Methods and compositions for producing inbred lines and f1 hybrids from self-incompatible species

Abstract

The invention provides constructs comprising: a) an expression cassette for disrupting self-incompatibility (SI), b) an expression cassette for disrupting the development of viable pollen, and c) an expression cassette for identifying seed carrying the genetic construct. The invention provides plants and plant parts comprising such constructs. The invention further provides methods for producing non-transgenic, self-incompatible, inbred seed and plants, plants, and inbred seed and plants produced by such methods. The invention also provides methods for producing an F1 hybrid seed by crossing such non-transgenic, self-incompatible, inbred plants with other plants which may also be non-transgenic, self-incompatible, inbred plants of the invention. The invention further provides F1 hybrid seed produced by the methods, and F1 hybrid plants grown from the seed.

Description

[0001] METHODS AND COMPOSITIONS FOR PRODUCING INBRED LINES AND Fl HYBRIDS FROM SELF-INCOMPATIBLE SPECIES

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] The contents of Australian provisional patent application number 2023902215 filed 11 July 2023, and Australian provisional patent application number 2024901883 filed 20 Jun 2024, are incorporated herein by reference in their entirety.

[0004] FIELD OF INVENTION

[0005] The invention is in the field of plant breeding.

[0006] BACKGROUND TO THE INVENTION

[0007] The production of Fl hybrids is an effective breeding tool, enabling the fixation of desirable traits, purging of deleterious traits, and exploitation of hybrid vigor, or heterosis. Fl hybrid breeding involves the production of inbred lines through several rounds of inbreeding to generate near-homozygous lines. Inbred lines are subsequently crossed to produce Fl hybrid seed which is sold to growers. The Fl hybrid seed contains many of the traits of the inbred parents but exhibits heterosis, resulting in a more vigorous growth habit and higher productivity than either parent. Such a breeding system has been widely exploited, particularly in maize and more recently in rice, as well as many vegetable crops.

[0008] There are two key facets of this breeding system, firstly, the ability to inbreed through self-fertilization, and secondly the ability to enforce outcrossing to ensure that only Fl hybrid seeds are produced in the final generation.

[0009] Inbreeding can be hampered by self-incompatibility (SI) systems, such as those found in Solanaceae, Poaceae, Brassicaceae and Papaveraceae. Similarly, the ability to enforce outcrossing is difficult in species with small, bisexual flowers, requiring the use of male-sterility in one parental line. In the case of maize this can be achieved by “de-tasseling” one parent and utilizing the other parent as a pollinator; however, more modem approaches use genetic tools to achieve the same result without the need for detasseling, which has an impact on plant productivity.

[0010] Several types of male -sterility exist, including cytoplasmic (CMS; through mitochondrial or plastid genomes), genetic (GMS: through the nuclear genome), environmental (EGMS; through environmental conditions), or chemical (by application of a sterility-inducing chemical).

[0011] For CMS and GMS a second “restorer” mutation is required during the inbreeding phase. In some cases, this restorer can take the form of a transgene, for example the “Seed Production Technology” system developed for maize, whereby a functional copy of the mutated (GMS) gene is linked to pollen lethality and seed fluorescence.

[0012] For EGMS, appropriate climatic conditions are required to inbreed a crop in one location, and outcross in a different location with different climatic conditions that induce sterility.

[0013] For most of these systems, a mutation must be identified that results in the desired phenotype, and a suitable restorer must also be found, making it difficult to engineer such a solution into a new species. Similarly, many of these systems are “leaky” allowing some portion of self-fertilized seed in the final generation.

[0014] Finally, these systems also rely on “pollinator rows” which are not able to be harvested as they contain no Fl hybrid seed, thus leading to inefficiency and / or wastage.

[0015] It is therefore an object of the invention to provide improved methods and compositions for producing inbred lines and Fl hybrids that overcome one or more of the deficiencies of the prior art and / or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION

[0016] The invention provides compositions and methods for producing inbred lines and Fl hybrid seed from self-incompatible plants. The applicants have named this system “Combined Inbreeding And Outcrossing” (CIAO). Utilizing the plants’ own SI system to enforce outcrossing enables Fl hybrid seed to be collected from both parents of the cross.

[0017] In addition, the system is fully transportable to other species as it relies on a transgene (the CIAO construct), during the inbreeding phase, as opposed to a chance mutation as in CMS, GMS or EGMS. The CIAO construct (Figure 1) comprises three expression cassettes: a cassette for disrupting the self-incompatible phenotype while it is present, that is maintained in a hemizygous state by the presence of a pollen disrupting cassette. A selectable marker cassette, encoding for example a fluorescent marker, enables the identification and selection of transgenic (self-fertile) and non-transgenic (selfincompatible) seeds at every generation.

[0018] Figure 2 shows an overview of a typical strategy for production of inbred seeds and plants from self-incompatible (SI) species using the CIAO system. Lines hemizygous for the CIAO construct produce only half of the normal amount of viable pollen, because of the pollen disrupting cassette. Such plants are self-fertilised to produce inbred seed. Half of the inbred seed produced is transgenic for the CIAO construct, the other half is non-transgenic (CIAO- / -). Hemizygous transgenic (CIAO+ / -) seed is selected based on fluorescence, and (CIAO+ / -) plants grown from the seed can be used for multiple (typically >8) further rounds of inbreeding, to ultimately generate near homozygous inbred lines. Hemizygosity of the CIAO construct is maintained by a pollen disrupting cassette, allowing transmission of the transgene through only the female gamete.

[0019] Figure 3 shows an overview of a typical strategy for production of Fl hybrids from selfincompatible (SI) species using the CIAO system, and the non-transgenic, selfincompatible inbred plants produced. To create an Fl hybrid, non-fluorescent (CIAO- / -) seed is selected from two inbred populations. The populations are mixed, and outcrossing is enforced through the (now active) SI machinery. As the lines are homozygous, all siblings within each population carry the same SI haplotypes, preventing intra-population fertilization. However, cross pollination between inbred populations is permitted as each population carries a different SI haplotype. Seed from both parents of the cross can be collected and will be genetically identical Fl hybrids.

[0020] Fl hybrids can of course also be produced crossing just one non-transgenic, selfincompatible inbred population produced using the CIAO system, with another population.

[0021] The system, its compositions and methods of use, are also as defined in the following aspects and embodiments of the invention.

[0022] Constructs

[0023] In one aspect the invention provides a genetic construct comprising: a) an expression cassette for disrupting self-incompatibility (SI), b) an expression cassette for disrupting the development of viable pollen, and c) an expression cassette for identifying seed carrying the genetic construct.

[0024] Expression cassette for disrupting self-incompatibility (SI)

[0025] In one embodiment the cassette in a) is designed to reduce or eliminate expression of at least one allele of an SI gene in a self-incompatible plant.

[0026] In a further embodiment the cassette in a) is designed to reduce or eliminate expression of both alleles of an SI gene present in a self-incompatible plant.

[0027] In a further embodiment the cassette in a) is designed to reduce or eliminate expression of multiple alleles of an SI gene present in a self-incompatible plant. In a further embodiment the cassette in a) is designed to reduce or eliminate expression of at least one positive modulator of SI in a self-incompatible plant.

[0028] In a further embodiment the cassette in a) is designed to increase expression of at least one negative modulator of SI in a self-incompatible plant.

[0029] In one embodiment the plant has a gametophytic SI system.

[0030] In a further embodiment the plant has a sporophytic SI system.

[0031] In a preferred embodiment the cassette in a) is designed to reduce or eliminate expression of at least one allele of an SI gene in a self-incompatible plant.

[0032] In one embodiment the SI gene encodes the female self-incompatibility determinant.

[0033] In one embodiment the plant has gametophytic SI system, and the SI gene to be disrupted encodes the female self-incompatibility determinant.

[0034] In one embodiment the plant has sporophytic SI system, and the SI gene to be disrupted encodes the female self-incompatibility determinant.

[0035] In a further embodiment the plant has sporophytic SI system, and the SI gene to be disrupted encodes the male self-incompatibility determinant.

[0036] Exemplary SI genes and plants are described further herein.

[0037] Expression cassette for disrupting the development of viable pollen

[0038] In one embodiment the cassette in b) is designed to express a component to disrupt the development of viable pollen. In one embodiment the cassette in b) is designed to disrupt the development of substantially half of the pollen of the plant.

[0039] In one embodiment the cassette in b) is designed to express the component to disrupt the development of viable pollen.

[0040] In a preferred embodiment the component is expressed after meiosis.

[0041] In this way, the development of substantially half of the pollen is disrupted, while the other substantial half is fully viable.

[0042] Expression cassette for identifying seed carrying the genetic construct.

[0043] In one embodiment the cassette in c) is designed to express the selectable marker in the seed of a self-incompatible plant.

[0044] In a further embodiment the cassette in c) is designed to express the selectable marker in the endosperm of a self-incompatible plant.

[0045] In a further embodiment the cassette in c) is designed to express the selectable marker in the endosperm of a self-incompatible monocotyledonous plant.

[0046] In a further embodiment the cassette in c) is designed to express the selectable marker in the endosperm of a self-incompatible cereal plant.

[0047] In a further embodiment the cassette in c) is designed to express the selectable marker in the embryo of a self-incompatible plant.

[0048] In a further embodiment the cassette in c) is designed to express the selectable marker in the embryo of a self-incompatible monocotyledonous plant. Preferably the selectable marker is expressed in the fertilization products (embryo and ensdosperm) of the self-incompatible plant.

[0049] Preferably the selectable marker is not expressed in the maternal tissues of the selfincompatible plant.

[0050] In one embodiment the maternal tissue is the seed coat.

[0051] In one embodiment the selectable marker is a fluorescent marker.

[0052] In a preferred embodiment this allows seed carrying the genetic construct to be non- invasively identified.

