HK1192109A - Canola germplasm exhibiting seed compositional attributes that deliver enhanced canola meal nutritional value having omega-9 traits - Google Patents

Description

Canola germplasm exhibiting seed compositional attributes providing enhanced canola meal nutritional value and having OMEGA-9traits

Priority requirement

The present application claims U.S. provisional application No.61/445,426 filed on 22/2/2011 for the benefit of 35u.s.c. § 119(e) on "CANOLA structural components THAT DELIVER ENHANCED CANOLA metallic VALUE bearing OMEGA-9 trains".

Technical Field

The present invention relates to canola (canola) germplasm and cultivars. In some embodiments, the present invention relates to canola germplasm having meal composition attributes (e.g., reduced anti-nutritional factor levels and increased protein levels) that are independent of seed coat color modifications. Particular embodiments relate to canola germplasm demonstrating dark seed color in combination with, for example, reduced levels of anti-nutritional factors (e.g., Acid Detergent Fiber (ADF) and polyphenolic compounds) and elevated levels of protein and phosphorus.

Background

"canola" refers to rapeseed (rapeseed) (Brassica species (Brassica spp.)) having an erucic acid (C22:1) content of up to 2% by weight (compared to the total fatty acid content of the seed) and (after crushing) producing an air-dried meal containing less than 30 micromoles (μmol) of glucosinolates (glucosinolates) per gram of defatted (oil-free) meal (meal). These types of rapeseed differ in their edibility compared to more traditional species varieties. Canola oil is considered to be a superior edible oil due to its low levels of saturated fatty acids.

Although rapeseed meal is relatively high in protein, its high fiber content reduces its digestibility and its value as animal feed. Canola and oilseed rape meal contain a higher dietary fiber value and a lower percentage of protein than soybean meal. Canola meal has about 20% less Metabolic Energy (ME) than soybean meal due to its high dietary fiber. Thus, the value of the meal remains low relative to other oil seed meals such as soybean meal, particularly in swine and poultry feeds. Rakow (2004a) Canola quality improvement through the weaving of yellow-continuous-an historical property in AAFC stable Production systems bulletin. In addition, the presence of glucosinolates in some canola meal also reduces its value due to the deleterious effects these compounds have on the growth and reproduction of livestock.

The varieties of brassica are partially distinguished by their seed coat color. Generally, seed coat color falls into two broad categories: yellow and black (or dark brown). Different hues of these colors are also observed, such as reddish brown and yellowish brown. It has been widely observed that brassica varieties with lighter seed coat colors have thinner hulls and thus less fiber and more oil and protein than varieties with dark seed coats. Stringam et al (1974) Chemical and hydraulic characteristics associated with seed coat color incorporated, in Proceedings of the 4th International ranked consistency, Giessen, Germany, pp.99-108, Bell and Shires (1982) Can.J.animal Science62:557-65, Shirzadegan and Shirpelog(1985)Fette Seifen antibiotic 87:235-7, Simbaya et al (1995) J.Agr.food chem.43:2062-6, Rakow (2004b) Yellow-seed Brassica napus canola for the Canadian canola Industry, in AAFC Sustainable production Systems Bulletin. One possible explanation for this is that the canola plant can expend more energy to produce protein and oil if it does not require the energy to produce the fibrous component of the seed coat. It has also been reported that yellow seed-bearing brassica lines have lower glucosinolate content than black seed-bearing brassica lines. Rakow et al, (1999b) Proc.10th int.Rapeeed Congress, Canberra, Australia, Sep.26-29,1999, Poster # 9. Thus, historically, the development of yellow-seeded brassica varieties has been tracked as a potential way to increase the feed value of canola meal. Bell (1995) mean and by-product utilization in animal number, in Brassica oillands, production and utilization in eds. Kimber and McGregor, Cab International, Wallingford, Oxon, OX108DE, UK, pp.301-37; Rakow (2004b), supra; Rakow&Raney(2003)。

Some yellow-set seed forms of Brassica (Brassica) species, which are closely related to Brassica napus (b.napus), e.g., turnip (b.rapa) and mustard (b.juncea), have been shown to have lower levels of fiber in their seeds and subsequent meal. The development of yellow seed bearing brassica napus germplasm has demonstrated that fiber can be reduced in brassica napus via the integration of genes controlling seed pigmentation from related brassica species. However, the integration of genes controlling seed pigmentation from related brassica species into valuable oilseed brassica varieties, such as brassica varieties, is complicated by the fact that multiple recessive alleles are involved in the inheritance of the yellow seed coat in currently available nodose seed lines. Furthermore, "pod curling" is also a problem commonly encountered during the integration of yellow seed coat colors from other brassica species, such as mustard (juncea) and brassica carinata (carinata).

