Solanaceae plants having altered morphology

Abstract

Plants, plant cells, and plant parts of the Solanaceae family having extreme structural dwarfism are provided that comprise a functionally inactivating mutation present within a cytochrome p450 class 85A1 (CYP85A1) gene and a Self Pruning 5G (SP5G) gene. The plants, plant cells and plant parts may further comprise at least one functionally inactivating mutation within an allele of a gene selected from: carotenoid cleavage dioxygenase 8 (CCD8); Gibberellin-insensitive dwarf 1a (GID1a); and Gibberellin- insensitive dwarf 1b1 (GID1b1). The plants, plant cells and plant parts are suitably from the species Solanum lycopersicum (tomato). Methods for making the plants, plant cells and plant parts are provided also.

Description

[0001] SOLANACEAE PLANTS HAVING ALTERED MORPHOLOGY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to improvements of tomato (Solatium lycopersicum) varieties utilising gene manipulation technologies, to generate plants having altered morphology in the form of significantly smaller stature and higher fruit yield per unit of non-edible vegetative biomass.

[0004] BACKGROUND OF THE INVENTION

[0005] The wild ancestor of the modern tomato plant (Solatium lycopersicum) originated in the Andes Mountains of South America. This plant is believed to have been domesticated in pre- Columbian Mexico with the name “tomato” derived from the Nahuatl (Aztec) word “tomatl”. Today, tomatoes are cultivated extensively around the world for their edible fruits, which are commonly eaten raw, used in cooking, pickled, or processed into diverse products like canned tomatoes, tomato juice, concentrates, sauces, passata and ketchup. It is an essential crop in many countries and has high nutritional value, being rich in antioxidants such as vitamin C and lycopene. Improved agronomy and biotechnology efforts have resulted in thousands of diverse cultivars and varieties that are able to generate improved yields, flavours as well as better adapt to environmental stresses.

[0006] Dwarf varieties of tomato offer several advantages for small or large scale cultivation. Dwarf tomato plants tend to be compact and bushy, making them ideal for urban areas, small gardens, balconies, vertical growing arrangements, or containers. They take up less space compared to taller counterparts. Their shorter stature can simplify tasks like staking, pruning, and harvesting. Dwarf tomatoes tend to mature more quickly than standard-sized plants and despite their smaller size, some dwarf varieties can produce a surprisingly abundant crop. Since they can be planted closer together, it is possible to maximize fruit harvest in limited spaces, such as in urban farms. Dwarf tomato plants can thrive in various conditions, including pots, raised beds, and hanging baskets. Their versatility allows for them to be raised indoors or outdoors, combining convenience, productivity, and adaptability, making them ideal for limited space or for increasing efficiency of harvest.

[0007] US-2023 / 0104872-A1 describes methods of increasing the proportion of edible biomass in a tomato plant by genetic modification of genes that encodes a poly (adenosine 5'-diphosphate (ADP)-Ribose) Polymerase (PARP) enzyme and plants generated using such methods. CRISPR / Cas9 gene editing was used to inactivate the PARP2 gene and resulted in dwarf phenotype with around 50% reduction in stem height compared to wild type. Marti et al. (Journal of Experimental Botany, Volume 57, Issue 9, June 2006, Pages 2037- 2047) describes a dwarf cultivar of tomato called Micro-Tom. This cultivar is shown to have mutations in the SELF-PRUNING (SP) and DWARF (D) genes. In addition to this, it is also shown that Micro-Tom harbours at least two independently segregating resistance loci to the plant pathogen Cladosporium fulvum. In Nagamine et al. (Breeding Sci, vol 74, 2024: 59-72) The and SP and D genes, were genome edited with CRISPR-Cas9 in a GABA hyperaccumulator genome-edited tomato cultivar, #87-17, which lacks the autoinhibitory domain of the SIGAD3 gene. The resultant plants were shorter than the unmodified cultivar although broadly similar in terms of plant morphology.

[0008] Choon-Tak et al. (Nature Biotech., vol 38, 2019: 182-188) devised a trait-stacking strategy to combine mutations for condensed shoots, rapid flowering (SP5G) together with precocious growth termination (SP) and also with with the tomato ERECTA (slER) gene, which is known to control internode length. The strategy used a one-step CRISPR-Cas9 genome editing approach that restructured vinelike tomato plants into more compact triple-determinate multistem, early yielding plants.

[0009] Despite the advantages of tomato plants of smaller stature, most conventional dwarf varieties such as Alaska are still around 40- 50% of the size of non-dwarf counterpart varieties. Hence, there exists a need to generate plants that have so-called extreme dwarfism in order to maximise on space utilisation and to offer even greater fruit yield compared to the non-edible vegetative biomass of the plant.

[0010] These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

[0011] SUMMARY OF THE INVENTION

[0012] A first aspect of the invention provides for a plant of the Solanaceae family comprising a functionally inactivating mutation present within a cytochrome p450 class 85A1 (CYP85A1) gene and a Self Pruning 5G (SP5G) gene.

[0013] In a specific embodiment of the invention the plant further comprises at least one functionally inactivating mutation within an allele of a gene selected from: carotenoid cleavage dioxygenase 8 (CCD8);

[0014] Gibberellin-insensitive dwarf 1a (GID 1a); and Gibberellin-insensitive dwarf 1b1 (GID1b1). A second aspect provides for a Solanum lycopersicum plant, the plant comprising functionally inactive CYP85A1 loci and SP5G loci.

[0015] In specific embodiments, the plant optionally comprises at least one or more functionally inactive alleles within a CCD8 locus, a GID1a locus, and a GID1b1 locus. In embodiments of the invention the inactivation of the said loci / alleles is associated with a phenotypic trait of structural dwarfism characterised by the vertical height of the plant being reduced by at least 70% compared to a wild-type plant of equivalent age. Suitably, the inactivation of the said loci / alleles is further associated with a phenotypic trait in which the plant is altered to exhibit reduced branching of the main stem compared to a wild-type plant of equivalent age, suitably not more than two main stems and optionally only a single main stem. In a further embodiment of the invention the inactivation of the said loci / alleles is further associated with a phenotypic trait that confers a full or partial involuting and / or revoluting morphology to the leaves compared to a wild-type plant.

[0016] A third aspect of the invention provides a plant cell of the Solanaceae family comprising at least one functionally inactivating mutation present within both the CYP85A1 gene and the SP5G gene. In embodiments, the plant cell may comprise at least one or more functionally inactivating mutations within alleles of a CCD8 locus, a GID1a locus, and a GID1b1 locus.

[0017] A fourth aspect provides a tissue culture of regenerable cells comprising plant cells of any of the embodiments described herein.

[0018] A fifth aspect provides a tomato fruit comprising a plant cell of any of the embodiments described herein.

[0019] A sixth aspect provides a plant part obtained or derived from a plant of any of the embodiments described herein.

[0020] A seventh aspect of the invention provides a method for modifying a plant cell from the Solanaceae family in order to introduce a phenotype of extreme dwarfism, the method comprising mutating one or more copies of both an CYP85A1 gene, or a homologue thereof, and a SP5G gene, within the genome of the plant cell, wherein the mutation comprises a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide or any combination thereof so as to induce functional inactivation of the said one or more genes.

[0021] In embodiments of the invention, all alleles of both the CYP85A1 gene and the SP5G gene are functionally inactivated. In particular embodiments, the method introduces one or more additional phenotypes selected from the group consisting of: determinate growth; reduced branching of the main stem; and / or full or partial involuting and / or revoluting morphology to the leaves.

[0022] An eighth aspect of the invention provides a method of generating a plant from the Solanaceae family, wherein the plant exhibits a phenotype of extreme dwarfism, the method comprising culturing a plant cell as described herein under conditions appropriate to facilitate proliferation of plant cells so as to form the plant.

[0023] In all aspects and embodiments of the invention a plant of the Solanaceae family may be selected from the species Solanum lycopersicum (tomato).

[0024] Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0027] Figure 1 are photographs of wildtype (WT) Ailsa Craig tomato plants (a) and mutants (b) to (d) at 7 weeks old - (d) shows a tomato variant having homozygous inactivation of both CYP85A1 and SP5G .

[0028] Figure 2 are photographs showing (a) and (b) close-ups of two tomato variants as per Figure 1 (d) at 12 weeks old; (c) shows Variant 1 plants at 14 weeks old with well-established fruiting trusses; and (d) shows comparison of shape and size of Variant 1 fruit (Mutant) versus wild-type fruit.

[0029] Figure 3 shows linear gene maps of (a) CYP85A1, (b) SP5G, (c) CCD8, (d) GID1a, (e) GID1b1. Shaded pointed boxes indicate genetic elements, either exons or introns. The location of guides RNAs (sgRNAs) used in generation of tomato variants via a CRISPR gene editing approach are labelled. Figure 4 shows sequence data of variant tomato plants for genes (a) CYP85A1, (b) SP5G, (c) CCD8, (d) GID1a, (e) GID1b1 with alignments of mutants versus wildtype (top line) to show the CRISPR induced mutations.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The invention relates to plants, fruits, cells and other propagatable material, and methods of making the same, of the Solanaceae family comprising at least one functionally inactivating mutation within the both of the cytochrome p450 class 85A1 (CYP85A1) gene, and the Self Pruning 5G (SP5G) gene; as well as optionally within at least one allele of the carotenoid cleavage dioxygenase 8 (CCD8) gene; the Gibberellin-insensitive dwarf 1a (GID1a) gene; and / or the Gibberellin-insensitive dwarf 1b1 (GID1b1) gene. A typical member of the Solanaceae family member is from the species Solanum lycopersicum (tomato).

[0032] In embodiments of the invention, the functionally inactivating mutations result in a phenotypic trait of extreme dwarfism compared to wild-type control plants. Other phenotypic traits that may be displayed include, but are not limited to, reduced branching of the main stem compared to a wild-type plant of equivalent age, as well as full or partial involuting and / or revoluting morphology of the leaves compared to a wild-type plant.

[0033] Plants having the phenotypic traits described herein advantageously fit into shelf-based production common in indoor, controlled environment agriculture (e.g. vertical farms) where standard vertical distance between a growing shelf floor and artificial illumination varies between 20 cm - 60 cm. Hence, plants having the phenotype of extreme dwarfism have a height as measured from base of the stem (typically where the soil surface would be) to highest apex that is typically reduced by at least 60%, at least 70%, at least 75%, and optionally by at least 80%, compared to a wild-type plant of equivalent age. In specific embodiments of the invention, the plants have a maximum height at maturity of typically not more than: 60 cm, 50 cm, 40 cm, 35 cm, and / or 30 cm.

[0034] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and chemical methods, which are within the capabilities of a person of ordinary skill in the art. Such techniques are also explained in the literature, for example, M.R. Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F. M. et al. (Current Protocols in Molecular Biology, John Wiley & Sons, Online ISSN:1934-3647); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Synthetic Biology, Part A, Methods in Enzymology, Edited by Chris Voigt, Volume 497, pages 2-662 (2011); Synthetic Biology, Part B, Computer Aided Design and DNA Assembly, Methods in Enzymology, Edited by Christopher Voigt, Volume 498, Pages 2-500 (2011); RNA Interference, Methods in Enzymology, David R. Engelke, and John J. Rossi, Volume 392, Pages 1-454 (2005). All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used herein, the term “comprising” means any of the recited elements are necessarily included and other elements may optionally be included as well. “Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. “Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

[0036] A “polynucleotide” is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA, and may be manufactured synthetically in vitro or isolated from natural sources. Sizes of polynucleotides are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called “oligonucleotides”.