[0053] In a further embodiment the selectable marker does not affect the viability of the seed.

[0054] Plants, plant parts, plant cells

[0055] In a further aspect the invention provides a plant, plant part, or plant cell comprising a genetic construct of the invention.

[0056] In one embodiment the plant, plant part, or plant cell is from a self-incompatible species.

[0057] In one embodiment although the plant is from a self-incompatible species, it is rendered self-fertile due to expression of the cassette for disrupting self-incompatibility (SI).

[0058] In one embodiment the plant is hemizygous for the construct.

[0059] In one embodiment the construct is maintained in a hemizygous state by the presence of the cassette for disrupting the development of viable pollen. Methods

[0060] Method for producing non-transgenic, self-incompatible, inbred seed

[0061] In a further aspect the invention provides a method for producing a non-transgenic, selfincompatible, inbred seed, the method comprising: a) self-fertilising a plant of the invention, that is hemizygous for a genetic construct of the invention, to produce inbred seed, wherein a proportion of the inbred seed produced is transgenic for the construct and a proportion of the inbred seed is non-transgenic for the construct, and b) identifying and the separating transgenic seed, based on expression of the selectable marker in the transgenic seed, to retain the non-transgenic, selfincompatible, inbred seed.

[0062] In one embodiment a transgenic seed from step b) is grown into a plant, and at least one further cycle of inbreeding performed using said plant.

[0063] Thus, in one embodiment and the steps a) and b) are repeated to produce further inbred non-transgenic, self-incompatible, inbred seed.

[0064] Preferably the method comprises at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10 cycles of inbreeding.

[0065] Preferably each further cycle of inbreeding is initiated with a plant, transgenic and hemizygous for the construct, wherein the plant is produced by the previous cycle of inbreeding.

[0066] Preferably the non-transgenic, self-incompatible, inbred seed, is at least 55%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% homozygous.

[0067] In some embodiments fluorescent transgenic seed is identified that appears approximately twice as bright as the typical hemizygous (CIAO+ / -) seed. The applicants postulate that this is homozygous (CIAO+ / +) resulting from incomplete activity of the component used to disrupt the development of viable pollen. In some embodiments, such homozygous (CIAO+ / +) seed can be conveniently screened out based on its increased (twice as bright) fluorescence.

[0068] In a further aspect the invention provides a non-transgenic, self-incompatible, inbred seed produced by a method of the invention.

[0069] Plant from seed

[0070] In a further embodiment the invention provides a method for producing a non-transgenic, self-incompatible, inbred plant, the method comprising growing a non-transgenic, selfincompatible, inbred seed of the invention, or produced by a method of the invention, into a non-transgenic, self-incompatible, inbred plant.

[0071] Preferably the non-transgenic, self-incompatible, inbred plant, is at least 55%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% homozygous.

[0072] In a further aspect the invention provides a non-transgenic, self-incompatible, inbred plant produced by a method of the invention, or a plant vegetatively or clonally propagated from a plant produced by the method. In a further embodiment the invention provides a plant part, propagule or progeny of a plant of the invention, or produced by a method of the invention.

[0073] Method for producing non-transgenic, self-incompatible, inbred plant

[0074] In a further aspect the invention provides a method for producing a non-transgenic, selfincompatible, inbred plant, the method comprising: a) self-fertilising a plant of the invention, that is hemizygous for a genetic construct of the invention, to produce inbred seed, wherein a proportion of the inbred seed produced is transgenic for the construct and a proportion of the inbred seed is non-transgenic for the construct, and b) identifying and the separating transgenic seed, based on expression of the selectable marker in the transgenic seed, and retaining the non-transgenic, selfincompatible, inbred seed, c) growing a non-transgenic seed from step b) to produce a non-transgenic, selfcompatible, inbred plant.

[0075] In one embodiment a transgenic seed from step b) is grown into a plant, and at least one further cycle of inbreeding performed using said plant.

[0076] Thus, in one embodiment and the steps a) to b) are repeated to produce a further inbred non-transgenic, self-incompatible, inbred seed.

[0077] Preferably the method comprises at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10 cycles of inbreeding.

[0078] Preferably each further cycle of inbreeding is initiated with a plant, transgenic and hemizygous for the construct, wherein the plant produced by the previous cycle of inbreeding. Preferably the non-transgenic, self-incompatible, inbred plant, is at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% homozygous.

[0079] The invention also encompasses plants vegetatively, or clonally, propagated from a plant produced by a method of the invention.

[0080] Thus, in one embodiment, the invention provides a further step: d) vegetatively, or clonally, propagating a non-transgenic, self-compatible, inbred plant from step c), to produce a further non-transgenic, self-compatible, inbred plant or plants.

[0081] Method for producing Fl hybrid seed

[0082] In a further aspect the invention provides a method for producing an Fl hybrid seed, the method comprising: a) crossing a first plant that is a non-transgenic, self-incompatible, inbred plant of the invention or produced by a method of the invention, with a second plant, b) collecting Fl hybrid seed produced from the crossing in a).

[0083] In one embodiment the second plant is also a non-transgenic, self-incompatible, inbred plant of the invention, or produced by a method of the invention.

[0084] In this embodiment the first plant or second plant has at least SI allele that is not common to both the first and second plant.

[0085] In a further embodiment the first and the second plant have different SI alleles. Fl hybrid seed produced by the method

[0086] In a further aspect the invention provides an Fl hybrid seed produced by the method.

[0087] Fl hybrid plant

[0088] In a further aspect the invention provides an Fl hybrid plant grown from an Fl hybrid seed of the invention, or produced by a method of the invention.

[0089] In one embodiment the invention provides a further Fl hybrid plant or plants, vegetatively, or clonally, propagated from an Fl hybrid plant of the invention.

[0090] In a further embodiment the invention provides a plant part, propagule or progeny of a plant of the invention, or produced by a method of the invention.

[0091] Method for producing Fl hybrid plant

[0092] In a further aspect the invention provides a method for producing an Fl hybrid plant, the method comprising: a) crossing a first plant that is a non-transgenic, self-incompatible, inbred plant of the invention or produced by a method of the invention, with a second plant, b) collecting Fl hybrid seed produced from the crossing in a), and c) growing the Fl hybrid seed to produce an Fl hybrid plant

[0093] In one embodiment the second plant is also a non-transgenic, self-incompatible, inbred plant of the invention, or produced by a method of the invention.

[0094] In this embodiment the first plant or second plant has at least SI allele that is not common to both the first and second plant.

[0095] In a further embodiment the first and the second plant have different SI alleles. The invention also encompasses plants vegetatively, or clonally, propagated from a plant produced by a method of the invention.

[0096] Thus, in one embodiment, the invention provides a further step: d) vegetatively, or clonally, propagating a non-transgenic, self-compatible, inbred plant from step c), to produce a further non-transgenic, self-compatible, inbred plant or plants.

[0097] Fl hybrid plant produced by the method

[0098] In a further aspect the invention provides an Fl hybrid plant produced by the method.

[0099] In one embodiment the invention provides a further Fl hybrid plant or plants, vegetatively, or clonally, propagated from an F 1 hybrid plant of the invention.

[0100] In a further embodiment the invention provides a plant part, propagule or progeny of a plant of the invention, or produced by a method of the invention.

[0101] DETAILED DESCRIPTION OF THE INVENTION

[0102] As discussed above, the invention provides compositions and methods for producing inbred lines and Fl hybrid seed from self-incompatible plants. The applicants have named this system “Combined Inbreeding And Outcrossing” (CIAO). Utilizing the plants’ own SI system to enforce outcrossing for production of Fl hybrid seed.

[0103] In addition, the system is fully transportable to other species as it relies on a transgene (the CIAO construct) comprising three expression cassettes, during the inbreeding phase. The CIAO construct contains a cassette for disrupting the self-incompatible phenotype while it is present, and is maintained in a hemizygous state by the presence of a pollen disrupting cassette. A selectable marker cassette, encoding for example a fluorescent marker, enables the identification and selection of transgenic (self-fertile) and non- transgenic (self-incompatible) seeds at every generation.

[0104] Self-incompatibility (SI)

[0105] Self-incompatibility exists in several plant families (Allen and Hiscock, 2008), and genetic components have been identified in Solanaceae, Brassicaceae, Poaceae and Papaveraceae.

[0106] In Solanaceae, a stigma-expressed RNAse (S-RNAse) interferes with self-pollen tube growth (Ai et al., 1990; Lee et al., 1994). The expression of a F-box (SLF) protein in non-self pollen results in the ablation of S-RNAse activity allowing fertilization (Wang et al., 2004). Modifiers of this system have been identified including 120k, HT-B, Sli and NaStEP which when mutated or suppressed allow self-fertilization (Eggers et al., 2021; Hancock et al., 2005; Jimenez-Duran et al., 2013; O'Brien et al., 2002).

[0107] In Brassicaceae a different system exists whereby a stigma-expressed receptor kinase protein (SRK) interacts with a pollen-secreted cysteine-rich protein (SLC / SP1 l)(Kachroo et al., 2001; Nasrallah, 2002; Schopfer et al., 1999). Upon self-recognition a signal is sent to the pollen arresting pollen-tube growth. A second stigma-expressed protein, SLG, may also act in the SI response in some S-haplotypes. Other members of the pathway, not involved in specificity, include ARC1, MLPK and Exo70Al (Indriolo et al., 2012;

[0108] Murase et al., 2004; Samuel etal., 2009).

[0109] In Papaver, the pollen determinant, PrpS is membrane bound and recognizes a secreted cysteine-rich stigmatic protein, PrsS, which rapidly results in programmed cell-death through influx of Ca2+ and K+ ions (Foote et al., 1994; Franklin-Tong et al., 2002; Walker et al., 1996; Wheeler et al., 2009).

