WO2022224146A1

WO2022224146A1 - Brassica napus line ome0001

Info

Publication number: WO2022224146A1
Application number: PCT/IB2022/053662
Authority: WO
Inventors: Antonio LEONFORTE; Nelson GORORO; Van RIPLEY
Original assignee: Nuseed Nutritional Australia Pty Ltd.
Priority date: 2021-04-20
Filing date: 2022-04-19
Publication date: 2022-10-27

Abstract

The present embodiments provide a novel DHA-producing Brassica napus line designated as OME0001 (DHA Canola OME0001). The embodiments also provide the seeds, the plants, and the plant parts (including DNA) of DHA Canola line OME0001, as well as to methods for producing a Brassica plant produced by crossing DHA Canola line OME0001 with itself or another Brassica line. These embodiments also provide methods for producing a DHA-producing Brassica plant containing in its genetic material one or more transgenes, and to the transgenic plants and plant parts produced by those methods. These embodiments further provide DHA production lines or breeding lines and plant parts derived from DHA Canola line OME0001, to methods for producing canola lines or plant parts derived from line OME0001, and to the DHA-producing Brassica plants, varieties, and their parts derived from use of those methods. These embodiments also provide hybrid seeds, plants, and plant parts produced by crossing the DHA Canola line OME0001 with another Brassica or canola line.

Description

Brassica napus Line OME0001

RELATED APPLICATION

[0001] This Application claims priority benefit of U.S. Provisional Application

No. 63/176,991 filed 20 April 2021, which is incorporated fully herein for all purposes.

FIELD

[0002] The present embodiments relate to a new and distinctive DHA-producing Bras sica napus line designated OME0001

BACKGROUND

[0003] Long-chain omega-3 polyunsaturated fatty acids (co3 LC PUFA) are well recognized for their critical role in human diets. The co3 LC PUFA (4Z,7Z,10Z,13Z,16Z,19Z)- docosa-4,7,10,13,16,19-hexaenoic acid (DHA) is a primary structural component of the human brain, cerebral cortex, skin, and retina. There remains a need for land-based sources of co3 LC PUFA including DHA.

SUMMARY

[0004] The present embodiments provide a plant, derivative or progeny plant, plant part, or seed of DHA Brassica napus line OME0001 (“DHA Canola OME0001”), representative sample of seed of which was deposited under ATCC Accession No. PTA-126906 (see Appendix). Line OME0001 produces DHA and other long chain omega 3 polyunsaturated fatty acids in its seed oil.

[0005] One aspect of the present embodiments provides a method for producing a DHA Canola seed comprising crossing two Brassica plants and harvesting the resultant seed, wherein at least one of the two Brassica plants is DHA Canola line OME0001 or progeny thereof.

[0006] Another aspect of the present embodiments provides methods of producing co3

LC PUFA comprising DHA from DHA Canola OME0001.

DETAILED DESCRIPTION

[0007] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. [0008] All patents and other publications identified are incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention, but are not to provide definitions of terms inconsistent with those presented herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0009] As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, “comprise,” “comprises,” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term “or” is inclusive unless modified, for example, by “either.” Thus, unless context indicates otherwise, the word “or” means any one member of a particular list and also includes any combination of members of that list.

[0010] All values are approximate as there is some fluctuation in fatty acid composition due to environmental conditions. Values are typically expressed as area percent, which approximates percent by weight, of total fatty acid or percent weight of the total seed. Accordingly, other than in the operating examples, or where otherwise indicated, all numbers expressing quantities or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0011] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0012] The present embodiments provide for Brassica lines that produces the LC co3 PUFA DHA in its seed oil, specifically a line embodying the trait of B. napus line OME0001. LC co3 PUFAs are known in the art and for a relevant discussion of these and other fatty acids, see e.g., WO 2017/218969 and WO 2017/219006.

