Herbicide tolerant lupin

Abstract

The present invention relates to genetically modified lupin plants that express a mutated acetolactate synthase 1 (ALS1) which confers upon the genetically modified plant increased tolerance to one or more Group 2 herbicides, which inhibit the enzymatic activity of ALS1, as compared to a level of tolerance to such herbicides in a corresponding wild-type variety of the plant expressing wild-type ALS1.

Description

[0001] HERBICIDE TOLERANT LUPIN

[0002] RELATED APPLICATION DATA

[0003] This application claims priority to Australian Provisional Patent Application 2025900104 filed on 14 January 2025, the contents of which are incorporated by reference in their entirety herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a genetically modified lupin, e.g., Lupinus angustifolius, expressing a mutated acetolactate synthase 1 (ALS1) conferring increased tolerance to Group 2 herbicides, which inhibit ALS1 enzymatic activity.

[0006] BACKGROUND OF THE INVENTION

[0007] Lupins, belonging to the legume family, are an important food crop and source of protein in animal feed. The most frequently cultivated species is Lupinus angustifolius. Unfortunately, a number of weed species have an impact on commercial production of lupin, such as annual ryegrass, wild oats, brome grass, barley grass, and wild radish. Thus, it is important for the commercial exploitation of lupins that such weed species be controlled by the use of herbicides. Thus, there remains an outstanding need for lupin variants that are herbicide tolerant in order to exploit the full commercial potential of lupins.

[0008] SUMMARY OF THE INVENTION

[0009] The present inventors have identified lupin plants genetically modified by chemical mutagenesis to be tolerant to Group 2 herbicides, which act by inhibiting the enzymatic activity of ALS1 and result in blocking the synthesis of branched chain amino acid. Examples of Group 2 herbicides to which the genetically modified lupin plants provided herein demonstrate tolerance include imidazolinone (IMI) herbicides or sulfonylurea (SU) herbicides.

[0010] Thus, in one aspect the present invention provides genetically modified lupin plant that expresses a mutated acetolactate synthase 1 (ALS1) which confers upon the genetically modified lupin plant increased tolerance to a Group 2 herbicide, as compared to a corresponding wild-type variety of the plant expressing wild-type ALS1, wherein the amino acid sequence of the mutated ALS1 has a serine to asparagine substitution at a position corresponding to residue 638 of the wild-type ALS1; an alanine to threonine at a position corresponding to residue 107 of the wild-type ALS1; a proline to serine substitution corresponding to residue 182 of the wild-type ALS1; or a proline to leucinesubstitution at a position corresponding to residue 182 of the wild-type ALS1, wherein the residue positions are relative to the amino acid positions of wild-type ALS1 from Lupinus angustifolius.

[0011] In some examples the Group 2 herbicide is an imidazolinone (IMI) herbicide or a sulfonylurea (SU) herbicide.

[0012] In some examples the genetically modified lupin plant exhibits increased tolerance to an IMI herbicide. In some examples the IMI herbicide includes one or more imidazolinones selected from the group consisting of: imazapic, imazamox, imazethapyr, imazamethabenz, imazaquin, imazapyr, and combinations thereof. In some examples the IMI herbicide comprises imazamox, imazapyr, or both.

[0013] In some examples the genetically modified lupin plant having increased tolerance to an IMI herbicide comprises the mutated ALS1 comprising the serine 638 asparagine substitution. In some examples the the amino acid sequence of the mutated ALS1 comprises the amino acid sequence of SEQ ID NO:2.

[0014] In some examples, where the genetic modification comprises the mutated ALS1 comprising the amino acid sequence of SEQ ID NO:2, the the genetically modified lupin plant is derived from a genetically modified lupin plant line selected from the group consisting of: LI, L9, L31, L32, L35, and L36.

[0015] In other examples the genetically modified lupin plant having increased tolerance to an IMI herbicide comprises the mutated ALS1 comprising the alanine 107 to threonine substitution. In some examples the amino acid sequence of the mutated ALS1 comprises the amino acid sequence of SEQ ID NO:3.

[0016] In some examples, where the genetic modification comprises the mutated ALS1 comprising the amino acid sequence of SEQ ID NO:3, the genetically modified lupin plant is derived from a genetically modified lupin plant line selected from the group consisting of: L2, L20, and L26.

[0017] In some examples a genetically modified lupin plant provided herein has increased tolerance to a SU herbicide. In some examples a genetically modified lupin plant provided herein that has increased tolerance to a SU herbicide also has increased tolerance to an IMI herbicide. In some examples a genetically modified lupin plant provided herein has increased tolerance to an IMI herbicide and increased tolerance to a SU herbicide. In some examples the SU herbicide comprises one or more SU herbicides selected from the group consisting of: Metsulfuron-methyl (Metsulfuron), Nicosulfuron, Chlorsulfuron, Rimsulfuron, Imazosulfuron, Flazasulfuron, Halosulfuron-methyl, Primisulfuron-methyl, Prosulfuron, lodosulfuron-methyl-Na, and Tribenuron-methyl.

[0018] In some examples, where the genetically modified lupin plant provided herein has increased tolerance to a SU herbicide, the amino acid sequence of the mutated ALS1 comprises the Proline 182 to Serine substitution. In some examples, where the aminoacid sequence of the mutated ALS1 comprises the Proline 182 to Serine substitution, the mutated ALS1 comprises the amino acid sequence of SEQ ID NO: 15. In some examples, where the genetically modified comprises the mutated ALS1 comprising the amino acid sequence of SEQ ID NO:3, the genetically modified lupin plant is derived from the genetically modified lupin plant line LI 2.

[0019] In some examples, where the genetically modified lupin plant provided herein has increased tolerance to a SU herbicide, the amino acid sequence of the mutated ALS1 comprises the Proline 182 to Leucine substitution. In some examples, where the amino acid sequence of the mutated ALS1 comprises the Proline 182 to Leucine substitution, the mutated ALS1 comprises the amino acid sequence of SEQ ID NO: 17. In some examples, where the genetically modified comprises the mutated ALS1 comprising the amino acid sequence of SEQ ID NO:17, the genetically modified lupin plant is derived from a genetically modified lupin plant line selected from the group consisting of: L15, L18, L21, L25, L29, and L30.

[0020] In other examples a genetically modified lupin plant provided herein is a transgenic plant comprising an exogenous nucleic acid encoding a mutated ALS1 that confers tolerance to a Group 2 herbicide. In some examples, the Group 2 herbicide is an IMI herbicide or a SU herbicide. In some examples the transgenic plant is resistant to both a IMI herbicide and a SU herbicide. In other examples a genetically modified lupin plant provided herein is not a transgenic plant.

[0021] In a related aspect provided herein is a plant cell of a genetically modified lupin plant provided herein.

[0022] In another related aspect provided herein is a plant part of a genetically modified lupin plant provided herein.

[0023] In a further related aspect provided herein is a seed produced by a genetically modified lupin plant provided herein.

[0024] In another related aspect provided herein is a food product prepared from a genetically modified lupin plant provided herein.

[0025] In another related aspect provided herein is consumer product prepared from or using a genetically modified lupin plant provided herein.

[0026] In another related aspect provided herein is an industrial product prepared from a genetically modified lupin plant provided herein.

[0027] In another related aspect provided herein is a veterinary product prepared from a genetically modified lupin plant provided herein.

[0028] In another related aspect provided herein is a method for producing a genetically modified lupin hybrid plant, comprising breeding a genetically modified lupin plant provided herein with a second plant to obtain a genetically modified lupin hybrid plant,wherein the genetically modified lupin hybrid plant exhibits increased tolerance to an IMI herbicide.

[0029] In another related aspect provided herein is a method for producing a genetically modified lupin hybrid plant, comprising breeding a genetically modified lupin plant provided herein with a second lupin plant to obtain a genetically modified lupin hybrid plant, wherein the genetically modified lupin hybrid plant exhibits increased tolerance to a SU herbicide. In some examples the method produces a genetically modified lupin hybrid plant that also has increased tolerance to a IMI herbicide, z.e., the genetically modified lupin hybrid plant has increased tolerance to both a SU herbicide and to an IMI herbicide.

[0030] In a further related aspect provided herein is a method for cultivation of a lupin plant, the method comprising planting a seed derived from a genetically modified lupin plant provided herein, whereby the planted seed germinates to generate a seedling. In some examples the cultivation method includes cultivating the seedling to obtain a mature genetically modified lupin plant and harvesting seeds from the mature plant. In some preferred examples the cultivation method also includes applying an IMI herbicide or a SU herbicide one or more times over a cultivation period beginning at a seedling stage, whereby growth of weeds in proximity of the seedling following the application is inhibited.

[0031] In a related aspect provided herein is an isolated nucleic acid encoding ALS1 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 17 In some examples provided herein is a plant expression vector comprising the just-mentioned nucleic acid and operable to expression of the encoded ALS1 in a lupin plant. In some examples the plant expression vector is a recombinant virus vector. In some examples the recombinant virus vector is a Cauliflower Mosaic Virus (CaMV) vector. In some examples the plant expression vector is a Ti plasmid.

[0032] In a related aspect provided herein is a recombinant cell comprising any of the above-mentioned plant expression vectors. In some examples the recombinant cell is a recombinant prokaryotic cell. In some examples the recombinant prokaryotic cell is a recombinant Agrobacterium tumefaciens cell.

[0033] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0034] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0035] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps orgroup of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0036] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0037] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0038] Figure 1 shows a picture of pots of 39 putative IMI herbicide resistant lupin (Coyote, L. angustifolius) lines and one control plant pot four weeks after application of the IMI herbicide Intercept® (a combination of IMI herbicides imazamox and imazapyr). Tolerant lines showed better growth and looked green compared to herbicide-sensitive lines.

[0039] Figure 2. shows a picture of nine putative IMI herbicide resistant tolerant lupin lines at 7 weeks following application of IMI herbicide Intercept®. No effect was observed on biomass of these plants compared to untreated plants.

[0040] Figure 3. shows a picture of 13 additional putative IMI herbicide tolerant lupin lines at 9 weeks following application of IMI herbicide Intercept®. These additional lines, although exhibiting partial sensitivity to Intercept® exhibited a delayed recovery of biomass relative to the nine lines shown in Figs. 1 and 2.

[0041] Figure 4. shows a picture of 15 mutant lupin line plants. Application of Priority® (a triazolopyrimidine-based ALS1 inhibitor) at 40 mL / ha, 9 weeks after its application resulted in 100% mortality. Untreated plants were at the flowering stage. Thus, these lines were shown to retain sensitivity to ALS1 inhibitor-based herbicides while being tolerant to the IMI herbicide Intercept®.

[0042] Figure 5. shows bar graphs illustrating the final survivorship (%) of the various lines compared with the standard CM4 line across eight herbicide treatments (T1-T8). The values represent survivorship within each line category averaged over IT and SU lines. Bars followed by the same letter within each line category do not differ significantly at p < 0.05.

[0043] KEY TO THE SEQUENCE LISTING SEQ ID NO:1 - Amino acid sequence of wild-type variety Lupinus angustifolius acetolactate synthase 1 (ALS1).SEQ ID NO:2 - Amino acid sequence of mutated Lupinus angustifolius ALS 1 including a Ser638→Asn638substitution.

[0044] SEQ ID NO:3 Amino acid sequence of mutated Lupinus angustifolius ALSI including an Ala107→Thr107substitution.

[0045] SEQ ID NO:4 - Amino acid sequence of wild-type variety Lupinus albus ALSI.

[0046] SEQ ID NO:5 - Amino acid sequence of mutated Lupinus albus ALSI including a Ser636→Asn636substitution.

[0047] SEQ ID NO:6 - Amino acid sequence of mutated Lupinus albus ALSI including an Ala107→Thr107substitution.

[0048] SEQ ID NO:7 - Amino acid sequence of wild-type variety Lupinus luteus acetolactate synthase 1 (ALSI).

[0049] SEQ ID NO:8 - Amino acid sequence of mutated Lupinus luteus ALSI including a Ser638→Asn638substitution.

[0050] SEQ ID NO:9 - Amino acid sequence of mutated Lupinus luteus ALSI including a Ala107→Thr107substitution.

[0051] SEQ ID NO: 10 - nucleic acid sequence from wild-type L. angustifolius encoding ALS 1.

[0052] SEQ ID NO:11 - nucleic acid sequence encoding mutated L. angustifolius encoding ALSI comprising a Ser638Asn substitution G→A transversion at nucleotide 1,913. SEQ ID NO:12 - nucleic acid sequence encoding mutated L. angustifolius encoding ALSI comprising a Ala107Thr substitution G→A transversion at nucleotide 319.

[0053] SEQ ID NO: 13 - exemplary, artificially generated, non-naturally occurring nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3, a mutated ALSI from Lupinus angustifolius comprising an Ala107Thr substitution. Sequence was generated by “reverse translation” from the amino acid sequence of SEQ ID NO:3 using the “Reverse Translate” function of the bioinformatics.org suite.

[0054] SEQ ID NO: 14 - nucleic acid sequence encoding mutated L. angustifolius encoding ALSI comprising a Pro182Ser substitution C→T transversion shown as bold and underlined; nucleotide 544).

[0055] SEQ ID NO: 15 amino acid sequence of ALS1 from of a mutated Lupinus angustifolius ALS1; Pro182Ser substitution is indicated in bold and underlined.

[0056] SEQ ID NO: 16 (nucleic acid sequence encoding mutated L. angustifolius encoding ALS1 comprising a Pro182Leu substitution C→T transversion shown as bold and underlined; nucleotide 545).

[0057] SEQ ID NO: 17 amino acid sequence of ALS1 from of a mutated Lupinus angustifolius ALS1; Pro182Leu substitution is indicated in bold and underlined.DETAILED DESCRIPTION OF THE INVENTION

[0058] General Techniques and Definitions

[0059] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, plant molecular biology, protein chemistry, and biochemistry).

[0060] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0061] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0062] As used herein, the term about, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, more preferably + / - 1%, more preferably + / - 0.5%, of the designated value.

[0063] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0064] As used herein, "tolerant" or "herbicide-tolerant" indicates a plant or portion thereof capable of growing in the presence of an amount of herbicide that normally causes growth inhibition in a non-tol erant fe.g, a wild-type) plant or portion thereof. Levels of herbicide that normally inhibit growth of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant.

[0065] As used herein, "recombinant" refers to an organism having genetic material from different sources.As used herein, "mutagenized" or “mutated” refers to an altered sequence in genetic material (e.g., a change in the sequence of one or more copies of a gene) as compared to the genetic material of a corresponding wild-type organism, wherein the alterations in genetic material were induced and / or selected by human action. Examples of human action-induced mutagenesis that can be used to produce a mutagenized or mutated organism having one or more mutated genes include, but are not limited to, treatment of plant cells (e.g., calli) with a chemical mutagen such as an alkylating agent, e.g., ethylene imine (El), diethyl sulfate (DES), ethyl nitrosourethane (ENU), ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS).

[0066] As used herein, a "genetically modified organism" (GMO) is an organism whose genetic characteristics have been altered by mutagenesis of at least one endogenous genetic locus; or by insertion of genetic material (e.g., a transgene) from another source organism or progeny thereof that retain the mutagenized or inserted genetic material.

[0067] As used herein, “acetolactate synthase 1” ALS1 (AKA “acetohydroxy acid synthase 1 (AHAS1)” refers to a plant enzyme that catalyzes the first step in the synthesis of the branched-chain amino acids (valine, leucine, and isoleucine).

[0068] As used herein, an amino acid substitution at a position “corresponding to residue ‘N’ of the wild-type ALS1” refers to an amino acid residue in an ALS1 sequence (or ortholog thereof) determined by sequence alignment to be at the position that best matches that of the reference (wild-type Lupinus angustifolius) ALSI sequence at the residue “N” position.

[0069] As used herein, "wild-type" or "corresponding wild-type plant" means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from mutagenized and / or recombinant forms.

[0070] For the present invention, the terms "herbicide-tolerant" and "herbicide-resistant" are used interchangeably and are intended to have an equivalent meaning.

[0071] As used herein in regard to herbicides useful in various embodiments hereof, terms such as Imidazolinone (IMI) herbicide and the like, refer to those agronomically acceptable herbicide active ingredients recognized in the art. ALSI “enzymatic activity” or “activity” refers to catalysis by ALSI of the first step in the synthesis of the branched-chain amino acids (valine, leucine, and isoleucine).

