested that they complex to suppress parthenocarpy. Therefore, to identify genes that are transcriptionally regulated by both S1MBP22 and S1AGL6 and potentially involved in parthenocarpy, the transcriptomes of slagl6CR'sgl(Gupta et al., 2021) and slmbp22CR'20unfertilized ovules were compared. To this end, the present inventors performed RNA-sequencing (RNA-seq) analysis on wild-type and slmbp22CR'20stage 18 isolated ovules. Three biological replicates were used for the construction of the wild-type and slmbp22CR'20RNA-seq libraries. Approximately 20 million clean pair-end sequences were generated from each library and around 92 % of them were mapped to the tomato IT AG 2.5 genome (not shown). Only transcripts that showed a significant (FDR < 0.01) 2-fold higher or lower expression difference between the wild type and slmbp22CR'20samples were considered as DEGs. Principal component analysis (PCA) with the RNA-seq data sets showed that the differences within the biological replicates are smaller than the differences between samples (Figure 5A). Applying the above-described criteria, 400 upregulated (up) and 704 downregulated (down) differentially expressed genes (DEGs) were identified between wildtype and slmbp22CR'20ovules (Figure 5B). Functional categorization of these DEGs revealed that the genes encoding components of signal transduction (transcription factors and kinases), transport, protein degradation and cell wall modification were mostly affected. Notably, 59% (236 DEGs) and 45% (319 DEGs) of slmbp22CR'20ovule up and down DEGs, respectively, were previously identified as DEGs in slagl6CR'sglstage 18 isolated ovules. Out of these DEGs, all the upregulated and 93% of the downregulated showed a similar trend in slagl6CR'sgl(designated common DEGs; Figure 5C), raising the possibility that their transcription is similarly regulated by S1MBP22 and S1AGL6. The common DEGs also include two parthenocarpy-associated genes, S1ARF5 and SIKLUH, which are downregulated and upregulated, respectively, in both slmbp22CR'20and slagl6CR~sglovules (Figure 5D). RT-qPCR analysis further confirmed the upregulation (~17 fold) of SIKLUH and downregulation (~5 fold) of S1ARF5 in the slmbp22CR'20ovules compared to wild-type ovules (Figure 5E-F). No other gene previously associated with parthenocarpy was identified among the common DEGs (Figure 5D). However, in slmbp22CR'20ovules, SlGA2oxl and SlGA2ox4 were downregulated by ~3 and ~7 fold, respectively (Figure 5D). Previously, silencing of all the five members of the tomato GA2ox family, which encodes GA catabolizing enzymes, induced the development of small puffy parthenocarpic fruits (Martinez-Bello et al., 2015). This suggests that SlGA2oxl and SlGA2ox4 play a minor, if any, role in slmbp22CR~20parthenocarpy. Annotation of other auxin and GA pathway-related slmbp22CR'20DEGs revealed three additional GA-related and 12 additional auxin-related DEGs of which 2 and 9, respectively, were common DEGs (Figure 5G), suggesting that they may facilitate both slagl6CR'sgland slmbp22CR'20mediated parthenocarpy.

[0305] Interestingly, while SIMBP22 was found to be significantly upregulated (by ~3.9 fold) in slagl6CR~sglstage 18 ovules (Gupta et al., 2021), the current RNA-seq results indicate that SIAGL6 expression is not modified in slmbp22CR'20ovules (log2FC = -0.023, FDR=0.87). These observations were confirmed by RT-qPCR analysis on wild-type and respective mutant isolated ovules (Figure 5H-I). The upregulation of SIMBP22 in slagl6CR'sglovules together with the finding that while S1AGL6 expression elevates during ovary maturation (between stage 15 and 20) that of SIMBP22 decrease, raises the possibility that S1AGL6 supresses SIMBP22 expression in maturing ovules.

[0306] EXAMPLE 6

[0307] The slmbp22CR'20ovule transcriptome undergoes post-fertilization-like reprogramming

[0308] To test whether post-fertilization transcriptional programs were, at least in part, precociously activated in slmbp22CR'20ovules, a classification of identified DEGs based on whether they were previously annotated as preferentially expressed in unfertilized or fertilized ovules (Pattison et al., 2015; Gupta et al., 2021) was performed. This analysis revealed that slmbp22CR'20ovule DEGs have substantial overlap with genes preferentially expressed in unfertilized ovules (cluster 23; 143 / 438) and 4 DPA fertilized ovule (seed) integument (seed coat) (cluster 14; 66 / 387). Like the slagl6CR'sglovule DEGs, the majority of cluster 23 DEGs (137 / 143) and cluster 14 DEGs (53 / 66) were downregulated and upregulated, respectively (Figure 6A). Moreover, 51% (70 / 137) and 68% (36 / 53) of the slmbp22CR~20cluster 23 and 14 DEGs, respectively, were also identified as similar trend DEGs in slagl6CR'sglovules (Figure 6B-C). However, only little overlap (16 / 299) was found between slmbp22CR'20DEGs and 4 DPA seed funiculus (cluster 21) detected genes. Nevertheless, the majority of cluster 21 DEGs (14 / 16) were upregulated in slmbp22CR'20as in slagl6CR'sglovules (Figure 6A, D). Collectively, the above data suggest that, similar to the slagl6CR'sglovules, slmbp22CR'20ovules underwent precocious postfertilization reprogramming of their integument.