There is little information available about how variable the fiber within the rape germplasm of dark coloured seeds is, and no report has been made on brassicas that have developed dark coloured seeds with reduced levels of anti-nutritional factors (e.g. fiber and polyphenolic compounds) and increased levels of protein.

Summary of The Invention

Described herein are canola (brassica napus) free-pollinated cultivars (CL044864, CL065620) and hybrids (CL166102H, CL121460H and CL121466H) comprising germplasm that provides a novel combination of canola meal compositional variations that have been shown to affect nutritional value. In some embodiments, canola plants comprising germplasm of the invention may produce seeds with novel combinations of, for example, protein, fiber, and phosphorus levels, such that these seed components are independent of seed coat color. In particular embodiments, such plants can produce higher protein and lower fiber than standard canola types, and phosphorus levels similar to or higher than phosphorus levels in standard canola types. In some embodiments, canola inbred lines and hybrids comprising the germplasm of the invention may provide nutritionally enhanced meal characteristics when utilized directly as a feed or food ingredient and/or when utilized as a feedstock to process protein isolates and concentrates. Such seeds may be dark (e.g., black, dark, and mottled) or light.

As such, described herein are canola germplasm that can be used to obtain a canola plant having desired seed composition traits in a seed color independent manner. In some embodiments, plants comprising such germplasm may be used to generate canola meal having a desired nutritional quality. In particular embodiments, inbred canola lines (and plants thereof) comprising germplasm of the invention are provided. In other embodiments, inbred canola lines (and plants thereof) having an inbred canola plant comprising the germplasm of the invention are provided as parents. Brassica varieties of the invention include, for example, but are not limited to: CL044864, CL065620, CL166102H, CL121460H, and CL 121466H.

Also described herein are plant commodity products obtained from an inbred canola plant or hybrid comprising the germplasm of the invention. Particular embodiments include canola meal or seeds obtained from such inbred canola plants or hybrids.

Particular embodiments of the invention include canola germplasm conferring high protein content and low fiber content traits to a canola seed, wherein the canola plant produces a seed having, on average, at least 68% oleic acid (C18:1) and less than 3% linolenic acid (C18: 3). In other embodiments, the canola plant comprises a canola germplasm. Seeds produced from the canola plants are also described. Additional embodiments include progeny plants produced from canola plant seeds. Also disclosed are methods of introducing at least one trait selected from the group consisting of: high protein content, low fiber content, at least 68% oleic acid (C18:1), and less than 3% linolenic acid (C18: 3).

Methods for improving the nutritional value of canola meal are also described. For example, methods are described for introgressing combinations of canola meal compositional characteristics into canola germplasm in a seed color independent manner. In particular embodiments, germplasm of the invention may be combined with canola germplasm characterized by yellow seed coats to produce germplasm capable of providing enhanced canola meal with desired characteristics conferred by each germplasm.

The above and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings.

Brief Description of Drawings

Figure 1 includes images of several brassica varieties with dark seed coat colors.

FIG. 2 includes data from a seed composition analysis of certain Brassica napus inbreds and hybrids. Seed samples were from duplicate trials in western canada. Seed composition data was predicted based on NIR and subsequently confirmed using reference chemistry.

Detailed Description

I. Brief summary of several embodiments

Canola meal is the canola seed fraction remaining after the oil extraction process. Canola meal is a protein source and therefore is utilized in several applications, including animal feed formulations and the separation of high value protein concentrates and isolates. The seed coat, cotyledon and fiber within the embryo that terminates in the meal limit the incorporation rate (inclusion rate) of canola meal in monogastric animal species, and as such, canola meal generally does not provide the same nutritional value as meals prepared from other sources (e.g., soybean). It has been shown that the yellow-set seed form in species closely related to brassica napus (e.g., turnip and mustard) has a lower level of fiber in its seeds and subsequent meal. This observation has motivated attempts to introduce low seed fiber traits into brassica napus in a yellow seed color-dependent manner. The development of the resulting yellow-pigmented seed brassica napus germplasm has demonstrated that fiber can be reduced in brassica napus via this method.