[0072] As used herein, “Group 2 herbicide”, refers to a herbicide of any chemical structural class that inhibits acetolactate synthase 1 (ALSI) activity.

[0073] When used in reference to a particular mutant ALSI, terms such as herbicide tolerant (HT) and herbicide tolerance refer to the ability of such enzyme or polypeptide to perform its physiological activity in the presence of an amount of an herbicide that would normally inactivate or inhibit the activity of the wild-type (non-mutant) version of said enzyme or polypeptide. In some examples, in the presence of an IMI herbicide or aSU herbicide at a concentration sufficient to substantially completely inhibit the enzymatic activity of a wild-type lupin ALS1, the level of enzymatic activity of a mutant lupin ALS1 disclosed herein is at least 30% of the level of enzymatic activity of a wildtype lupin ALS1 (SEQ ID NO:1) activity in the absence of the IMI herbicide or the SU herbicide, e.g., 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, or another level of enzymatic activity from at least 30% of that of a wild-type lupin ALS1 in the absence of an IMI herbicide or a SU herbicide to 100% of that of a wild-type lupin ALS1 in the absence of an IMI herbicide or SU herbicide. In some examples, a genetically modified lupin plant provided herein has increased tolerance to both an IMI herbicide and to a SU herbicide. In some examples the level of tolerance to an IMI herbicide is greater than the level of tolerance to SU herbicide. In other examples the level of tolerance to a SU herbicide is greater than the level of tolerance to an IMI herbicide. In other examples the level of tolerance to an IMI herbicide is similar to the level of tolerance to a SU herbicide. In some preferred examples the wild-type lupin ALS1 is wild-type lupin ALS1 from L. angustifolius.

[0074] As used herein, "descendant" refers to any generation plant.

[0075] As used herein, "progeny" refers to a first generation plant.

[0076] Genetically Modified and Transgenic Lupin Plants

[0077] As referred to herein, lupin plants refer to plants belonging to the genus Lupinus. Lupinus is a large and diverse genus in the legume family (Fabaceae). Its common name used in Europe and Australia is lupin for both native and domesticated species, while the common name for native Lupinus in North America is lupine. Taxonomically, lupins are classified within order Fabales, family Fabaceae, tribe Genisteae and genus Lupinus L. In some examples, a genetically modified lupinus plant, as referred to herein, refers to a plant of a species selected from among L. angustifolius (narrow leafed lupin), L. albus (white lupin), and L. luteus (yellow lupin) that expresses a mutated ALS1 which confers upon the genetically modified plant increased tolerance to an imidazolinone (IMI) herbicide, as compared to a corresponding wild-type variety of the plant expressing wildtype ALS1, wherein the amino acid sequence of the mutated ALS1 has a serine to asparagine substitution at a position corresponding to residue 638 of the wild-type ALS1 of L. angustifolius (residue 636 in L. albus),' a threonine substitution of an alanine at a position corresponding to residue 107 of the wild-type ALS1 of L. angustifolius.

[0078] In some examples a genetically modified lupinus plant, as referred to herein, refers to a plant of a species selected from among L. angustifolius (narrow leafed lupin), L. albus (white lupin), and L. luteus (yellow lupin) that expresses a mutated ALS1 which confers upon the genetically modified plant increased tolerance to an IMI herbicide and also tolerance to a SU herbicide as compared to a corresponding wild-type variety of theplant expressing wild-type ALS1, wherein the amino acid sequence of the mutated ALS1 has a proline to serine substitution at a position corresponding to residue 182 of the wildtype ALS1 of / .. angustifolius (residue 636 in I.. albus, or a proline to leucine substitution at a position corresponding to residue 182 of the wild-type ALS1 of / .. angustifolius.

[0079] In some examples a genetically modified lupinus plant provided herein has increased tolerance to both an IMI herbicide and a SU herbicide.

[0080] Suitable examples of IMI herbicides include, but are not limited to a imidazolinone herbicide selected from among: imazapic (CAS 104098-48-8), imazamox (CAS 114311-32-9), imazethapyr (CAS 81335-77-5), imazamethabenz (CAS 81405-85-8), imazaquin (CAS 81335-37-7), imazapyr (CAS 81334-34-1), and combinations thereof.

[0081] Suitable examples of SU herbicides include, but are not limited to, a SU herbicide selected from the group consisting of: Metsulfuron-methyl (Metsulfuron; CAS 74223-64-6), Nicosulfuron (CAS 111991-09-4), Chlorsulfuron (CAS 64902-72-3), Rimsulfuron (122931-48-0), Imazosulfuron (CAS 122548-33-8), Flazasulfuron (CAS 104040-78-0), Halosulfuron-methyl (CAS 100784-20-1), Primisulfuron-methyl (CAS 86209-51-0), Prosulfuron (CAS 94125-34-5), lodosulfuron-methyl-Na (CAS 144550-36-7), Tribenuron-methyl (CAS 144550-36-7), and combinations thereof.

[0082] In some examples the genetically modified lupin plants provided herein are transgenic plants. In some examples provided herein is a transgenic lupin plant that expresses an exogenously introduced nucleic acid encoding a mutated ALS1 as disclosed herein. In these instances, the plants and cells produce a recombinant ALS1 polypeptide. The term "recombinant" in the context of a polypeptide refers to the polypeptide encoded by an exogenous polynucleotide when produced by a cell, which polynucleotide has been introduced into the cell or a progenitor cell by recombinant DNA or RNA techniques such as, for example, transformation. Typically, the cell comprises a non-endogenous gene that causes an altered amount of the polypeptide to be produced. In an embodiment, a "recombinant polypeptide" is a polypeptide made by the expression of an exogenous (recombinant) polynucleotide in a plant cell.

[0083] The term "plant" as used herein as a noun refers to whole plants and refers to any member of the Kingdom Plantae, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. Plantlets and germinated seeds from which roots and shoots have emerged are also included within the meaning of "plant". The term "plant parts" as used herein refers to one or more plant tissues or organs which are obtained from a plant and which comprises genomic DNA of the plant. Plant parts include vegetative structures (for example, leaves, stems), roots, floral organs / structures, seed (including embryo,cotyledons, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same. The term "plant cell" as used herein refers to a cell obtained from a plant or in a plant and includes protoplasts or other cells derived from plants, gamete-producing cells, and cells which regenerate into whole plants. Plant cells may be cells in culture. By "plant tissue" is meant differentiated tissue in a plant or obtained from a plant ("explant") or undifferentiated tissue derived from immature or mature embryos, seeds, roots, shoots, fruits, tubers, pollen, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as calli. Exemplary plant tissues in or from seeds are cotyledon, embryo and embryo axis. The invention accordingly includes plants and plant parts and products comprising these.

[0084] As used herein, the term "seed" refers to "mature seed" of a plant, which is either ready for harvesting or has been harvested from the plant, such as is typically harvested commercially in the field, or as "developing seed" which occurs in a plant after fertilisation and prior to seed dormancy being established and before harvest.

[0085] A "transgenic plant" as used herein refers to a plant that contains a nucleic acid construct not found in a wild-type plant of the same species, variety or cultivar. That is, transgenic plants (transformed plants) contain genetic material (a transgene) that they did not contain prior to the transformation. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The transgene may include genetic sequences obtained from or derived from a plant cell, or another plant cell, or a non-plant source, or a synthetic sequence. Typically, the transgene has been introduced into the plant by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes. The genetic material is preferably stably integrated into the genome of the plant. The introduced genetic material may comprise sequences that naturally occur in the same species but in a rearranged order or in a different arrangement of elements, for example an antisense sequence. Plants containing such sequences are included herein in "transgenic plants".

[0086] The term "genetically modified" includes introducing genes into cells by transformation or transduction, mutating genes in cells (e.g., by chemical mutagenesis) and altering or modulating the regulation of a gene in a cell or organisms to which these acts have been done or their progeny.

[0087] A "non-transgenic plant" is one which has not been genetically modified by the introduction of genetic material by recombinant DNA techniques. In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype.

[0088] " Wild-type", as used herein, refers to a gene (e.g., a wildtype ALS1 gene), protein, cell, tissue or plant that has not been modified according to the invention. Wildtype genes, proteins, cells, tissue or plants may be used as controls to compare levels ofexpression or activity of a nucleic acid or encoded protein or the extent and nature of trait modification of genes, proteins, cells, tissue or plants modified as described herein.

[0089] Transgenic plants, as defined in the context of the present invention include progeny of the plants which have been genetically modified using recombinant techniques, wherein the progeny comprise the transgene of interest. Such progeny may be obtained by self-fertilisation of the primary transgenic plant or by crossing such plants with another plant of the same species. This would generally be to modulate the production of at least one protein defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of said plants comprising the transgene such as, for example, cultured tissues, callus and protoplasts.

[0090] Transgenic plants, as defined in the context of the present disclosure include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide of the present invention in the desired plant or plant organ. Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

[0091] In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype. The transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in Fl progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.

[0092] Four general methods for direct delivery of a gene into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87 / 06614, US 5,472,869, 5,384,253, WO 92 / 09696 and WO 93 / 21335); and the gene gun (see, for example, US 4,945,050 and US 5,141,131); (3) viral vectors; and (4) receptor-mediated mechanisms.

[0093] Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. A particle delivery system suitablefor use with the present invention is the helium acceleration PDS-1000 / He gun is available from Bio-Rad Laboratories. For the bombardment, immature embryos or derived target cells such as scutella or calli from immature embryos may be arranged on solid culture medium.

[0094] In another alternative embodiment, plastids can be stably transformed. Method disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (US 5, 451,513, US 5,545,818, US 5,877,402, US 5,932479, and WO 99 / 05265.

[0095] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome.

[0096] Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., Plant DNA Infectious Agents, Hohn and Schell, (editors), Springer-Verlag, New York, (1985): 179-203). Moreover, technological advances in vectors for Agrobacterium -mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant varieties where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

[0097] A transgenic plant formed using Agrobacterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, Breeding Methods for Cultivar Development, J. Wilcox (editor) American Society of Agronomy, Madison Wis. (1987).

[0098] Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are well known in the art.

[0099] Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.

[0100] The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., Methods for Plant Molecular Biology, Academic Press, San Diego, (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

[0101] The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.

[0102] To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way toquantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and / or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

[0103] Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art. Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining variation in any base pair of a particular sequence.

[0104] In some preferred examples the genetically modified lupin plants provided herein are not transgenic plants (ie., non-transgenic plants). Also encompassed by the present disclosure are any of the following obtained or derived from the genetically modified lupin plants disclosed herein or their use as a component or ingredient: a plant part, a plant cell, a seed, a food product prepared from a genetically modified lupin plant, a consumer product prepared from a genetically modified lupin plant, or a veterinary product prepared from a genetically modified lupin plant.

[0105] For example, a food product may be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, an English muffin, a muffin, a pita bread, a quickbread, a refrigerated / frozen dough product, dough, baked beans, a burrito, chili, a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a ready to eat meal, stuffing, a microwaveable meal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll, a candy bar, a pie crust, pie filling, baby food, a baking mix, a batter, a breading, a gravy mix, a meat extender, a meat substitute, a seasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup, sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo mein noodles, an ice cream inclusion, an ice cream bar, an ice cream cone, an ice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, an extruded snack, a fruit and grain bar, a microwaveable snack product, a nutritional bar, a pancake, a par-baked bakery product, a pretzel, a pudding, a granola-based product, a snack chip, a snack food, a snack mix, a waffle, a pizza crust, animal food or pet food.

[0106] In alternative examples, a lupin flour may be a component of a nutritional supplement. For instance, the nutritional supplement may be a product that is added to the diet containing one or more additional ingredients, typically including: vitamins, minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber. The lupin flour, refined flour or coarse fraction thereof includes protein, vitamins, minerals, amino acids, enzymes, and fiber. For instance, the coarse fraction contains a concentrated amount of dietary fiber as well as other essential nutrients, such as B-vitamins, selenium, chromium, manganese, magnesium, and antioxidants, which are essential for a healthy diet. A nutritional supplement may includeany known nutritional ingredients that will aid in the overall health of an individual, examples include but are not limited to vitamins, minerals, other fiber components, fatty acids, antioxidants, amino acids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and / or other nutritional ingredients. The supplement may be delivered in, but is not limited to the following forms: instant beverage mixes, ready-to-drink beverages, nutritional bars, wafers, cookies, crackers, gel shots, capsules, chews, chewable tablets, and pills.

[0107] In some examples a consumer product prepared from or using a genetically modified lupin plant is, e.g., a heating or cooling pack that utilizes lupin seeds as filler, a textile made from lupin fibers, or a moisturizing lotion.

[0108] In some examples an industrial produced prepared from a genetically modified lupin plant is, e.g., a bioplastic, a dye, or a pesticide.

[0109] In some examples a veterinary product prepared from a genetically modified lupin plant provided herein is an an animal feed, a pet food supplement, or a fishmeal replacement.

[0110] Also disclosed herein are recombinant cells comprising a host cell transformed with a nucleic acid encoding a mutated lupin ALSI disclosed herein. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred host cells are lupin plant cells. In other examples, a recombinant cell is a prokaryotic cell.

[0111] Acetolactate Synthase 1 (ALSI) enzymes

[0112] Disclosed herein are invention provides genetically modified lupin plants expressing mutated ALS1 enzyme comprising an amino acid sequence that differs from the amino acid sequence of the ALS 1 enzyme found in the corresponding wild-type plant.

[0113] For ease of understanding, the amino acid numbering system used herein will be the numbering system used for the ALSI from Lupinus angustifolius (also referred to as narrow-leaf lupin), which corresponds to the amino acid sequence listed under GenBank Accession No. XP_019425280 provided herein as SEQ ID NO:1.

[0114] SEQ ID NO:1; amino acid sequence of ALSI from Lupinus angustifolius'.

[0115] MAATTTPNPTFTPLPSSSSKQILRFTLPVTSFPSIRRRSLRITSSLSSNPKIPAPRAT STAAAAPTLTTEPFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGASMEIHQALTRSNTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADALLDSVPI VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRP GPVLIDVPKDIQQQLAVPNWDQPIRLTGYVSRLPKSPDEKHLHQIVRLISESKKPVLY VGGGSLDSSEELRKFVELTGIPVASTLMGLGSYPLGDEKSLQMLGMHGTVYANYAVDK SDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKVALEG INRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRISPQYAIQVLDELTNGNA IVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIWDIDG DGS FIMNVQELAT IRVENLPVKVLLLNNQHLGMWQWEDRFYKANRAHT YLGNPANE I EIFPNMLKFADACGIPAARVTKKDEVTAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPS NGSFQDVITEGDGRISY

[0116] In some examples a mutated ALSI expressed in a genetically modified lupin plant disclosed herein comprises a serine 638 to asparagine 638 substitution, as shown in the amino acid sequence corresponding to SEQ ID NO:2.

[0117] SEQ ID NO:2; amino acid sequence of a mutated ALSI from Lupinus angiislifolius Ser638Asn substitution is indicated in bold and underlined:

[0118] MAATTTPNPTFTPLPSSSSKQILRFTLPVTSFPSIRRRSLRITSSLSSNPKIPAPRAT STAAAAPTLTTEPFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGTSMEIHQALT RSNTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADALLDSVPI VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRP GPVLIDVPKDIQQQLAVPNWDQPIRLTGYVSRLPKSPDEKHLHQIVRLISESKKPVLY VGGGSLDSSEELRKFVELTGIPVASTLMGLGSYPLGDEKSLQMLGMHGTVYANYAVDK SDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKVALEG INRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRISPQYAIQVLDELTNGNA IVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIWDIDG DGS FIMNVQELAT IRVENLPVKVLLLNNQHLGMWQWEDRFYKANRAHT YLGNPANE I EIFPNMLKFADACGIPAARVTKKDEVTAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPN NGSFQDVITEGDGRISY

[0119] In some examples a mutated ALSI expressed in a genetically modified lupin plant disclosed herein comprises an alanine 107 to threonine 107 substitution, as shown in the amino acid sequence corresponding to SEQ ID NO:3.