[0110] In Poaceae, SI is regulated by two loci, named “S” and “Z” which work together to prevent self-pollen tube growth (Baumann, 2000). Upon recognition of both S and Z pollen tube growth is rapidly arrested. The genes underlying this phenomenon include two pollen-expressed “DUF247” genes at each of S and Z locus, and a single, small stigma-expressed secreted protein called “SP” or “ZP” at each locus (Herridge et al., 2022; Rohner et al., 2023; Lian et al., 2021; Manzanares et al., 2016; Thorogood et al., 2017).

[0111] The invention may be applied to plant species with both gametophytic and sporophytic self-incompatibility systems.

[0112] Gametophytic self-incompatibility (GSI)

[0113] In gametophytic self-incompatibility (GSI), the SI phenotype of the pollen is determined by its own gametophytic haploid genotype. This is the most common type of SI.

[0114] Sporophytic self-incompatibility (SSI)

[0115] In sporophytic self-incompatibility (SSI), the SI phenotype of the pollen is determined by the diploid genotype of the anther (the sporophyte) in which it was created. This form of SI was identified in the Brassicaceae.

[0116] In one embodiment the SI species to which the present invention is applied is selected from one of the following families: Acanthaceae, Amaryllidaceae, Annoneaceae, Asclepidaceae, Asphodelaceae, Asteraceae, Austrobaileyaceae, Betulaceae, Bignoniaceae, Bixaceae, Boranginaceae, Brassicaceae, Bromeliaceae, Campanulaceae, Capparidaceae, Caryophyllaceae, Chenopodiaceae, Chloranthaceae, Cistaceae, Clusiaceae, Commelinaceae, Pontederiaceae, Connaraceae, Convolvulaceae, Degeneriaceae, Ericaceae, Erythroxylaceae, Fabaceae, Fagaceae, Gentainaceae, Gesneriaceae, Grossulariaceae, Hemerocallidaceae, Illiciaceae, Iridaceae, Lauraceae, Liliaceae, Loganiaceae, Lythraceae, Magnoliaceae, Magnoliaceae, Malvaceae, Melanthiaceae, Menyanthaceae, Myrtaceae, Nelumbonaceae, Nothofagaceae, Olacaceae, Oleaceae, Onagraceae, Orchidaceae, Oxalidaceae, Papaveraceae, Passifloraceae Linaceae, Nymphaeaceae Plantaginaceae, Plumbaginaceae, Poaceae, Polemoniaceae, Polygonaceae, Primulaceae, Ranunculaceae, Resedaceae, Rosaceae, Rubiaceae, Rubiaceae, Santalaceae, Saururaceae, Saxifragaceae, Solanaceae, Sterculiaceae, Trimeniaceae, Tumeraceae and Winteraceae.

[0117] In a further embodiment the SI species to which the present invention is applied is selected from one of the following families: Asteraceae, Brassicaceae, Fabaceae, Lillaceae, Papaveraceae, Poaceae, Rosaceae and Solanaceae.

[0118] In a further embodiment the SI species to which the present invention is applied is selected from one of the following families: Brassicaceae, Papaveraceae, Poaceae and Solanaceae.

[0119] Asteraceae

[0120] In one embodiment the SI species in the Asteraceae is from the genus Carthamus.

[0121] In one embodiment the SI species from the Carthamus genus is Carthamus tinctorium.

[0122] Brassicaceae

[0123] In one embodiment the SI species in the Brassicaceae is selected from the genera: Brassica, Raphanus and Camelina.

[0124] In one embodiment the SI species from the Brassica genus is selected from: Brassica oleracea. Brassica napus, Brassica rapa and Brassica juncea.

[0125] In one embodiment the SI species from the Raphanus genus is selected from: Raphanus sativa and Raphanus raphanistrum.

[0126] In one embodiment the SI species from the Camelina genus is Camelina sativa. Fabaceae

[0127] In one embodiment the SI species in the Fabaceae is selected from the genera: Medicago and Trifolium.

[0128] In one embodiment the SI species from the Medicago genus is Medicago sativa.

[0129] In one embodiment the SI species from the Trifolium genus is selected from the species: Trifolium repens, Trifolium pratense, Trifolium hybridum and Trifolium granduliferum .

[0130] Solanaceae

[0131] In one embodiment the SI species in the Solanaceae is selected from the genera: Solanum. Nicotiana and Physalis .

[0132] In one embodiment the SI species from the Solanum genus is selected from: Solanum tuberosum, Solanum chilense, Solanum melongena and Solanum okadae.

[0133] In one embodiment the SI species from the Nicotiana genus is Nicotiana alata.

[0134] In one embodiment the SI species from the Physalis genus is selected from: Physalis crassifolia and Physalis philadelphica .

[0135] Papaveraceae.

[0136] In one embodiment the SI species in the Papaveraceae is from the genus Papaver.

[0137] In one embodiment the SI species from the Papaver genus is Papaver rhoeas. Poaceae

[0138] In one embodiment the SI species in the Poaceae is selected from the genera: Lolium.

[0139] Fe ilica. Secale, Oryza, Hordeum, Thinopyrum. Paspalum, Panicum and Eragrostis.

[0140] In one embodiment the SI species from the Lolium genus is selected from: Lolium perenne. Lolium multiflorum, Lolium rigidum and Lolium temulentum

[0141] In one embodiment the SI species from the Festuca genus is selected from: Festuca arundinacea, Festuca ovina. Festuca pratensis and Festuca rubra.

[0142] In one embodiment the SI species from the Oryza genus is Oryza longistaminata.

[0143] In one embodiment the SI species from the Hordeum genus is Hordeum bulbosum.

[0144] In one embodiment the SI species from the Secale genus is Secale cereale.

[0145] In one embodiment the SI species from the Thinopyrum genus is Thinopyrum intermedium.

[0146] In one embodiment the SI species from the Paspalum genus is Paspalum vaginatum.

[0147] In one embodiment the SI species from the Panicum genus is Panicum virgatum.

[0148] In one embodiment the SI species from the Eragrostis genus is Eragrostis curvula.

[0149] Non-SI crops

[0150] Non-SI crops can be targeted through the introduction of SI machinery, e.g. those from Papaver or from Brassicas (as demonstrate herein with naturally SC A.thaliana (de Graaf et al., 2012; Nasrallah et al., 2002)). Similar results may be achieved through hybridization of SI and SC species. Further non-limiting useful examples of SI genes and proteins are described below.

[0151] Vegetatively and clonally propagated plants

[0152] The terms “plant of the invention” and “plant produced by a method of the invention” include plants clonally vegetatively, or clonally propagated, from a plant directly produced by a method of the invention.

[0153] Self-incompatibility (SI) genes and proteins

[0154] The SI gene for which the present invention can be applied may be from any SI species.

[0155] In one embodiment the SI gene is from a plant discussed above.

[0156] Non-limiting examples of SI genes and proteins are shown in Table 1 below and their sequences are provided in the Sequence Listing.

[0157] Table 1. Examples of SI genes and proteins known in the art

[0158] In one embodiment the SI protein has at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to an SI protein in Table 1.

[0159] In one embodiment the SI protein is another SI protein encoded by an SI gene at the same S-locus as that of an SI protein in Table 1.

[0160] In one embodiment the SI gene has a coding sequence with at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to an SI gene coding sequence in Table 1.

[0161] In one embodiment the SI gene is another SI gene at the same S-locus as that of an SI gene (DNA [cds]) / protein in Table 1.

[0162] Modulators of self-incompatibility

[0163] Self-incompatibility can also be disrupted in accordance with the invention by altering expression of modulators of self-incompatibility.

[0164] Positive modulators of SI should be down-regulated or silenced.

[0165] Negative modulators of SI should be up-regulated or over-expressed. The modulator of SI for which the present invention can be applied may be from any SI species.

[0166] In one embodiment the modulator is from a plant discussed above.

[0167] Non-limiting examples of SI modulator genes and proteins are shown in Table 2 below and their sequences are provided in the Sequence Listing. The table also indicates whether the modulator is a positive or negative modulator of SI. Table 2. Examples of SI modulator genes and proteins known in the art

[0168] In one embodiment the SI modulator protein has at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to a SI modulator protein in Table 2. In one embodiment the SI modulator gene has a coding sequence with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to an SI modulator gene coding sequence in Table 2.

[0169] Disrupting Self-incompatibility (SI)

[0170] Down-regulating SI genes / proteins and positive modulators of SI

[0171] Self-incompatibility can be disrupted by targeted disruption of the expression of one or more SI genes / proteins, or positive modulator of SI, as described above.

[0172] Strategies designed to reduce, or eliminate, expression of a genes / proteins in a plant cell, tissue, organ, or at a particular developmental stage which / when it is normally expressed, are known as gene silencing strategies.

[0173] Gene silencing strategies may be focused on the gene itself or regulatory elements which affect expression of the encoded polypeptide. "Regulatory elements" is used here in the widest possible sense and includes other genes which interact with the gene of interest.

[0174] Genetic constructs designed to decrease or silence the expression of a target polynucleotide / polypeptide may include an antisense copy of all or part of a polynucleotide described herein. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator. An "antisense" polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,

[0175] 5'GATCTA 3' (coding strand) 3'CTAGAT 5' (antisense strand) 3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA

[0176] Genetic constructs designed for gene silencing may also include an inverted repeat. An 'inverted repeat' is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,

[0177] 5'-GATCTA TAGATC-3'

[0178] 3'-CTAGAT ATCTAG-5'

[0179] The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually, a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.

[0180] Such constructs are used in RNA interference (RNAi) approaches.

[0181] Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to a target polynucleotide is expressly contemplated.

[0182] Transformation with a sense expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5' or 3' untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3) : 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the target polynucleotides / genes is also contemplated. The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and / or non-coding sequence, such as promoter and / or intron and / or 5' or 3'-UTR sequence, or the corresponding gene.