[0037] The term “isolated”, when applied to a polynucleotide sequence, denotes that the sequence has been removed from its natural organism of origin and is, thus, free of extraneous or unwanted coding or regulatory sequences. Isolated oligonucleotides may be fully or partially of synthetic origin, that is, synthesised chemically as opposed to derived from natural sources.

[0038] The adjective “isolated,” when applied to a polypeptide or a ribonucleoprotein complex refers to a substantially purified composition, or in the case of a ribonucleoprotein complex, at least one component being a substantially purified component. In further respect to an isolated ribonucleoprotein complex, preferably all components are substantially purified.

[0039] As used herein, the terms 3' (‘3 prime’) and 5' (‘5 prime’) take their usual meanings in the art, i.e. to distinguish the ends or directionality within polynucleotide sequences. A polynucleotide has a 5' and a 3' end and polynucleotide sequences are conventionally written in a 5' to 3' direction. The 5’ end is suitably considered to be upstream of the 3’ end of a polynucleotide sequence. Hence, a sequence referred to as upstream of a given reference point in a gene, such as the ATG transcription start codon of an open reading frame (ORF), is a sequence that is 5’ to the reference point. Likewise a sequence denoted as downstream is 3’ to the reference point, e.g. downstream to the ATG start codon.

[0040] The term “gene expression control sequence” comprises regulatory sequences, sometimes referred to as a cis- regulatory element (CRE) and includes promoters, ribosome binding sites, enhancers, silencers and insulators and other control elements which regulate transcription of a gene or translation of a resultant mRNA. In particular embodiments of the invention, the gene expression control sequences confer plant tissue or cell-type specificity that assist in determining the phenotype of the cell. Gene expression control sequences may also contribute to regulation of gene expression levels. For example, the expression level of a particular gene can be considered as the amount of mRNA and / or polypeptide produced from that particular gene. Gene expression levels can referto an absolute (e.g., molar or gram-quantity) abundance of mRNA or polypeptide, or a relative (e.g., the amount relative to a standard, reference, calibration, or to another gene expression level). Hence, according to embodiments of the present invention inactivating mutations of at least one allele of two or more of the genes selected from: cytochrome p450 class 85A1 (CYP85A1); Self Pruning 5G (SP5G) carotenoid cleavage dioxygenase 8 (CCD8); Gibberellin-insensitive dwarf 1a (GID1a) and / or Gibberellininsensitive dwarf 1b1 (GID1b1), may occur within non-coding regions such as within one or more gene expression control sequences.

[0041] As used herein, the terms "plant" and "plant part" refer to cells, tissues, organs, seeds, and severed parts (e.g., roots, leaves, and flowers) that retain the distinguishing characteristics of the parent plant. "Tuber" refers to a thickened underground part of a stem or rhizome, serving as a food reserve and bearing buds from which new plants arise. "Seed" refers to any plant structure that is formed by continued differentiation of the ovule of the plant, following its normal maturation point at flower opening, irrespective of whether it is formed in the presence or absence of fertilization and irrespective of whether or not the seed structure is fertile or infertile. Other propagatable parts of a plant may include tissue cultures of regenerable cells, calli, cuttings, or root segments. Suitably, a “plant cell” may be selected from a gametophyte, a reproductive cell, a vegetative cell and / or a meristematic cell. In embodiments, suitable plant tissue is selected from: leaf, stem, root, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, scion, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, or leaf sheath. In specific embodiments plant parts and cellular material may include root tissue, leaf mesophyll and / or cultured callus. In relation to the plant, the term “nonedible vegetative biomass” refers to non-fruiting or non-yielding parts of a Solanaceae plant, such as the leaves, stem, and roots. In certain embodiments the plant cell is in the form of a protoplast. As used herein, the term “plant protoplasts” (also referred to simply as “protoplast”, throughout this disclosure) refers to a plant cell that has had its cell wall completely or partially removed. Removal of cell wall can be effected by mechanical, chemical or enzymatic means. In embodiments, protoplasts are obtained from suitable plant material using cell wall digestive enzymes. For example, enzymes such as cellulase, macerozyme, pectinase, hemicellulase, pectolyase, driselase, xylanase and combinations thereof may be suitable for use in the context of the invention. In embodiments, cellulase may be used at a concentration of 1w% - 1.5w%. In embodiments, macerozyme may be used at a concentration of 0.2w% - 0.4w%. In embodiments, hemicellulase may be used at a concentration of 2w% - 5w%. In embodiments, pectolyase may be used at a concentration 0.01w% - 0.5w%. In embodiments, driselase may be used at a concentration of 0.5w% - 2w%. Protocols for obtaining protoplasts from plant tissues are known in the art, for example in Yoo, Cho, & Sheen (2007) Nature Protocols volume 2, pages 1565-1572.

[0042] The term "allele(s)" means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus on a chromosome, with one allele being present on each chromosome of the pair of homologous chromosomes. Similarly, in a tetrapioid cell of an organism, one allele is present on each chromosome of the group of four homologous chromosomes. "Heterozygous" alleles are different alleles residing at a specific locus, positioned individually on corresponding homologous chromosomes. "Homozygous" alleles are identical alleles residing at a specific locus, positioned individually on corresponding homologous chromosomes in the cell. "Wild type" as used herein refers to a typical form of a plant or a gene as it most commonly occurs in nature or as an unmodified control. In embodiments of the invention “copies” of a gene refers to all alleles of that gene that are functionally equivalent.

[0043] The term “allelic variant” is used herein to denote any two or more alternative forms of a gene occupying the same chromosomal locus and controlling the same inherited characteristic. Allelic variation arises naturally through mutation and may result in phenotypic polymorphism within populations. Gene mutations typically result in an altered nucleic acid sequence and in some cases an altered polypeptide sequence also. As used herein, the term “allelic variant” is additionally used to refer to the protein or polypeptide encoded by the allelic variant of a gene.

[0044] In embodiments of the present invention a functionally inactivating mutation is present in at least one allele of a least two genes selected from of at least one allele of two or more of the genes selected from: cytochrome p450 class 85A1 (CYP85A1) Self Pruning 5G (SP5G) carotenoid cleavage dioxygenase 8 (CCD8); Gibberellin-insensitive dwarf 1a (GID1a) and / or Gibberellininsensitive dwarf 1b1 (GID1b1). Typically, the inactivating mutation is present in all alleles at least of CYP85A1 and also SP5G. This is intended to indicate that at least one allele within each of at least two genes selected in combination is functionally inactivated so that at least two of the genes listed are at least heterozygous or homozygous for the inactivated allele, optionally more than two of the listed genes may be heterozygous or homozygous for the inactivated allele. In specific embodiments of the invention at least: cytochrome p450 class 85A1 (CYP85A1); and Self Pruning 5G (SP5G) are both heterozygous and / or homozygous each comprising at least one functionally inactivated allele, suitably wherein all alleles are functionally inactivated; cytochrome p450 class 85A1 (CYP85A1) Self Pruning 5G (SP5G) and carotenoid cleavage dioxygenase 8 (CCD8) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; cytochrome p450 class 85A1 (CYP85A1) Self Pruning 5G (SP5G) and Gibberellin-insensitive dwarf 1a (GID1a) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; cytochrome p450 class 85A1 (CYP85A1) Self Pruning 5G (SP5G) and Gibberellin-insensitive dwarf 1b1 (GID1b1) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; cytochrome p450 class 85A1 (CYP85A1) carotenoid cleavage dioxygenase 8 (CCD8) and Gibberellin-insensitive dwarf 1a (GID1a) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; cytochrome p450 class 85A1 (CYP85A1) carotenoid cleavage dioxygenase 8 (CCD8) Gibberellin-insensitive dwarf 1a (GID1a) and Gibberellin-insensitive dwarf 1b (GID1b) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; cytochrome p450 class 85A1 (CYP85A1) Self Pruning 5G (SP5G) carotenoid cleavage dioxygenase 8 (CCD8) and Gibberellin-insensitive dwarf 1a (GID1a) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; cytochrome p450 class 85A1 (CYP85A1) Self Pruning 5G (SP5G) carotenoid cleavage dioxygenase 8 (CCD8) Gibberellin-insensitive dwarf 1a (GID1a) and Gibberellin-insensitive dwarf 1b1 (GID1b1) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; (GID1b1) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele;

[0045] Self Pruning 5G (SP5G) carotenoid cleavage dioxygenase 8 (CCD8) and Gibberellin-insensitive dwarf 1a (GID1a) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele;

[0046] Self Pruning 5G (SP5G) carotenoid cleavage dioxygenase 8 (CCD8) and Gibberellin-insensitive dwarf 1b1 (GID1b1) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; carotenoid cleavage dioxygenase 8 (CCD8) and Gibberellin-insensitive dwarf 1a (GID1a) are both heterozygous and / or homozygous each comprising at least one functionally inactivated allele; carotenoid cleavage dioxygenase 8 (CCD8) Gibberellin-insensitive dwarf 1a (GID1a) and Gibberellin-insensitive dwarf 1b1 (GID1b1) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele; or

[0047] Gibberellin-insensitive dwarf 1a (GID1a) and Gibberellin-insensitive dwarf 1b (GID1b) are all heterozygous and / or homozygous each comprising at least one functionally inactivated allele.

[0048] A "wild type CYP85A1 allele" is a naturally occurring CYP85A1 allele (e.g., as found within naturally occurring S. lycopersicum plants, Gene ID: Solyc02g089160) that encodes a functional 6-deoxocastasterone oxidase protein, while a "functionally inactive CYP85A1 allele" is a CYP85A1 allele that does not encode or express a functional 6-deoxocastasterone oxidase protein. Such a "functionally inactive CYP85A1 allele" can include one or more mutations in the nucleic acid sequence of a CYP85A1 gene, where the mutation(s) result in reduced or even no detectable amount of functional 6-deoxocastasterone oxidase protein in the plant or plant cell in vivo or in extracts taken from mutant plant cells and tested in vitro. The one or more mutations in the nucleic acid sequence of a CYP85A1 gene may be located within coding (e.g. exon) or non-coding parts of the gene.

[0049] A "wild type SPG5 allele" is a naturally occurring SPG5 allele (e.g., as found within naturally occurring S. lycopersicum plants, Gene ID: Solyc05g053850, also referred to as flowering locus T) that encodes a phosphatidylethanolamine-binding protein called SELF PRUNING 5G, while a "functionally inactive SPG5 allele" is a SPG5 allele that does not encode or express a functional SELF PRUNING 5G protein. Such a "functionally inactive SPG5 allele" can include one or more mutations in the nucleic acid sequence of a SPG5 gene, where the mutation(s) result in reduced or even no detectable amount of SELF PRUNING 5G protein in the plant or plant cell in vivo or in extracts taken from mutant plant cells and tested in vitro. The one or more mutations in the nucleic acid sequence of a SPG5 gene may be located within coding (e.g. exon) or non-coding parts of the gene.