[0013] Brassica napus belongs to the Cruciferae ( Brassicaceae ) plant family, typically referred to as rapeseed or canola. More specifically, rapeseed ( Brassica napus subsp. napus) is a bright-yellow flowering member of the family Brassicaceae (mustard or cabbage family), cultivated mainly for its oil-rich seed. Rapeseed is the third-largest source of vegetable oil and second-largest source of protein meal in the world. Rapeseed oil that often contains appreciable amounts of toxic erucic acid, however, rendering the oil inedible. Canola are a group of rapeseed cultivars that were bred to have very low levels of erucic acid and are especially prized for use for human and animal food. More specifically, “canola oil” must contain less than 2% erucic acid; and canola meal is defined as one gram of air-dry, oil-free meal that must contain less than 30 pmoles of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, 2-hydroxy-4-pentenyl glucosinolate, or a mixture thereof. See, e.g.. CODEX ALIMENTARIUS: FATS, OILS & RELATED PRODUCTS, Vol. 8 (2nd ed., Food & Agriculture Org. United Nations, Rome, Italy, 2001). Brassica napus is a digenomic amphidiploid resulting from interspecific hybridization between Brassica oleracea and Brassica rapa. It is a self-compatible pollinating species like the other amphidiploid brassica species. Notably, wild-type B. napus does not produced DHA or other very long chain PUFA in seed oil.

[0014] A “line” is a group of plants that displays very little overall variation among individuals sharing that designation. “Line” also refers to a homogeneous assemblage of plants carrying substantially the same genetic material that display little or no genetic variation between individuals for at least one trait, , in particular the transgenes present in line OME0001. “Variety” or “cultivar” may be used interchangeably with “line,” but in general the former two terms refer to a line that is suitable for commercial production.

[0015] “Genetically derived” as used for example in the phrase “genetically derived from the parent lines” means that the characteristic in question is dictated wholly or in part by an aspect of the genetic makeup of the plant in question.

[0016] “Progeny” means all descendants including offspring and derivatives of a plant or plants and includes the first, second, third, and subsequent generations; and may be produced by self-pollination or crossing with plants with the same or different genotypes, and may be modified by a range of suitable genetic engineering techniques. Cultigen generally relates to plants that have been deliberately altered and selected by human. “TO” refers to the first generation of transformed plant material, “Tl” refers to the seed produced on TO plants, T1 seed gives rise to Tl plants that produce T2 seed, etc., to subsequent Tx progeny.

[0017] “Breeding” includes all methods of developing or propagating plants and includes both intra- and inter-species and into- and inter-line crosses as well as all suitable conventional breeding and artificial breeding techniques. Desired traits (e.g., the OME0001 DHA trait) may be transferred to other canola or B. napus lines, cultivars, or cultigens; or through conventional breeding methods and can also be transferred to other Brassica species, such as B. rapa, through inter-specific crossing. Both conventional breeding methods and inter specific crossing methods, as well as other methods of transferring genetic material between plants, are well-known in the art. For pure line selection, the progeny of single plant selections (self-pollinated), are kept separate and not bulked together. The expected result is a set of genetically homogeneous lines. Accordingly, a pure line may be the progeny of a single self- fertilized homozygous plant.

[0018] “Plant part” includes plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, pods, leaves, flowers, branches, fruit, stalks, roots, root tips, anthers, cotyledons, hypocotyls, radicles, single cells, gametes, cell cultures, tissue cultures, and the like. A cotyledon is a type of seed leaf; a small leaf contained on a plant embryo. A cotyledon contains the food storage tissues of the seed. The embryo is a small plant contained within a mature seed. “Plant cells” also encompasses non- regenerable plant cells. Accordingly, the embodiments include plants and plant parts from line OMEOOOl line, as well as other plants produced by the described methods. Progeny, derivatives, variants, and mutants of regenerated plants are also included within the scope of the present embodiments, provided that these parts comprise event OMEOOOl nucleic acid molecules. The present embodiments are also directed to line OMEOOOl transgenes, and to their use, e.g., in plant cell culture and tissue culture, and for methods of identifying line OMEOOO 1. The embodiments include plants and plant parts from the elite event OMEOOO 1 line, as well as other plants produced by the described methods. Progeny, derivatives, variants, and mutants of regenerated plants are also included within the scope of the present embodiments, provided that these parts comprise event OMEOOOl nucleic acid molecules.