[0309] EXAMPLE 7

[0310] Characterization of the slmbp22CR'20slagl6CR'S81double mutant

[0311] To assess the functional relationships between SIMBP22 and S1AGL6 we crossed slmbp22CR'20and slagl6CR'sglsingle mutants and compared their phenotypes to the phenotypes of the resulting F2 double mutant (henceforth will be designated DM). Like the slmbp22CR'20and slagl6CR'sglmutants, the DM mutant did not exhibit any pleiotropic phenotypes except that its flowers exhibited long sepals and pale petals (Figure 7A), a characteristic slagl6CR'sglflower phenotype (Gupta et al., 2021). As shown in Figure 7B, the DM red fruits had wild-type morphology and size but, a fraction of them were seedless and contained tiny soft structures instead of seeds, similar to slagl6CR'sgland slmbp22CR'20parthenocarpic fruits. Concerning seed bearing, under pollination permissive conditions, 37% of the DM fruits were seedless, 52% contained <10 seeds, and only -11% contained >10 seeds (Figure 7C). Manual dissection of DM seeds revealed that, unlike slagl6CR'sglor slmbp22CR'20single mutant seeds, they did not contain discernible embryos (Figure 7D). In line with that, compared to 83% germination efficiency of MP-1 seeds, none of the DM seeds germinated

[0312] (Figure 7E). The DM seedless fruits implied that it is parthenocarpic. The efficiency of parthenocarpy in DM was determined by by emasculating stage 18 DM flowers and testing for fruit set in the absence of fertilization. Compared to the 15% and 62.5% parthenocarpic fruit formation following flower emasculation in slmbp22CR'20and slagl6CR'sgl, respectively, 69.2% (18 / 26) of the emasculated DM flowers could set parthenocarpic fruits. The present inventors showed that slmbp22CR'20and slagl6CR'sglmutations perturbed the differentiation of the ovule endothelium (Figure 4A). Consistent with that, histology of wild-type and DM stage 18 ovaries revealed that unlike the integument of the wild-type ovules, a characteristic strongly stained endothelium layer was not present in the DM ovules (Figure 7F).

[0313] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0314] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A Solanaceous plant exhibiting a facultative parthenocarpy and at least 80 % of fruit yield being devoid of homeotic aberrations and comprising a loss-of-function mutation in a MBP22 gene.

2. The plant of claim 1, being a tomato.

3. The plant of claim 1, being a tomato, eggplant or pepper.

4. The plant of claim 1, further exhibiting at least one of:(i) a fruit yield / plant at least the same as that of a non-parthenocarpic plant of the same genetic background under fertilization permissive conditions;(ii) an average fruit weight / plant at least the same as that of a non-parthenocarpic plant of the same genetic background under fertilization permissive conditions;(iii) when the plant is tomato the fruit comprises a jelly fill;(iv) enlarged ovules within the seedless fruits developed from non-fertilized ovaries;(v) petals color and sepals length of a flower having wild type AGL6 alleles.

5. The plant of any one of claims 1-4, wherein said fruit yield is mature fruit yield.

6. The plant of claim 2 or 4, being a processing tomato.

7. The plant of claim 2 or 4, being a determinate tomato.

8. The plant of claim 2 or 4, being an indeterminate tomato.

9. The plant of claim 2 or 4, being a semi-determinate tomato.

10. The plant of any one of claims 1-9, being of an elite line.

11. The plant of any one of claims 1-10, being transgenic.

12. The plant of any one of claims 2-10, wherein said tomato is of a species selected from the group consisting of Lycopersicon esculentum, Lycopersicon cerasiforme, Lycopersicon pimpinellifolium, Lycopersicon cheesmanii, Lycopersicon parviflorum, Lycopersicon chmielewskii, Lycopersicon hirsutum, Lycopersicon penellii, Lycopersicon peruvianum, Lycopersicon chilense and Solanum lycopersicoides.

13. The plant of any one of claims 6-10 and 12, wherein said tomato is selected from the group consisting of a single fruit per truss, branched tomato and cherry tomato.

14. The plant of any one of claims 1-13, wherein said facultative parthenocarpy is manifested under heat or cold stress.

15. The plant of any one of claims 1-14 being an inbred.

16. The plant of any one of claims 1-15, wherein said loss-of-function mutation is in a homozygous form.

17. The plant of any one of claims 1-16, further comprising a loss-of-function mutation in an A GL6 gene.

18. The plant of any one of claims 1-15, comprising a silencing agent for suppressing expression of an MBP22 gene and optionally an AGL6 gene.

19. The plant of claim 16, exogenously expressing a nuclease selected from the group consisting of a meganuclease, an RNA-guided DNA endonuclease, a zinc-finger nuclease and a TALEN.

20. A fruit of the plant of any one of claims 1-19.

21. A seed of the plant of any one of claims 1-19.

22. A hybrid seed produced of the plant of claim 1.

23. An edible processed product of the plant or fruit of any one of claims 1-20.

24. The processed product of claim 23 selected from the group consisting of a tomato paste, a ketchup, a tomato sauce a tomato soup, a tomato juice, a tomato powder, a tomato dice, a crushed tomato, a chopped tomato and a tomato concentrate.

25. A method of producing a processed product, the method comprising processing the fruit of claim 20 to produce an edible processed product.

26. A method of producing the plant of any one of claims 1-19, the method comprising down-regulating expression or activity of MBP22 gene in the plant and detecting a mutation in MBP22 gene in the plant.

27. The method of claim 26, wherein said down-regulating is effected by treating the plant with an RNA silencing agent.

28. The method of claim 26, wherein said down-regulating is effected by treating the plant with an DNA editing agent.

29. The method of any one of claims 26-28 further comprising down-regulating expression or activity of AGL6 gene in the plant and detecting a mutation in AGL6 gene in the plant.

30. A method of breeding comprising selfing or crossing the plant of any one of claims1-19.

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