[0050] A "wild type CCD8 allele" is a naturally occurring CCD8 allele (e.g., as found within naturally occurring S. lycopersicum plants, Gene ID: Solyc08g066650 also referred to as carotenoid cleavage dioxygenase 8) that encodes a carotenoid cleaving deoxygenase, an enzyme that cleaves beta-carotene, while a "functionally inactive CCD8 allele" is a CCD8 allele that does not encode or express a functional CCD8 protein. Such a "functionally inactive CCD8 allele" can include one or more mutations in the nucleic acid sequence of a CCD8 gene, where the mutation(s) result in reduced or even no detectable amount of CCD8 protein in the plant or plant cell in vivo or in extracts taken from mutant plant cells and tested in vitro. The one or more mutations in the nucleic acid sequence of a CCD8 gene may be located within coding (e.g. exon) or non-coding parts of the gene.

[0051] A "wild type GID1a allele" is a naturally occurring GID1a allele (e.g., as found within naturally occurring S. lycopersicum plants, Gene ID: Solyc01g098390, also referred to as Gibberellininsensitive dwarf 1a) that encodes a gibberellin receptor protein, while a "functionally inactive GID1a allele" is a GID1a allele that does not encode or express a functional GID1a protein. Such a "functionally inactive GID1a allele" can include one or more mutations in the nucleic acid sequence of a GID1a gene, where the mutation(s) result in reduced or even no detectable amount of GID1a protein in the plant or plant cell in vivo or in extracts taken from mutant plant cells and tested in vitro. The one or more mutations in the nucleic acid sequence of a GID1a gene may be located within coding (e.g. exon) or non-coding parts of the gene.

[0052] A "wild type GID1b1 allele" is a naturally occurring GID1b1 allele (e.g., as found within naturally occurring S. lycopersicum plants, Gene ID: Solyc09g074270, also referred to as Gibberellininsensitive dwarf 1 b1) that encodes a gibberellin receptor protein, while a "functionally inactive GID1b1 allele" is a GID1b1 allele that does not encode or express a functional GID1 b1 protein. Such a "functionally inactive GID1b1 allele" can include one or more mutations in the nucleic acid sequence of a GID1b1 gene, where the mutation(s) result in reduced or even no detectable amount of GID1 b1 protein in the plant or plant cell in vivo or in extracts taken from mutant plant cells and tested in vitro. The one or more mutations in the nucleic acid sequence of a GID1b1 gene may be located within coding (e.g. exon) or non-coding parts of the gene.

[0053] The terms “functionally inactivating”, “suppressing” or “reducing” when used in reference to a gene(s), refers to a lowering, reduction, or elimination of the expression level of a mRNA and / or protein product encoded by the gene(s), and / or a lowering, reduction, or elimination of the functional activity of a protein encoded by the gene(s) in a plant, plant cell or plant tissue, at one or more stage(s) of plant development, as compared to the expression level of such target mRNA and / or protein, and / or the activity of such encoded protein in a wild-type or control plant, cell or tissue at the same stage(s) of plant development. Lowering, reduction, or elimination may be determined functionally such as by testing the activity of the gene product, or via various quantitative measures such as those that assess reduction in production of protein product (e.g. Western blot, mass spectrometry, ELISA).

[0054] The genetically modified or genetically edited tomato cells or protoplasts described herein can be prepared using conventional gene editing methods or those described more specifically herein to edit one or more of target genes. Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease- independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence, such as within targeted regions of at least one allele of a CYP85A1, SPG5, CCD8, GID1a and / or GID1b1 gene, or any combination thereof. The mutations to these genes may serve to knock out the gene function entirely and / or may result in allelic variants that have reduced or impaired functional activity. In an embodiment of the invention, functionally inactivating mutation comprises a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide or any combination thereof. In embodiments of the invention, a deletion may be from about one nucleotide to more than 2000 nucleotides, typically up to 1000 nucleotides, suitably up to 500 nucleotides, optionally up to 100 nucleotides.

[0055] Alternatively, a nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare- cutting nucleases (e.g., endonucleases). Nuclease-dependent targeted editing of this type also utilizes host DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to these DSBs. DNA repair by NHEJ can lead to random insertions or deletions (so called “indels”) of a small number of endogenous nucleotides at the cleavage site. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material. Gene Editing

[0056] In some embodiments, gene disruption may occur by deletion of a genomic sequence using one or two guide RNAs. Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell, such as to knock out a gene in a plant cell, are described in Nekrasov et al. Sci Rep 7, 482 (2017).

[0057] Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas nucleases (CRISPR-Cas; Clustered Regular Interspaced Short Palindromic Repeats Associated). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases may also be used for targeted integration.

[0058] CRISPR-Cas systems are widespread prokaryotic adaptive immune systems that are best known as components of a next generation of genome-editing tools. Cas9 recognizes 3' G-rich PAMs; it has become the most widely used CRISPR-Cas system and has been adapted for genome editing in various contexts, including in plants. Unlike Cas9, Cas12a recognizes 5' T- rich PAMs and self-processes its CRISPR RNA (crRNA). According to certain embodiments of the present invention an RNA-guided CRISPR-Cas12a nuclease (CRISPR / Cas12a) nuclease is utilised, suitably a ErCas12a (MAD7) (US patent number US9982279B1) (https: / / www.inscripta.com / madzymes / faq / ). MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a / Cpf1) family with a low level of homology to canonical Cas12a nucleases, that is particularly effective at generating indel mutations in plant cells. Other Nonlimiting examples of RNA-guided endonuclease systems include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Casio, Csy1 , Csy2, Csy3, Cse1 , Cse2, Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, and Csf4, as well as homologs thereof, or modified versions thereof. Alternatively, some RNA-guided endonucleases are modified versions of the wildtype form, for example, comprising an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof, relative to a wild-type version of the protein. In some embodiments, the endonuclease comprises a region exhibiting at least 70% identity over at least 70% of its residues to a Cas9 domain or a Cpfl domain. In particular embodiments, the Cas9 is selected from the group consisting of SpCas9 SaCas9, StCas9, NmCas9, FnCas9, and CjCas9. In embodiments of the invention the endonuclease comprises an endonuclease having a Cas9 activity or a variant or derivative thereof.

[0059] CRISPR-Cas gene editing technology involves the use of a genome-targeting nucleic acid that can direct the endonuclease to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be an RNA. A genome- targeting RNA is referred to as a “guide RNA” or “gRNA” herein. A gRNA typically comprises a guide sequence having sufficient complementarity with a target nucleic acid to hybridize via Watson-Crick base pairing interactions with the target nucleic acid molecule at a given sequence and to direct sequence-specific binding of the endonuclease complex to the target nucleic acid sequence. Typical gRNA molecules include a targeting sequence, which binds to the complementary DNA sequence, and a Cas protein binding scaffold region, which interacts with the Cas enzyme (or equivalent or derivative thereof). The guide molecule or guide RNA may encompass RNA-based molecules having one or more chemical modifications, including synthetic bases, or by chemical linking two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides). For example, chemical modifications such as 2'-O-methyl or phosphorothioate modifications can be introduced to increase gRNA stability. The present disclosure provides a guide nucleic acid suitable for use in a CRISPR / Cas system. A gRNA binds to a Cas protein via the scaffold region and targets the Cas protein to a specific location within a target nucleic acid. In some cases, a guide nucleic acid comprises a single nucleic acid molecule, referred to as a single guide nucleic acid (sgRNA). Alternatively, a guide nucleic acid comprises two separate nucleic acid molecules, referred to as a double guide nucleic acid.

[0060] In Type V gRNAs, of the type that is utilised by Cas12a nucleases (e.g. ErCas12a / MAD7), the crRNA forms a duplex that binds to the endonuclease, such that the guide RNA and endonuclease form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the endonuclease. The genome-targeting nucleic acid thus directs the activity of the endonuclease to a specific target site within the genome of a host cell. Hence, it will be understood by the person of ordinary skill in the art, that each gRNA is designed to include a spacer sequence complementary to its genomic target sequence. The term guide RNA (gRNA) is used synonymously with single guide RNA, “sgRNA”.

[0061] In embodiments of the present invention, the spacer sequence comprised within the gRNA is an oligonucleotide sequence, typically around 20 to 25 nucleotides in length, that defines a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of a target gene of interest. In embodiments of the present invention the targets are within at least one allele of each of two or more genes selected from the: CYP85A1, SPG5, CCD8, GID1a, and GID1b1 genes. In some embodiments, the spacer sequence ranges from 15 to 30 nucleotides in length. For example, the spacer sequence may contain at least 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence contains at least 22 nucleotides.

[0062] In embodiments of the present invention, the selected “target sequences” are in target alleles, suitably within the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 genes, that is adjacent to a PAM sequence. The sequences are modified by an RNA-guided endonuclease (e.g., Cas12a, or MAD7).

[0063] In specific embodiments of the present invention the region of the CYP85A1 gene is identified as exon 1 and exon 2. In specific embodiments of the present invention the region of the SPG5 gene is identified as exon 1 and exon 2. In specific embodiments of the present invention the region of the CCD8 gene is identified as exon 2. In specific embodiments of the present invention the region of the GID1a gene is identified as exon 2. In specific embodiments of the present invention the region of the GID1b1 gene is identified as exon 2. The “target sequences” are on the so-called PAM-strand in a “target nucleic acid,” which is a double-stranded DNA molecule containing the PAM-strand and a complementary non-PAM strand. One skilled in the art recognizes that the gRNA spacer sequence hybridizes to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. The spacer sequence of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via Watson- Crick base pairing. The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.

[0064] The gRNAs disclosed herein may target any sequence of interest via the spacer sequence comprised within it. In some embodiments, the amount of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene is 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene may contain up to 5 mismatches, e.g., up to 4, up to 3, up to 2, or up to 1 mismatch. Typically, the mismatches are not consecutive and may be distributed across the spacer sequence. Hence according to specific embodiments, gRNAs corresponding to any one of SEQ ID Nos: 1-18 may be used in gene editing methods, such as for the production of any of the tiny tomato variants as disclosed herein.

[0065] Any of the gRNAs disclosed herein may be chemically unmodified - i.e. utilising naturally occurring nucleotides. Alternatively, the gRNAs may contain one or more modified nucleotides and / or modified backbones. For example, a modified gRNA can comprise one or more 2'-O- methyl phosphorothioate nucleotides, which may be located at either the 5' end, the 3' end, or both. Alternatively, the gRNAs may comprise at least one non-naturally occurring nucleotide such as any one of those described in US-2020 / 0224234-A1 .