[0019] At least one embodiment described herein provides a new B. napus line, designated OMEOOOl (DHA Canola OMEOOOl), that produces DHA in its seed oil. Other aspects of the present embodiments provide seed, plants, and plant parts of DHA Canola OMEOOOl; progeny of OMEOOOl; methods for producing a canola plant produced by crossing the DHA Canola OMEOOOl with itself or another canola genotype; and the creation of variants by mutagenesis or transformation of DHA Canola OMEOOOl. Thus, the present embodiments include any such methods using the line OMEOOOl, including selfing, backcrossing, hybrid production, and crosses to populations. All plants produced using line OMEOOOl as a parent are within the scope of the present embodiments. In at least one embodiment, DHA Canola OMEOOOl is used in crosses with other different canola plants to produce first generation (Fi) hybrid seeds and plants with superior characteristics. Accordingly, in addition to B. napus, examples of members of the Brassica genus useful in practicing the present embodiments include but are not limited to B. napus, B. napobrassica, B. oleracea, B. carinata, B. rapa,

B. juncea, and B. campestris, as well as any other plants belonging to the genus Brassica that permit breeding between Brassica species. Generally, “oilseed plant” refers to any one of the species B. napus, B. rapa (or campestris), or B. juncea.

[0020] DHA Canola OMEOOOl is open pollinated, produces medium to tall sized plants, flowers mid-season, and matures early. Plants have high early plant vigor, are branched, and produce long pods. The seed of OMEOOOl contains co3 LC PUFA including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) characteristic of the Omega 3 NS-B500274 trait, and low saturated and erucic fatty acid content, and low glucosinolate content characteristic of Canola.

[0021] Phenotypic characterizations of DHA Canola OMEOOOl, in addition to production of DHA in seed oil, include:

[0022] In one embodiment, the DHA Canola OMEOOOl provided herein is a B-line. Alternatively, DHA Canola derived from OMEOOOl may be an A-line, a B-line, or a R-line. An A-line is an Ogura cytoplasmic male sterile line (CMS). A B-line is similar to an A-line in all features, but it is a male fertile version (a line of similar nuclear genotype but carrying a male fertile cytoplasm). A R-line (Ogura Rf) carries male sterile cytoplasm but is fixed for a nuclear restorer factors and restores fertility and is used to produce an FI hybrid seed. Because a B-line is used to maintain the fertility it may also be referred to as the maintainer line. A restorer line is a pollinator variety used to pollinate a CMS or A-line to produce FI progeny which are male fertile and thus produce seeds on selfing. An inbred line may permit restoration of fertility to the progeny of male sterile lines to which it is crossed. Thus, hybrid lines may be grown from FI seed produced by crossing two inbred (pure) lines, one of which may be male sterile. OMEOOOl, being a B-line, can be grown as an open pollinated variety or used in the Ogura system to produce FI hybrid variety.

[0023] Accordingly, the present embodiments provide a method of introducing a desired trait into DHA Canola line OMEOOOl, wherein the method comprises crossing an OMEOOOl plant with a plant of another Brassica genotype that comprises a desired trait to produce progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide tolerance, insect resistance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, modified lodging resistance, and resistance to bacterial disease, fungal disease, or viral disease; selecting one or more progeny plants that have the desired trait to produce selected progeny plants; crossing the selected progeny plants with the OMEOOOl plants to produce backcross progeny plants; selecting for backcross progeny plants that have the desired trait and essentially all of the physiological and morphological characteristics of the OME0001 line to produce selected backcross progeny plants; and repeating these steps three or more times to produce selected fourth or higher backcross progeny plants that comprise the desired trait and essentially all of the physiological and morphological characteristics of line OMEOOOl as described herein. Included in this aspect of the embodiments is the plant produced by the method wherein the plant has the desired trait (i.e., DHA production) and essentially all the physiological and morphological characteristics of line OME0001. [0024] Line OMEOOOl was bred from parental line B. napus, NS-B50027-4, which was developed by Agrobacterium tumefaciens -mediated transformation with the genetic construct pJP3416_GA7-modB (“modB”), that includes seven genes capable of promoting the accumulation of co3 fatty acids in seed. See Petrie et al., Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels ofDHA in the Seed Oil, 11 Frontiers Plant Sci. 727 (2020); U.S. Patents No. 9,718,759 and No. 9,932,541. Briefly, the modB construct contains genes encoding a Dό-dcsaturasc cloned from the microalga Micromonas pusilla, a Dό-elongase and a A5-elongase cloned from the microalga Pyramimonas cordata, a A5-desaturase cloned from the microalga Pavlova salina, a D 15/o)3-desaturase cloned from the yeast Pichia pastoris, a D 12-desaturase cloned from the yeast Lachancea kluyveri, and a A4-desaturase cloned from Pavlova salina, each with suitable transcription promoters and terminators to provide expression in seed. See, e.g., WO 2017/219006. An intensive breeding and selection program resulted in a DHA canola line designated NS-B50027-4, (see U.S. Patents No. 10,570,405 and No. 10,563,218).