[0183] Preferably the insert sequence for use in a construct (e.g. an antisense, sense suppression or RNAi construct) for silencing of a target gene, comprises an insert sequence of at least 20 nucleotides in length corresponding to, or complementary, to the target gene.

[0184] Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257).

[0185] Targeted silencing can also be achieved using CRISPR interference (CRISPRi), wherein expression of a catalytically inactive Cas9 protein (or dCas9) and a single guide RNA (sgRNA) designed to bind to a target transcript in a cell results in a block of transcript elongation, resulting in repression of the target gene (Qi, et ah, Cell 152: 1173-1183, 2013; Larson, et al, Nat. Protoc. 8:2180-2196, 2013). Another example of targeted silencing utilizes the Class 2 type VI-A CRISPR-Cas effector C2c2 RNase function, which is guided by a single crRNA that can be designed to bind to a target transcript (ssRNA), leading to cleavage of the target transcript and repression of the target gene (Abudayyeh, et al, 2016, Science, 353(6299).

[0186] Several further methods known in the art may be employed to alter, reduce or eliminate expression of a gene / protein according to the invention. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called "Deletagene" technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors, (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally, antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally, peptides interacting with a polypeptide of the invention may be identified through technologies such as phase-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a target polypeptide. Use of each of the above approaches in alteration of expression of a target nucleotide and / or polypeptide is specifically contemplated.

[0187] Up-regulating negative modulators of SI

[0188] Self-incompatibility can also be disrupted by increasing the expression of one or more negative modulator of SI, as described above.

[0189] Strategies designed to increase the expression of a genes / proteins in a plant cell, tissue, organ, or at a particular developmental stage, are known as well-known to those skilled in the art, and are discussed further herein.

[0190] Expression cassette for disrupting self-incompatibility (SI),

[0191] The CIAO construct of the invention includes an expression cassette for disrupting selfincompatibility (SI).

[0192] Targeting SI genes and positive modulators of SI

[0193] An expression cassette for disrupting self-incompatibility (SI), in accordance with the invention typically comprises a promoter, and element for disrupting expression of at least one SI gene or positive modulator of SI, and optionally a terminator sequence.

[0194] In one embodiment the element for disrupting self-incompatibility (SI), comprises a sequence of at least 15 nucleotides of, or complementary to, a gene encoding an SI protein with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to an SI protein in Table 1. In a further embodiment the element for disrupting self-incompatibility (SI), comprises a sequence of at least 15 nucleotides of, or complementary to, a sequence with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a DNA sequence in Table 1.

[0195] In one embodiment the element for disrupting self-incompatibility (SI), comprises a sequence of at least 15 nucleotides of, or complementary to, a gene encoding a positive modulator of SI with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to a positive modulator of SI in Table 2.

[0196] In a further embodiment the element for disrupting self-incompatibility (SI), comprises a sequence of at least 15 nucleotides of, or complementary to, a sequence with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% , preferably at least 99% sequence identity to a positive modulator of SI DNA sequence in Table 2.

[0197] In one embodiment the element includes a sequence complementary to the 15 nucleotides, such that the transcript formed may undergo complementary base pairing to form a hairpin structure.

[0198] In one embodiment a spacer of at least 3 bp is included between the 15 nucleotide sequence and the complementary sequence. Upregulating negative modulators of SI

[0199] An expression cassete for disrupting self-incompatibility (SI), in accordance with the invention may also comprise a promoter, and polynucleotide encoding a negative modulator of SI, and optionally a terminator sequence.

[0200] In one embodiment the negative modulator of SI comprises a sequence of with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% amino acid sequence identity to a negative modulator of SI in Table 2.

[0201] In a further embodiment the polynucleotide comprises a sequence with at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a DNA sequence of a negative modulator of SI in Table 2.

[0202] Promoters for disrupting self-incompatibility (SI)

[0203] Non-limiting examples of promoters useful for directing expression of components to target silencing of SI genes, or positive modulators of SI, are shown in Table 3 below.

[0204] Table 3. Examples of promoters useful for silencing SI gene or positive modulators of SI

[0205] Disrupting the development of viable pollen Pollen development is a post-meiotic process that produces immature pollen grains from microspores. The maturation process can be dissected into various stages. During microspore mitosis, the pollen first develops a large vacuole that mediates nuclear migration to produce polarized cells. Concomitantly, starch begins to accumulate and the vacuolar volume decreases in the maturing pollen. Continued intrinsic polarization of the pollen grains must be maintained before germination. During the final stage of maturation, pollen grains may exhibit developmental arrest, which involves programmed dehydration (Franchi et al., 2011. J. Exp. Bot.62, (15), 5267-5281). Development of pollen is tightly controlled by dynamic changes in gene expression (Motomura et al., 2020, Plant and Cell Physiology, 61 (4), 712-721). Methods for disrupting the development of viable pollen are well known to those skilled in the art. Such methods typically involve transforming a plant with an expression cassette for disrupting the development of viable pollen.

[0206] An expression cassette for disrupting the development of viable pollen, in accordance with the invention typically comprises a promoter, an element for disrupting the development of viable pollen, and optionally a terminator sequence.

[0207] Targeted expression of pollen-disrupting genes / proteins

[0208] Methods for disrupting the development of viable pollen may involve employing a tissuespecific and / or developmental stage-specific promoters to control the targeted expression of a pollen-disrupting gene / protein to inhibit development or ablate pollen.

[0209] Thus, in one embodiment the element for disrupting the development of viable pollen is a pollen-disrupting gene, and its expression is targeted by a suitable promoter.

[0210] Pollen-disrupting proteins useful for disrupting the development of viable pollen

[0211] Non-limiting examples of pollen-disrupting genes useful for disrupting the development of viable pollen are shown in Table 4 below.

[0212] Table 4. Examples of pollen-disrupting genes / proteins known in the art

[0213] Promoters useful for targeting pollen disrupting proteins to disrupt the development of viable pollen

[0214] Promoters active in the vegetative cell or sperm cells may be used. Promoters may be active at any stage after the formation of a tetrad (post-meiosis), through the microspore stage, bi-cellular, tri-cellular and mature pollen, as well as during pollen tube germination and extension.

[0215] For the present invention, development of only half of the pollen should be disrupted. Thus, in one embodiment the promoter is expressed after meiosis in the target plant.

[0216] Non-limiting examples of promoters useful for targeting cytotoxic proteins to disrupt the development of viable pollen are shown in Table 5 below.

[0217] Table 5. Examples of promoters useful for targeting cytotoxic genes / proteins Silencing genes to disrupt pollen development

[0218] Alternatively, the silencing of genes required for the development of viable pollen can be targeted to disrupt the development of viable pollen. Silencing approaches are discussed above.

[0219] Pollen developmental genes that are suitable targets for silencing

[0220] For the present invention, development of only half of the pollen should be disrupted.

[0221] Thus, in one embodiment the pollen developmental gene to be silenced is expressed after meiosis.

[0222] Non-limiting examples of pollen developmental genes that are suitable targets for silencing are shown in Table 6 below.

[0223] Table 6. Examples of pollen development genes / proteins known in the art

[0224] Promoters for disrupting the development of viable pollen by silencing pollen developmental genes For the present invention, development of only half of the pollen should be disrupted.

[0225] Thus, in one embodiment the targeted gene is expressed after meiosis in the target plant.

[0226] Non-limiting examples of promoters useful for disrupting the development of viable pollen by silencing pollen developmental genes are shown in Table 4 above.

[0227] Selection of seed harboring the CIAO construct

[0228] The present invention involves methods and expression cassettes for facilitating identification of seed that is transgenic for the CIAO construct. In this way, transgenic (CIAO+ / -) and non-transgenic (CIAO- / -) seed can be readily identified and separated.

[0229] Thus, the CIAO construct comprises an expression cassette for identifying seed carrying the construct.

[0230] In one embodiment the expression cassette is designed to express a selectable marker for identifying seed carrying the construct.

[0231] Such selectable markers are well known to those skilled in the art.

[0232] Preferably expression of the selectable marker can facilitate identify seeds carry the construct without impacting the viability of the seeds, or plant grown from the seeds.

[0233] In a preferred embodiment, the selectable marker is a fluorescent marker.

[0234] Fluorescent markers suitable for use in the CIAO construct include: GFP (Chalfie et al., 1994), dsRed (Matz et al., 1999), mCherry (Shaner et al. 2004) and CFP (Goedhart et al., 2012).

[0235] In a preferred embodiment the fluorescent marker is dsRed (Matz et al., 1999). Non-limiting examples of promoters useful for driving expression of fluorescent markers in accordance with the invention are shown in Table 6 below.

[0236] Table 6. Promoters for useful for driving expression of fluorescent markers.

[0237] In some embodiments fluorescent transgenic seed is identified that appears approximately twice as bright as the typical hemizygous (CIAO+ / -) seed. The applicants postulate that this is homozygous (CIAO+ / +) resulting from incomplete activity of the component used to disrupt the development of viable pollen. In some embodiments, such homozygous (CIAO+ / +) seed can be conveniently screened out based on its increased (twice as bright) fluorescence.

[0238] Polypeptide sequence identity

[0239] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www.ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

[0240] Methods for producing polynucleotides

[0241] Methods for producing polynucleotides (that can be used in accordance with the invention) are well known to those skilled in the art, and include use of cloning and recombinant DNA technologies. These technologies may involve modification of an existing polynucleotide encoding a dairy protein. Alternatively, the polynucleotide can be synthesised in its entirety by methods commonly used by those skilled in the art, and available commercially as a service from numerous well-known providers (e.g. GeneArt, Thermo Fisher Scientific).