[0066] Exemplary spacer sequences suitable for inclusion in gRNAs targeting an CYP85A1 gene are provided in Table 1 below. In embodiments of the invention the spacer sequences hybridize with an antisense strand of the CYP85A1 gene at a location 107 nucleotides downstream of the start codon (ATG). In further embodiments, spacer sequences hybridize with an antisense strand of the CYP85A1 gene at a location 383 downstream of the start codon (ATG). The exemplary spacer sequences are particularly suitable for inclusion within a gRNA intended for use with a Cas12a based gene editing system, most typically with ErCas12a / MAD7. However, it will be understood by the skilled person that alternative sequences may be utilised for other Cas based systems, such as those that utilise Cas9. The exemplary spacer sequences show unexpectedly good utility in effecting a gene editing event resulting in a mutation in the genome of more than one distinct variety of Solanum lycopersicum.

[0067] Table 1 - gRNAs targeting an CYP85A1 gene [SEQ ID NOs: 1-6]

[0068] Exemplary spacer sequences suitable for inclusion in gRNAs targeting an SPG5 gene are provided in Table 2 below. In embodiments of the invention the spacer sequences hybridize with an antisense strand of the SPG5 gene at a location 118 nucleotides downstream of the start codon (ATG). In further embodiments, spacer sequences hybridize with an antisense strand of the SPG5 gene at a location 657 downstream of the start codon (ATG). The exemplary spacer sequences are particularly suitable for inclusion within a gRNA intended for use with a Cas12a based gene editing system, most typically with ErCas12a / MAD7. However, it will be understood by the skilled person that alternative sequences may be utilised for other Cas based systems, such as those that utilise Cas9. The exemplary spacer sequences show unexpectedly good utility in effecting a gene editing event resulting in a mutation in the genome of more than one distinct variety of Solanum lycopersicum.

[0069] Table 2 - gRNAs targeting an SPG5 gene [SEQ ID Nos: 7-12] Exemplary spacer sequences suitable for inclusion in gRNAs targeting an CCD8 gene are provided in Table 3 below. In embodiments of the invention the spacer sequences hybridize with an antisense strand of the CCD8 gene at a location 1200 nucleotides downstream of the start codon (ATG). In further embodiments, spacer sequences hybridize with an antisense strand of the CCD8 gene at a location 1655 downstream of the start codon (ATG). The exemplary spacer sequences are particularly suitable for inclusion within a gRNA intended for use with a Cas12a based gene editing system, most typically with ErCas12a / MAD7. However, it will be understood by the skilled person that alternative sequences may be utilised for other Cas based systems, such as those that utilise Cas9. The exemplary spacer sequences show unexpectedly good utility in effecting a gene editing event resulting in a mutation in the genome of more than one distinct variety of Solanum lycopersicum.

[0070] Table 3 - gRNAs targeting a CCD8 gene

[0071] Exemplary spacer sequences suitable for inclusion in gRNAs targeting an GID1a gene are provided in Table 4 below. In embodiments of the invention the spacer sequences hybridize with an antisense strand of the GID1a gene at a location 702 nucleotides downstream of the start codon (ATG). In further embodiments, spacer sequences hybridize with an antisense strand of the GID1a gene at a location 832 downstream of the start codon (ATG). The exemplary spacer sequences are particularly suitable for inclusion within a gRNA intended for use with a Cas12a based gene editing system, most typically with ErCas12a / MAD7. However, it will be understood by the skilled person that alternative sequences may be utilised for other Cas based systems, such as those that utilise Cas9. The exemplary spacer sequences show unexpectedly good utility in effecting a gene editing event resulting in a mutation in the genome of more than one distinct variety of Solanum lycopersicum.

[0072] Table 4 - gRNAs targeting an GID1a gene

[0073] Exemplary spacer sequences suitable for inclusion in gRNAs targeting an GID1B1 gene are provided in Table 5 below. In embodiments of the invention the spacer sequences hybridize with an antisense strand of the GID1B1 gene at a location 345 nucleotides downstream of the start codon (ATG). In further embodiments, spacer sequences hybridize with an antisense strand of the GID1B1 gene at a location 857 downstream of the start codon (ATG). The exemplary spacer sequences are particularly suitable for inclusion within a gRNA intended for use with a Cas12a based gene editing system, most typically with ErCas12a / MAD7. However, it will be understood by the skilled person that alternative sequences may be utilised for other Cas based systems, such as those that utilise Cas9. The exemplary spacer sequences show unexpectedly good utility in effecting a gene editing event resulting in a mutation in the genome of more than one distinct variety of Solanum lycopersicum.

[0074] Table 5 - gRNAs targeting an GID1b1 gene

[0075] Cas12a RNA guided endonucleases, such as ErCas12a / MAD7, will bind to genomic regions matching the designed RNA spacer sequence that are adjacent to a suitable immediate upstream to a PAM site. In this location, the MAD7 endonuclease enzyme uses a single RuvC-like endonuclease domain to cut the DNA in a staggered manner, leaving 4-nucleotide 5'-overhangs at the PAM distal end of the genomic target site. The two resulting cleavage sites are located 19 bases after the PAM on the PAM-strand (sense strand) and after 23 bases on the non-PAM strand (antisense strand). In contrast to Cas 12a endonucleases, such as MAD7, the canonical Streptococcus pyogenes Cas9 protein uses two nuclease domains (HNH and RuvC-like) to break each DNA strand and generate a nearly synchronous blunt-ended DSB proximal to the PAM. The ability of Cas12a RNA guided endonucleases to produce sticky ends upon cleavage of the target DNA enhances the generation of indels, thus, facilitating knock out type frame-shift mutations at the target site. The mutations to any one of the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 genes may serve to knock out the gene function entirely or may result in an allelic variant that has substantially reduced or impaired functional activity. In certain embodiments of the invention only one allele may be subject to gene editing resulting in a heterozygous mutant that nonetheless exhibits functional inhibition resulting in the desired phenotypic trait.

[0076] Base Editing

[0077] In an alternative embodiment of the invention a base editing approach may be adopted to introduce mutations to the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 genes that may serve to knock out or reduce activity of the gene product function. Cytidine deaminase base editing enables the direct, irreversible conversion of a C:G base pair to a T: A base pair in a programmable manner without requiring HDR or the introduction of a double strand break. In contrast to conventional gene editing which relies on a canonical sequence-guided endonuclease such as a Cas9 protein that creates a double strand break, base editors contain a single-stranded DNA- specific cytidine deaminase enzyme tethered to a catalytically impaired Cas9 protein and a base excision repair inhibitor (see . Y. B. Kim et al, Nat Biotechnol 35, 371 - 376 (2017)). In an embodiment of the present invention, the Cas9 variant is targeted to a CYP85A1, SPG5, CCD8, GID1a, and GID1b1 locus of interest, programmed by a corresponding guide RNA that may comprise a sequence of nucleotides that correspond to any one of those set out herein or that could be determined by the skilled person. This leads to the formation of a protein-RNA-DNA ternary “R- loop” complex that exposes a small (~5-nt) bubble of single-stranded DNA that serves as a substrate for the tethered cytidine deaminase enzyme. Cytidines within this bubble may be hydrolytically deaminated to uracils, resulting in G:U intermediates. The cell’s primary response to the presence of G:U mismatches is to initiate a base excision repair mechanism to replace the uracils with cytidines. This process is initiated by excision of the uracil by uracil N-glycosylase. To protect the edited G:U intermediate from excision by uracil N-glycosylase, an uracil glycosylase inhibitor (UGI) can be incorporated into the base editing complex, such as via fusion with the C-terminus of catalytically impaired Cas9. Manipulation of the cellular DNA mismatch repair systems into preferentially replacing the G in the G:U mismatch with an A, requires that Cas9 is modified to have nickase activity rather than catalysing a double strand break. This enables the Cas9 protein to nick the DNA strand opposite the newly formed uracil in the G:U mismatch, resulting in much more efficient conversion of the G:U intermediate to sequence altered A:U and A:T products.

[0078] An alternative to cytidine deaminase base editing utilises an adenosine deaminase base editor complex to convert A:T base pairs to G:C base pairs at a given target location. The deamination of adenine yields inosine (I), which is read and replicated as guanine (G) by polymerases. The approach is broadly similar to that described in relation to cytidine deamination base editing (see Gaudelli et al. Nature. (2017) Nov 23; 551 (7681): 464-471).

[0079] Prime Editing

[0080] Prime editing is a powerful editing tool that is an alternative to the base editing approaches described previously. Prime editing also allows precise modifications to genomic DNA utilising a protein editing complex that is guided to a locus within a gene via a guide RNA sequence. It consists of two key components: nCas9, modified Cas9 endonuclease having nickase activity, which nicks the DNA strand, and an associated Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT), which facilitates the editing process (Anzalone AV et al. Nature 576, 149-157 (2019)). The RT component may be comprised within the prime editing complex or present in solution. Guide RNAs (called pegRNAs) direct the prime editor complex to the target locus in the genome. These pegRNAs have an extended structure at their 3’ end compared to single-guide RNAs (sgRNAs) which includes a primer binding site (PBS): complementary to a portion of the DNA protospacer and an RT template that encodes the intended edit. Once nicking occurs, the PBS sequence pairs with the complementary target DNA sequence to start priming reverse transcription from the RT template, which enables the desired editing. However, unlike cytidine / adenosine deaminase containing base editors, prime editing can induce a relatively high frequency of indels. Hence, prime editing represents an alternative mechanism to introduce mutations to any of the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 genes that may serve to knock out or reduce activity of the gene product function.

[0081] Delivery of genetic modification complex

[0082] The genetic modification systems disclosed herein, comprising one or more gRNAs and at least one RNA-guided nuclease, such as Cas9 and Cas12a, can be delivered to a target cell or protoplast (e.g., a tomato cell or protoplast) for genetic editing of the target gene, via any conventional method. In some embodiments, components of an endonuclease system as disclosed herein may be delivered to a target cell separately, either simultaneously or sequentially. In other embodiments, the components of the endonuclease system may be delivered into a target together, for example, as a complex. In some instances, gRNA and the RNA-guided endonuclease can be pre-complexed together to form a ribonucleoprotein (RNP), which can be delivered into a target cell or protoplast using conventional techniques. Hence, as used herein the phrase “ribonucleoprotein complex” or “RNP” refers to a ribonucleoprotein complex having CRISPR-associated endonuclease activity.

[0083] In alternative embodiments, genetic modification systems may be introduced via transformation protocols that rely on use of Agrobacterium tumefaciens to transfer a binary vector with a gene or genes that encoding genetic modification system and selectable markers and / or reporter genes placed between T-DNA borders, into leaf epidermal cells. The genetic modification systems may include the gene, prime or base editing complex as well as nucleotides encoding gRNAs that enable targeting of the genetic modification system to two or more of the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 genes within the cells. Protocols using Agrobacterium tumefaciens are known to the skilled person, exemplary approaches are described, for example in Sparkes, I., Runions, J., Kearns, A. et al. Nat Protoc 1 , 2019-2025 (2006).