[0025] Line OMEOOOl was bred from an advanced breeding line NX 140, a parental line developed by Nuseed via a pedigree and recurrent selection based breeding program that included a complex crossing using Nuseed Canola Genetic Resources and targeted selection strategy using field nurseries to combine: high grain yield potential, traits that provide general agronomic adaptation (e.g., spring type canola, aerial branching habit, and pod set characteristics), blackleg disease resistance, and a canola quality seed oil profile.

[0026] The DHA canola line OME0001 was derived from a marker assisted backcross (BC) program were the “final cross” was from the third cross between NX0140 and NS-B50027-4. BC3F2 generation plants homozygous for NS-B50027-4 event construct inserts were identified using molecular markers. OMEOOOl was derived from BC3F2 seed from and individual F2 plant. Reselection was undertaken in the glasshouse and in single row progeny experimental trials to F4 generation. At each cycle, single plant selections were bagged with a perforated plastic bag prior to flowering and then selfed. At each cycle of reselection, the optimal sib genotypes were advanced on the basis of fatty acid profile (e.g., LC PUFA %, specifically DHA% as a % of total fatty acids in the seed), agronomic traits (e.g., vigor, fertility, standability, blackleg disease tolerance, oil content, maturity, flowering time, plant and pod structures) and other seed quality traits (e.g., seed oil %). The BC3-derived F4 selection was tested in replicated field experiments from 2018 for grain yield, agronomic traits, fatty acid profile, and seed oil traits (e.g., seed oil%). Based on this assessment, the selection was advanced for analysis as a potential commercial cultivar in the United States. OMEOOOl is thus a transgenic Brassica that is genetically stable, uniform, and no off-type plants have been exhibited in evaluation. The OMEOOOl line has shown genetic stability and uniformity as described in the phenotypic description information. The line has been increased with continued observation for uniformity. Accordingly, an embodiment provides a seed of B. napus line designated OMEOOOl, a representative sample of seed of which was deposited under ATCC Accession No. PTA-126906.

[0027] The primary phenotypic characteristic of OMEOOO 1 is the production of noticeable amounts of DHA in its seed oil. As shown herein, the bulk seed of OMEOOOl typically contains about 7.5% DHA (as % of total fatty acids in seed), or more.

[0028] At least one embodiment is directed to methods for producing a DHA Canola Brassica plant by crossing a first parent plant with a second parent plant, wherein the first or second plant is the plant from the Brassica line OMEOOO 1. In at least one embodiment, both first and second parent plants may be from the line OMEOOO 1. Any breeding methods using the line OMEOOOl are envisioned: selfing, backcrosses, hybrid breeding, and crosses to populations.

Any plants produced using line OMEOOO 1 as a parent are within the scope of the present embodiments.