[0120] SEQ ID NO:3; amino acid sequence of a mutated ALSI from Lupinus anguslifolius AlalO7Thr substitution is indicated in bold and underlined:

[0121] MAATTTPNPTFTPLPSSSSKQILRFTLPVTSFPSIRRRSLRITSSLSSNPKIPAPRAT STAAAAPTLTTEPFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGTSMEIHQALT RSNTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADALLDSVPI VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRP GPVLIDVPKDIQQQLAVPNWDQPIRLTGYVSRLPKSPDEKHLHQIVRLISESKKPVLY VGGGSLDSSEELRKFVELTGIPVASTLMGLGSYPLGDEKSLQMLGMHGTVYANYAVDK SDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKVALEG INRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRISPQYAIQVLDELTNGNA IVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIWDIDG DGS FIMNVQELAT IRVENLPVKVLLLNNQHLGMWQWEDRFYKANRAHT YLGNPANE IEIFPNMLKFADACGIPAARVTKKDEVTAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPS NGSFQDVITEGDGRISY

[0122] In some examples a genetically modified lupin plant comprising one the above-mentioned mutated ALSI sequences is of the species Lupinus angustifolius. In other examples a genetically modified lupin plant is of the species Lupinus albus. The amino acid sequence of wild-type Lupinus albus (GenBank Accession No. KAE9591745.1) is shown below as SEQ ID NO:4 In some examples a genetically modified Lupinus albus plant comprises a mutated Lupinus albus ALSI sequence corresponding to SEQ ID NO:5 or SEQ ID NO:6 as shown below.

[0123] SEQ ID NO:4 amino acid sequence of ALSI from wild-type Lupinus albus),' from GenBank KAE9591745.1:

[0124] MAATTVNPAFTPLPSSSSKQIPRFTLPFSTFPSLRRRSLRITSSLSGNPKLPTSRATS AAAAAPTLTTEHFVSRFAPDEPRKGSDILVEALERQGVTNVFAYPGGASMEIHQALTR SNTIRNILPRHEQGGIFAAEGYARSSGRPGVCMATSGPGATNLVSGLADALLDSVPIV AITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRPG PVLIDVPKDIQQQLAVPNWDQPIRLTGYMSRLPKSPDEKLLHQIVRLISESKKPVLYV GGGSLDSSVELRKFVELTGIPVASTLMGLGSYPVGDDKSLQMLGMHGTVYANYAVDKS DLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALEGI NRILESKGIKDKVDFRNWREELNEQKVKFPLSFKTFEDRICPQYAIQVLDELTNGNAI ISTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAVWDIDGD GSFIMNVQELATIRVENLPVKVLLLNNQHLGMWQWEDRFYKANRAHTYLGNPSNENE IFPNMLKFADACGIPAARVTKKDEVTAAIQKMLDTPGPYLLEVIVPHQEHVLPMIPSN GSFQDVITEGDGRISY

[0125] SEQ ID NO:5 amino acid sequence of ALSI from of a mutated Lupinus albus ALSI; Ser636Asn substitution is indicated in bold and underlined:

[0126] MAATTVNPAFTPLPSSSSKQIPRFTLPFSTFPSLRRRSLRITSSLSGNPKLPTSRATS AAAAAPTLTTEHFVSRFAPDEPRKGSDILVEALERQGVTNVFAYPGGASMEIHQALTR SNTIRNILPRHEQGGIFAAEGYARSSGRPGVCMATSGPGATNLVSGLADALLDSVPIV AITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRPG PVLIDVPKDIQQQLAVPNWDQPIRLTGYMSRLPKSPDEKLLHQIVRLISESKKPVLYV GGGSLDSSVELRKFVELTGIPVASTLMGLGSYPVGDDKSLQMLGMHGTVYANYAVDKS DLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALEGI NRILESKGIKDKVDFRNWREELNEQKVKFPLSFKTFEDRICPQYAIQVLDELTNGNAI ISTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAVWDIDGD GSFIMNVQELATIRVENLPVKVLLLNNQHLGMWQWEDRFYKANRAHTYLGNPSNENE IFPNMLKFADACGIPAARVTKKDEVTAAIQKMLDTPGPYLLEVIVPHQEHVLPMIPNN GSFQDVITEGDGRISY

[0127] SEQ ID NO:6 amino acid sequence of ALSI from of a mutated Lupinus albus ALSI; AlalO7Thr substitution is indicated in bold and underlined:

[0128] MAATTVNPAFTPLPSSSSKQIPRFTLPFSTFPSLRRRSLRITSSLSGNPKLPTSRATS AAAAAPTLTTEHFVSRFAPDEPRKGSDILVEALERQGVTNVFAYPGGTSMEIHQALTR SNTIRNILPRHEQGGIFAAEGYARSSGRPGVCMATSGPGATNLVSGLADALLDSVPIV AITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRPGPVLIDVPKDIQQQLAVPNWDQPIRLTGYMSRLPKSPDEKLLHQIVRLISESKKPVLYV GGGSLDSSVELRKFVELTGIPVASTLMGLGSYPVGDDKSLQMLGMHGTVYANYAVDKS DLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALEGI NRILESKGIKDKVDFRNWREELNEQKVKFPLSFKTFEDRICPQYAIQVLDELTNGNAI ISTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAVWDIDGD GSFIMNVQELATIRVENLPVKVLLLNNQHLGMWQWEDRFYKANRAHTYLGNPSNENE IFPNMLKFADACGIPAARVTKKDEVTAAIQKMLDTPGPYLLEVIVPHQEHVLPMIPSN GSFQDVITEGDGRISY

[0129] In other examples a genetically modified lupin plant is of the species Lupinus luteus. The amino acid sequence of wild-type Lupinus albus (GenBank Accession No. KAE9591745.1) is shown below as SEQ ID NO:7. In some examples a genetically modified Lupinus luteus plant comprises a mutated Lupinus luteus ALSI sequence corresponding to SEQ ID NO:8 or SEQ ID NO:9 as shown below.

[0130] SEQ ID NO:7 amino acid sequence of ALSI from wild-type Lupinus luteus),' from GenBank CAL0309352.1 MAATTTPNPAFTPLPSSSSKQIPRFTLPFSTFPSLRRRSLRITSSISSNPKLPAPRAT STAAAAPTLTTESFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGASMEIHQALT RSSTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADALLDSVPI VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRP GPVLIDIPKDIQQQLAVPNWDQPIRLTGYMSRLPKSPDEKLLHQIVRLISESKKPVLY VGGGSLDSSKELRKFVELTGIPVASTLMGLGSYPVGDDKSLQMLGMHGTVYANYAVDK SDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALEG INRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRISPQYAIQVLDELTNGNA IVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIWDIDG DGSFIMNVQELATIRVENLPVKILLLNNQHLGMWQWEDRFYKANRAHTYLGNPSNEN EIFPNMLMFADACGIPAARVTKKDEVSAAIQKMLDTPGPYLLEVIVPHQEHVLPMIPS NGSFQDVITEGDGRIMLLPTETDCIFELNFHLF

[0131] SEQ ID NO:8 amino acid sequence of ALSI from of a mutated Lupinus luteus ALSI; Ser638Asn substitution is indicated in bold and underlined MAATTTPNPAFTPLPSSSSKQIPRFTLPFSTFPSLRRRSLRITSSISSNPKLPAPRAT STAAAAPTLTTESFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGASMEIHQALT RSSTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADALLDSVPI VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRP GPVLIDIPKDIQQQLAVPNWDQPIRLTGYMSRLPKSPDEKLLHQIVRLISESKKPVLY VGGGSLDSSKELRKFVELTGIPVASTLMGLGSYPVGDDKSLQMLGMHGTVYANYAVDK SDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALEG INRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRISPQYAIQVLDELTNGNA IVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIWDIDG DGSFIMNVQELATIRVENLPVKILLLNNQHLGMWQWEDRFYKANRAHTYLGNPSNEN EIFPNMLMFADACGIPAARVTKKDEVSAAIQKMLDTPGPYLLEVIVPHQEHVLPMIPN NGSFQDVITEGDGRIMLLPTETDCIFELNFHLFSEQ ID NO:9 amino acid sequence of ALSI from of a mutated Lupinus albus ALS1; AlalO7Thr substitution is indicated in bold and underlined MAATTTPNPAFTPLPSSSSKQIPRFTLPFSTFPSLRRRSLRITSSISSNPKLPAPRAT STAAAAPTLTTESFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGTSMEIHQALT RSSTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADALLDSVPI VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRWNEAFFLATSGRP GPVLIDIPKDIQQQLAVPNWDQPIRLTGYMSRLPKSPDEKLLHQIVRLISESKKPVLY VGGGSLDSSKELRKFVELTGIPVASTLMGLGSYPVGDDKSLQMLGMHGTVYANYAVDK SDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADLKLALEG INRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRISPQYAIQVLDELTNGNA IVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIWDIDG DGSFIMNVQELATIRVENLPVKILLLNNQHLGMWQWEDRFYKANRAHTYLGNPSNEN EIFPNMLMFADACGIPAARVTKKDEVSAAIQKMLDTPGPYLLEVIVPHQEHVLPMIPS NGSFQDVITEGDGRIMLLPTETDCIFELNFHLF

[0132] Also disclosed herein are nucleic acids encoding any of the mutated amino acid ALSI sequences disclosed herein.

[0133] For reference the nucleic acid encoding ALSI from L. angustifolius corresponds to SEQ ID NO: 10

[0134] SEQ ID NO:10 (nucleic acid sequence from wild-type L. angustifolius encoding ALSI) ATGGCGGCCACTACCACTCCAAATCCCACATTTACCCCTCTCCCTTCTTCTTCTTCAAAG CAAATTCTCCGTTTTACCCTCCCTGTAACCTCATTCCCCTCTATACGACGTCGTTCTCTT CGAATCACCAGTTCCCTCTCCAGTAACCCTAAAATACCTGCACCACGCGCCACCTCCACC GCCGCCGCCGCTCCAACCCTAACAACTGAGCCTTTCATTTCCCGGTTTGCCCCTGACGAG CCACGTAAGGGCTCCGACATCCTCGTCGAAGCTCTTGAGCGGCAAGGTGTCACCAATGTC TTCGCTTACCCTGGCGGCGCGTCGATGGAGATTCACCAGGCCCTCACGCGCTCCAATACC ATCCGCAATATCCTCCCTCGCCATGAACAGGGTGGAATCTTCGCCGCCGAGGGCTACGCG CGCTCCTCCGGCCTTCCTGGTGTCTGCATGGCGACCTCTGGACCTGGCGCCACCAATCTC GTCAGCGGTCTCGCCGACGCGCTCCTCGACAGTGTTCCGATCGTGGCAATCACCGGACAA GTTCCGCGGCGAATGATCGGTACGGACGCTTTTCAAGAAACTCCGATTGTTGAGGTAACG AGATCAATCACTAAGCATAATTATCTTGTTCTTGATATTGATGATATTCCTAGGGTTGTG AATGAGGCATTTTTTTTAGCAACTTCTGGAAGGCCTGGTCCTGTGTTGATTGATGTTCCT AAAGATATTCAGCAACAACTTGCTGTTCCAAATTGGGATCAACCAATTAGGTTAACTGGA TATGTGTCTAGATTGCCTAAATCCCCTGATGAGAAACATTTACACCAAATTGTGAGGTTG ATTTCGGAATCTAAGAAACCGGTTTTGTATGTTGGTGGTGGTAGTTTGGATTCGAGTGAG GAATTGAGGAAATTCGTAGAGCTTACTGGGATACCTGTTGCTAGTACCTTGATGGGTTTA GGATCATACCCTCTCGGTGATGAGAAGTCACTTCAAATGCTTGGAATGCATGGGACCGTG TATGCGAATTATGCTGTTGATAAGAGTGATTTGCTGCTTGCATTCGGGGTTAGGTTTGAT GATCGTGTGACAGGGAAGCTTGAGGCTTTTGCTAGTCGTGCGAAAATTGTTCACATTGAT ATTGATTCGGCTGAGATTGGGAAAAACAAACAGCCACATGTGTCTGTTTGTGCGGACTTG AAGGTGGCTTTGGAAGGGATTAATAGGATATTGGAGAGCAAAGGGATCAAGGATAAAGTT GATTTTCGAGGTTGGAGAGAAGAGCTGAATGAGCAAAAGGTCAAATTTCCATTGAGTTTT AAGACATTCGAAGATCGTATTTCTCCACAGTATGCTATTCAGGTTTTGGATGAGCTGACG AATGGAAATGCTATCGTAAGTACTGGTGTTGGACAGCATCAGATGTGGGCTGCTCAGTTT TACAAATACAAGAGACCTAGGCAGTGGTTAACATCTGGTGGTCTTGGTGCTATGGGTTTT GGATTGCCTGCTGCCATTGGAGCTGCCGTAGCTAACCCGGGCGCTATTGTAGTTGACATT GACGGGGATGGAAGTTTTATAATGAATGTTCAAGAGCTAGCCACCATAAGGGTGGAGAAT CTCCCTGTTAAGGTATTATTGTTGAATAATCAACACTTGGGTATGGTTGTTCAGTGGGAG GACCGCTTTTACAAGGCTAATAGAGCTCACACCTATCTGGGAAACCCAGCAAATGAGATT GAGATTTTCCCAAATATGTTGAAGTTTGCAGATGCGTGTGGAATACCAGCAGCTCGTGTG ACGAAGAAAGACGAAGTCACTGCAGCAATTCAGAAAATGTTGGACACCCCTGGCCCCTAC CTTCTTGATGTCATTGTACCCCATCAAGAGCATGTCTTGCCTATGATTCCTAGTAACGGA TCCTTCCAGGACGTGATAACCGAGGGCGATGGTAGAATAAGTTATSEQ ID NO: 11 (nucleic acid sequence encoding mutated L. angustifolius encoding ALS1 comprising a Ser638Asn substitution G^A transversion shown as bold and underlined; nucleotide 1,913) ATGGCGGCCACTACCACTCCAAATCCCACATTTACCCCTCTCCCTTCTTCTTCTTCAAAG CAAATTCTCCGTTTTACCCTCCCTGTAACCTCATTCCCCTCTATACGACGTCGTTCTCTT CGAATCACCAGTTCCCTCTCCAGTAACCCTAAAATACCTGCACCACGCGCCACCTCCACC GCCGCCGCCGCTCCAACCCTAACAACTGAGCCTTTCATTTCCCGGTTTGCCCCTGACGAG CCACGTAAGGGCTCCGACATCCTCGTCGAAGCTCTTGAGCGGCAAGGTGTCACCAATGTC TTCGCTTACCCTGGCGGCGCGTCGATGGAGATTCACCAGGCCCTCACGCGCTCCAATACC ATCCGCAATATCCTCCCTCGCCATGAACAGGGTGGAATCTTCGCCGCCGAGGGCTACGCG CGCTCCTCCGGCCTTCCTGGTGTCTGCATGGCGACCTCTGGACCTGGCGCCACCAATCTC GTCAGCGGTCTCGCCGACGCGCTCCTCGACAGTGTTCCGATCGTGGCAATCACCGGACAA GTTCCGCGGCGAATGATCGGTACGGACGCTTTTCAAGAAACTCCGATTGTTGAGGTAACG AGATCAATCACTAAGCATAATTATCTTGTTCTTGATATTGATGATATTCCTAGGGTTGTG AATGAGGCATTTTTTTTAGCAACTTCTGGAAGGCCTGGTCCTGTGTTGATTGATGTTCCT AAAGATATTCAGCAACAACTTGCTGTTCCAAATTGGGATCAACCAATTAGGTTAACTGGA TATGTGTCTAGATTGCCTAAATCCCCTGATGAGAAACATTTACACCAAATTGTGAGGTTG ATTTCGGAATCTAAGAAACCGGTTTTGTATGTTGGTGGTGGTAGTTTGGATTCGAGTGAG GAATTGAGGAAATTCGTAGAGCTTACTGGGATACCTGTTGCTAGTACCTTGATGGGTTTA GGATCATACCCTCTCGGTGATGAGAAGTCACTTCAAATGCTTGGAATGCATGGGACCGTG TATGCGAATTATGCTGTTGATAAGAGTGATTTGCTGCTTGCATTCGGGGTTAGGTTTGAT GATCGTGTGACAGGGAAGCTTGAGGCTTTTGCTAGTCGTGCGAAAATTGTTCACATTGAT ATTGATTCGGCTGAGATTGGGAAAAACAAACAGCCACATGTGTCTGTTTGTGCGGACTTG AAGGTGGCTTTGGAAGGGATTAATAGGATATTGGAGAGCAAAGGGATCAAGGATAAAGTT GATTTTCGAGGTTGGAGAGAAGAGCTGAATGAGCAAAAGGTCAAATTTCCATTGAGTTTT AAGACATTCGAAGATCGTATTTCTCCACAGTATGCTATTCAGGTTTTGGATGAGCTGACG AATGGAAATGCTATCGTAAGTACTGGTGTTGGACAGCATCAGATGTGGGCTGCTCAGTTT TACAAATACAAGAGACCTAGGCAGTGGTTAACATCTGGTGGTCTTGGTGCTATGGGTTTT GGATTGCCTGCTGCCATTGGAGCTGCCGTAGCTAACCCGGGCGCTATTGTAGTTGACATT GACGGGGATGGAAGTTTTATAATGAATGTTCAAGAGCTAGCCACCATAAGGGTGGAGAAT CTCCCTGTTAAGGTATTATTGTTGAATAATCAACACTTGGGTATGGTTGTTCAGTGGGAG GACCGCTTTTACAAGGCTAATAGAGCTCACACCTATCTGGGAAACCCAGCAAATGAGATT GAGATTTTCCCAAATATGTTGAAGTTTGCAGATGCGTGTGGAATACCAGCAGCTCGTGTG ACGAAGAAAGACGAAGTCACTGCAGCAATTCAGAAAATGTTGGACACCCCTGGCCCCTAC CTTCTTGATGTCATTGTACCCCATCAAGAGCATGTCTTGCCTATGATTCCTAATAACGGA TCCTTCCAGGACGTGATAACCGAGGGCGATGGTAGAATAAGTTAT SEQ ID NO: 12 (nucleic acid sequence encoding mutated L. angustifolius encoding ALS1 comprising a AlalO7Thr substitution G^A transversion shown as bold and underlined; nucleotide 319) ATGGCGGCCACTACCACTCCAAATCCCACATTTACCCCTCTCCCTTCTTCTTCTTCAAAG CAAATTCTCCGTTTTACCCTCCCTGTAACCTCATTCCCCTCTATACGACGTCGTTCTCTT CGAATCACCAGTTCCCTCTCCAGTAACCCTAAAATACCTGCACCACGCGCCACCTCCACC GCCGCCGCCGCTCCAACCCTAACAACTGAGCCTTTCATTTCCCGGTTTGCCCCTGACGAG CCACGTAAGGGCTCCGACATCCTCGTCGAAGCTCTTGAGCGGCAAGGTGTCACCAATGTC TTCGCTTACCCTGGCGGC CGTCGATGGAGATTCACCAGGCCCTCACGCGCTCCAATACC ATCCGCAATATCCTCCCTCGCCATGAACAGGGTGGAATCTTCGCCGCCGAGGGCTACGCG CGCTCCTCCGGCCTTCCTGGTGTCTGCATGGCGACCTCTGGACCTGGCGCCACCAATCTC GTCAGCGGTCTCGCCGACGCGCTCCTCGACAGTGTTCCGATCGTGGCAATCACCGGACAA GTTCCGCGGCGAATGATCGGTACGGACGCTTTTCAAGAAACTCCGATTGTTGAGGTAACG AGATCAATCACTAAGCATAATTATCTTGTTCTTGATATTGATGATATTCCTAGGGTTGTG AATGAGGCATTTTTTTTAGCAACTTCTGGAAGGCCTGGTCCTGTGTTGATTGATGTTCCT AAAGATATTCAGCAACAACTTGCTGTTCCAAATTGGGATCAACCAATTAGGTTAACTGGA TATGTGTCTAGATTGCCTAAATCCCCTGATGAGAAACATTTACACCAAATTGTGAGGTTG ATTTCGGAATCTAAGAAACCGGTTTTGTATGTTGGTGGTGGTAGTTTGGATTCGAGTGAG GAATTGAGGAAATTCGTAGAGCTTACTGGGATACCTGTTGCTAGTACCTTGATGGGTTTA GGATCATACCCTCTCGGTGATGAGAAGTCACTTCAAATGCTTGGAATGCATGGGACCGTG TATGCGAATTATGCTGTTGATAAGAGTGATTTGCTGCTTGCATTCGGGGTTAGGTTTGATGATCGTGTGACAGGGAAGCTTGAGGCTTTTGCTAGTCGTGCGAAAATTGTTCACATTGAT ATTGATTCGGCTGAGATTGGGAAAAACAAACAGCCACATGTGTCTGTTTGTGCGGACTTG AAGGTGGCTTTGGAAGGGATTAATAGGATATTGGAGAGCAAAGGGATCAAGGATAAAGTT GATTTTCGAGGTTGGAGAGAAGAGCTGAATGAGCAAAAGGTCAAATTTCCATTGAGTTTT AAGACATTCGAAGATCGTATTTCTCCACAGTATGCTATTCAGGTTTTGGATGAGCTGACG AATGGAAATGCTATCGTAAGTACTGGTGTTGGACAGCATCAGATGTGGGCTGCTCAGTTT TACAAATACAAGAGACCTAGGCAGTGGTTAACATCTGGTGGTCTTGGTGCTATGGGTTTT GGATTGCCTGCTGCCATTGGAGCTGCCGTAGCTAACCCGGGCGCTATTGTAGTTGACATT GACGGGGATGGAAGTTTTATAATGAATGTTCAAGAGCTAGCCACCATAAGGGTGGAGAAT CTCCCTGTTAAGGTATTATTGTTGAATAATCAACACTTGGGTATGGTTGTTCAGTGGGAG GACCGCTTTTACAAGGCTAATAGAGCTCACACCTATCTGGGAAACCCAGCAAATGAGATT GAGATTTTCCCAAATATGTTGAAGTTTGCAGATGCGTGTGGAATACCAGCAGCTCGTGTG ACGAAGAAAGACGAAGTCACTGCAGCAATTCAGAAAATGTTGGACACCCCTGGCCCCTAC CTTCTTGATGTCATTGTACCCCATCAAGAGCATGTCTTGCCTATGATTCCTAGTAACGGA TCCTTCCAGGACGTGATAACCGAGGGCGATGGTAGAATAAGTTAT