[0084] Other targeted genomic modification techniques

[0085] In contrast to genome editing approaches based around the CRISPR platform described above, zinc finger nucleases (ZFNs) can be made by combining two different protein domains: engineered Cys2-His2 DNA-binding ZF and a DNA-cutting tool — or restriction endonuclease — called Fok1 . The binding of Cys2-His2 ZF to DNA involves the insertion of an alpha helix of the protein into the major groove of double stranded DNA. Each ZF recognizes and binds to three tandem nucleotides. This allows the liberty to link a ZF to a DNA sequence of interest. In theory, ZFs can be programmed to bind to almost any specific DNA sequence, facilitating a Fok1- induced double-stranded DNA (dsDNA) break. Another approach to genome editing uses DNA- binding transcription activator-like (TAL) effectors from the plant bacterial pathogen Xanthomonas. TAL effectors have sets of repeating amino acids, and small variations in these amino acid repeats determine DNA-binding specificity. These variations can be programmed to target specific DNA sequences. TAL effector nucleases (TALENs), combining the TAL effector DNA-binding domains and the catalytic domain of Fok1 , are also able to make dsDNA breaks at directed genomic loci. Since both the ZFN and TALEN based approaches, like CRISPR, rely upon introducing targeted double strand breaks in the genome, this can be utilised to introduce indel mutations to generate knock outs of targeted genes, such as within alleles of any of the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 loci.

[0086] Non-targeted genomic modification techniques

[0087] In addition to the sequence directed approaches described previously, a range of induced mutagenesis techniques can be utilised to drive genetic variation that results in novel phenotypic traits in Solanaceae family members, such as S. lycopersicum. Exposing the genetic material within plant cells to mutagenic agents brings changes in nuclear DNA which results in genomic or chromosomal mutations enabling plant breeders to select useful mutants. Mutants can be ascertained by evaluation of phenotypes determined from progeny of mutated plants, as well as through techniques such as NGS. Tomato behaves like a basic diploid, hence, mutations of many types can be clearly identified in its phenotype. Exemplary mutagenesis approaches, according to embodiments of the present invention, that may result in functional mutation of CYP85A1, SPG5, CCD8, GID1a, and GID1b1 and the corresponding phenotype may include the following:

[0088] Chemical Mutagenesis: Chemical mutagens, such as ethyl methane sulfonate (EMS) and N- methyl-N-nitrosourea (MNU), are used to induce point mutations in the target genome by alkylating DNA bases. Chemical mutagenesis allows for the introduction of specific nucleotide changes, facilitating the generation of allelic diversity within plant populations. EMS is considered as one of the most effective chemical mutagenic agents to induce genetic variability in a number of crop plants, including tomato, through primarily G / C- to A / T transitions (Greene at al. Genetics, Volume 164, Issue 2, 1 June 2003, Pages 731-740).

[0089] Radiation Mutagenesis: Ionizing radiation sources, including gamma rays and X-rays, induce random DNA damage, including base substitutions, deletions, and insertions. Radiation mutagenesis provides a broad spectrum of genetic alterations that can produce useful mutants due to the property of large-scale deletions and occasionally, chromosome reconstitution. Typically plant material can be irradiated with doses of ionising radiation, such as X-rays, in an amount of between 20 and 100 Grays (Gy) in order to induce mutations within the genome. Insertional Mutagenesis: Transposable elements, such as maize Ac / Ds and En / Spm transposon systems, T-DNA from Agrobacterium tumefaciens and retrotransposons, can be utilized to disrupt gene function or alter gene expression patterns, in Solanaceae family members. Insertional mutagenesis offers the advantage of easier characterisation of mutants as the inserted element acts as a tag for gene Identification, for example, see Memelink, J. (2003). T- DNA Activation Tagging. In: Grotewold, E. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 236.

[0090] In specific embodiments of the present invention, the tomato (S. lycopersicurri) plants, cells, plant parts, seeds, other propagatable material and progeny thereof that are provided herein can have a mutation in the endogenous alleles of one or more the CYP85A1, SPG5, CCD8, GID1a, and GID1b1 loci, such that expression of a functional protein product is reduced or completely inhibited. Thus, in some embodiments, the plants, cells, plant parts, seeds, other propagatable material and progeny exhibit substantially reduced or even totally eliminated detectable levels of functional protein activity. Hence, in other embodiments of the invention the level of any one of CYP85A1 , SPG5, CCD8, GID1 a, and / or GID1 b1 functional activity in the plants, cells, plant parts, seeds, other propagatable material and progeny thereof is sufficiently reduced by at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30% and 20% when compared to a plant from the same variety that has not been subjected to a genetic modification by the methods as described herein.

[0091] In another embodiment of the present invention, the plant exhibits determinate growth habits. Indeterminate growth is characterized by a continuously growing vegetative meristem that allows the plant to grow taller throughout the growing season, with the main stem developing in a vine-like manner that can reach heights exceeding two meters under favorable conditions. Such varieties produce flower clusters laterally along the stems, enabling them to simultaneously flower, set fruit, and ripen fruit over an extended harvest period until terminated by environmental factors such as frost. Plants, such as those of embodiments of the present invention, that exhibit determinate growth patterns show finite growth wherein the main stem terminates in a flower cluster, effectively halting vertical growth once a predetermined genetic height is reached. This allows for generation of more compact varieties that tend to produce the majority of their fruit within a concentrated time period of approximately two to four weeks. Tomato (S. lycopersicurri) plants that show determinate growth are particularly suitable for mechanical harvesting applications and cultivation in space-limited environments. The determinate or indeterminate growth habit substantially influences cultivation practices, including spacing requirements, staking methodologies, pruning protocols, and harvest scheduling considerations. In one embodiment of the present invention, the plant, suitably a tomato (S. lycopersicum) plant, exhibits revolute leaf morphology. Revolute leaf morphology refers to a phenotypic characteristic wherein the edges or margins of the leaf blade curl or roll downward and under, toward the abaxial (lower) surface of the leaf. This downward rolling creates a partial cylinderlike structure, with the adaxial (upper) surface of the leaf remaining predominantly exposed while the abaxial surface is partially concealed by the curled margins. The degree of revolution may vary from slight curling at the leaf edge to significant rolling that substantially reduces the apparent width of the leaf blade when viewed from above. Revolute leaf morphology may be observed throughout the entire margin of the leaf or may be limited to specific portions and can occur in varying degrees of severity from the base to the apex of the plant.

[0092] In another embodiment of the present invention, the plant, suitably a tomato (S. lycopersicum) plant, exhibits involute leaf morphology. Involute leaf morphology refers to a phenotypic characteristic wherein the edges or margins of the leaf blade curl or roll upward and over, toward the adaxial (upper) surface of the leaf. This upward rolling creates a partial cylinder-like structure, with the abaxial (lower) surface of the leaf remaining predominantly exposed while portions of the adaxial surface are partially concealed by the curled margins. The degree of involution may range from minimal curling at the edges to pronounced rolling that significantly reduces the apparent width of the leaf blade when viewed from below. Involute leaf morphology may be exhibited uniformly across the entire leaf margin or may be restricted to particular regions, and can manifest with varying intensity from the base to the apex of the plant.

[0093] In embodiments of the invention, the plant, suitably a tomato (S. lycopersicum) plant, exhibits both revolute and involute leaf morphology. Examples of such leaf morphologies are evident in plants depicted in Figure 2.

[0094] Alternative morphologies and coloration of leaves may be deemed to the grower or consumer as distinctive and desirable from an aesthetic perspective within the fields of horticulture and agriculture. Additionally, more compact leaf morphology, derived from the revoluted / involuted phenotypes described above, can facilitate manual or automated post-harvest processing of waste non-edible biomass by making clean up and handling much easier.

[0095] According to embodiments of the invention, methods are provided for efficiently engineering and recovering plants, plant cells, or other propagatable plant material - e.g. seeds, cuttings, calli, protoplasts or any other tissue culture of regenerable cells - comprising a non-wild type CYP85A1, SPG5, CCD8, GID1a, and GID1b1 gene expression patterns, particularly effective knock out of two or more of CYP85A1, SPG5, CCD8, GID1a, and GID1b1 gene expression. Typically, the plants, plant cells, or other propagatable plant material are from the Solanaceae family, more suitably varieties and cultivars of Solanum lycopersicum - i.e. tomato varieties, of which several thousands are known to exist; Solanum melongena - i.e. aubergine, or egg plant; Solanum tuberosum - the potato, a key staple food; and Capsicum annuum - i.e. sweet, chilli or bell pepper.

[0096] Tomato varieties and cultivars may be classed according the main shapes of the fruit produced. These shapes include: globe (regular-sized slicer tomatoes); beefsteak (large slicer tomatoes); cherry (mini tomatoes); plum (paste tomatoes) and oxheart (heart-shaped tomatoes). Although typically red in colour, the fruits of certain varieties of tomato may vary from green, pink, yellow, to darker plum and purple, even black. It will be appreciated that the phenotypic traits as described herein may be applied to any variety or cultivar of Solanum lycopersicum.