[0029] Additional methods of the present embodiments include introduction of expression vectors introduced into plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation or by using Agrobacterium- mediated transformation. In some embodiments, a transgenic variant of OMEOOOl may contain at least one additional transgene (in addition to those present in OMEOOOl), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional transgenes. Accordingly, the present invention also relates to super- transformed variants of the claimed line OMEOOOl, or variants obtained by breeding OMEOOOl with one or more other transgenic Brassica. Such transgenes are expressed under control of regulatory sequences (e.g., promoters, enhancers, intervening sequences, terminators) well- known in the art. In one embodiment, the desired trait may be one or more of herbicide tolerance, insect resistance, disease resistance, modified seed yield, modified oil percent, modified protein percent, modified lodging resistance, or modified fatty acid or carbohydrate metabolism. The specific gene may be any gene known in the art or listed herein, including but not limited to a marker gene, a polynucleotide (nucleic acid molecule) conferring resistance or tolerance to imidazolinone, sulfonylurea, glyphosate, glufosinate, 2,4-D, Dicamba, L- phosphinothricin, triazine, hydroxyphenylpyruvate dioxygenase inhibitor, protoporphyrinogen oxidase inhibitor, phenoxy proprionic acid, cyclohexone, or benzonitrile; a polynucleotide encoding a Bacillus thuringiensis polypeptide, a polynucleotide encoding phytase, a polynucleotide encoding a 1 -acyl-glycerol-3 -phosphate acyltransferase (LPAAT), a galactinol synthase, or a raffmose synthetic enzyme; or a polynucleotide conferring resistance to blackleg, white rust, or other common canola diseases. Such transgenes and related techniques are well- known in the art, see, e.g.. U.S. Patents No. 9,718,759, No. 8,143,488. Alternatively, the DHA trait of such transgenic embodiments may be obtained by breeding a canola comprising at least one transgene with OMEOOO 1. For example, the trait of producing DPA as well as DHA may be obtained by introgressing chromosomes from DPA JunceaNUBJ1207 (see W02021041570) into OMEOOO 1.

[0030] Accordingly, transgenes or other phenotypic traits can be introduced into OMEOOO 1 or the DHA-producing trait of OMEOOO 1 introduced into other Brassica or canola lines using traditional breeding techniques, such as introgression or backcrossing, well-known in the art. Canola plant breeding techniques that may be employed in generating progeny of OMEOOO 1 include, for example, recurrent selection, bulk selection, mass selection, mutation breeding, backcrossing, pedigree breeding, tissue culture, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, producing doubled haploids. Often combinations of these techniques are used. The development of Brassica varieties in a plant breeding program requires, in general, the development and evaluation of homozygous varieties. See, e.g., U.S. Patents No. 10,570,405, No. 10,563,218, and No. 8,143,488; Downey et ah, Rapeseed & Mustard, at 437 in PRINCIPLES OF CULTIVAR DEVEL. (Fehr (ed.), Macmillan & Co., NY, 1987); Thompson, Breeding winter oilseed rape Brassica napus, 7 Adv. Appl. Biol. 1-104 (1983); Ward et ah, Oilseed Rape (Farming Press Ltd., Wharefedale Road, Ipswich, Suffolk, 1985).

[0031] In another aspect of the present embodiments, OMEOOO 1 and its progeny can be identified not only by its phenotypic trait (i.e., production of DHA), but by identification of its genotype. Methods and kits are useful for identifying in biological samples the presence of plant material comprising specifically the transgenes in DHA Canola OMEOOO 1, as well as transgenic Brassica plants, plant materials, and seeds containing such event. The elite event DHA Canola OMEOOO 1 described herein can be identified by genotype, which can be characterized through a genetic marker profile that can identify plants of the same cultivar or a related cultivar or be used to determine or validate a pedigree. Genetic marker profiles can be obtained by techniques such as Restriction Fragment Length Polymorphisms (RFLP), Randomly Amplified Polymorphic DNAs (RAPD), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCAR), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR) (also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNP).

[0032] For example, the DHA Canola OMEOOO 1 described herein can be identified by generation of a genetic map from a sample of plant material. A genetic map can be generated by conventional RFLP, Polymerase Chain Reaction (PCR) analysis, or SSR which identifies the approximate chromosomal location of the integrated DNA molecule coding for the foreign protein. See Glick & Thompson, METHODS IN PLANT MOLEC. BIOL. & BIOTECHNOL. (CRC Press, Boca Raton, 1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. For example, the map of the integration region can be compared to similar maps for suspect plants to determine if the latter have a common parentage with the subject plant. Map comparisons can involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques.

[0033] For identification of DEIA Canola OMEOOO 1 , primers to identify the junctions can be any suitable length derived from a larger junction sequence. The length of the junction sequences used to identify DEIA Canola OMEOOO 1 may be any suitable length that provides identification of a unique junction. These sequences are described for the parent donor of the transgenes required for DEIA synthesis, NS-B50027-4 in, for example, U.S. Patents No. 10,570,405 and No. 10,563,218. Further markers useful for the identification of this trait may be found in PCT/ US2019/050243 and U.S. Appl. No. 17/198,064.