[0135] While the above-referenced nucleic acid mutated sequences encoding ALS1 reflect mutations to naturally occurring ALS1 sequences, the skilled person will appreciate that, due to the redundancy of the genetic code, many non-naturally occurring nucleic acid sequences encoding the amino acid sequences of the mutated ALS1 proteins disclosed herein can envisioned readily. For example, in one example, by reverse translation the amino acid sequence corresponding to SEQ ID NO:3 is encoded by the nucleic acid sequence corresponding to:

[0136] SEQ ID NO: 13 (variant, non-naturally occurring nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3, a mutated ALS I from Lupinus angustifolius comprising an Ala107Thr substitution.

[0137] ATGGCGGCGACCACCACCCCGAACCCGACCTTTACCCCGCTGCCGAGCAGCAGCAGCAAA CAGATTCTGCGCTTTACCCTGCCGGTGACCAGCTTTCCGAGCATTCGCCGCCGCAGCCTG CGCATTACCAGCAGCCTGAGCAGCAACCCGAAAATTCCGGCGCCGCGCGCGACCAGCACC GCGGCGGCGGCGCCGACCCTGACCACCGAACCGTTTATTAGCCGCTTTGCGCCGGATGAA CCGCGCAAAGGCAGCGATATTCTGGTGGAAGCGCTGGAACGCCAGGGCGTGACCAACGTG TTTGCGTATCCGGGCGGCACCAGCATGGAAATTCATCAGGCGCTGACCCGCAGCAACACC ATTCGCAACATTCTGCCGCGCCATGAACAGGGCGGCATTTTTGCGGCGGAAGGCTATGCG CGCAGCAGCGGCCTGCCGGGCGTGTGCATGGCGACCAGCGGCCCGGGCGCGACCAACCTG GTGAGCGGCCTGGCGGATGCGCTGCTGGATAGCGTGCCGATTGTGGCGATTACCGGCCAG GTGCCGCGCCGCATGATTGGCACCGATGCGTTTCAGGAAACCCCGATTGTGGAAGTGACC CGCAGCATTACCAAACATAACTATCTGGTGCTGGATATTGATGATATTCCGCGCGTGGTG AACGAAGCGTTTTTTCTGGCGACCAGCGGCCGCCCGGGCCCGGTGCTGATTGATGTGCCG AAAGATATTCAGCAGCAGCTGGCGGTGCCGAACTGGGATCAGCCGATTCGCCTGACCGGC TATGTGAGCCGCCTGCCGAAAAGCCCGGATGAAAAACATCTGCATCAGATTGTGCGCCTG ATTAGCGAAAGCAAAAAACCGGTGCTGTATGTGGGCGGCGGCAGCCTGGATAGCAGCGAA GAACTGCGCAAATTTGTGGAACTGACCGGCATTCCGGTGGCGAGCACCCTGATGGGCCTG GGCAGCTATCCGCTGGGCGATGAAAAAAGCCTGCAGATGCTGGGCATGCATGGCACCGTG TATGCGAACTATGCGGTGGATAAAAGCGATCTGCTGCTGGCGTTTGGCGTGCGCTTTGAT GATCGCGTGACCGGCAAACTGGAAGCGTTTGCGAGCCGCGCGAAAATTGTGCATATTGAT ATTGATAGCGCGGAAATTGGCAAAAACAAACAGCCGCATGTGAGCGTGTGCGCGGATCTG AAAGTGGCGCTGGAAGGCATTAACCGCATTCTGGAAAGCAAAGGCATTAAAGATAAAGTG GATTTTCGCGGCTGGCGCGAAGAACTGAACGAACAGAAAGTGAAATTTCCGCTGAGCTTT AAAACCTTTGAAGATCGCATTAGCCCGCAGTATGCGATTCAGGTGCTGGATGAACTGACC AACGGCAACGCGATTGTGAGCACCGGCGTGGGCCAGCATCAGATGTGGGCGGCGCAGTTT TATAAATATAAACGCCCGCGCCAGTGGCTGACCAGCGGCGGCCTGGGCGCGATGGGCTTT GGCCTGCCGGCGGCGATTGGCGCGGCGGTGGCGAACCCGGGCGCGATTGTGGTGGATATT GATGGCGATGGCAGCTTTATTATGAACGTGCAGGAACTGGCGACCATTCGCGTGGAAAAC CTGCCGGTGAAAGTGCTGCTGCTGAACAACCAGCATCTGGGCATGGTGGTGCAGTGGGAA GATCGCTTTTATAAAGCGAACCGCGCGCATACCTATCTGGGCAACCCGGCGAACGAAATTGAAATTTTTCCGAACATGCTGAAATTTGCGGATGCGTGCGGCATTCCGGCGGCGCGCGTG ACCAAAAAAGATGAAGTGACCGCGGCGATTCAGAAAATGCTGGATACCCCGGGCCCGTAT CTGCTGGATGTGATTGTGCCGCATCAGGAACATGTGCTGCCGATGATTCCGAGCAACGGC AGCTTTCAGGATGTGATTACCGAAGGCGATGGCCGCATTAGCTAT

[0138] In some examples, a mutated ALS1 comprises a mutation that confers tolerance to IMI herbicides and to SU herbicides. Nucleic acids encoding such mutated ALS1 and the nucleic acids encoding them are set out below.

[0139] SEQ ID NO: 14 (nucleic acid sequence encoding mutated L. angustifolius encoding ALS1 comprising a Prol82Ser substitution C^T transversion shown as bold and underlined; nucleotide 544) ATGGCGGCCACTACCACTCCAAATCCCACATTTACCCCTCTCCCTTCTTCTTCTTCAAAG CAAATTCTCCGTTTTACCCTCCCTGTAACCTCATTCCCCTCTATACGACGTCGTTCTCTT CGAATCACCAGTTCCCTCTCCAGTAACCCTAAAATACCTGCACCACGCGCCACCTCCACC GCCGCCGCCGCTCCAACCCTAACAACTGAGCCTTTCATTTCCCGGTTTGCCCCTGACGAG CCACGTAAGGGCTCCGACATCCTCGTCGAAGCTCTTGAGCGGCAAGGTGTCACCAATGTC TTCGCTTACCCTGGCGGCGCGTCGATGGAGATTCACCAGGCCCTCACGCGCTCCAATACC ATCCGCAATATCCTCCCTCGCCATGAACAGGGTGGAATCTTCGCCGCCGAGGGCTACGCG CGCTCCTCCGGCCTTCCTGGTGTCTGCATGGCGACCTCTGGACCTGGCGCCACCAATCTC GTCAGCGGTCTCGCCGACGCGCTCCTCGACAGTGTTCCGATCGTGGCAATCACCGGACAA GTTTCGCGGCGAATGATCGGTACGGACGCTTTTCAAGAAACTCCGATTGTTGAGGTAACG AGATCAATCACTAAGCATAATTATCTTGTTCTTGATATTGATGATATTCCTAGGGTTGTG AATGAGGCATTTTTTTTAGCAACTTCTGGAAGGCCTGGTCCTGTGTTGATTGATGTTCCT AAAGATATTCAGCAACAACTTGCTGTTCCAAATTGGGATCAACCAATTAGGTTAACTGGA TATGTGTCTAGATTGCCTAAATCCCCTGATGAGAAACATTTACACCAAATTGTGAGGTTG ATTTCGGAATCTAAGAAACCGGTTTTGTATGTTGGTGGTGGTAGTTTGGATTCGAGTGAG GAATTGAGGAAATTCGTAGAGCTTACTGGGATACCTGTTGCTAGTACCTTGATGGGTTTA GGATCATACCCTCTCGGTGATGAGAAGTCACTTCAAATGCTTGGAATGCATGGGACCGTG TATGCGAATTATGCTGTTGATAAGAGTGATTTGCTGCTTGCATTCGGGGTTAGGTTTGAT GATCGTGTGACAGGGAAGCTTGAGGCTTTTGCTAGTCGTGCGAAAATTGTTCACATTGAT ATTGATTCGGCTGAGATTGGGAAAAACAAACAGCCACATGTGTCTGTTTGTGCGGACTTG AAGGTGGCTTTGGAAGGGATTAATAGGATATTGGAGAGCAAAGGGATCAAGGATAAAGTT GATTTTCGAGGTTGGAGAGAAGAGCTGAATGAGCAAAAGGTCAAATTTCCATTGAGTTTT AAGACATTCGAAGATCGTATTTCTCCACAGTATGCTATTCAGGTTTTGGATGAGCTGACG AATGGAAATGCTATCGTAAGTACTGGTGTTGGACAGCATCAGATGTGGGCTGCTCAGTTT TACAAATACAAGAGACCTAGGCAGTGGTTAACATCTGGTGGTCTTGGTGCTATGGGTTTT GGATTGCCTGCTGCCATTGGAGCTGCCGTAGCTAACCCGGGCGCTATTGTAGTTGACATT GACGGGGATGGAAGTTTTATAATGAATGTTCAAGAGCTAGCCACCATAAGGGTGGAGAAT CTCCCTGTTAAGGTATTATTGTTGAATAATCAACACTTGGGTATGGTTGTTCAGTGGGAG GACCGCTTTTACAAGGCTAATAGAGCTCACACCTATCTGGGAAACCCAGCAAATGAGATT GAGATTTTCCCAAATATGTTGAAGTTTGCAGATGCGTGTGGAATACCAGCAGCTCGTGTG ACGAAGAAAGACGAAGTCACTGCAGCAATTCAGAAAATGTTGGACACCCCTGGCCCCTAC CTTCTTGATGTCATTGTACCCCATCAAGAGCATGTCTTGCCTATGATTCCTAGTAACGGA TCCTTCCAGGACGTGATAACCGAGGGCGATGGTAGAATAAGTTAT SEQ ID NO: 15 amino acid sequence of ALS I from of a mutated Lupinus angustifolius ALS1; Prol82Ser substitution is indicated in bold and underlined MAATTTPNPTFTPLPSSSSKQILRFTLPVTSFPSIRRRSLRITSSLSSNPKIPA PRATSTAAAAPTLTTEPFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGAS MEIHQALTRSNTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVS GLADALLDSVPIVAITGQVSRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDI PRVVNEAFFLATSGRPGPVLIDVPKDIQQQLAVPNWDQPIRLTGYVSRLPKSPD EKHLHQIVRLISESKKPVLYVGGGSLDSSEELRKFVELTGIPVASTLMGLGSYP LGDEKSLQMLGMHGTVYANYAVDKSDLLLAFGVRFDDRVTGKLEAFASRAKIVH IDIDSAEIGKNKQPHVSVCADLKVALEGINRILESKGIKDKVDFRGWREELNEQ KVKFPLSFKTFEDRISPQYAIQVLDELTNGNAIVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPGAIVVDIDGDGSFIMNVQELATIRVEN LPVKVLLLNNQHLGMVVQWEDRFYKANRAHTYLGNPANEIEI FPNMLKFADACG IPAARVTKKDEVTAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPSNGSFQDVITE GDGRISY SEQ ID NO: 16 (nucleic acid sequence encoding mutated L. angustifolius encoding ALS1 comprising a Prol82Leu substitution C^T transversion shown as bold and underlined; nucleotide 545) ATGGCGGCCACTACCACTCCAAATCCCACATTTACCCCTCTCCCTTCTTCTTCTTCAAAG CAAATTCTCCGTTTTACCCTCCCTGTAACCTCATTCCCCTCTATACGACGTCGTTCTCTT CGAATCACCAGTTCCCTCTCCAGTAACCCTAAAATACCTGCACCACGCGCCACCTCCACC GCCGCCGCCGCTCCAACCCTAACAACTGAGCCTTTCATTTCCCGGTTTGCCCCTGACGAG CCACGTAAGGGCTCCGACATCCTCGTCGAAGCTCTTGAGCGGCAAGGTGTCACCAATGTC TTCGCTTACCCTGGCGGCGCGTCGATGGAGATTCACCAGGCCCTCACGCGCTCCAATACC ATCCGCAATATCCTCCCTCGCCATGAACAGGGTGGAATCTTCGCCGCCGAGGGCTACGCG CGCTCCTCCGGCCTTCCTGGTGTCTGCATGGCGACCTCTGGACCTGGCGCCACCAATCTC GTCAGCGGTCTCGCCGACGCGCTCCTCGACAGTGTTCCGATCGTGGCAATCACCGGACAA GTTCTGCGGCGAATGATCGGTACGGACGCTTTTCAAGAAACTCCGATTGTTGAGGTAACG AGATCAATCACTAAGCATAATTATCTTGTTCTTGATATTGATGATATTCCTAGGGTTGTG AATGAGGCATTTTTTTTAGCAACTTCTGGAAGGCCTGGTCCTGTGTTGATTGATGTTCCT AAAGATATTCAGCAACAACTTGCTGTTCCAAATTGGGATCAACCAATTAGGTTAACTGGA TATGTGTCTAGATTGCCTAAATCCCCTGATGAGAAACATTTACACCAAATTGTGAGGTTG ATTTCGGAATCTAAGAAACCGGTTTTGTATGTTGGTGGTGGTAGTTTGGATTCGAGTGAG GAATTGAGGAAATTCGTAGAGCTTACTGGGATACCTGTTGCTAGTACCTTGATGGGTTTA GGATCATACCCTCTCGGTGATGAGAAGTCACTTCAAATGCTTGGAATGCATGGGACCGTG TATGCGAATTATGCTGTTGATAAGAGTGATTTGCTGCTTGCATTCGGGGTTAGGTTTGAT GATCGTGTGACAGGGAAGCTTGAGGCTTTTGCTAGTCGTGCGAAAATTGTTCACATTGAT ATTGATTCGGCTGAGATTGGGAAAAACAAACAGCCACATGTGTCTGTTTGTGCGGACTTG AAGGTGGCTTTGGAAGGGATTAATAGGATATTGGAGAGCAAAGGGATCAAGGATAAAGTT GATTTTCGAGGTTGGAGAGAAGAGCTGAATGAGCAAAAGGTCAAATTTCCATTGAGTTTT AAGACATTCGAAGATCGTATTTCTCCACAGTATGCTATTCAGGTTTTGGATGAGCTGACG AATGGAAATGCTATCGTAAGTACTGGTGTTGGACAGCATCAGATGTGGGCTGCTCAGTTT TACAAATACAAGAGACCTAGGCAGTGGTTAACATCTGGTGGTCTTGGTGCTATGGGTTTT GGATTGCCTGCTGCCATTGGAGCTGCCGTAGCTAACCCGGGCGCTATTGTAGTTGACATT GACGGGGATGGAAGTTTTATAATGAATGTTCAAGAGCTAGCCACCATAAGGGTGGAGAAT CTCCCTGTTAAGGTATTATTGTTGAATAATCAACACTTGGGTATGGTTGTTCAGTGGGAG GACCGCTTTTACAAGGCTAATAGAGCTCACACCTATCTGGGAAACCCAGCAAATGAGATT GAGATTTTCCCAAATATGTTGAAGTTTGCAGATGCGTGTGGAATACCAGCAGCTCGTGTG ACGAAGAAAGACGAAGTCACTGCAGCAATTCAGAAAATGTTGGACACCCCTGGCCCCTAC CTTCTTGATGTCATTGTACCCCATCAAGAGCATGTCTTGCCTATGATTCCTAGTAACGGA TCCTTCCAGGACGTGATAACCGAGGGCGATGGTAGAATAAGTTAT