[0242] The polynucleotides may be codon optimised to resemble the codon usage of the target cell or organism in which the sequences are expressed. Codons may be optimised using known tools (such as www.genewiz.com). This may result in improved gene expression and increased the translational efficiency, by accommodating the codon bias of the host.

[0243] Methods for producing constructs and vectors

[0244] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al. , Current Protocols in Molecular Biology, Greene Publishing, 1987).

[0245] Methods for producing host cells comprising polynucleotides, constructs or vectors

[0246] The invention provides cells which comprises a genetic construct or vector of the invention.

[0247] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

[0248] Methods for producing plant cells and plants comprising constructs and vectors

[0249] The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide in accordance with the invention. Plants comprising such cells also form an aspect of the invention.

[0250] Methods for transforming plant cells, plants and portions thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

[0251] Methods for genetic manipulation of plants

[0252] A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297; Hellens et al., 2000, Plant Mol Biol 42: 819-32; Hellens et al., Plant Meth 1: 13). For example, strategies may be designed to increase expression of a polynucleotide / polypeptide in a plant cell, organ and / or at a particular developmental stage where / when it is normally expressed or to ectopically express a polynucleotide / polypeptide in a cell, tissue, organ and / or at a particular developmental stage which / when it is not normally expressed. The expressed polynucleotide / polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

[0253] Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.

[0254] The promoters suitable for use in the constructs of this invention are functional in plant cells that contain oil bodies. Preferably the promoters drive expression that is spatially and / or developmentally coordinated with the biosynthesis of oil bodies. In one embedment the promoters are from genes encoding oil body associated proteins. Suitable oil body- associated genes include those encoding oleosins, caleosins and steroleosins as described herein

[0255] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.

[0256] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance. Plant transformation protocols

[0257] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial No. 5, 159, 135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Ui et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent Nos. 5, 187, 073 and 6. 020, 539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci.104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, Oi l ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant Cell Rep. 24( l):45-51); Primus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink 2005 Plant Cell Rep. 2006 ;25(2): 117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep.22(l):38-45); strawberry (Oosumi et al., 2006 Planta. 223(6): 1219-30; Folta et al., 2006 Planta Apr 14; PMID: 16614818), rose (Ui et al., 2003), Rubus (Graham etal., 1995 Methods Mol Biol. 1995;44: 129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407-412), Canola (Brassica napus L.). (Cardoza and Stewart, 2006 Methods Mol Biol. 343:257-66), safflower (Orlikowska et al, 1995, Plant Cell Tissue and Organ Culture 40:85-91), ryegrass (Altpeter et al., 2004 Developments in Plant Breeding 11 (7):255-250), rice (Christou et al., 1991 Nature Biotech. 9:957-962), maize (Wang et al., 2009 In: Handbook of Maize pp. 609- 639) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25,5: 425-31). Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature. Identification of transgenic seed expressing the fluorescent marker

[0258] Seeds can be sorted based on fluorescence using an automated seed sorting machine, for example the Satake ScanMaster!! 200 high-volume colour sorter. Methods for the optimization of this process are presented in Wu et al., 2016 and Weber et al., 2009.

[0259] Methods for production of Fl hybrids

[0260] The present invention involves using the CIAO system to produce inbred lines, und use of the inbred lines to produce Fl hybrids.

[0261] Agronomic methods for producing Fl hybrids using inbred lines as parents are well known in the art.

[0262] General comments

[0263] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[0264] Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[0265] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

[0266] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0267] The term “comprising” as used in this specification means “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that orthose prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. In some embodiments, the term "comprising" (and related terms such as "comprise and "comprises") can be replaced by "consisting of' (and related terms "consist" and "consists").

[0268] BRIEF DESCRIPTION OF THE DRAWINGS

[0269] Various embodiments of the invention are now illustrated with the following non-limiting examples.

[0270] Figure 1 shows three key components of the CIAO transgene. The “SI disruptor cassette” encodes a gene that reversibly disrupts self-incompatibility, e.g. through RNAi, or miRNA targeting a key SI gene. The “pollen disruptor’ prevents transmission through the male gamete, ensuring 50% CIAO+ / - and 50% CIAO- / - seeds are produced each generation. The “Selectable marker cassette’ allows rapid sorting of the progeny depending on the desired genotype.

[0271] Figure 2 shows an overview of the strategy for production inbred seeds and plants from self-incompatible (SI) species using the CIAO system. Lines carrying the CIAO construct, described herein, are created. In plants hemizygous for the CIAO construct only half of the normal amount of viable pollen is produced, because of the pollen inhibiting cassette. Such plants are self-fertilised to produce inbred seed. Half of the seed produced is transgenic for the CIAO construct. Transgenic (CIAO+ / -) seed is selected based on fluorescence, and (CIAO+ / -) plants grown from the seed can be used for multiple (typically >8) further rounds of inbreeding, to ultimately generate near homozygous inbred lines. Hemizygosity of the CIAO construct is maintained by a pollen- lethal-cassette, allowing transmission of the transgene through only the female gamete. To create an Fl hybrid, non-fluorescent (CIAO- / -) seed is selected from two inbred populations. The populations are mixed, and outcrossing is enforced through the (now active) SI machinery. As the lines are homozygous, all siblings within each population carry the same SI haplotypes, preventing intra-population fertilization. However, cross pollination between inbred populations is permitted as each population carries a different SI haplotype. Seed from both parents of the cross can be collected and will be genetically identical Fl hybrids.

[0272] Figure 3 shows an overview of the strategy for Fl hybrid production from selfincompatible (SI) species using the CIAO system. Lines carrying the CIAO construct are created and inbred (selecting for fluorescent (CIAO+ / -) seed) for multiple (typically >8) generations to generate homozygous inbred lines. Hemizygosity of the CIAO construct is maintained by a pollen-lethal-cassette, allowing transmission of the transgene through only the female gamete. To create an Fl hybrid, non-fluorescent (CIAO- / -) seed is selected from two inbred populations. The populations are mixed, and outcrossing is enforced through the (now active) SI machinery. As the lines are homozygous, all siblings within each population carry the same SI haplotypes, preventing intra-population fertilization. However, cross pollination between inbred populations is permitted as each population carries a different SI haplotype. Seed from both parents of the cross can be collected and will be genetically identical Fl hybrids.

[0273] Figure 4 shows CIAO constructs created for Arabidopsis ihaliana. Solcinum tuberosum or Lolium pereme. TN OS = nopaline synthase terminator; T35S = 35S cauliflower mosaic virus terminator; LB / RB = left / right border for Agrobacterium integration; p35S = 35S cauliflower mosaic virus promoter; pNOS = nopaline synthase promoter;

[0274] HygR / KanR / BAR = hygromycin / kanamycin / BASTA resistance; Other components are described in Table 7. Numbering (left) is used throughout manuscript in the format CIAO #. The sequences of each construct in the Sequence Listing is as follows: 1 = SEQ ID NO:61, 2 = SEQ ID NO:62, 3 = SEQ ID NO:63, 4 = SEQ ID NO:64, 5 = SEQ ID NO:65, 6 = SEQ ID NO:66, 7 = SEQ ID NO:67, 8 = SEQ ID NO:68, 9 = SEQ ID NO:69, 10 = SEQ ID NO:70, 11 = SEQ ID NO:71, 12 = SEQ ID NO:72, 13 = SEQ ID NO:73, and 14 = SEQ ID NO:74.

[0275] Figure 5 shows seed fluorescence from CIAO+ / - plants self (A) or outcrossed (B). A rate of 50% in selfed seeds, and 0% in outcrossed seeds indicates functionality of the pollen- lethal-cassette.

[0276] Figure 6 shows example images of fluorescent seed. A and B selfed seed from CIAO_4 construct showing -50% fluorescent seed, indicating activity of the pLAT52::EcoRI PLC and pNAPIN::dsRed. C and D, outcrossed seed from CIAO_4 plant showing single escaped seed, most lines show 0% escape. E and F Selfed seed from CIAO_1 construct showing -75% fluorescent seed, indicating low activity of the pLAT52:Alpha-amylase PLC in this line, but good activity of pOLE-OLE: :GFP.

[0277] Figure 7 shows alignment of amiR(SRKb / 6) to SRKb and SRK-6. The 1stand 17thbase is mismatched in both alignments.

[0278] Figure 8 (A) shows exemplary kanamycin-resistant offspring from a cross of SRKb / SCRb and CIAO+ / - parents. Seeds were sorted prior to sowing based on fluorescence. Non-fluorescent SRKb / SCRb+ / -; CIAO- / - plant (left) produced little / no seed while fluorescent SRKb / SCRb+ / -; CIAO+ / - plants (right) produced seed. (B) shows close-up showing main inflorescence of each plant in (A). The CIAO construct in this plant is pLAT52::amiR(BCPl)+pUBI::amiR(SRKb6)+pNAPIN::dsRed.

[0279] Figure 9 shows exemplary kanamycin-resistant offspring from a cross of SRKb / SCRb and CIAO+ / - parents. Seeds were sorted prior to sowing based on fluorescence. Non- fluorescent SRKb / SCRb+ / -;CIAO- / - plant (left) produced little / no seed while fluorescent SRKb / SCRb+ / -; CIAO+ / - plants (right) produced seed. The CIAO construct in this plant is pLAT52::Alpha-amylase+pUBI::RNAi(SRKb6)+pOLE-OLE::GFP. Figure 10 shows seed production from SRKb / SCRb+ / - plants carrying CIAO constructs. Seeds were sorted based on red (R) fluorescence or non-red (NR) to indicate presence of CIAO construct.

[0280] Figure 11 shows inflorescences of Arabdopsis plants carrying SRKb / SCRb with or without the CIAO_5 construct as judged by seed fluorescence (A). Seed weight from CIAO+ / - and CIAO- / - plants was measured by collecting seeds from mature plants in a tube and weighing the tube+seeds. Seed weight was estimated by subtracting the weight of the lightest tube (B).