[0034] Canola is recognized as an increasingly important oilseed crop and a source of meal in many parts of the world. DEIA Canola line OMEOOO 1 or its progeny can be used in the production of oil, meal, or other food or feed products in accordance with known techniques.

[0035] In accordance with the Budapest Treaty, Applicants have deposited seed of DHA B. napus OMEOOO 1 with the American Type Culture Collection (ATCC®) located at 10801 University Blvd., Manassas, Va., 20110-2209 U.S.A., under Accession No. PTA-126906. Applicants have satisfied the requirements of 37 C.F.R. §§ 1.801-1.809. Applicants have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicants do not waive any rights granted under patent laws or plant breeders’ rights.

EXAMPLES

Example 1. Cultivation trials, Australia

[0036] DHA Canola OMEOOO 1 was field trialed in replicated plot experiments in 2018, in the South West (Location A) and Wimmera (location B) cereal and broadleaf cropping zones of Victoria, Australia. These cropping zones are characterized by autumn sowing, winter dominant rainfall (i.e., Mediterranean-like) and basalt based acid sodosols for location A, and clay black cracking alkaline vertosols soils for location B. Seed obtained from OMEOOO 1 had the following fatty acid profile as determined by gas chromatography (Table 2):

[0037] Additional data on oil yield, seed protein, meal protein, and glucosinolates from the two trials are shown in Table 3 :

[0038] The fatty acid content profde of line OME0001 seed oil was also compared with that of parent NS-B50027-4 and commercial lines, Nuseed Diamond and Nuseed Quartz, grown in other plots in these two Australian trials. The data was averaged and is presented in Table 4:

Example 2. Cultivation trials (Canada)

[0039] DHA-producing B. napus line OMEOOO 1 was grown at two test sites in Canada in 2018, in Canadian plant hardiness zone 3A (Saskatchewan); and the data on oil yield, seed protein, meal protein, and glucosinolates from the two trials are shown in Table 5:

Example 3. Cultivation trials (Australia)

[0040] B. napus line OMEOOO 1 was grown in Australian plot experiments in 2019, in two locations in the Wimmera cropping zone described above. Seed obtained from OMEOOO 1 had the following fatty acid profde as determined by gas chromatography as shown in Table 6:

[0041] Further, for one location of the Australian 2019 trial, line OMEOOO 1 pooled seed contained 7.88% total saturated fatty acid. Additional data on yield, oil yield, seed protein, meal protein, and glucosinolates from the two trials are shown in Table 7 :

[0042] The fatty acid content profile of DHA canola line OMEOOO 1 was also compared with that of parent NS-B50027-4 and Nuseed commercial cultivars, Nuseed Diamond and Nuseed Quartz, grown as comparators in these two Australian field trials. The data for the two locations were averaged and are presented in Table 8:

Example 5. Cultivation in Canada

[0043] DHA-producing B. napus line OMEOOO 1 was field tested at two sites in Canada in 2019, and the data on grain yield, oil yield, seed protein, meal protein, and glucosinolates from the two trials are shown in Table 9:

Example 6. Cultivation in the North America

[0044] DHA-producing B. napus line OMEOOO 1 was field trialed with comparators at two sites in Canada (Site A, zones 3a; and Site B, zones 3b, respectively) and two sites in the United States (C and D, each zone 4a) in 2019. The mean grain yield across all four sites was 2.10 t/ha. The amount of DHA (as area %) produced by DHA Canola OMEOOO 1 ranged from 5.5% DHA to 8.6% DHA, and the mean across the sites was 7.0% DHA. Further yield at the two Canadian sites is shown in Table 10:

[0045] The faty acid content for DHA Canola OMEOOO 1 tested at Canadian Site A and

Site B are shown in Table 11 :

[0046] The North American trials included comparator plots growing B. napus line NS-B50027-4 and commercial varieties Nuseed Diamond and Nuseed Quartz. Data from one of the two Canadian sites are presented in Table 12:

Example 4. Field trials in North America and Australia

[0047] DHA-producing B. napus line OMEOOO 1 was field trialed with comparators at two sites in the United States (Sites E and F), one site in Canada (Site G), and two sites in Australia (Sites H and I) in 2020. The seed content for the 2020 experiment are presented in Table 13.