[0140] SEQ ID NO: 17 amino acid sequence of ALS I from of a mutated Lupinus angustifolius ALS1; Prol82Leu substitution is indicated in bold and underlined MAATTTPNPTFTPLPSSSSKQILRFTLPVTSFPSIRRRSLRITSSLSSNPKIPAPR ATSTAAAAPTLTTEPFISRFAPDEPRKGSDILVEALERQGVTNVFAYPGGASMEIH QALTRSNTIRNILPRHEQGGIFAAEGYARSSGLPGVCMATSGPGATNLVSGLADAL LDSVPIVAITGQVLRRMIGTDAFQETPIVEVTRSITKHNYLVLDIDDIPRVVNEAF FLATSGRPGPVLIDVPKDIQQQLAVPNWDQPIRLTGYVSRLPKSPDEKHLHQIVRL ISESKKPVLYVGGGSLDSSEELRKFVELTGIPVASTLMGLGSYPLGDEKSLQMLGM HGTVYANYAVDKSDLLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQP HVSVCADLKVALEGINRILESKGIKDKVDFRGWREELNEQKVKFPLSFKTFEDRIS PQYAIQVLDELTNGNAIVSTGVGQHQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPA AIGAAVANPGAIVVDIDGDGSFIMNVQELATIRVENLPVKVLLLNNQHLGMVVQWE DRFYKANRAHTYLGNPANEIEI FPNMLKFADACGIPAARVTKKDEVTAAIQKMLDT PGPYLLDVIVPHQEHVLPMIPSNGSFQDVITEGDGRISYThe skilled person in the art will appreciate that a myriad of such nucleotide sequence variations encoding the same amino acid sequence are possible due to the redundancy of the genetic code and are thereby encompassed by the present disclosure.

[0141] As used herein, a "nucleic acid" or "nucleic acid molecule" means a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA, and cDNA. It may be DNA or RNA of cellular, genomic or synthetic origin, for example made on an automated synthesizer, and may be combined with carbohydrate, lipids, protein or other materials, labelled with fluorescent or other groups, or attached to a solid support to perform a particular activity defined herein, or comprise one or more modified nucleotides not found in nature, well known to those skilled in the art. The polymer may be single-stranded, essentially double-stranded or partly double-stranded. Basepairing as used herein refers to standard basepairing between nucleotides, including G: U basepairs.

[0142] By "isolated nucleic acid" is meant a nucleic acid which has generally been separated from the nucleic acid sequences with which it is associated or linked in its native state, if the nucleic acid is found in nature. Preferably, the isolated nucleic acid is at least 90% free from other components with which it is naturally associated, if it is found in nature.

[0143] As used herein, the term "gene" includes any deoxyribonucleotide sequence which includes a protein coding region or which is transcribed in a cell but not translated, as well as associated non-coding and regulatory regions. Such associated regions are typically located adjacent to the coding region or the transcribed region on both the 5’ and 3’ ends for a distance of about 2 kb on either side. In this regard, the gene may include control signals such as promoters, enhancers, termination and / or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals in which case the gene is referred to as a "chimeric gene". The sequences which are located 5’ of the coding region and which are present on the mRNA are referred to as 5’ nontranslated sequences. The sequences which are located 3’ or downstream of the coding region and which are present on the mRNA are referred to as 3’ non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene.

[0144] A genomic form or clone of a gene containing the transcribed region may be interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences", which may be either homologous or heterologous with respect to the “exons” of the gene. An "intron" as used herein is a segment of a gene which is transcribed as part of a primary RNA transcript but is not present in the mature mRNA molecule. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA). Introns may contain regulatory elements such as enhancers. " Exons" as used herein refer to the DNA regionscorresponding to the RNA sequences which are present in the mature mRNA or the mature RNA molecule in cases where the RNA molecule is not translated. An mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above. A gene may be introduced into an appropriate vector for extrachromosomal maintenance in a cell or, preferably, for integration into the host genome.

[0145] The term "endogenous" is used herein to refer to a substance that is normally present or produced in an unmodified plant at the same developmental stage as the plant under investigation. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, "recombinant nucleic acid molecule" or variations thereof refer to a nucleic acid molecule which has been constructed or modified by recombinant DNA technology. The terms "exogenous nucleic acid" and the like refer to any nucleic acid which is introduced into the genome of a cell by experimental manipulations.

[0146] In some examples an isolated nucleic acid disclosed herein is a genomic DNA comprising an exon comprising a mutation disclosed herein or a cDNA. In other examples the nucleic acid is an RNA.

[0147] In some examples a nucleic acid disclosed herein is a provided as a plant expression vector. Suitable examples of plant expression vectors include, but are not limited to, a recombinant virus vector or a Ti plasmid vector. In some examples a recombinant virus vector is a Cauliflower Mosaic Virus (CaMV) vector. Such vectors can be propagated in recombinant prokaryotic or eukaryotic hosts cells. In some examples a recombinant prokaryotic cell is a recombinant Agrobacterium tumefaciens cell.

[0148] With reference to plant expression vectors, the present disclosure refers to elements which are operably connected or linked. " Operably connected" or "operably linked" and the like refer to a linkage of polynucleotide elements in a functional relationship. Typically, operably connected nucleic acid sequences are contiguously linked and, where necessary to join two protein coding regions, contiguous and in reading frame. A coding sequence is "operably connected to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single RNA, which if translated is then translated into a single polypeptide having amino acids derived from both

[0149] The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.As used herein, the term "cis-acting sequence", "cis-acting element" or "cis-regulatory region" or "regulatory region" or similar term shall be taken to mean any sequence of nucleotides, which when positioned appropriately and connected relative to an expressible genetic sequence, is capable of regulating, at least in part, the expression of the genetic sequence. Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and / or cell-type-specificity and / or developmental specificity of a gene sequence at the transcriptional or post-transcriptional level. In preferred embodiments of the present invention, the cis-acting sequence is an activator sequence that enhances or stimulates the expression of an expressible genetic sequence.

[0150] " Operably connecting" a promoter or enhancer element to a transcribable polynucleotide means placing the transcribable polynucleotide (e.g., protein-encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription of that polynucleotide. In the construction of heterologous promoter / structural gene combinations, it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide which is approximately the same as the distance between that promoter and the protein coding region it controls in its natural setting; i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer etc) with respect to a transcribable polynucleotide to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived.

[0151] " Promoter" or "promoter sequence" as used herein refers to a region of a gene, generally upstream (5') of the RNA encoding region, which controls the initiation and level of transcription in the cell of interest. A "promoter" includes the transcriptional regulatory sequences of a classical genomic gene, such as a TATA box and CCAAT box sequences, as well as additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) that alter gene expression in response to developmental and / or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily (for example, some PolIII promoters), positioned upstream of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and / or to alter the timing or inducibility of expression of a structural gene to which it is operably connected." Constitutive promoter" refers to a promoter that directs expression of an operably linked transcribed sequence in many or all tissues of an organism such as a plant. The term constitutive as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in level is often detectable. " Selective expression" as used herein refers to expression almost exclusively in specific organs of, for example, the plant, such as, for example, endosperm, embryo, leaves, fruit, tubers or root. In a preferred embodiment, a promoter is expressed selectively or preferentially in leaves and / or stems of a plant, preferably a cereal plant. Selective expression may therefore be contrasted with constitutive expression, which refers to expression in many or all tissues of a plant under most or all of the conditions experienced by the plant.

[0152] Selective expression may also result in compartmentation of the products of gene expression in specific plant tissues, organs or developmental stages. Compartmentation in specific subcellular locations such as the plastid, cytosol, vacuole, or apoplastic space may be achieved by the inclusion in the structure of the gene product of appropriate signals, eg. a signal peptide, for transport to the required cellular compartment, or in the case of the semi-autonomous organelles (plastids and mitochondria) by integration of the transgene with appropriate regulatory sequences directly into the organelle genome.

[0153] A "tissue-specific promoter" or "organ-specific promoter" is a promoter that is preferentially expressed in one tissue or organ relative to many other tissues or organs, preferably most if not all other tissues or organs in, for example, a plant. Typically, the promoter is expressed at a level 10-fold higher in the specific tissue or organ than in other tissues or organs.

[0154] In an embodiment, the promoter is a stem-specific promoter or a promoter which directs gene expression in an aerial part of the plant (green tissue specific promoter) such as a ribulose- 1,5 -bisphosphate carboxylase oxygenase (RUBISCO) promoter.

[0155] Examples of stem-specific promoters include, but are not limited to those described in US 5,625,136.

[0156] The promoters contemplated by the present invention may be native to the host plant to be transformed or may be derived from an alternative source, where the region is functional in the host plant. Other sources include the Agrobacterium T-DNA genes, such as the promoters of genes for the biosynthesis of nopaline, octapine, mannopine, or other opine promoters, tissue specific promoters (see, e.g., US 5,459,252 and WO 91 / 13992); promoters from viruses (including host specific viruses), or partially or wholly synthetic promoters. Numerous promoters that are functional in dicotyledonous plants such as lupins are well known in the art; including various promoters isolated from plants and viruses such as the cauliflower mosaic virus promoter (CaMV 35S, 19S). Non-limiting methods for assessing promoter activity are disclosed in US 5,164,316.Alternatively or additionally, the promoter may be an inducible promoter or a developmentally regulated promoter which is capable of driving expression of the introduced polynucleotide at an appropriate developmental stage of the, for example, plant. Other cv.s-acting sequences which may be employed include transcriptional and / or translational enhancers. Enhancer regions are well known to persons skilled in the art, and can include an ATG translational initiation codon and adjacent sequences. When included, the initiation codon should be in phase with the reading frame of the coding sequence relating to the foreign or exogenous polynucleotide to ensure translation of the entire sequence if it is to be translated. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from a foreign or exogenous polynucleotide. The sequence can also be derived from the source of the promoter selected to drive transcription, and can be specifically modified so as to increase translation of the mRNA.

[0157] The nucleic acid construct of the present invention may comprise a 3' nontranslated sequence from about 50 to 1,000 nucleotide base pairs which may include a transcription termination sequence. A 3' non -translated sequence may contain a transcription termination signal which may or may not include a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing. A polyadenylation signal functions for addition of polyadenylic acid tracts to the 3' end of a mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. Transcription termination sequences which do not include a polyadenylation signal include terminators for Poll or PolIII RNA polymerase which comprise a run of four or more thymidines. Examples of suitable 3' non-translated sequences are the 3' transcribed non-translated regions containing a polyadenylation signal from an octopine synthase (ocs) gene or nopaline synthase (nos) gene of Agrobacterium tumefaciens. Suitable 3' non-translated sequences may also be derived from plant genes such as the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene, although other 3' elements known to those of skill in the art can also be employed.

[0158] As the DNA sequence inserted between the transcription initiation site and the start of the coding sequence, i.e., the untranslated 5’ leader sequence (5’UTR), can influence gene expression if it is translated as well as transcribed, one can also employ a particular leader sequence. Suitable leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence.

[0159] Also provided herein are oligonucleotides that may be used as hybridization probes, sequencing primers, and / or PCR primers. Such oligonucleotides may be used, for example, to determine a codon sequence at a particular position in a nucleic acid moleculeencoding an ALS1, for example, by allele specific PCR. Such oligonucleotides may be from about 15 to about 30, from about 20 to about 30, or from about 20-25 nucleotides in length.