[0281] Figure 12 shows seeds derived from an Arabidopsis plant carrying the CIAO_5 construct. A subset of very bright seeds (right) were identified as putative homozygous “escapes” (A). Brightness of the seeds was measured using the “Measure” Function in hnageJ showing that bright seeds are ~2x as bright as regular seeds (B).

[0282] Figure 13 shows the design of a CIAO construct including two amiRNAs targeting SRKb and SRK6 as well as the pAt2S3::mCherry seed-marker (A). Fluorescent seed was observed in plants carrying the CIAO construct in A (B). Independent transgenic lines carrying the CIAO construct in A were assessed for their rate of seed fluorescence to determine if the pollen-lethal cassette was functional (C).

[0283] Figure 14 shows the resulting Fl hybrid seedlings produced when a CIAO- / - ;SRKb / SCRb+ / - plant was pollinated with Sha pollen (A). The sequence of the TUBULIN gene was examined in these seedlings showing 10 / 10 Fl hybrid plants carry both the Sha and C24 sequence, confirming they are Fl hybrids (B).

[0284] Figure 15 shows Sanger sequencing of two alleles derived from Andean Sunside genomic DNA reveals alignment to known S-RNAse coding sequences.

[0285] Figure 16 shows alignment of Andean Sunside S-RNAse alleles and their respective amiRNAs. Figure 17 shows flower and berries in Andean Sunside potatoes transformed with CIAO l 1. (A) healthy flowers used for manual pollination. (B) healthy pollen from a dissected anther. (C) Berries produced on manually-pollinated CIAO l 1 transgenic plant. (D) Previously healthy flowers dropped from wild-type plant.

[0286] Figure 18 shows the resulting seeds from a cross of Andean Sunside potato harboring a CIAO transgene (CIAO 13) with Scapa pollen visualized under white light and fluorescence to detect GFP derived from the p35S::GFP marker (A). A cross using Aztec Gold as the maternal plant and Scapa harboring CIAO 13 was performed and GFP observed as in A, showing no seed fluorescence, indicative of a functional pollen-lethal cassette (pLAT52::EcoRI; B).

[0287] Figure 19 shows the sequence of S-RNAse from Aztec Gold potatoes, aligned to the two miRNAs designed to target it.

[0288] Figure 20 shows the sequence of S-RNAse from Scapa potatoes, aligned to the two miRNAs designed to target it.

[0289] Figure 21 shows the design of three CIAO constructs for use in Aztec Gold and Scapa potatoes. All three contain pLAT52::EcoRI and pAt2S3: :mCherry as the pollen-lethal cassette and seed-fluorescent marker, respectively. (1) Shows a construct with pSLG driving an RNAi construct designed to target the S-RNAse gene of Scapa and Aztec Gold SEQ ID 142. (2 and 3) show dual-amiRNA constructs designed to target the S-RNAse genes of Aztec Gold (2; SEQ ID 143) or Scapa (3; SEQ ID 144).

[0290] EXAMPLES

[0291] Various embodiments of the invention are now illustrated with the following non-limiting example. Example 1: Producing inbred lines and fl hybrids from self-incompatible species

[0292] Materials and methods

[0293] Plant growth conditions

[0294] A.thaliana plants of ecotype C24 were grown at 22°C in 16h / 8h light / dark cycles. Seeds of A.thaliana C24 harboring the SCR / SRKb allele were obtained from June Nasrallah (Nasrallah et al., 2002).

[0295] Andean Sunside potatoes were obtained from Eurogrow NZ, and grown on MSB5 tubs at 22°C in 24h light prior to transformation. After transformation potatoes were grown at 20°C in 16h / 8h light / dark cycles.

[0296] Lolium perenne plants of ecotype ONE50 can be grown at 22°C in 16h / 8h light / dark cycles.

[0297] Plasmid constructs

[0298] Constructs containing the various components of the system were cloned in two stages. The first stage involved either synthesizing, or cloning via PCR, relevant genes of interest (Table 7) with adapters at the 5’ and 3’ enabling insertion into a custom vector pUCR4-Ll, pUCLl-L2 or pUCR2-L3. PCR products were generated using PrimeStarMax DNA polymerase mastermix, 2.5uL of each F and R primer and luL of appropriate template (Table 8). Cycling conditions were 5s at 98C, 15s at 60C and 15s / kb at 72C. PCR products were cleaned using Zymo Clean and Concentrate kit to remove primers and any other substance that may interfere with downstream reactions. Each component “Pollen Lethal Cassette”, “Self-fertility-inducer”, or “Seed-fluorescent-marker” was constructed in pUCR4-Ll, pUCLl-L2, or pUCR2-L3 respectively. The backbone (pUC) vectors were digested with Ncol and Kpnl and cleaned using Zymo Clean and concentrate. The cleaned PCR products (promoter + gene) and appropriate vector were combined in a Gibson assembly reaction with NEB DNA HiFi Assembly Mastermix. The 20uL reaction was incubated at 50°C for Ih, after which 4pL was diluted in 20pL and used to transform competent DH5 Alpha E. coli. In the case of RNAi constructs NEB Stable3 cells were used to avoid rearrangements of the hairpin. Transformed bacteria were selected on Ampicillin LB plates overnight, and subsequently cultured the next day in 2-4mL of LB+Ampicillin. Plasmids were extracted from overnight cultures using Zymo plasmid miniprep kit. To combine components into a binary vector (pB7m34GW, pH7m35GW+35S::GFP, or pAGM4673) Gateway LR Clonase II plus was used. 15ng of each plasmid (PLC, SFI and SFM) plus 75ng of the binary vector were combined in an LR reaction with 2pL of clonase to a total volume of 5pL and incubated O / N at 25°C. 4pL was transformed into either NEBlO-beta or NEB-Stable3 cells (in the case of RNAi constructs).

[0299] Table 7 : Components used in CIAO construct design Table 8: Primers used for CIAO cloning A. thaliana transformation

[0300] A. thaliana (ecotype C24) was transformed using the floral drop method with Agrobacterium strain GV3101. Transformed seedlings were selected based on activity of the fluorescent marker under a stereomicroscope fitted with a mercury bulb and filterset for GFP (excitation 460-490 emission 510-) or RFP (excitation 540-580, emission 610-). Seeds were sterilized and sown on ! > Murashige Skoog medium.

[0301] Potato transformation

[0302] Potatoes of variety Andean Sunside were grown on MSB5 + 20g / L sucrose under (light / dark) 22°C. Leaf and stem segments were transformed by inoculating with Agrobacterium strain EHA 105 containing the plasmids of interest. Leaf and stem explants were first treated for 2-3 days with High Hormones (HH) media (Kumar, 1995). Agrobacterium was streaked from a frozen stock onto LB plates containing Rif+Spec. Agrobacterium liquid cultures were made in two stages, first a 24h culture from a freshly streaked plate in LB+Rif+Spec, then another overnight culture in LB+Rif+Spec and acetosyringone (20pM). After overnight culture, Agrobacterium were collected at the bottom of a 50mL falcon tube by centrifugation for 5minutes at lOOOxg prior to resuspending in MSB5 liquid media containing acetosyringone (Kumar, 1995). On the day of transformation HH media was drained from the potato explants, and they were submerged for ~lh in Agrobacterium culture. Explants were placed onto MGC media for 3 days, followed by MGC media + timentin (250mg / L) + hygromycin (5mg / L) (Banerjee et al., 2006). After 10 days on MGC media explants were moved to MGS media (Baneqee et al., 2006), and freshly transferred every 10-14 days to new MGS media until shoots appeared. Shoots were transplanted onto MSB5+Timentin media to produce roots. 35S::GFP was used as a control to detect true transformants and monitor the status of the transformation. L. perenne transformation

[0303] L. perenne can be transformed by Agrobacterium treatment as described in Patel et al. (2013).

[0304] Identifying seeds transgenic for the CIAO construct

[0305] For small scale identification transgenic seeds containing the CIAO construct were selected using a dissecting microscope (Olympus SZX12) fitted with a coaxial fluorescence attachment (Olympus SZX-RFL2). For observation of GFP the SZX-GFP filter combination was used (Excitation 460nm-490nm, emission 510nm-) for dsRed the SZX- RFP2 filter was used (Excitation 540nm-580nm, emission 610nm-).

[0306] Results:

[0307] Arabidopsis

[0308] Production of non-transgenic self-incompatible, inbred plants

[0309] Plasmids containing CIAO machinery were transformed into Arabidopsis thaliana to assess the key features of each construct. To assess pollen lethality, transformed plants were crossed in both directions to the self-incompatible model C24-SRKb / SCRb (Nasrallah et al., 2002). Pollen lethality and seed fluorescence was assessed by examining the number of fluorescent seeds produced when the transformed plants were outcrossed, or self-fertilized (Figure 5 and 6). When outcrossing, 0% of seeds should be fluorescent if the PLC is active, and when selfing 50% should be fluorescent assuming a single insertion of the binary construct. pLAT52::amiR(BCPl) and pLAT52:: Alpha-amylase showed minimal inhibition of transmission of the transgene, whereas pLAT52: :EcoRI frequently showed -50% fluorescent seeds upon selfing, and 0% fluorescent seeds when outcrossed (Figure 5). BCP1 has been shown to be essential for pollen tube growth in A.thaliana so it is possible that the amiRNA the Applicants selected was not sufficiently potent to repress BCP1 expression (Tehseen et al., 2010). Alpha Amylase degrades starch which is required for pollen tube growth, however, it’s expression in A.thaliana pollen was not sufficient to prevent fertilization, suggesting that the Alpha-Amylase was either not sufficiently active, or that in the absence of starch A. thaliana pollen is still capable of fertilization (Wu et al., 2016). The restriction enzyme, EcoRI was a potent disruptor of pollen transmission, likely due to degradation of mitochondrial or plastid DNA, as the EcoRI protein does not contain a nuclear localization signal, although there is also the possibility that small fractions of EcoRI enter the nucleus and disrupt the nuclear genome (Millwood et al., 2015).