[0097] In general, well-known cultivated tomato varieties to which the genetic modifications described herein may be made include, but are not limited to: 42 days; 506 Bush; A Grappoli D'lnverno; Abracazebra; Ace; Ailsa Craig; Alicante; Amai; Amana Orange; Amarillo; Amelia; Amish Gold Slicer; Amish Paste; Amsterdam; Ananas Noire; Andiamo; Andrew; Arvento; Rahart's Jumbo Red; Andrina; Anna Aasa; Apero; Applause; Apple Yellow; Apricot Zebra Hybrid; Arbason; Argentina Cherry; Arkansas Traveler; Armenian; Artic Rose; Attention; Aubry's Special Pink; Aunt Gertie's Gold; Aunt Ginny's; Aunt Molly's Ground Cherry; Aunt Ruby's German Cherry; Aunt Ruby's German Green; Austin's Red Pear; Azoychka Azoychka; Baby Bottle; Baby Bottle Red Pear; Baby Cakes; Baby Grape; Bali; Ball's Beefsteak; Banana Legs; Barnes Mountain Yellow; Bartelly; Basinga; Basket Vee; Basrawya; Baxter's Early Bush Cherry; Beall's Gourmet; Beam's Yellow Pear; Bear Creek; Beauty King; Beauty Queen; Beefmaster; Beefsteak; Believe It Or Not; Bella Rosa Bella Rosa; Bellestar; Bellini; Best Boy; Betalux; Better Boy Better Boy; Better Bush; Betty; BHN 785; BHN 1021 BHN 1021 ; BHN 189 BHN 189; BHN 268 BHN 268; BHN 444; BHN 543 BHN 543; BHN 589 BHN 589; BHN 602; BHN 624; BHN 762 BHN 762; BHN 826 BHN 826; BHN 871 ; BHN 901 ; BHN 961 BHN 961 ; BHN 964; BHN YC1 ; Bi-Color Cherry; Big Beef; Big Boy; Big Brandy; Big Bunch; Big League; Big Pink; Big Rainbow; Big Raspberry; Big Red; Big Tiger; Big White; Big White Pink Stripes; Big Yummy; Big Zebra; Bison; Black; Black Cherry; Black Icicle; Black Krim; Black Mauri; Black Opal; Black Pear; Black Pearl; Black Plum; Black Prince; Black Sea Man; Black Strawberry; Black Velvet; Black Vernissage; Black Zebra; Bliss; Blondkopfchen; Bloody Butcher; Blue Beauty; Blue Beech; Blue Ribbon; Blush; Blushing Star; Bobcat; Bolseno; Boondocks; Booty; Box Car Willie; Bradley; Brandymaster Pink; Brandymaster yellow; Brandysweet Plum; Brandywine; Brandywine Black; Brandywine OTV; Brandywine Pink; Brandywine Red; Brave General; Braveheart; Bronze Torch; Brown Berry; Buckbees New Fifty Day; Buffalo Steak; Buffalosun Hybrid; Bulgarian #7; Bulgarian Triumph; Burbank; Burgess Stuffing Tomato; Burpee's Big Boy; Burpee's Burger; Burpee's Summer Choice; Burrell's Special; Bush Beefsteak; Bush Big Boy; Bush Blue Ribbon; Bush Early Girl II; Bush Goliath; Cabernet; Cabo; Cacady's Folly; Caiman; Calypso; Camaro; Camelia; Campbell's 1327; Campbell's 33; Candyland; Canestrino; Cannellino; Capaya Capaya; Captain Lucky; Carbon; Carmelita; Carmello; Caro Rich; Carolina Gold; Casa del Sol; Caspian Pink; Cedro; Celano; Celebration; Celebrity; Celebrity Supreme; Centiflor Red; Cerise Orange; Ceylon; Chadwick Cherry; Chalk's Early Jewel; Champion; Chancha; Chapman; Charger; Chefs Choice Black; Chefs Choice Green; Chefs Choice Orange; Chefs Choice Pink; Chefs Choice Purple; Chefs Choice Red; Chefs Choice Striped; Chello; Cherokee Carbon; Cherokee Chocolate; Cherokee Green; Cherokee Purple; Cherries Jubilee; Cherry Baby; Cherry Blossom; Cherry Bomb; Cherry Brandywine; Cherry Buzz; Cherry Ember; Cherry Pink; Cherry Princess Sweetie Surprise; Cherry Roma; Cherry Sweetie; Chianti Rose; Chile Verde; Chiquita; Chocolate; Chocolate Cherry; Chocolate Pear; Chocolate Sprinkles; Chocolate Stripes; Christmas Grapes; Church; Cipolla's Pride; Classica; Clear Pink Early; Clementine; Clermon; Cloudy Day; Cluster Grande; Colonial; Conestoga; Copia Copia; Corbarino; Cordova; Corona; Cosmonaut Volkov Red; Costoluto Fiorentino; Costoluto Genovese; Council Bluffs; Country Taste; Cour Di Bue; Coustralee; Coyote; Cream Sausage; Creme Brulee; Creole Original; Crimson Cushion Beefsteak; Crimson Sprinter; Crista; Crnkovic Yugoslavian; Crokini; Csiko Botermo; Cupid; Dacquiri Dacquiri; Dads Sunset; Dafel Dafel; Dagma's Perfection; Damsel; Dark Galaxy; David Davidson's; Daytona; Debaro; Debut; Defiant PhR; Delizia; Dester; Dixie Red; Djena Lee's Golden Girl; Dona; Dorothy's Green; Double Rich; Dr. Carolyn; Dr. Wyche's Yellow; Druzba; Dwarf Golden Tipsy; Dwarf Tennessee Suited; Earliana; Earl's Faux; Early Blue Ribbon; Early Boy Bush; Early Cherry; Early Choice; Early Doll; Early First Prize; Early Girl; Early Goliath; Early Harvest; Early Treat; Early Wonder; Egg Yolk; El Dorado; El Fresco Hybrid; Elberta Girl; Elfin; Ella Bella; Emerald; Emmy; Emmylou; Empire; Enchantment; Esterina; Estiva; Eva Purple Ball; Evil Olive; Fabulous; Fantastic; Fantastico; Fantome du Laos; Favorita; Fenda; Ferline; Finishline; Firecracker; Fireworks; First Light; First Prize; Five Star Grape; FLA 47R; FLA 7514; Flaming Burst; Floradade; Floralina; Florida 47; Florida 91 ; Fourth of July; Fox Cherry; Frazier's Gem; Fresh Salsa; Fried Green Tomato; Front Runner; Frosted Green Doctors; Fruity Cherry; Gabrielle; Galina; Garden Gem; Garden Leader Monster; Garden Peach; Garden Treasure; Gardener's Delight; Garnet; Garnet; Genuwine; Georgia Streak; Geranium Kiss; German Giant; German Head; German Johnson Pink; German Lunchbox; German Pink; German Queen; German Red Strawberry; Geronimo; Get Stuffed!; Giallo De Summer; Giant Belgium; Giant Syrian; Giant Tree Giant Tree; Gill's All Purpose; Gin Fiz; Glacier; Glamour; Glitter; Gold Medal; Gold Nuggets; Gold Spark; Golden Delight; Golden Gem; Golden Girl; Golden Jubilee; Golden Mama; Golden Pearl; Golden Ponderosa; Golden Queen USDA Strain; Golden San Marzano; Golden Sunburst; Golden Sunshine; Golden Sweet; Goldene Konigin; Goldie; Golova Negra; Grandaddy; Grandero Plum; Grandeur; Grandma's Little Girl; Grandma's Pick; Grandpap's Rose Wax; Granny Cantrell's; Granny Smith; Great White; Greater Baltimore; Green Bell Pepper; Green Berkeley Tie-Dye; Green Doctors; Green Envy; Green Giant; Green Grape; Green Pear; Green Sausage; Green Tiger; Green Zebra; Green Zebra Cherry; Gremlin; Grinch Dwarf; Grushovka; Gulf State Market; Gum Drop; Gypsy; Halley 3155; Hard Rock; Harlequin; Harless Creek Gold; Hartman's Yellow Gooseberry; Hawaiian Pineapple; Health Kick; Heinz 1350; Heinz 1370; Heinz 1439; Heirloom Green; Heirloom Orange; Helix; Heritage; High Carotene; Hillbilly; Holland; Homestead; Homesweet; Honey Bunch; Honey Bunch Yellow; Honey Delight; Honey Drop; Honey Hybrid; Honeybee; Honeycomb; Hugh's; Huichol; Hungarian Heart; Husky Gold; Husky Pink; Hybrid 46; Hybrid Beef 9904; Hy-Brix; Igleheart Yellow Cherry; I Idi; lllini Star; Illinois Beauty; Indian Stripe; Indigo Cream Berries; Indigo Gold Berries; Indigo Kumquat; Indigo Rose; Indigo Ruby; Invincible; Iron Lady; Isis Candy; Italian Giant Beefsteak; Italian Goliath; Italian Heirloom; Italian Ice; Ivory Pear; Janet's Jewel; Japanese Trifele Black; Jasper; Jaune Flamme; Jazzy; Jelly Bean Red; Jersey Boy; Jersey Devil; Jet Star Jet Star; Jetsonic; Joker; Jolly; Jolly Elf; Jolly Girl; Jubilee; Jujube Cherry; Juliet; Jung's Wayahead; Kalman's Hungarian Pink; Kanner Hoell; Katana; KC 146; Kellogg's Breakfast; Kimberly; Kobe Beefsteak; Kolb; Koralik; La Roma III; Lady Finger; Ladybug; Lake; Large Barred Boar; Legend; Lemon Boy; Lemon Cherry; Lemon Drop; Lemon Tree; Lime Green Salad; Limmony; Lisa King; Lizziebelle; Lollipop; Longkeeper; Lost Marbles; Lovertino; Lucky Cross; Lucky Tiger; Lunch Box; Lyn's Mahogany Garnet; Madame Marmande; Maglia Rosa; Magnum; Maiden's Gold; Malakhitovaya Shkatulka; Malinowski; Mama Leone; Mamie Brown's Pink; Mandarin Cross; Manitoba; Manyel; Margherita; Marglobe Improved; Margo; Mariana; Marion; Marizol Magic; Marizol Purple; Marmande; Marmara; Martian Giant; Martin; Martino's Roma; Marvel Stripe; Marzinera; Matchless; Mater Sandwich; Matina; Matthew; Maya; Medford; Medium Rare; Mega Tom Giant; Megabite; Mexico; Micado Violettor; Midnight Pear; Mighty Sweet; Mikado; Mingle Mix; Mini Charm; Minibel; Mint Julep; Mirabelle Blanche; Miroma; Missouri Pink Love Apple; Momotaro; Moneymaker; Montesino; Moonbeam; Moonglow; Moonshadow; Moravsky Div; Moreton; Morning Light; Mortgage Lifter; Mortgage Lifter, bi-color strain; Mosaico; Moskvich; Mountain Delight; Mountain Fresh; Mountain Fresh Plus; Mountain Gem; Mountain Glory; Mountain Gold; Mountain Magic; Mountain Majesty; Mountain Man; Mountain Merit; Mountain Pride; Mountain Spring; Mountain Vineyard; Mr. Stripey; Mr. Ugly; Mrs. Maxwell's Big Italian Hr; Napa Grape; Napa Rose Blush; Napoli; Nature Bites; Nature's Riddle; Nebraska Wedding; Nectar; Nectarine; Neves Azorean Red; New Big Dwarf; New Girl; New Hampshire Red Pickling; New Yorker; Northern Lights; Nova; Nugget; Nyagous; Oaxacan Jewel; Oh Happy Day; Old Brooks; Old Fashioned Goliath; Old German; Old Ivory Egg; Old Yellow Candystripe; Olivade; Orange Banana; Orange Blossom; Orange Brandywine; Orange Fizz; Orange Icicle; Orange Jazz; Orange King; Orange Minsk; Orange Oxheart; Orange Panuche; Orange Peach; Orange Queen; Orange Roma; Orange Russian 117; Orange Santa; Orange Slice; Orange Strawberry; Orange Sunshine; Orange Wellington; Orange Zinger; Oregon Spring; Oroshan; Out Damn Spot; Oxheart Pink; Pamella; Pantano Romanesco; Park's Beefy Boy; Park's Early Challenge; Park's Season Starter; Patsy; Patty's Yellow Striped Beefsteak; Paul Robeson; Peacevine; Peach Blow Sutton; Pearly Pink; Pellicore; Peppermint; Perfect Flame; Perfectly Pink; Peron; Persimmon; Phoenix; Phuket Egg; Piccolo Carina; Picus; Pilcer Vesy; Pineapple; Pineapple Pig; Pink Accordion; Pink Beauty; Pink Berkeley Tie-Dye; Pink Boar; Pink Bumble Bee; Pink Champagne; Pink Cupcake; Pink Girl; Pink Peach; Pink Ping Pong; Pink Pounder; Pink Stuffer; Pink Tiger; Pink Wonder; Pink-a- Licious; Piriform; Pixie Stripe; Placero; Plum Crimson; Plum Lemon; Plum Regal; Polar Beauty; Polar Star; Polbig; Polish Dwarf; Poma Amoris Minora Lutea; Pony Express; Pork Chop; Porter; Porterhouse; Poseidon 43; Power Pops; Prairie Fire; Premio; Prime Beef Goliath; Primo Red; Princess; Principe Borghesi; Pritchard; Prize of the Trials; Pruden's Purple; Purple Boy; Purple Brandy; Purple Bumble Bee; Purple Russian; Purple Smudge; Quali T 23; Quarter Century; Quedlinburger Fruhe Liebe; Queen Aliquippa; Queen of the Night; Queens; Querida; Quick Pick; Quimbaya; Rally; Ramapo; Rambler; Rambling Gold Stripe; Rambling Red Stripe; Ranger; Rapunzel; Raspberry Lyanna; Ravello; Razzle Dazzle; Rebekah Allen; Red Anjou; Red Brandywine; Red Candy; Red Cherry Large Fruited; Red Cup; Red Defender; Red Eclipse; Red Fig; Red Grape; Red House Free Standing; Red Lightning; Red Morning; Red Mountain; Red Pear; Red Pearl; Red Plum; Red Pride; Red Rave; Red Robin; Red Rocket; Red Rose; Red Star; Red Zebra; Redfield Beauty; Reisetomate; Ridge Runner; Riesentraube; Rio Grande; Riviera; Roadster; Rocket; Rojita; Roma; Roman Candle; Rosalita; Rose; Rose De Berne; Rosella; Rosso Sicilian; Rostova; Rowdy Red; Royal Hillbilly; Royal Mountie; Royesta; RuBee Dawn; RuBee Prize; Rugged Boy; Russian Persimon; Russian Rose; Rutgers; Rutgers 250; Rutgers 39; Rutgers Improved PS; Rutgers Select; S 151496; Sakura Honey; Salt Spring Sunrise; Sanibel; Santa Clara Canner; San Marzano; Santiam; Sapho; Sara's Galapagos; Sasha's Pride; Saybrook; Scarlet Red; Scarlet Sunrise; Schimmeig Striped Hollow; Sean's Yellow; Seattle's Best of All; Seminis 0172-1432; Seminis 1236; Seminis Grape 9137; Serrat; Shady Lady; Shasta; Sheboygan; Shelby; Shilling Giant; Sicilian Saucer; Siletz; Silvery Fir Tree; Sioux; Skorospelka; Skyreacher; Skyway; Slava; Sleeping Lady; Small Fry; Smarty; Snacker's Delight; Snow White; Snowberry; Solar Fire; Solar Flare; Solar Power; Solid Gold; Sophie's Choice; Sophya; Southern Night; Sparky XSL; Spear's Tennesse Green; Speckled Roman; Spike; Spitfire; Sprite; St. Nick; St. Pierre; Steak House; Steak Sandwich; Stellar; Stone; Striped Cavern; Striped German; Striped Roman; Striped Stuffer; Subarctic; Sugar Lump; Sugar Plum; Sugar Rush; Sugar Snack; Sugary; Summer Girl; Summer Pick; Summer Pink; Summer Sunrise; Sun Cherry; Sun Gold; Sun King; Sun Orange; Sunbrite; Sunchocola; Sungold Select II; Sungreen 4029; Sungreen Garden; Sunkist; Sunleaper; Sunlemon; Sunny Blue Ribbon; Sunny Boy; Sunny Goliath; Sunpeach; Sunray; Sunrise; Sunrise Bumble Bee; Sunrise Sauce; Sunset Falls; Sunshine Heirloom; Sunstart; SunSugar; Super Boy 785; Super Bush; Super Fantastic; Super Marmande; Super Snow White; Super Sweet 100; Supernova; SuperSauce; Supersonic; Supersteak; Supertasty; Supremo; SVR 1400; Sweet 100; Sweet Aperitif; Sweet Aroma; Sweet Baby Girl; Sweet Canary; Sweet Carnernos Pink; Sweet Chelsea; Sweet Cheri; Sweet Cluster; Sweet Elite; Sweet Gold; Sweet Golden Roma; Sweet Hearts; Sweet Million; Sweet Olive; Sweet Orange; Sweet Quartz; Sweet Seedless; Sweet Snax; Sweet Tangerine; Sweet Treats; Sweet Zen; Sweethearts; Sweetie; Talladega; Tami G; Tamina; Tangella; Tangerine Mama; Tappy's Hertitage; Tasmanian Blushing; Tasmanian Chocolate; Tasti-Lee; Tasty Evergreen; Tasty Treat; Taxi; Ten Fingers of Naples; Tennessee Britches; Thai Pink Egg; Think Pink; Thunderbird; Tiesto; Tidwell German; Tiffen Mennonite; Tiger Like; Tiger Tom; Tigerella; Tinkerbell; Tip-Top; Tocan; Tolstoi; Tomatoberry Garden; Tom Thumb; Tommy Toe; Tonopah; Top Gun; Topaz; Torbay; Toronjina; Tough Boy; Tribeca; Tribute; Trophy; Tropic; Trucker's Favorite; Tsungshigo Chinese; Two Tasty Hybrid; Tye-Dye; Tygress; Ukrainian Purple; Ultimate Opener; Ultra Pink; Ultra Sweet; Uluru Ochre; Umamin; Umberto; Valencia; Valley Girl; Valleycat; Varsity; Velvet Red; Vintage Wine; Violaceum Krypni-Rozo; Virginia Sweets; Viva Italia; Vivacious; Volante; Wapsipinicon Peach; Washington Cherry; Watermelon Beefsteak; Weissbehaarte; Wes; Wherokowhai; White Beauty; White Cherry; White Currant; White Potato Leaf; White Queen; White Tomesol; White Wax; White Wonder; Whittemore; Wild Cherry; Wild Fred; Willamette; Wins All; Wonder Light; Woodie Orange; Yaqui; Yellow Belgium; Yellow Bell; Yellow Brandywine; Yellow Cherry; Yellow Fire; Yellow Magic; Yellow Mini; Yellow Peach; Yellow Pear; Yellow Perfection; Yellow Stuffer; Yellow Vernissage; Yukon Quest; Zapotec Oaxacan Ribbed; Zapotec Pink Ribbed; Zebra Cherry; and / or Zenzei.