[0160] Methods

[0161] Also provided herein are methods for for producing a genetically modified lupin hybrid plant, comprising breeding a genetically modified lupin plant disclosed herein with a second plant to obtain a genetically modified lupin hybrid plant, wherein the genetically modified lupin hybrid plant exhibits increased to an IMI herbicide or a SU herbicide.

[0162] In some examples such methods include selecting contacting progeny plants or plant cells with an IMI herbicide or a SU herbicide to verify tolerance of the lupin progeny plants as compared to the corresponding wild-type variety of the lupin plant.

[0163] Also provided herein are methods for cultivation of a genetically modified lupin plant that includes planting a seed obtained from a genetically modifed lupin plant disclosed herein, whereby the planted seed germinates to generate a seedling. Preferably, the seedling is further cultivated to obtain a mature genetically modified lupin plant, and harvesting the the mature genetically modified lupin plant and / or its seeds.

[0164] In some examples the method also includes applying an IMI herbicide or a SU herbicide one or more times over a cultivation period. In some examples, an IMI herbicide or a SU herbicide is applied up to two times over the total cultivation period, as needed to eliminate or reduce weed growth in proximity of cultivated genetically modified herbicide tolerant lupin plants. In some examples, a single application is applied and is sufficient to eliminate or reduced weed growth. In some examples the total cultivation period (dated from emergence) is about 28 weeks to about 33 weeks, e.g., 29 weeks, 30 weeks, 31 weeks, 32 weeks, or another total cultivation period from about 28 weeks to about 33 weeks. In some examples a IMI herbicide or a SU herbicide is first applied at about 1, 2, 3, or 4 weeks post-emergence. In some examples an IMI herbicide or a SU herbicide is first applied from around the time a lupin three leaf stage up to the beginning of stem elongation.

[0165] Suitable examples of IMI herbicides include, but are not limited to, imazapyr, imazapic (Plateau®), imazethapyr (Cheetah®), imazamox (Raptor®), imazamethabenz (Assert®), imazaquin (Scepter®), and combinations thereof. In some examples a suitable herbicide for a lupin cultivation method disclosed herein is Intercept®. Intercept® contains a combination of imazamox (CAS 114311-32-9) at 33 g / L and imazapyr (CAS 81334-34-1) at 15 g / L.

[0166] In some examples an IMI herbicide is Intercept® applied at a rate of about 300 ml / hectare to about 3000 ml / hectare, c.g, 400 ml / hectare, 500 ml / hectare, 600ml / hectare, 700 ml / hectare, 800 ml / hectare, 1000 ml / hectare, 1200 ml / hectare, 1300 ml / hectare, 1400 ml / hectare, 1500 ml / hectare, 1600 ml / hectare, 1700 ml / hectare, 1800 ml / hectare, 2000 ml / hectare, 2250 ml / hectare, 2500 ml / hectare, 2750 ml / hectare, or another application rate from about 750 ml / hectare to about 3000 ml / hectare. Intercept® contains a combination of imazamox (CAS 114311-32-9) at 33 g / L and imazapyr (CAS 81334-34-1) at 15 g / L. Herbicide tolerance will typically be evident by about four weeks post-treatment.

[0167] In some examples, genetically modified lupin plant having increased tolerance to an IMI herbicide or a SU herbicide refers to a lupin plant from a lupin plant line that exhibits at least 30% survivorship at 7 seeks post-application of an IMI herbicide or a SU herbicide, e.g., 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or another percentage survivorship from at least 30% to 100% survivorship, where survivorship is defined as (Number of plants post-treatment / Number of plants pre-treatment) x 100 at 7 weeks post application.

[0168] Suitable examples of SU herbicides include, but are not limited to, Metsulfuron-methyl (Metsulfuron), Nicosulfuron, Chlorsulfuron, Rimsulfuron, Imazosulfuron, Flazasulfuron, Halosulfuron-methyl, Primisulfuron-methyl, Prosulfuron, lodosulfuron-methyl-Na, and Tribenuron-methyl, and combinations thereof. In some examples the SU herbicide is Metsulfuron.

[0169] In some examples Metsulfuron is applied at a rate of about 5 g / hectare to about 10 g / hectare, e.g., 5.5 g / hectare, 6.0 g / hectare, 6.5 g / hectare, 7 g / hectare, 8 g / hectare, 8.5 g / hectare, 9.0 g / hectare, 9.5 g / hectare, or another application rate from about 5 g / hectare to about 10 g / hectare. Herbicide tolerance will typically be evident by about four weeks post-treatment.

[0170] EXAMPLES

[0171] Example 1 - Mutagenesis of Lupin seedlings

[0172] A forward mutagenesis screen was undertaken to identify Lupin seedlings tolerant to an IMI herbicide. Lupin seeds (100) were added to 75 ml of a 0.3% (v / v) ethylmethane sulfonate (EMS) solution and were shaken (100 RPM) for 16 hours. The seeds were then washed in 200 ml of water with shaking for 60 minutes for a total of five washes. Seeds were then planted and seedlings were screened for IMI tolerance as described in the Examples below.Example 2 - Confirmation of herbicide tolerance of field selected lupin lines in a controlled environment

[0173] The aim of the trial was to confirm the tolerance of field-selected single plant lupin lines, to Intercept® and Priority® herbicides under screenhouse conditions.

[0174] Materials and Methods

[0175] Lupin lines and herbicide treatments

[0176] The trial included 39 lupin lines for IMI herbicide (Intercept®) testing and 15 lines for Priority® testing. The testing was conducted in a screenhouse at DPIRD Northam fitted with overhead sprinklers for irrigation, during autumn-winter. Coyote M4 bulk (CM4 Lupin line) was used as a standard / control line for survivorship and biomass comparison for both the herbicides.

[0177] Table 1 - Herbicide Treatments

[0178] No. Herbicide Rate / Ha Adjuvant

[0179] 1 Intercept® 1500 ml Hasten 0.5% 2 Priority® 40 ml Uptake oil 0.5% 3 Untreated

[0180] Only for L41

[0181] (Coyote M4 Bulk)

[0182] Intercept® composition: imazamox 33g + imazapyr 15g / L

[0183]

[0184] Priority®composition: florasulam 200g / L

[0185] Lupin Herbicide rate selection

[0186] Due to a limited seed supply of the mutant lupin lines, only one rate of each herbicide (Intercept® and Priority®) was used in the trial. For Intercept®, the maximum registered rate for IMI-tolerant crops is 750 mL / ha, but no Coyote plants survived at this rate in a pilot pot trial. In the AGT field trial, Intercept® was applied at 650 mL / ha on 6 July and 800 mL / ha on 3 September 2023. A few plants survived, and their seeds were collected, making 39 tolerant lines part of trial described in this example. To better differentiate tolerance, the herbicide rate was doubled to 1500 mL / ha.

[0187] For Priority®, the label rate range is 15-25 mL / ha. At the maximum label rate (25 mL / ha) in the pilot pot trial, no Coyote plants survived. In a subsequent field trial, Priority® was applied once at 25 mL / ha. The surviving plants were stunted but produced some seeds. To avoid killing all plants in the subsequent trial described in this example, the herbicide rate was set at 40 mL / ha (1.6 times the maximum label rate) to help differentiate tolerance.Trial setup

[0188] Pots with a capacity of 2.4-liters were filled with loamy sand soil (pH CaCh, 4.7), suitable for growing lupins, sourced from a paddock in Northam. Five lupin seeds from each line were sown at a depth of 3 cm per pot on 24 May. Pots were irrigated three times daily for 3-5 minutes each time using automatic sprinklers. By 3 June, most of the lupin plants had emerged. Plant counts were recorded on 21 June, four weeks after sowing, prior to herbicide application.

[0189] Herbicide application

[0190] On 24 June, the pots were watered thoroughly to avoid the need for irrigation for 24-36 hours. Herbicide treatments were applied using an indoor cabinet sprayer with a moving boom, calibrated to deliver a spray volume of 100 L / ha at 300 kPa pressure, at the 4-leaf stage of the lupin plants. For optimal herbicide performance, the pots were kept in a glasshouse at 18-20°C for a day before being transferred to the screenhouse. Granular fertilizer was applied to all pots one week after herbicide application.

[0191] Observations and measurements

[0192] Visual assessments were conducted one week after herbicide treatment on 1 July 2024. Nine lupin lines that exhibited good tolerance to Intercept® had their plants counted per pot four weeks after treatment on 26 July 2024. For the 13 lines showing delayed recovery, plant counts were taken seven weeks after treatment on 16 August 2024. Survivorship (%) was calculated using the following formula:

[0193] Survivorship (%) =

[0194] (Number of plants post-treatment / Number of plants pre-treatment) x 100

[0195] Intercept® data was subjected to analysis of variance using GenStat 22ndedition.

[0196] Results and discussion:

[0197] One week after herbicide treatment, all lupin lines displayed symptoms on newly emerging leaves (5th to 7th leaves), including yellowing, shortening, and twisting of leaflets. Older leaves remained unaffected, while internodes on the main stem appeared shortened. These symptoms were consistent across both Intercept® and Priority® treatments.

[0198] By four weeks post-treatment, nine lupin lines showed a clear distinction of higher tolerance to Intercept® (Figure 1 and Table 2 ). By the 9th week, these lines had similar biomass and flowering levels to the untreated control (Figure 2).By the 7th (Table 3) and 9th week (Figure 3) after Intercept® treatment, an additional 13 lines showed good recovery, with approximately 50% biomass reduction compared to the untreated control. These lines experienced delayed flowering, but the majority are expected to set seeds. Line 38 (L38) appeared particularly promising.

[0199] All lupin lines treated with Priority® at 40 mL / ha died by the 7th week posttreatment (Figure 4 and Table 4). Priority® application at lower rates of 15-25 mL / ha might have resulted in survivors in some lupin lines.

[0200] Table 2 - Survivorship of nine lupi n mutant lines 4 weeks after intercept application as compared to untreated Coyote control (CM4 line.

[0201] Lupin Lupin Pre-treatment Post- treatment Survivorship Line Herbicide Rate / ha growth plant no per plant no per pot

[0202] (%) No stage pot 4 WAT

[0203] Date 21-Jun 26-Jul

[0204] L 1 Intercept® 1500 mL 4 leaves 5.0 3.8 75 b L2 Intercept® 1500 mL 4 leaves 5.0 4.8 95 ab L9 Intercept® 1500 mL 4 leaves 4.0 4.0 100 a L20 Intercept® 1500 mL 4 leaves 4.3 4.0 94 ab L26 Intercept® 1500 mL 4 leaves 4.8 4.8 100 a L31 Intercept® 1500 mL 4 leaves 4.8 4.8 100 a L32 Intercept® 1500 mL 4 leaves 4.3 4.0 95 ab L35 Intercept® 1500 mL 4 leaves 4.5 3.8 84 ab L36 Intercept® 1500 mL 4 leaves 4.8 4.3 90 ab Coyote Intercept® 1500 mL 4 leaves 4.5 0.0 0 c control

[0205] (CM4)

[0206] Coyote Untreated control 4 leaves 4.3 4.3 100 a control

[0207] (CM4)

[0208] Isd (0.05) 0.8 1.3 24.4

[0209] P value 0.24 <0.001 <0.001 “WAT” = weeks after treatment application, the figures followed by the same letters are not significantly

[0210]

[0211] differentTable 3: Survivorship of 13-lupin mutant lines 7 weeks after intercept application as compared to untreated Coyote M4 (CM4) bulk line.

[0212] PostPretreatme

[0213] Lupin Lupin

[0214] Line No Herbicide Rate / ha growth treatment nt plant Survivorship (%) stage plant no no per

[0215] per pot pot 7

[0216] WAT

[0217] Date 21 -Jun 16-Aug

[0218] L6 Intercept® 1500 mL 4 leaves 5.0 4.0 80 ab L11 Intercept® 1500 mL 4 leaves 5.0 4.5 90 ab L15 Intercept® 1500 mL 4 leaves 5.0 4.3 85 ab L18 Intercept® 1500 mL 4 leaves 3.5 3.3 95 ab L19 Intercept® 1500 mL 4 leaves 5.0 1.0 20 cd L21 Intercept® 1500 mL 4 leaves 3.8 3.8 100 a L24 Intercept® 1500 mL 4 leaves 4.5 4.0 89 ab L25 Intercept® 1500 mL 4 leaves 3.0 2.0 63 abc L29 Intercept® 1500 mL 4 leaves 4.5 4.3 92 ab L30 Intercept® 1500 mL 4 leaves 3.8 3.8 100 a L33 Intercept® 1500 mL 4 leaves 4.8 2.0 41 bed L38 Intercept® 1500 mL 4 leaves 4.8 4.5 95 ab L40 Intercept® 1500 mL 4 leaves 3.00 1.5 43 bed Coyote Intercept® 1500 mL 4 leaves 4.5 0 0 d control

[0219] (CM4)

[0220] Coyote Untreated 4 leaves 4.3 4.3 4.3 100 a control control

[0221] (CM4)

[0222] Isd (0.05) 1.4 1.6 29

[0223] P value 0.0 0.0 <0.001 <0.001 WAT = weeks after treatment application, the figures followed by same letters are not significantly different.Table 4: Effect of Priority® on 15 mutant lupin lines as compared to untreated Coyote M4 (CM4) bulk line.

[0224] Lupin Pre-treatment Post- treatmentT.XTHerbicide Rate / ha growth plant no per plant no per Line 1 0

[0225] stage pot pot 7 WAT Date 21 -Jun 16-Aug

[0226] L1P Priority® 40 mL 4 leaves 4.8 0 L2P Priority® 40 mL 4 leaves 3.8 0 L3P Priority® 40 mL 4 leaves 4.3 0 L4 P Priority® 40 mL 4 leaves 4.5 0 L7 P Priority® 40 mL 4 leaves 5.0 0 L8 P Priority® 40 mL 4 leaves 3.8 0 L9 P Priority® 40 mL 4 leaves 4.0 0 L10 P Priority® 40 mL 4 leaves 3.8 0 L12 P Priority® 40 mL 4 leaves 4.8 0 L13 P Priority® 40 mL 4 leaves 4.5 0 L14 P Priority® 40 mL 4 leaves 4.5 0 L19 P Priority® 40 mL 4 leaves 5.0 0 L20 P Priority® 40 mL 4 leaves 4.8 0 L22 P Priority® 40 mL 4 leaves 1.5 0 L23 P Priority® 40 mL 4 leaves 4.8 0 Coyote Priority® 40 mL 4 leaves 3.8 0 control

[0227] (CM4)

[0228] Coyote Untreated control 4 leaves 4.8 4.8 control

[0229] (CM4)

[0230] WAT = weeks after treatment application

[0231] Example 3 - Sequencing of Lupin field lines identified in a IMI tolerance screen This project aimed to analyse putative narrow-leafed lupin (L. angustifolius) mutants to determine potential mutations in the acetolactate synthase (ALS) genes, which could confer herbicide tolerance. Mutant plants were obtained by screening the EMS mutagenized population in the Coyote variety background as described in Example 2.Materials and Methods

[0232] Plant materials

[0233] Dried leaf samples from were obtained from 33 individual mutagenized plants. Control leaf samples were obtained from of cultivars Tanjil and Coyote narrow-leaf lupin.

[0234] ALS gene identification

[0235] The lupin acetolactate synthase (ALS) genes were determined using two Arabidopsis ALS protein sequences from the NCBI database and Lupinus angustifolius V2 reference genome (CSIRO in-house database). This resulted in identification of two potential ALS genes from the narrow-leafed lupin genome.

[0236] Primer design for PCR and sequencing of ALS genes

[0237] Five pairs of primers for ALS1 and four pairs for ALS2 were designed for PCR amplification and sequencing of both ALS genes in the 33 test samples and two control samples of Coyote and Tanjil.

[0238] Genomic DNA was isolated from individual samples using Qiagen DNeasy kit, following the manufacturer’s protocol. The Sanger sequencing was achieved using an Applied Biosystems 3730x1 DNA capillary sequencer.

[0239] Data analysis workflow

[0240] The steps below were followed to analyse data and identify genetic variants in the two ALS genes:

[0241] • Aligned all returned sequence files per gene per individual with reference sequence. • Confirmed no discrepancy between Tanjil (reference) and Coyote (population background) for both ALS genes.

[0242] • Investigated conflicts and resolved individually where sequencing trace data was clear.

[0243] • Assembled completed contigs together against Tanjil reference genome, and extracted list of SNP mutations.Results

[0244] The genomic sequences of the two ALS genes in the control wild-type Coyote plants are shown below. EMS produces random mutations by nucleotide substitution; particularly through G: C to A: T transitions.