[0310] The subsequent generation of plants crossed with SRKb / SCRb were selected based on seed fluorescence and Kanamycin Resistance (to select for the SRKb / SCRb construct). Selffertility of each class (fluorescent or not) was assessed by examining silique development on the plants, and collecting seeds (Figure 8-10). CIAO_1 plants restored seed production / silique length in the SRKb / SCRb background, indicating activity of the pUBQ::RNAi(SRKb / 6) construct (Figure 10). CIAO 2 and CIAO 3 plants also restored seed production in the SRKB / SCRb plants to a small degree, indicating activity of the amiR(SRKb / 6) construct under the pUBQ and pSLG13 promoters. Seeds derived from CIAO 2 and CIAO 3 plants containing SRKb / SCRb were weighed indicating a number of plants in which seed production had increased (Figure 10). As a large amount of non-seed material was also collected, this impacted the ability to accurately weigh seeds in cases where only a few were produced. Therefore, seeds were germinated from these plants to confirm that more seeds had been produced from CIAO+ than CIAO- plants. The amiR(SRKb / SRK6) is therefore capable of inducing self-fertility, however, is not a potent inducer. The amiR(SRKb / SRK6) sequence was the only predicted amiRNA that matched to both SRKb and SRK6 similarly, and also had a high predicted free-energy. A multiplexed amiRNA approach, allowing independent selection of SRKb- and SRK6- targeting amiRNAs can also be used, and may be more effective.

[0311] Another CIAO construct was tested, containing an RNAi cassette fused to the pSLG stigma-specific promoter. Transgenic A.thaliana carrying a CIAO construct (CIAO_5; Fig 4) were crossed with transgenic A.thaliana carrying the SI cassette (SRKb / SCRb). Seeds from this cross were sorted based on seed fluorescence, and grown on Kanamycin selective mediate ensure the presence of SRKb / SCRb. The resulting plants (n=15) were assessed for self fertility by examining siliques and weighing seeds (Fig 14), showing a significant increase in seed production from CIAO+ / - (red seed) plants over CIAO- / - (not red seed). Seed from these plants was observed, revealing -50% fluorescent seed as expected. Interestingly, a small number of seed were approximately twice as bright as other seed (Fig 12) the applicant postulates that there are homozygous seeds resulting from pollen grains that escape the effects of the pollen-lethal cassette. These seed can be differentiated by their brightness and removed from the breeding pipeline.

[0312] Arabidopsis carrying a CIAO construct utilizing the pAt2S3 promoter (Kroj et al., 2003; SEQ ID 137) driving mCherry (Campbell et al., 2004; SEQ ID 138) were created and assessed for seed fluorescence. The construct also contained pLat52: :EcoRI and two miRNA genes expressed under the pUBQlO promoter targeting SRKb and SRK6 alleles (SEQ ID 139; Fig. 17). Seed fluorescence was observed in these lines (Fig 13), confirming the activity of the pAt2S3 promoter, line 1, 2 and 5 showed 50% seed fluorescence indicating activity of the pLAT52: :EcoRI construct. These plants were crossed to A.thaliana lines carrying the SI cassette (SRKb / SCRb), seed was sorted based on fluorescence and sown on Kanamycin media to select for the SI cassette. These plants can be assessed for self-fertility to determine the efficacy of the pUBQ::dual-amiRNA construct.

[0313] Production of Fl hybrids

[0314] Next the Applicants sought to create exemplary Fl hybrid plants from the offspring of SRKb / SCRb / CIAO+ / - plants, however, this required creation of a second SI line. The S6 allele from A. lyrata (containing the native genomic fragment of SRK6 and SCR6 from A.lyrata), which is reported to produce a strong SI response (Boggs et al., 2009) was utilised. Generation of SI plants from this construct is in development.

[0315] An alternative method of production is to use only one SI parent to produce Fl hybrid seed. Null-segregant plants generated from the cross of CIAO_5+ / - plants with SRKb / SCRb plants (CIAO- / -, not red seed) were used as female parents in a cross with a different ecotype (Shahdara / Sha) to produce Fl hybrid seed. CIAO- / - plants carrying SRKb / SCRb were pollinated without emasculation using pollen from open flowers of Sha and the resulting seeds were collected. Fl hybrid seed was planted and compared to the parental lines (C24 and Sha; Fig 14). Sequencing of the TUBULIN gene from C24, Sha and 10 Fl hybrids was performed. The TUBULIN sequence contains a “G” insertion in Sha that is read as “G” or “T” in hybrids. All 10 hybrids showed evidence of the C24 and Sha alleles, confirming they were hybrids of the parental genotypes (no self-fertilization had occurred; Fig 14).

[0316] Potato

[0317] To apply the system for potato, the Applicant used clonal Andean Sunside material from Eurogrow NZ. Firstly, the S-RNAse alleles present in this genotype were identified. The Applicants expected to find two alleles, and indeed, found two sequences matching closely to reported S-RNAse alleles (Figure 15)(Dzidzienyo et al., 2016; Ma et al., 2021). The Applicants then designed RNAi constructs targeting ~1 lObp of each allele, or constructs containing two amiRNAs targeting each allele, separated by tRNA linkers to produce the amiRNAs from a single transcript (Figure 16)(Ossowski et al., 2008; Zhang et al., 2018). CIAO constructs from Figure 4 were created and transformed into leaf and stem material, prior to plant regeneration.

[0318] Potato plants harboring CIAO constructs were regenerated and planted into soil to grow to maturity. Under the growth conditions used, Andean Sunside potatoes rarely set high quality flowers, typically buds would fall of before maturity, or immature flowers would form but abort prior to producing viable pollen. Nevertheless, it was possible to identify instances of quality flowers and these were utilized to manually collect pollen from the anthers (which did not appear to naturally dehisce) and apply this to the stigma (Figure 17A and B). In at least one instance, berries were formed on CIAO+ plants (containing CIAO l 1 pLat52:: Alpha-amylase + pUBQ::2xamiRNA + pOLE-OLE: :GFP), berries were not formed on similar high-quality wild-type flowers (Figure 17C and D). Andean Sunside potato plants harboring CIAO_13 (Fig. 4) were used as female parents in a cross with Scapa potatoes. The resulting seeds were examined for fluorescence of GFP and dsRed. No dsRed fluorescence was observed, indicating inactivity of the pNAPIN promoter in potato seeds, however transgenic seeds could be identified by activity of the p35S::GFP promoter active in the embryo (Fig. 18A). Scapa plants harboring CIAO 13 were used as male parents in a cross with Aztec Gold potatoes acting as the female parent. The seeds were observed for fluorescence of the 35 S: :GFP marker and none was observed, indicating the activity of the pLAT52::EcoRI cassette which prevents transmission of the CIAO transgene through the pollen (Fig. 18B).

[0319] Due to the poor pollen production from Andean Sunside potatoes, the applicants designed further CIAO constructs tailored to Aztec Gold and Scapa potato varieties. The S-RNAse alleles of Aztec Gold and Scapa potato varieties were sequenced, revealing one allele from each genotype (SEQ ID 140, SEQ ID 141). Constructs were designed based on these sequences, including a dual-amiRNA construct targeting the Aztec Gold S-RNAse allele, a dual-amiRNA construct targeting the Scapa S-RNAse allele, and an RNAi construct containing inverted repeats of both the Aztec Gold and Scapa S-RNAse alleles (Fig. 19-21; SEQ ID 142-144). These constructs were transformed into Aztec Gold and Scapa leaves, and regenerated into plants. The resulting plants can be tested for self-fertility, seed fluorescence, and pollen-transmission.

[0320] Lolium perenne

[0321] Seed derived calli of L.perenne can be transformed with CIAO constructs by treatment with Agrobacterium. Plants can be regenerated from this callus, and subsequently vernalized and allowed to flower. Upon flowering, the degree of self-fertilization can be measured by isolating flowering heads to prevent pollen contamination. Successful selffertilization will allow assessment of the seed fluorescence and transmission frequency of the CIAO construct. Simultaneously, CIAO+ plants can be outcrossed to wild-type L.perenne for confirmation that the CIAO construct is not transmitted through the pollen. Discussion

[0322] The Applicants have demonstrated the use of a novel system for production of non- transgenic, inbred lines and Fl hybrid breeding that can be applied to a large number of SI crops.

[0323] Non-SI crops can be targeted through the introduction of SI machinery, e.g. those from Papaver or from Brassicas (as demonstrated herein with naturally SC A. thaliana (de Graaf et al., 2012; Nasrallah et al., 2002)).

[0324] During inbreeding there is a chance of occasional homozygous CIAO+ / + seeds forming due to incomplete activity of the PLC. These seeds appear twice as bright as CIAO+ / - seeds and can be screened out of the breeding pipeline (Fig. 12).

[0325] In the case of seed crops, for seed production in the field, two different Fl hybrids would need to be planted, as the Fl hybrid generated from two inbred SI lines would itself be SI and lead to little / no seed production.

[0326] This also leads to the ability to produce double-cross hybrids in the instance that seed production is poor from early Fl hybrids (as was seen in Fl hybrid maize produced in the 1930s). Double cross hybrids could easily be produced by creating four Fl hybrid lines and pair crossing them - outcrossing would be enforced through the Si-mechanism in the same manner when crossing inbred lines - as all Fl hybrids are genetically identical the SI machinery makes no distinction between siblings.