Example 5. Field trials in North America and Australia

[0048] DHA-producing B. napus line OMEOOOl was field trialed with NS-B50027-4 as a comparator at four sites in Canada (Sites J, K, L, and M), one site in the United States (Site N), and two sites in Australia (Sites O and P) in 2021. The seed content data for the 2021 experiments are presented in Table 14 (nm = not measured):

[0049] Although the preceding embodiments have been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of skill in the art that certain changes and modifications, such as single gene modifications and mutations, somaclonal variants, variant individuals selected from large populations of the plants of the instant inbred line, and the like, may be practiced within the scope of the invention which is limited solely by the appended claims.

Claims

CLAIMS We claim:

1. A seed of transgenic Brassica napus line designated OMEOOOl, a representative sample of seed of which was deposited under ATCC Accession No. PTA- 126906.

2. A transgenic Brassica plant, or part thereof, comprising the genome of the seed of claim 1, produced by growing the seed of claim 1.

3. Pollen of the transgenic plant of claim 2.

4. An ovule of the transgenic plant of claim 2.

5. A transgenic Brassica plant, or parts thereof, having all the physiological and morphological characteristics of the plant of claim 2.

6. A tissue culture of regenerable cells produced from the Brassica plant of claim 2.

7. A tissue culture according to claim 6, the cells or protoplasts being of a tissue selected from the group consisting of leaves, pollen, embryos, roots, root tips, pods, flowers, ovules, and stalks.

8. A tissue culture of regenerable cells of the Brassica plant according to claim 2, wherein the tissue regenerates plants capable of expressing all the morphological and physiological characteristics of the plant according to claim 2.

9. A method of producing Brassica seed, comprising growing the plant of claim 2 to produce seed and harvesting said seed.

10. The method of claim 9, wherein said growing is done in a greenhouse.

11. The method of claim 9, wherein said growing is done in a tent.

12. The method of claim 9, wherein said growing is done in an open field.

13. A method of producing oil, comprising obtaining seed of the plant of claim 2 and processing the seed to obtain oil.

14. The Brassica plant, or parts thereof, of claim 5, wherein the plant or parts thereof have been transformed so that its genetic material contains one or more trans genes, operably linked to one or more regulatory elements, in addition to the transgenes present in B. napus line OMEOOOl.

15. A method for producing a Brassica plant that contains in its genetic material at least one transgene in addition to the transgenes of line OMEOOOl, comprising crossing the plant of claim 14 with either a second plant of another Brassica line, or a plant of line OMEOOOl, so that the genetic material of the progeny that result from the cross expresses the additional at least one transgene.

16. The method of claim 15, wherein the additional transgene confers herbicide tolerance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility.

17. The Bras sica plant, or parts thereof, of claim 5, wherein the plant or parts thereof is the product of traditional breeding techniques and has not been modified by recombinant, mutagenic or other genetic modification other than the elite event associated with OMEOOO 1.

18. A method for producing a Bras sica plant disposed with at least one desired trait in addition to the trait of inbred Bras sica line OMEOOO 1, comprising crossing the plant of claim 17 with either a plant of another Bras sica line, or a plant of inbred line OMEOOO 1 , so that the genetic material of progeny that result from the cross expresses the desired additional trait.

19. The method of claim 18, wherein the additional transgene confers herbicide tolerance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility.

20. A method for producing a Bras sica line OMEOOO 1 -derived canola plant comprising:

(a) crossing Bras sica napus line OMEOOO 1 with a second canola plant to yield progeny canola seed; and

(b) growing said progeny canola seed, under plant growth conditions, to yield said Bras sica line OMEOOO 1 -derived canola plant.

21. The method of claim 20, further comprising:

(c) crossing said OMEOOO 1 -derived canola plant with itself or another canola plant to yield additional OMEOOO 1 -derived progeny canola seed;

(d) growing said progeny canola seed of step (a) under plant growth conditions, to yield additional OMEOOO 1 -derived canola plants; and

(e) repeating the crossing and growing steps of (a) and (b) from zero (0) to seven (7) times to generate further OMEOOO 1 -derived canola plants, wherein said OMEOOO 1 -derived canola plants express least two OMEOOO 1 -traits selected from DHA production and at least one additional agronomically desirable trait.