[0245] Summary of the SNPs identified from 33 genotypes

[0246] ALS1 - 17 SNPs identified from 32 genotypes analysed (individual L27 excluded due to poor sequencing quality).

[0247] ALS2 - no SNPs detected in any of the 32 genotypes analysed (individual L27 excluded due to poor sequencing quality).

[0248] ALS1 - 7 unique SNP types: 53% (9 / 17) G— > A (4 unique positions); 41% (7 / 17) C--> T (2 unique positions); 6% (1 / 17) T— > G (1 unique position; unsure if it was an EMS mutation). No individual plant has more than one SNP identified. In all cases other than one (L20, noted in table below) mutations were observed to be homozygous.

[0249] Table 5 - Summary of SNP Variations Identified

[0250] Sample / Line SNP location SNP observed Notes

[0251] Bold and underlined (corresponds to AlalO7Thr

[0252] L2 319 G-> A

[0253] substitution)

[0254] Bold and underlined (corresponds to AlalO7Thr L20 319 G-> A

[0255] substitution)

[0256] Bold and underlined (corresponds to AlalO7Thr L26 319

[0257] G-> A substitution)

[0258] L14 327 G-> A underlined

[0259] L11 545 C-> T underlined (corresponds to Pro182Leu substitution) L12 544 C-> T underlined (corresponds to Prol82Ser substituion L15 545 C-> T underlined (corresponds to Prol82Leu substitution) L18 545 C-> T underlined (corresponds to Prol82Leu substitution) L21 545 C-> T underlined (corresponds to Prol82Leu substitution) L25 545 C-> T underlined (corresponds to Prol82Leu substitution) L29 545 C-> T underlined (corresponds to Prol82Leu substitution) L30 545 C-> T underlined (corresponds to Prol82Leu substitution) L24 568 G-> A underlined

[0260] L6 568 G-> A underlined

[0261] L19 568 G-> A underlined

[0262] L33 568 G-> A underlined

[0263] L23 1211 T-> G underlined

[0264] Bold and underlined (corresponds to Ser638Asn

[0265] L1 1913

[0266] G-> A substitution)

[0267] Bold and underlined (corresponds to Ser638Asn L31 1913

[0268] G-> A substitution)

[0269] Bold and underlined (corresponds to Ser638Asn L32 1913

[0270]

[0271] G-> A substitution)Bold and underlined (corresponds to Ser638Asn L35 1913

[0272] G-> A substitution)

[0273] Bold and underlined (corresponds to Ser638Asn L36 1913

[0274] G-> A substitution)

[0275] Bold and underlined (corresponds to Ser638Asn

[0276]

[0277] L9 1913 G-> A substitution)

[0278] SEQ ID NO:10 (nucleic acid sequence from wild-type L. angustifolius encoding ALS1) ATGGCGGCCACTACCACTCCAAATCCCACATTTACCCCTCTCCCTTCTTCTTCTTCAAAG CAAATTCTCCGTTTTACCCTCCCTGTAACCTCATTCCCCTCTATACGACGTCGTTCTCTT CGAATCACCAGTTCCCTCTCCAGTAACCCTAAAATACCTGCACCACGCGCCACCTCCACC GCCGCCGCCGCTCCAACCCTAACAACTGAGCCTTTCATTTCCCGGTTTGCCCCTGACGAG CCACGTAAGGGCTCCGACATCCTCGTCGAAGCTCTTGAGCGGCAAGGTGTCACCAATGTC TTCGCTTACCCTGGCGGCGCGTCGATGGAGATTCACCAGGCCCTCACGCGCTCCAATACC ATCCGCAATATCCTCCCTCGCCATGAACAGGGTGGAATCTTCGCCGCCGAGGGCTACGCG CGCTCCTCCGGCCTTCCTGGTGTCTGCATGGCGACCTCTGGACCTGGCGCCACCAATCTC GTCAGCGGTCTCGCCGACGCGCTCCTCGACAGTGTTCCGATCGTGGCAATCACCGGACAA GTTCCGCGGCGAATGATCGGTACGGACGCTTTTCAAGAAACTCCGATTGTTGAGGTAACG AGATCAATCACTAAGCATAATTATCTTGTTCTTGATATTGATGATATTCCTAGGGTTGTG AATGAGGCATTTTTTTTAGCAACTTCTGGAAGGCCTGGTCCTGTGTTGATTGATGTTCCT AAAGATATTCAGCAACAACTTGCTGTTCCAAATTGGGATCAACCAATTAGGTTAACTGGA TATGTGTCTAGATTGCCTAAATCCCCTGATGAGAAACATTTACACCAAATTGTGAGGTTG ATTTCGGAATCTAAGAAACCGGTTTTGTATGTTGGTGGTGGTAGTTTGGATTCGAGTGAG GAATTGAGGAAATTCGTAGAGCTTACTGGGATACCTGTTGCTAGTACCTTGATGGGTTTA GGATCATACCCTCTCGGTGATGAGAAGTCACTTCAAATGCTTGGAATGCATGGGACCGTG TATGCGAATTATGCTGTTGATAAGAGTGATTTGCTGCTTGCATTCGGGGTTAGGTTTGAT GATCGTGTGACAGGGAAGCTTGAGGCTTTTGCTAGTCGTGCGAAAATTGTTCACATTGAT ATTGATTCGGCTGAGATTGGGAAAAACAAACAGCCACATGTGTCTGTTTGTGCGGACTTG AAGGTGGCTTTGGAAGGGATTAATAGGATATTGGAGAGCAAAGGGATCAAGGATAAAGTT GATTTTCGAGGTTGGAGAGAAGAGCTGAATGAGCAAAAGGTCAAATTTCCATTGAGTTTT AAGACATTCGAAGATCGTATTTCTCCACAGTATGCTATTCAGGTTTTGGATGAGCTGACG AATGGAAATGCTATCGTAAGTACTGGTGTTGGACAGCATCAGATGTGGGCTGCTCAGTTT TACAAATACAAGAGACCTAGGCAGTGGTTAACATCTGGTGGTCTTGGTGCTATGGGTTTT GGATTGCCTGCTGCCATTGGAGCTGCCGTAGCTAACCCGGGCGCTATTGTAGTTGACATT GACGGGGATGGAAGTTTTATAATGAATGTTCAAGAGCTAGCCACCATAAGGGTGGAGAAT CTCCCTGTTAAGGTATTATTGTTGAATAATCAACACTTGGGTATGGTTGTTCAGTGGGAG GACCGCTTTTACAAGGCTAATAGAGCTCACACCTATCTGGGAAACCCAGCAAATGAGATT GAGATTTTCCCAAATATGTTGAAGTTTGCAGATGCGTGTGGAATACCAGCAGCTCGTGTG ACGAAGAAAGACGAAGTCACTGCAGCAATTCAGAAAATGTTGGACACCCCTGGCCCCTAC CTTCTTGATGTCATTGTACCCCATCAAGAGCATGTCTTGCCTATGATTCCTAGTAACGGATCC TTCCAGGACGTGATAACCGAGGGCGATGGTAGAATAAGTTAT

[0279] Example 4 - Confirmation of herbicide tolerance in mutant lupin lines under controlled conditions

[0280] Background and aims

[0281] A pot-based screening experiment was conducted on nine mutant lupin lines, selected from 39 single-plant field selections, that exhibited tolerance to twice the registered rate of an imidazolinone (IMI) herbicide (e.g., Intercept® 1.5 L / ha). Genomic characterisation revealed that an additional seven of the 39 lines (L12, L15, L18, L21, L25, L29, L30) carried putativemutations that may be associated with tolerance to sulfonylurea (SU) herbicides, so these lines were also included in the experiment. Both IMI and SU herbicides belong to the Group 2 of herbicides mode-of-action class.

[0282] The present study was undertaken to validate IMI and SU tolerance of these lines under controlled glasshouse conditions by treating plants to a range of herbicide rates and application timings.

[0283] Materials and methods

[0284] Plant material and experimental design

[0285] Table 6 - Lupin mutant lines evaluated in the trial No Code Mutant line

[0286] 1 CM4 Susceptible check (Coyote M4

[0287] bulk)

[0288] 2 L1 PM01-B21-B22-H02-INT001

[0289] 3 L2 PM01-B21-B22-H02-INT002

[0290] 4 L9 PM01-B21-B22-H02-INT009

[0291] 5 L20 PM01-B21-B22-H02-INT020

[0292] 6 L26 PM01-B21-B22-H02-INT026

[0293] 7 L31 PM01-B21-B22-H02-INT031

[0294] 8 L32 PM01-B21-B22-H02-INT032

[0295] 9 L35 PM01-B21-B22-H02-INT035

[0296] 10 L36 PM01-B21-B22-H02-INT036

[0297] 11 L12 PM01-B21-B22-H02-INT012

[0298] 12 L15 PM01-B21-B22-H02-INT015

[0299] 13 L18 PM01-B21-B22-H02-INT018

[0300] 14 L29 PM01-B21-B22-H02-INT029

[0301] 15 L21 PM01-B21-B22-H02-INT021

[0302] 16 L25 PM01-B21-B22-H02-INT025

[0303]

[0304] 17 L30 PM01-B21-B22-H02-INT030

[0305] Pots (3.3 L) were filled with loamy sand soil suitable for growing lupins, sourced from a paddock near Northam, WA. Five seeds were sown per pot on 30-31 January. Seedling emergence commenced on 10 February, with most lines emerging within a narrow window.Glasshouse conditions and management

[0306] The trial was conducted in a temperature-controlled glasshouse with a daytime setpoint of 20°C (06:00-18:00 h); night temperatures remained slightly below the target. To reduce light intensity, the glasshouse walls were whitewashed and the roof covered with a white curtain.

[0307] Plants were maintained using a drip irrigation system to deliver consistent and uniform moisture. A controlled-release fertiliser (PowerFeed® All Purpose) was applied twice during the trial at approximately 25 g per pot.

[0308] Herbicide treatments

[0309] The following eight herbicide treatments were evaluated in the trial referred to in Table 6.

[0310] Table 7 - Herbicide treatments used in the tria

[0311] Treatment Active No of No Herbicide Ingredient Rate / ha Adjuvant Timing Lines lines Untreated

[0312] 1 control (UTC) All 17 CanDo 4 -6 leaf

[0313] 2 Intercept 750 mL 0.5% stage All 17 L1, L2, L9,

[0314] L20, L26,

[0315] CanDo 4 -6 leaf L31, L32,

[0316] 3 Intercept 1500 mL 0.5% stage L35, L36 10 Imazamox LI, L2, L9,

[0317] 33g + L20, L26, imazapyr CanDo 4 -6 leaf L31, L32,

[0318] 4 Intercept 15g / L 3000 mL 0.5% stage L35, L36 10 CanDo 8-12 leaf

[0319] 5 Intercept 750 mL 0.5% stage All 17 LI, L2, L9,

[0320] L20, L26,

[0321] CanDo 8-12 leaf L31, L32,

[0322] 6 Intercept 1500 mL 0.5% stage L35, L36 10 Genfarm BS1000 4 -6 leaf

[0323] 7 Metsulfuron 5 g 0.1% stage All 17 Genfarm Metsulfuron BS1000 4 -6 leaf

[0324]

[0325] 8 Metsulfuron 600g / kg 10 g 0.1% stage All 17 CanDo® oil = 500 g / L ethyl esters of canola oil fatty acids

[0326] Herbicide application

[0327] Applications for the 4-6 leaf treatments (T1-T4, T7, T8) were made on 20 February, corresponding to 20 days after sowing (2.9 weeks). Treatments for the 8-12 leaf stage (T5 and T6) were applied on 6 March, 14 days after the first set.

[0328] Herbicides were applied using an indoor cabinet sprayer fitted with a moving boom and calibrated to deliver 100 L / ha at 300 kPa. The enclosed system ensured uniform spray deposition across pots.Assessments and measurements

[0329] Pre-treatment plant counts

[0330] Baseline plant counts were recorded on 20 February for all treatments except T5 and T6, which were counted on 6 March before application.

[0331] Yellowing (Visual Injury)

[0332] Early yellowing

[0333] Assessed one week after application, focusing on yellowing of newly emerging leaves, typically at the shoot apex. A 15% score represented noticeable yellowing across all plants in a pot.

[0334] • First treatment set: 28 February

[0335] • T5 and T6: 17 March

[0336] Extended yellowing

[0337] Yellowing was again recorded at:

[0338] • 4 weeks after treatment (WAT) for the first set (21 March), and

[0339] • 2 WAT for T5 and T6 (21 March).

[0340] Symptoms were scored as the proportion of yellowed leaves per plant and the proportion of affected plants within the pot.

[0341] Visual biomass reduction

[0342] Biomass reduction was visually estimated (% relative to untreated controls) on 21 March, considering both live and dead plants.

[0343] Survivorship

[0344] Survival was assessed on:

[0345] • 21 March for the first set,

[0346] • 11 April for T5 and T6.

[0347] Final plant counts were recorded on 7 July (20 and 18 WAT). Plants were classified as dead if the growing point was severally bleached or necrotic.

[0348] Number of plants post-treatment

[0349] Survivorship (%) = ( - ) x 100

[0350] Number of plants pre-treatmentData analysis

[0351] Statistical analysis of yellowing and biomass reduction was not conducted due to the extremely high variability in these parameters, with a large proportion of pots showing either 0% or 100% response.

[0352] For survivorship, the total number of plants present at the start of each treatment (sum of four replicates) was calculated for every line. The total number of plants surviving at 4 weeks after treatment (4 WAT) and at the final assessment was also calculated. Survival proportions were obtained by dividing the number of surviving plants at each assessment time by the corresponding number of starting plants.

[0353] For each lupin line, treatment differences in survivorship were evaluated using chi-square tests, comparing survival proportions among treatments. Treatments that did not differ significantly (P < 0.05) were assigned the same significance letter, enabling pairwise comparison within each line.

[0354] Results

[0355] The results are summarized in Table 8 below.

[0356] CM4 (standard line)

[0357] The untreated control (Tl) showed no yellowing or biomass reduction and 100% survivorship. All herbicide treatments (T2-T8) caused visible yellowing 1 week after treatment (“1 WAT”; 7-15%) and substantial biomass reduction (53-100%). Treatments T4, T6, and T8 resulted in complete mortality (0% survivorship; P<0.05).

[0358] Moderate survivorship (40-42%) occurred under T3, T5, and T7, whereas T2 recorded only 13% survivorship. All treatments reduced survivorship significantly compared with the control. Overall, the pattern of survivorship suggests that this population segregated for herbicide tolerance, with individual plants exhibiting differential responses in some treatments and replications.

[0359] Mutant lines

[0360] LI

[0361] The control and treatments T2-T6 maintained 100% survivorship (no significant difference; P<0.05). Moderate injury was observed under T7 and T8, resulting in reduced survivorship (80-67%). T7 and T8 were significantly lower than Tl, confirming partial susceptibility.L2

[0362] Treatments T1-T6 caused no yellowing and maintained 100% survivorship. Severe yellowing and biomass reduction occurred under T7 and T8, resulting in sharp reductions in survivorship (44% and 0%, respectively). These two treatments differed significantly from the untreated control.

[0363] L9

[0364] Treatments T1-T6 resulted in no injury and maintained 100% survivorship. Treatment T8 caused complete mortality (0% survivorship), representing a significant deviation from Tl.

[0365] L20

[0366] T1-T6 maintained 93-100% survivorship with no measurable injury. Strong phytotoxicity under T7 and T8 led to 0% survivorship.

[0367] L26

[0368] All treatments from T1-T6 maintained 100% survivorship. T7 reduced survivorship to 29%, while T8 resulted in complete mortality. Both differed significantly from Tl.

[0369] L31

[0370] T1-T6 maintained high survivorship (78-100%). Treatment T7 reduced survival to 63% and T8 to 7%, both significantly lower than the control.

[0371] L32

[0372] T1-T6 resulted in 83-100% survivorship with no yellowing. Moderate to severe phytotoxicity under T7 and T8 led to survivorship of 38% and 0%, respectively, significantly lower than Tl.

[0373] L35

[0374] No visible yellowing or loss in survivorship under treatments T1-T6. Treatments T7 and T8 resulted in complete mortality (0% survivorship), differing significantly from Tl.