[0327] In the case of potatoes, inbred lines, and their Fl hybrids would be capable of being propagated as tubers if desired. This may be the case if seed sorting is laborious; in this case the identification of non-transgenic inbred lines through sorting a minimal amount of seed, followed by “bulking” the inbred lines via tubers prior to Fl hybrid production on a large scale, may be desirable. RNAi and amiRNA constructs can be multiplexed to target a wide-range of SI allele haplotypes. For RNAi constructs, including multiple sequences within a hairpin (or antisense or bi-directionally transcribed sequence) can be used to target multiple different genes. In the case of amiRNAs, tRNA sequences can be used to exploit the natural processing machinery of tRNAs to generate a single transcript that is cleaved into multiple miRNAs targeting a number of genes (Zhang et al., 2018). Alternative approaches to knock down genes may include CRISPRi or Cas 13 -targeting or any other reversible mechanism to knock-down a gene (Abudayyeh et al., 2017; Larson et al., 2013). Targeting the pollen-expressed components to disrupt SI may also be possible. In the case of Brassicas, the pollen components are expressed in the (paternal) tapetum, making them suitable to a standard knock-down (Schopfer et al., 1999). In the case of systems that act gametophytically (ie. the pollen Si-components are expressed in the gametes) it is necessary to knock down these genes through transfer from the paternal parent. In this case an engineered-phasiRNA construct may be applicable, or alternatively, a highly expressed miRNA in the tapetum may be capable of transmission to the developing pollen (Zhai et al., 2015).

[0328] Example 2. Using a CIAO construct targeting modulators of SI

[0329] The invention can also involve producing and utilising CIAO constructs in which the SI disruptor cassette is designed to manipulate expression of SI modulators to disrupt SI.

[0330] HT-B and 120k RNAi are positive modulators of SI and can therefore be silenced to disrupt SI (O’Brien, et al., 2002; Hancock, etal., 2005).

[0331] Materials and methods

[0332] Cloning

[0333] A fragment containing nucleotides corresponding to the messenger RNA of HT-B and 120k was ordered from Twist Biosciences. The fragment contained overhangs allowing it to be amplified by PCRwith existing primers (SEQ ID 91, SEQ ID 92, SEQ ID 93 and SEQ ID 94) to produce two fragments (“forward” and “reverse”) for insertion as an inverted repeat. The forward and reverse PCR products (5ng each) were combined with PCR products of Arabidopsis pUBI (5ng) an intron sequence (10 ng) and digested NcoI+KpnI-digested pUC-LlL2 (20ng) in a Gibson reaction using NEB DNA Assembly HiFi master mix and incubated at 50°C for 1 hour. The assembly was transformed into NEB Stbl3 cells and plated on LB media containing Ampicillin. Individual colonies were picked and miniprepped using Zymo Plasmid Miniprep kit according to manufacturer’s instructions. The resulting pUBI::HT-B+120k RNAi construct was combined with a cassette encoding pLAT52::EcoRI and pOLE-OLE: :GFP into the vector pH7M34GW+35S::GFP using Invitrogen LR Clonase II plus according to manufacturer’s instructions. The reaction was incubated overnight before transformation into NEB Stbl3 cells which were plated on LB media containing Spectinomycin. The resulting construct containing SEQ ID 74 can be used for potato transformation.

[0334] Plant transformation

[0335] Potatoes of variety Scapa and Aztec Gold were obtained from EuroGrow NZ and can be transformed as described for Andean Sunside above using methods described in Kumar (1995) and Banerjee et al. (2006).

[0336] Results

[0337] Transformed varieties of S.tuberosum harboring the CIAO_14 construct can be assessed for self-fertility, production of fluorescent seeds and segregation of the CIAO construct as described above for A. thaliana.

[0338] Two Aztec Gold plants harboring the CIAO 14 construct targeting HT-B and 120kD protein were created and tested for self-fertility. No fruits were bom from these plants suggesting that either: the plants were not self-fertile, or Aztec Gold is not capable of producing pollen of high enough quality for fertilization in our growth conditions. The second explanation is further supported by the lack of berries produced on Andean Sunside or Scapa individuals when pollinated by Aztec Gold pollen in our growth conditions. Further transgenic lines have been produced to substantiate this observation. REFERENCES

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Claims

CLAIMS1. A genetic construct comprising: a) an expression cassette for disrupting self-incompatibility (SI), b) an expression cassette for disrupting the development of viable pollen, and c) an expression cassette for identifying seed carrying the genetic construct.

2. The genetic construct of claim 1 in which cassette in a) is designed to at least one of: i. reduce or eliminate expression of at least one allele of an SI gene in a selfincompatible plant, ii. reduce or eliminate expression of at least one positive modulator of SI, and iii. increase expression of at least one negative modulator of SI.

3. The genetic construct of claim 2 in which SI gene encodes the female selfincompatibility determinant.

4. The genetic construct of claim 2 in which SI gene encodes the male selfincompatibility determinant.

5. The genetic construct of any preceding claim in which cassette in b) is designed to inhibit or ablate pollen substantially half of the pollen of the plant.

6. The genetic construct of any preceding claim in which cassette in b) is designed to express a component to inhibit or ablate pollen.

7. The genetic construct of claim 6 in which the component is expressed after meiosis.

8. The genetic construct of any preceding claim in which the cassette in c) is designed to express the selectable marker in the seed of a self-incompatible plant.

9. The genetic construct of claim 8 in which the selectable marker is a fluorescent marker.

10. A plant, plant part, or plant cell comprising a genetic construct any preceding claim.

11. The plant, plant part, or plant cell is from a self-incompatible species.

12. The plant of claim 11 that is rendered self-fertile due to expression of the cassette for disrupting self-incompatibility (SI).

13. The plant of claim 11 or that is hemizygous for the construct.

14. The plant of claim 13 that is maintained in a hemizygous state by the presence of the cassette for disrupting the development of viable pollen.

15. A method for producing a non-transgenic, self-incompatible, inbred seed, the method comprising: a) self-fertilising a plant of the any preceding claim, that is hemizygous for the genetic construct, to produce inbred seed, wherein a proportion of the inbred seed produced is transgenic for the construct and a proportion of the inbred seed is non-transgenic for the construct, and b) identifying and the separating transgenic seed, based on expression of the selectable marker in the transgenic seed, to retain the non-transgenic, selfincompatible, inbred seed.

16. The method of claim 15 in which a transgenic seed from step b) is grown into a plant, and at least one further cycle of inbreeding performed using said plant.

17. The method of claim 15 or 16 in which the non-transgenic, self-incompatible, inbred seed, is at least 55% homozygous.

18. A non-transgenic, self-incompatible, inbred seed produced by a method of any one of claims 15 to 17.

19. A method for producing a non-transgenic, self-incompatible, inbred plant, the method comprising growing a non-transgenic, self-incompatible, inbred seed of anypreceding claim, or produced by a method of any preceding claim, into a non- transgenic, self-incompatible, inbred plant.

20. The method of claim 19 in which the non-transgenic, self-incompatible, inbred plant is at least 55% homozygous.

21. A method for producing a non-transgenic, self-incompatible, inbred plant, the method comprising: a) self-fertilising a plant of the invention, that is hemizygous for a genetic construct of the invention, to produce inbred seed, wherein a proportion of the inbred seed produced is transgenic for the construct and a proportion of the inbred seed is non- transgenic for the construct, and b) identifying and the separating transgenic seed, based on expression of the selectable marker in the transgenic seed, and retaining the non-transgenic, selfincompatible, inbred seed, c) growing a non-transgenic seed from step b) to produce a non-transgenic, selfcompatible, inbred plant.

22. The method of claim 21 in which a transgenic seed from step b) is grown into a plant, and at least one further cycles of inbreeding performed using said plant.

23. The method of claim 21 or 22 in which in which the non-transgenic, selfincompatible, inbred plant is at least 55% homozygous.

24. A non-transgenic, self-incompatible, inbred plant produced by a method of any preceding claim.

25. A method for producing an Fl hybrid seed, the method comprising: a) crossing a first plant that is a non-transgenic, self-incompatible, inbred plant of any preceding claim, or produced by a method of any preceding claim, with a second plant, b) collecting Fl hybrid seed produced from the crossing in a).

26. The method of claim 25 in which the second plant is also a non-transgenic, selfincompatible, inbred plant of any preceding claim, or produced by a method of any preceding claim.

27. The method of claim 26 in which the first plant or second plant has at least SI allele that is not common to both the first and second plant.

28. The method of claim 26 in which the first and the second plant have different SI alleles.

29. An Fl hybrid seed produced by the method of any one of claims 25 to 28.

30. A method for producing an Fl hybrid plant, the method comprising growing an Fl hybrid seed of claim 29 to produce an Fl hybrid plant.

31. A method for producing an Fl hybrid plant, the method comprising: a) crossing a first plant that is a non-transgenic, self-incompatible, inbred plant of any preceding claim, or produced by a method of any preceding claim, with a second plant, b) collecting Fl hybrid seed produced from the crossing in a), and c) growing the Fl hybrid seed to produce an Fl hybrid plant.

32. The method of claim 31 in which the second plant is also a non-transgenic, selfincompatible, inbred plant of any preceding claim, or produced by a method of any preceding claim.

33. The method of claim 31 or 32 in which the first plant or second plant has at least SI allele that is not common to both the first and second plant.

34. The method of claim 31 or 32 in which the first and the second plant have different SI alleles.

35. An Fl hybrid plant produced by the method of any preceding claim.

36. A non-transgenic, self-incompatible, inbred plant vegetatively or clonally propagated from a non-transgenic, self-incompatible, inbred plant of any preceding claim.

37. An Fl hybrid plant vegetatively or clonally propagated from an Fl hybrid plant of any preceding claim.

38. A plant part, propagule or progeny of a plant of any preceding claim.

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