[0098] In further embodiments, the present invention provides for uses of plants, plant parts, cells or seed of as described herein in agriculture and / or in the production of a human and / or animal food products. In additional embodiments, the present invention provides for uses of plants, plant parts, cells or seed of as described herein in a plant breeding method and / or in the production of hybrid seed or other propagatable material.

[0099] In embodiments of the invention, RNP complexes comprising any of the gRNAs described herein may be introduced into plant cells or protoplasts via techniques known to the person of skill in the art. For instance, RNP complexes may be formed in vitro and mixed directly with recipient protoplasts. Alternatively, plant cells or protoplasts may be transformed with one or more plasmid or viral vectors that express the Cas endonuclease and the gRNA in the cell.

[0100] The invention is further illustrated by the following non-limiting examples.

[0101] EXAMPLES

[0102] Tomato and other large Solanaceae e.g. Aubergine, Potato, Pepper and others, are not easy to grow in enclosed physical environments such as indoor farms, because their general size does not allow it. While these crops can be grown in other more conventional forms of agriculture (field, greenhouse), these pose their own challenges, such as weather and diseases impacting yield, and geopolitical situations impacting transport logistics price and availability of supply. In addition, the move towards reducing carbon miles in food production favours local rearing of crops which may not be practical for some Solanaceae species, like tomato, that generally thrive in warmer climates. Hence, indoor intensive rearing where climate conditions are more controllable may suit year-round growing in more temperate latitudes.

[0103] Hence, modifying plant architecture so that tomato and other Solanaceae crops are enabled to be produced in indoor farms, can provide a number of advantages: mainly food security in form of removal of weather, disease and geopolitical risks associated with food production, stabilising supply and prices, and benefit to the average consumer as well as society at large. Selection of genes of interest:

[0104] Academic literature was searched to identify candidate genes that, when knocked out in combination, could change the plant architecture to more suitable to small, low-maintenance, growing environments. After screening multiple potential candidates, five genes were identified, CYP85A1, SPG5, CCD8, GID1a, and GID1b1, that surprisingly have drastic and causal impact on the phenotype of tomato plants, when knocked-out in combination using CRISPR gene editing approaches. More surprisingly the effects were evident when various combinations of these genes were modified and even when only one allele was modified - i.e. the heterozygous mutants exhibited the desired phenotypic trait.

[0105] Gene editing:

[0106] To test which gene knockout combinations would create the desired architectural changes, target genes were selected for CRISPR knock out experiment, and transformation of the Ailsa Craig tomato lines with the desired CRISPR targets were carried out.

[0107] Two sgRNAs for each target gene were designed and tested against tomato genome SL2.5 to ensure no fewer than two mismatches against potential off targets.

[0108] Gene editing was performed using Agrobacterium-mediated gene transfer (Sparkes et al., 2006). In this process, the Agrobacterium introduces Transfer-DNA (T-DNA) which contains the CRISPR cassette, which expresses both the CRISPR nuclease and the guide RNAs. The cassette is stably integrated into the host genome alongside an antibiotic selection marker.

[0109] The tomato plants of the line Ailsa Craig were inoculated with Agrobacterium containing the CRISPR Cassette. Figure 3 (a) to (e) show linear gene maps of CYP85A1, SPG5, CCD8, GID1a, and GID1b1. Shaded pointed boxes indicate genetic elements, either exons or introns within the genes. Guide RNAs including spacer sequences used in generation of tomato variants are labelled, SG3 and SG4 for CYP85A1, SG15 and SG16 for SP5G, SG1 and SG2 for CCD8, SG17 and SG18 for GID1a, and SG19 and SG20 for GID1 b1. The spacer sequences (SEQ ID Nos: 1 , 2, 7, 8, 13-18) are as described previously in Tables 1 to 5 above. Alternative guide RNAs are provided for generation of tomato variants are labelled as SG3a,b and SG4a,b in Table 1 for CYP85A1, and SG15a,b and SG16a,b in Table 2 for SP5G.

[0110] Inoculated plant material was selected on medium containing kanamycin to select for successful integration of the T-DNA constructs in shoots. Shoots were then genotyped for editing of the target genes. Edited lines were grown to maturity, self-pollinated, seed selected and grown to maturity. The individual plants were genotyped to confirm presence of edited genes (indels within target genes) and presence of the Agrobacterium T-DNA carrying the CRISPR construct, antibiotic selection marker as well as Left and Right Borders. Figure 4 (a) to (e) show sequencing data of tiny tomato plants for genes CYP85A1, SP5G, CCD8, GID1a, and GID1b (lower line) showing the CRISPR induced mutations compared to the wild type sequences (upper line). A range of inactivating mutations were generated in the target alleles and these are shown in Table 6.

[0111] Table 6

[0112] Following subsequent rounds of self-pollination, seed selection based on genotyping and growing up of the individual plants, the T-DNA inserted by the transformation step was bred out, but the inactivating indel mutations maintained in the selected genes of interest. This way it was possible to derive non-GMO genetically edited lines from T-DNA-containing GMO plants.

[0113] Phenotypinq:

[0114] Candidate knock out lines were grown and genotyped for candidate gene knock outs and T- DNA absence at 7 days old. Plants with confirmed edits were grown up to fruiting and phenotyped for height, architecture, fruit size, and fruit BRIX at appropriate ages.