22. A Bras sica line OMEOOO 1 -derived canola plant, or parts thereof, produced by the method of claim 20.

23. A canola OMEOOO 1 -derived canola plant, or parts thereof, produced by the method of claim 21.

24. A method for producing Bras sica seed comprising crossing a first parent Bras sica plant with a second parent Brassica plant and harvesting the resultant first generation Brassica seed, wherein said first or second parent Brassica plant is the Brassica plant of claim 2.

25. The method according to claim 24, wherein said first parent Brassica plant is different from said second parent Brassica plant, and wherein said resultant seed is a first generation (FI) hybrid Brassica seed.

26. A method for obtaining a canola inbred line comprising:

(a) planting a collection of seed comprising seed of a hybrid, one of whose parent is the Brassica plant according to claim 2, said collection also comprising seed of said inbred line;

(b) growing canola plants from said collection of seeds;

(c) identifying an inbred plant from said inbred line;

(d) selecting said inbred plant; and

(e) controlling pollination in a manner that preserves the homozygosity of said inbred plant.

27. The method according to claim 26, wherein said one parent has all the physiological and morphological characteristics of inbred Brassica line OMEOOOl, seed of said line having been deposited under ATCC Accession No. PTA- 126906

28. The method of claim 24, wherein the canola inbred line is B. napus or B. juncea.

29. A cell of Brassica napus line OMEOOOl, representative sample of seed of which was deposited under ATCC accession number PTA- 126906.

30. Genomic DNA of inbred canola line OMEOOOl, representative seed of said inbred canola line having been deposited under ATCC Accession Number PTA- 126906.

31. A seed of OMEOOOl comprising about 8%, about 9%, about 10%, about 11 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%, inclusive, DHA (as % of total fatty acid in seed oil).

32. A collection of seed comprising at least 95% seed of Brassica napus line OMEOOOl, wherein said seed comprises about 8%, about 9%, about 10%, about 11 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%, inclusive, DHA + EPA + DPA (as % of total fatty acids in seed oil).

33. A method of obtaining DHA-containing canola grain comprising the steps of

(a) introgressing the DHA trait of OMEOOOl (deposited under ATCC Accession Number PTA-126906) into an elite Brassica line that is male sterile;

(b) introgressing the DHA trait of OmeOOOl into a second elite Brassica line that is fertile;

(c) crossing the two lines (a) and (b) to obtain a hybrid progeny;

(d) cultivating the seed of the hybrid progeny; and

(e) harvesting the grain produced by the cultivated hybrid progeny.

34. The method of claim 33, wherein said hybrid progeny comprises at least one trait selected from herbicide tolerance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility.

35. A method of producing docosahexaenoic acid (DHA) -containing canola oil, comprising processing seed from transgenic Brassica napus line designated OMEOOO 1 or its progeny, representative sample of seed of which was deposited under ATCC Accession No. PTA-126906, to obtain DHA-canola oil.

36. A method of producing a seed oil comprising docosahexaenoic acid (DHA), said method comprising the steps of:

(a) obtaining the seed of the transgenic Brassica napus line designated OMEOOO 1 or its progeny, representative sample of seed of which was deposited under ATCC Accession

No. PTA-126906, and

(b) extracting the seed oil from said seed.

37. The method of claim 36, further comprising incorporating said seed oil into a foodstuff for human consumption.

38. The method of claim 37, wherein said foodstuff is cooking oil or salad oil

39. The method of claim 36, further comprising incorporating said seed oil into a foodstuff for animal consumption.

40. The method of claim 39, wherein said foodstuff is an aquaculture feed.

41. The method of claim 36, further comprising incorporating said seed oil into a nutritional supplement.

42. The method of claim 41, further comprising the steps of refining said seed oil and incorporating said refined seed oil into a nutritional supplement.

43. A method of producing a seed meal obtained from seeds of the transgenic Brassica napus line designated OMEOOO 1 or its progeny, representative sample of seed of which was deposited under ATCC Accession No. PTA-126906, said method comprises,

(a) crushing the seeds of said transgenic Brassica napus line designated OMEOOO 1 or its progeny;

(b) extracting the oil from the crushed seeds; and

(c) obtaining the seed meal from the crushed seeds.

44. The method of claim 43, further comprising processing the seed meal into foodstuff for animal consumption.

45. The method of claim 44, wherein said foodstuff is aquaculture feed.