[0375] L36

[0376] All treatments except T7 and T8 maintained >88% survivorship. T7 caused yellowing and reduced survivorship to 50%, while T8 caused complete mortality (0%). Both differed significantly from Tl.L12

[0377] Tl, T2, T5, and T7 maintained high survivorship (93-100%). Treatment T8 caused greater yellowing and reduced survivorship to 63%, significantly lower than the control. Biomass reduction was higher with application of Intercept than metsulfuron.

[0378] L15

[0379] All treatments (Tl, T5, T7, T8) maintained high survivorship (81-100%) and did not differ significantly from the control. Intensity of yellowing and biomass reduction was higher with metsulfuron than with Intercept.

[0380] L18

[0381] Survivorship remained 89-100% across all treatments, with no significant reduction relative to Tl. Intensity of yellowing and biomass reduction was higher with metsulfuron than with Intercept, especially at higher rate (T8).

[0382] L21

[0383] T1-T7 maintained 100% survivorship with limited phytotoxicity. Treatment T8 significantly reduced survival to 0%. Intensity of yellowing and biomass reduction was higher with metsulfuron than with Intercept.

[0384] L25

[0385] T1-T8 all maintained high survivorship (83-100%), with no significant differences relative to the control. Intensity of yellowing and biomass reduction was higher with metsulfuron than with Intercept.

[0386] L29

[0387] T1-T7 maintained 100% survivorship except for a moderate reduction under T5 (43%). Treatment T8 resulted in 0% survivorship. Intensity of yellowing and biomass reduction was higher with metsulfuron than with Intercept.

[0388] L30

[0389] All treatments (T1-T8) maintained 60-100% survivorship with no statistically significant differences among treatments. Intensity of yellowing and biomass reduction was higher with metsulfuron than with Intercept.Table 8: Yellowing, biomass reduction and survivorship as affected by herbicide treatments as compared to untreated control within each lupin line.

[0390] Biomass

[0391] Line Treatment Sum of Yellowing (%) Survivorship (%)

[0392] Pre-treatment reduction (%) plant Number 1 WAT* 21-Mar 21-Mar 21-Mar 7-Jul- CM4 T1 7 0 0 0 100 a 100 a T2 8 15 98 86 13 be 13 be T3 10 15 100 91 40 b 40 b T4 10 15 100 100 0 c 0 c T5 5 7 100 30 40 b 40 b T6 12 8 95 53 0 c 0 c T7 12 15 83 68 42 b 42 b T8 15 15 100 90 0 c 0 c LI T1 14 0 0 0 100 a 100 a T2 12 0 0 0 100 a 100 a T3 12 0 0 5 100 a 100 a T4 15 4 0 10 100 a 100 a T5 12 0 0 0 100 a 83 ab T6 16 0 4 8 100 a 88 ab T7 10 15 26 74 80 ab 70 b T8 9 15 43 80 67 b 56 b L2 T1 12 0 0 0 100 a 100 a T2 13 0 0 0 100 a 100 a T3 12 0 0 0 100 a 100 a T4 10 0 0 17 100 a 100 a T5 11 0 4 10 100 a 100 a T6 14 1 4 8 100 a 100 a T7 9 15 85 84 44 b 0 b T8 10 15 87 50 0 c 0 b L9 T1 6 0 0 0 100 a 100 a T2 7 0 0 0 100 a 100 a T3 7 0 0 0 100 a 100 a T4 8 14 8 16 100 a 88 a T5 5 0 0 0 100 a 100 a T6 4 0 0 0 100 a 100 a T7

[0393] T8 7 0 100 100 0 b 0 b L20 T1 9 0 0 0 100 a 100 a

[0394]

[0395] T2 15 0 0 0 100 a 100 aT3 10 0 0 0 100 a 100 a T4 10 0 0 16 100 a 100 a T5 16 0 0 0 100 a 100 a T6 15 0 0 0 100 a 93 a T7 9 15 100 100 0 b 0 b T8 10 15 100 97 0 b 0 b L26 T1 7 0 0 0 100 a 100 a T2 7 0 0 0 100 a 100 a T3 10 0 0 0 100 a 100 a T4 9 0 0 0 100 a 100 a T5 6 0 0 0 100 a 100 a T6 9 0 0 0 100 a 100 a T7 7 15 77 85 29 b 29 b T8 14 16 98 93 0 c 0 c L31 T1 6 0 0 0 100 a 100 a T2 11 0 0 0 100 a 100 a T3 11 0 0 0 100 a 100 a T4 9 0 0 0 100 a 78 a T5 11 0 0 0 100 a 82 a T6 17 1 0 0 100 a 100 a T7 16 15 100 90 63 b 19 b T8 15 16 100 91 7 c 0 b L32 T1 7 0 0 0 100 a 100 a T2 12 0 0 1 100 a 100 a T3 15 0 0 0 100 a 93 a T4 13 2 0 0 100 a 100 a T5 13 0 0 0 100 a 100 a T6 12 0 0 0 100 a 83 a T7 8 15 60 75 38 b 0 b T8 15 15 75 83 0 c 0 b L35 T1 16 0 0 0 100 a 100 a T2 12 0 0 0 100 a 100 a T3 12 0 0 0 100 a 100 a T4 15 0 0 0 100 a 100 a T5 12 0 0 0 100 a 100 a T6 16 0 0 0 100 a 100 a T7 16 14 60 84 0 b 0 b T8 13 15 83 85 0 b 0 b

[0396]

[0397] L36 T1 14 0 0 0 100 a 100 a T2 12 0 0 0 100 a 100 a T3 17 0 0 0 100 a 100 a T4 10 5 5 4 100 a 90 a T5 16 0 0 0 100 a 88 a T6 7 2 0 0 100 a 100 a T7 2 15 100 80 50 b 0 b T8 9 15 100 85 0 c 0 b L12 T1 17 0 0 0 100 a 100 a T2 14 14 100 64 93 a 93 ab T5 16 20 20 15 94 a 69 be T7 12 14 100 33 100 a 100 a T8 16 18 90 38 63 b 63 c L15 T1 6 0 0 0 100 a 100 a T2

[0398] T5 12 0 0 0 100 a 92 a T7 13 15 93 22 100 a 77 a T8 16 15 100 45 81 a 81 a L18 T1 15 0 0 0 100 a 100 a T2 11 4 0 50 100 a 100 a T5 9 3 18 24 100 a 89 a T7 11 14 100 58 100 a 100 a T8 9 10 100 70 100 a 100 a L21 T1 13 0 0 0 100 a 100 a T2 9 0 0 15 100 a 100 a T5 6 0 17 18 100 a 100 a T7 4 10 50 40 100 a 100 a T8 1 15 80 60 100 a 0 b L25 T1 7 0 0 0 100 a 100 a T2 6 0 0 10 100 a 100 a T5 10 0 16 16 100 a 100 a T7 7 15 100 37 100 a 100 a T8 6 15 100 77 83 a 83 a L29 T1 6 0 0 0 100 a 100 a T2 2 15 0 15 100 a 100 ab T5 7 0 0 0 100 a 43 ab

[0399]

[0400] T7 2 15 80 40 100 a 100 abT8 1 15 80 50 100 a 0 b L30 Tl 1 0 0 0 100 a 100 a T2 10 0 0 0 100 a 60 a T5 5 0 15 20 100 a 100 a T7 5 15 73 18 100 a 100 a

[0401]

[0402] T8 3 15 80 40 100 a 100 a *WAT = Week(s) after treatment application.

[0403] Differences in figures followed by same letters within a lupin line are not statistically significant (P<0.05)

[0404] Conclusions

[0405] The data clearly demonstrated differences in herbicide tolerance across lupin lines (Fig.

[0406] 5). CM4, the standard susceptible line, exhibited severe phytotoxicity and complete mortality under several treatments (T4, T6, T8), confirming sensitivity to both Intercept and metsulfuron. In contrast, lines LI, L2, L9, L20, L26, L31, L32, L35, L36 generally maintained high survivorship under most Intercept treatments (T2-T6), confirming robust tolerance to IMI herbicide. Late timing of application at both rates (T5 and T6) had significantly lower survivorship than untreated control (Tl) and label rate of Intercept (T2). This warrants further investigation.

[0407] Metsulfuron treatments (T7 and T8) consistently caused substantial injury and mortality across lines LI, L2, L9, L20, L26, L31, L32, L35, L36, demonstrating poor cross-tolerance of these lines to SU herbicides.

[0408] Lines L12, L15, L18, L21, L25, L29, L30 showed broad tolerance, maintaining high survivorship especially at a lower rate of metsulfuron. A few of this group of lines (e.g., L12, L21, L29) showed sensitivity to a higher rate, indicating poor tolerance or safety margins.

[0409] Overall, the results confirm that lines LI, L2, L9, L20, L26, L31, L32, L35, L36 are selectively tolerant to IMI chemistry and lines L12, L15, L18, L21, L25, L29, L30 are broadly tolerant to SU chemistry at lower rate, whereas CM4 is highly sensitive.

[0410] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0411] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0412] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Claims

CLAIMS1. A genetically modified lupin plant that expresses a mutated acetolactate synthase 1 (ALS1) which confers upon the genetically modified lupin plant increased tolerance to a Group 2 herbicide, as compared to a corresponding wild-type variety of the plant expressing wild-type ALS1, wherein the amino acid sequence of the mutated ALS1 has a serine to asparagine substitution at a position corresponding to residue 638 of the wild-type ALS1; an alanine to threonine at a position corresponding to residue 107 of the wild-type ALS1; a proline to serine substitution corresponding to residue 182 of the wild-type ALS1; or a proline to leucine substitution at a position corresponding to residue 182 of the wild-type ALS1, wherein the residue positions are relative to the amino acid positions of ALS1 from wild-type Lupinus angustifolius.

2. The genetically modified lupin plant according to claim 1, wherein the Group 2 herbicide is an imidazolinone (IMI) herbicide or a sulfonylurea (SU) herbicide.

3. The genetically modified lupin plant according to claim 1 or claim 2, wherein the genetically modified lupin plant has increased tolerance to an imidazolinone (IMI) herbicide.

4. The genetically modified lupin plant according to claim 3, wherein the IMI herbicide comprises one or more imidazolinones selected from the group consisting of imazapic, imazamox, imazethapyr, imazamethabenz, imazaquin, imazapyr, and combinations thereof.

5. The genetically modified lupin plant according to any one of claims 2 to 4, wherein the IMI herbicide is imazamox or imazapyr.

6. The genetically modified lupin plant according to any one of claims 1 to 5, wherein the amino acid sequence of the mutated ALS1 comprises the Ser 638 to Asn substitution.

7. The genetically modified lupin plant according to claim 6, wherein the amino acid sequence of the mutated ALS1 comprises the amino acid sequence of SEQ ID NO:2.

8. The genetically modified lupin plant according to any one of claims 1 to 7, wherein the genetically modified lupin plant is derived from a genetically modified lupin plant line selected from the group consisting of LI, L9, L31, L32, L35, and L36.

9. The genetically modified lupin plant according to any one of claims 1 to 5, wherein the amino acid sequence of the mutated ALS1 comprises the Ala 107 to Thr substitution.

10. The genetically modified lupin plant according to claim 9, wherein the amino acid sequence of the mutated ALS1 comprises the amino acid sequence of SEQ ID NO:3.

11. The genetically modified lupin plant according to claim 9 or claim 10, wherein the genetically modified lupin plant is derived from a genetically modified lupin plant line selected from the group consisting of L2, L20, and L26.

12. The genetically modified lupin plant according to any one of claims 1 to 5, wherein the genetically modified lupin plant has increased tolerance to: (i) a SU herbicide or (ii) both to a SU herbicide and to an IMI herbicide.

13. The genetically modified lupin plant according to claim 12, wherein the SU herbicide comprises one or more SU herbicides selected from the group consisting of Metsulfuron-methyl (Metsulfuron), Nicosulfuron, Chlorsulfuron, Rimsulfuron, Imazosulfuron, Flazasulfuron, Halosulfuron-methyl, Primisulfuron-methyl, Prosulfuron, lodosulfuron-methyl-Na, and Tribenuron-methyl.

14. The genetically modified lupin plant according to claim 12 or claim 13, wherein the amino acid sequence of the mutated ALS1 comprises the Pro 182 to Ser substitution.

15. The genetically modified lupin plant according to claim 14, wherein the amino acid sequence of the mutated ALS1 comprises the amino acid sequence of SEQ ID NO: 15.

16. The genetically modified lupin plant according to any one of claims 12 to 15, wherein the genetically modified lupin plant is derived from the genetically modified lupin plant line L12.

17. The genetically modified lupin plant according to claim 12 or claim 13, wherein the amino acid sequence of the mutated ALS1 comprises the Pro 182 to Leu substitution.

18. The genetically modified lupin plant according to claim 17, wherein the amino acid sequence of the mutated ALS1 comprises the amino acid sequence of SEQ ID NO: 17.

19. The genetically modified lupin plant according to claim 17 or claim 18, wherein the genetically modified lupin plant is derived from a genetically modified lupin plant line selected from the group consisting of L15, L18, L21, L25, L29, and L30.

20. The genetically modified lupin plant according to claim 1 or claim 2, wherein the genetically modified lupin plant is not a transgenic plant.

21. The genetically modified lupin plant according to claim 1 or claim 2, wherein the genetically modified plant is a transgenic plant comprising an exogenous nucleic acid encoding the mutated ALS1.

22. A plant cell of the genetically modified lupin plant according to any one of claims 1 to 21.

23. A plant part of the genetically modified lupin plant according to any one of claims 1 to 21.

24. A seed produced by the genetically modified lupin plant according to any one of claims 1 to 21.

25. A food product prepared from the genetically modified lupin plant according to any one of claims 1 to 21.

26. A consumer product prepared from the genetically modified lupin plant according to any one of claims 1 to 21.

27. An industrial product prepared from the genetically modified lupin plant according to any one of claims 1 to 21.

28. A veterinary product prepared from the genetically modified lupin plant according to any one of claims 1 to 21.

29. A method for producing a genetically modified lupin hybrid plant, comprising breeding the genetically modified lupin plant according to any one of claims 1 to 21 with a second plant to obtain a genetically modified lupin hybrid plant, wherein the genetically modified lupin hybrid plant exhibits increased to an IMI herbicide.

30. A method for producing a genetically modified lupin hybrid plant, comprising breeding the genetically modified lupin plant according to any one of claims 12 to 21 with a second plant to obtain a genetically modified lupin hybrid plant, wherein the genetically modified lupin hybrid plant exhibits increased to an IMI herbicide and a SU herbicide.

31. A method for cultivation of a genetically modified lupin plant, the method comprising planting the seed according to claim 24, whereby the planted seed germinates to generate a seedling.

32. The method according to claim 31, further comprising cultivating the seedling to obtain a mature genetically modified lupin plant and harvesting seeds from the mature plant.

33. The method according to claim 31 or claim 32, further comprising applying an IMI herbicide or a SU herbicide one or more times over a cultivation period beginning at a seedling stage, whereby growth of weeds in proximity of the seedling following the application is inhibited.

34. An isolated nucleic acid encoding an acetolactate synthase 1 (ALS1) comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 1735. The isolated nucleic according to claim 34, wherein the encoded ALS comprises the amino acid of SEQ ID NO:

236. The isolated nucleic according to claim 34, wherein the encoded ALS comprises the amino acid sequence of SEQ ID NO:3.

37. The isolated nucleic according to claim 34, wherein the encoded ALS comprises the amino acid sequence of SEQ ID NO:15.

38. The isolated nucleic according to claim 34, wherein the encoded ALS comprises the amino acid sequence of SEQ ID NO:17.

39. A plant expression vector comprising the isolated nucleic according to any one of claims 35 to 38, and operable to expression of the encoded ALS1 in a lupin plant.

40. The plant expression vector according to claim 39, wherein the plant expression vector is a recombinant virus vector.

41. The plant expression vector according to claim 40, wherein the recombinant virus vector is a Cauliflower Mosaic Virus (CaMV) vector.

42. The plant expression vector according to claim 39, wherein the plant expression vector is a Ti plasmid.

43. A recombinant cell comprising the plant expression vector according to any one of claims 39 to 42.

44. The recombinant cell according to claim 43, wherein the recombinant cell is a recombinant prokaryotic cell.

45. The recombinant cell according to claim 44, wherein the recombinant prokaryotic cell is a recombinant Agrobacterium tumefaciens cell.

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