[0115] Figure 1 shows photographs of mutants of SP5G and / or CYP85A1 and wildtype control at 7 weeks old. The SP5G and CYP85A1 homozygous mutant gives rise to a structurally tiny (extreme dwarfism) phenotypic trait, that provides a basis for a group of variants referred to as Variant 1. Figure 2 shows close-up photographs of two variant lines at 12 weeks old having the SP5G and CYP85A1 homozygous mutation . The individuals display reduced lateral branching, enlarged stems, and dark, thickly-ridged leaves. Furthermore, iconically, all the trusses develop on one side of the plant. Fruits are slightly reduced in size, but yield of fruit per unit of vegetative growth (i.e. non-edible biomass) is disproportionally greater than the wildtype. Vertical height data was obtained for the homozygous Variant lines compared to wildtype and individual mutants. The height data was collected at week 7 and week 12 (mutants of CYP85A1 were not maintained to week 12 due to normal height). The wildtype progenitor of the mutants is included as a control. The CYP85A1 mutant showed similar height to wild-type, whereas SP5G mutant showed minor dwarfing. However, when these mutations were combined, the combinatorial effect is extreme dwarfism.

[0116] Table 7 - Vertical height comparisons of Variant mutants versus wild-type at weeks seven (W7) and twelve (W12)

[0117] An alternative genotype of Variant 1 , comprises either heterozygous (i.e. one recessive nonfunctional allele and one functional allele) in genes CYP85A1, SP5G, GID1a and GID1b1 together; or in genes CYP85, SP5G and CCD8 together. Figure 2 (c) shows photographs of Variant 1 Ailsa Craig plants at 14 weeks with characteristic short stocky stature, much reduced lateral branching and wrinkled foliage (i.e. involuted / revoluted leaves). However, it is readily apparent that the large amount of fruit is produced relative to the overall biomass of the plant. Even more surprising is the observation that harvested fruit from Variant 1 is only marginally smaller in size compared to wild type (see Figure 2(d)) and are comparable in terms of shape and colour. This indicates that the mutations made in these variants are preferentially affecting non-edible biomass rather than the size or quality of fruit.

[0118] Alignments:

[0119] To investigate whether the genes CYP85A1 and SP5G conferring drastic height reduction are conserved among tomato relatives, and to quantify this conservation of candidate genes, similar sequences were compared across different species, starting with closely related species to tomato. The comparisons were performed with multiple sequence alignment and sequences of candidate genes from Solanaceae species were obtained from publicly available databases (https: / / solgenomics.net / and https: / / plants.ensembl.org / index.html). These sequences were then aligned using MAFFT (ebi.ac.uk) to analyse sequence similarity see Tables 8 and 9 below. Table 8 - CYP85A1 Solanaceae sequence alignments Table 9 - SP5G Solanaceae sequence alignments

[0120] Table 10 - CCD8 Solanaceae sequence alignments

[0121]

[0122] Where no alignment score is indicated the genes are simply identified as being homologous Table 11 - GID1a Solanaceae sequence alignments

[0123] Where no alignment score is indicated the genes are simply identified as being homologous Table 12 - GID1b1 Solanaceae sequence alignments

[0124] Where no alignment score is indicated the genes are simply identified as being homologous

[0125] High levels of sequence conservation was displayed in CYP85A1, as evidenced by the high nucleotide identity between Solanum lycopersicum CYP85A1 and other Solanaceae species (99.43% to 80.1%). Furthermore, the Solanum lycopersicum homolog CYP85A3 has nucleotide similarity of 78.22%, showing conservation. SP5G has multiple homologs and these are conserved and spread across the Solanaceae and further. For example, conservation between S. lycopersicum CYP85A1 and coffee homologs (Coffee canephora, family Rubiaceae) is very high at ~70%. The combination of non-functional alleles for all of these genes that have been demonstrated to confer extreme dwarf phenotype in tomato are, therefore, credible as targets for production of extreme dwarf phenotypes in other related species.

[0126] Genetic resources used in the inception of the present invention were sourced within the United Kingdom. Traditional or indigenous knowledge, whether or not associated with the genetic resources described herein, has not been used in the invention.

[0127] Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. The choice of nucleic acid starting material, the clone of interest, or type of libraries used are believed to be a routine matter for the person of skill in the art with knowledge of the presently described embodiments. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims

CLAIMS:1 . A plant of the Solanaceae family comprising a functionally inactivating mutation present within a cytochrome p450 class 85A1 (CYP85AT) gene and a Self Pruning 5G (SP5G) gene.

2. The plant of claim 1 , wherein the plant further comprises at least one functionally inactivating mutation within an allele of a gene selected from: carotenoid cleavage dioxygenase 8 (CCD8)Gibberellin-insensitive dwarf 1a (GID 1a) andGibberellin-insensitive dwarf 1b1 (GID1b1).

3. The plant of claim 1 or claim 2, wherein the plant is from the species Solanum lycopersicum (tomato).

4. The plant of any one of claims 1 to 3, wherein the functionally inactivating mutation comprises a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide or any combination thereof.

5. The plant of claim 4, wherein the deletion is from about 1 to about 2000 nucleotides in length.

6. The plant of any one of claims 1 to 5, wherein the plant exhibits a determinate growth phenotype.

7. The plant of any one of claims 1 to 6, wherein the plant has a phenotype of reduced height compared to a wild-type plant of equivalent age.

8. The plant of claim 7, wherein the height of the plant is reduced by at least 60%, typically at least 70%, optionally by at least 75%, compared to a wild-type plant of equivalent age.

9. The plant of claim 8, wherein the plant has phenotype such that the height does not exceed around 50 cm, suitably around 40 cm, optionally around 30 cm when the plant has reached maturity.

10. The plant of any one of claims 1 to 9, wherein the plant has a phenotype in which it exhibits reduced branching of the main stem compared to a wild-type plant of equivalent age.

11. The plant of claim 10, wherein the reduced branching of the main stem compared to a wildtype plant of equivalent age is defined by having not more than two main stems.

12. The plant of claim 10, wherein the reduced branching of the main stem compared to a wildtype plant of equivalent age is defined by having a single main stem.

13. The plant of any one of claims 1 to 12, wherein the plant has a phenotype confers a full or partial involuting and / or revoluting morphology to the leaves compared to a wild-type plant.

14. The plant of any one of claims 1 to 13, wherein the plant maintains a level of fruit yield to vegetative growth that is comparable to a wild-type plant of equivalent age.

15. The plant of any one of claims 1 to 14, wherein the plant maintains a level of fruit yield to nonedible vegetative biomass that is greater than that of a wild-type plant of equivalent age.

16. A Solanum lycopersicum plant, the plant comprising functionally inactive alleles of both a CYP85A1 locus and a SP5G locus, wherein the inactivation of the said alleles is associated with a determinate growth phenotype as well as exhibiting a phenotypic trait of structural dwarfism characterised by the vertical height of the plant being reduced by at least 70% compared to a wildtype plant of equivalent age.

17. The Solanum lycopersicum plant of claim 16, wherein the plant has a phenotype such that the height does not exceed around 50 cm, suitably around 40 cm, optionally around 30 cm when the plant has reached maturity.

18. The Solanum lycopersicum plant of any one of claims 16 or 17, wherein the plant has a phenotype in which it exhibits reduced branching of the main stem compared to a wild-type plant of equivalent age.

19. The Solanum lycopersicum plant of claim 18, wherein the reduced branching of the main stem compared to a wild-type plant of equivalent age is defined by having not more than two main stems.

20. The Solanum lycopersicum plant of claim 18, wherein the reduced branching of the main stem compared to a wild-type plant of equivalent age is defined by having a single main stem.21 . The Solanum lycopersicum plant of any one of claims 16 to 20, wherein the plant has a phenotype confers a full or partial involuting and / or revoluting morphology to the leaves compared to a wild-type plant.

22. The Solanum lycopersicum plant of any one of claims 16 to 21 , wherein the plant maintains a level of fruit yield to vegetative growth that is comparable to a wild-type plant of equivalent age.

23. The Solanum lycopersicum plant of any one of claims 16 to 22, wherein the plant further comprises at least one functionally inactivating mutation within an allele of a gene selected from: carotenoid cleavage dioxygenase 8 (CCD8);Gibberellin-insensitive dwarf 1a (GID 1a) andGibberellin-insensitive dwarf 1b1 (GID1b1).

24. A plant cell of the Solanaceae family comprising at least one functionally inactivating mutation present within both the CYP85A1 gene and the SP5G gene.

25. The plant cell of claim 24, wherein the plant cell is from the species Solanum lycopersicum (tomato).

26. The plant cell of claims 24 or claim 25, wherein the functionally inactivating mutation comprises a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide or any combination thereof.

27. The plant cell of claim 26, wherein the deletion is from about 1 to about 1000 nucleotides in length.

28. A tissue culture of regenerable cells comprising plant cells of any one of claims 24 to 27.

29. A tomato (Solatium lycopersicum) fruit comprising a plant cell of any one of claims 24 to 27.

30. A plant part obtained or derived from the plant of any one of claims 1 to 15.31 . A Solatium lycopersicum plant part obtained or derived from the plant of any one of claims 16 to 23.

32. The plant part of either of claims 30 or 31 wherein the plant part is selected from the group consisting of: a leaf; pollen; an ovule; a fruit; a rootstock; a scion; a flower; and a seed.

33. A method for modifying a plant cell from the Solanaceae family in order to introduce a phenotype of extreme dwarfism within a plant grown from the cell, the method comprising mutating one or more copies of an CYP85A1 gene, or a homologue thereof, and a SP5G gene, or a homologue thereof, within the genome of the plant cell, wherein the mutation comprises a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide or any combination thereof so as to induce functional inactivation of the said one or more genes.

34. The method of claim 33, wherein all alleles of both the CYP85A1 gene and the SP5G gene are functionally inactivated.

35. The method of any one of claims 33 or 34, wherein the method introduces one or more additional phenotypes selected from the group consisting of: determinate growth; reduced branching of the main stem; and / or full or partial involuting and / or revoluting morphology to the leaves.

36. The method of any one of claims 33 to 35, wherein the method comprises mutating at least one or more copies of: a CCD8 gene, a GID1a gene, and / or a GID1b gene.

37. The method of claim 36, wherein the mutation results in functional inactivation.

38. The method of any one of claims 33 to 37, wherein mutating of one or more copies is via a gene editing technique.

39. The method of claim 38, wherein the gene editing technique comprises use of a CRISPR / Cas gene editing complex.

40. The method of claim 39, wherein the CRISPR / Cas gene editing complex comprises a Cas endonuclease selected from: Cas9 (including derivatives and homologues thereof) and Cas12a / Cpf1 (including derivatives and homologues thereof).41 . The method of any of claims 39 to 40, wherein the gene editing technique is selected from: gene editing; base editing; prime editing and combinations thereof.

42. The method of any of claims 33 to 37, wherein mutating of one or more copies is via: trans- genetic technology; cis-genetic technology; mutagenesis; and / or spontaneous mutation.

43. The method of any one of claims 33 to 42, wherein the plant cell is from the species Solanum lycopersicum (tomato).

44. A method of generating a plant from the Solanaceae family, wherein the plant exhibits a phenotype of extreme dwarfism, the method comprising culturing a plant cell of any one of claims 33 to 42 under conditions appropriate to facilitate proliferation of plant cells so as to form the plant.

45. The method of claim 44, wherein the plant is from the species Solanum lycopersicum (tomato).

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