Prior to the present invention, it was not believed that dark seed canola varieties would exhibit as low a seed fiber content as observed in yellow seed varieties. Furthermore, dark seed canola lines containing reduced levels of anti-nutritional factors (e.g., fiber and polyphenolic compounds), and elevated levels of protein and phosphorus have not been described, which would represent an improved source of canola meal. In some embodiments, the canola germplasm described herein provides a combination of several key enhanced meal composition attributes that are independent of seed coat color expression. In particular embodiments, canola meal prepared from canola seeds comprising germplasm of the invention may achieve higher rates of dietary intake, for example in pig and poultry diets.

The germplasm of the invention may be used (e.g., via selective breeding) to develop brassicas with desirable seed component traits and one or more additional desirable traits (e.g., improved oil composition, increased oil yield, modified protein composition, increased protein content, disease, parasite resistance, herbicide resistance, etc.). The germplasm of the invention may be used as a starting germplasm into which additional variations in seed composition may be introduced, such that canola lines and hybrids may be developed that provide improved elevated canola meals of the type described herein.

Abbreviation II

ADF acid detergent fiber

ADL acid detergent lignin

Apparent ileal digestibility of AID

Apparent metabolizable energy of AME

Brassica campestris with black seeds formed by BSC

Percentage of CP crude protein

DM Dry matter concentration

ECM enhanced canola meal of the invention

FAME fatty acid/fatty acid methyl ester

GE Total energy

HT "high temperature" processing

LT "Low temperature" processing

NDF neutral detergent fiber

NMR nuclear magnetic resonance

NIR near infrared spectroscopy

SAE erucic acid ester

SBM Soy meal

SER soluble extraction residue

SID normalized ileal digestibility

TAAA true amino acid availability

TDF Total dietary fiber

True metabolic energy of TME

WF white flake (white flake)

Term of

Backcrossing: backcrossing methods can be used to introduce nucleic acid sequences into plants. Backcrossing techniques have been widely used for decades to introduce new traits into plants. Jensen, N., ed.plant Breeding method, John Wiley & Sons, Inc.,1988. In a typical backcrossing scheme, an initial variety of interest (the recurrent parent) is crossed to a second variety (the non-recurrent parent) that carries the gene of interest to be transferred. The resulting progeny from this cross are then crossed again with the recurrent parent and the process is repeated until a plant is obtained in which, in addition to the transgene from the non-recurrent parent, substantially all of the desired morphological and physiological characteristics of the recurrent plant are restored in the transformed plant.

Canola oil: canola oil refers to oil extracted from a commercial variety of rapeseed. To produce canola oil, the seeds are typically fractionated and mixed on a grain elevator to produce a consistent and acceptable product. The mixed seeds are then crushed and the oil is extracted, usually with hexane, followed by refining. The resulting oil can then be sold for use. Oil content is usually measured as a percentage of fully dried seeds, and a particular oil content is characteristic of different brassica varieties. Oil content can be readily and routinely determined using a variety of analytical techniques, such as, but not limited to: NMR, NIR, Soxhlet extraction. See Bailey, Industrial Oil & Fat Products (1996),5th Ed.Wiley Interscience Publication, New York, New York. Typically, the percent composition of total fatty acids is determined by extracting an oil sample from seeds, generating fatty acid methyl esters present in the oil sample, and analyzing the proportion of the various fatty acids in the sample using gas chromatography. The fatty acid composition may also be a distinctive feature of a particular variant.

Commercially useful: as used herein, the term "commercially useful" refers to plant lines and hybrids that have sufficient plant vigor and fertility such that farmers can use conventional farming equipment to produce plant lines or hybrid crops. In particular embodiments, plant commodity products having the described components and/or qualities can be extracted from commercially useful varieties of plants or plant materials. For example, oil containing the desired oil component can be extracted from commercially useful plant lines or hybrid seeds using conventional grinding and extraction equipment. In certain embodiments, the commercially useful plant line is an inbred line or a hybrid line. "commercial elite" lines and hybrids often have desirable characteristics; such as but not limited to: improved yield of at least one plant commodity; the maturity; disease resistance; and standability.

The good strain: any plant line that results from breeding and selection for superior agronomic performance. An elite plant is any plant from the elite line.

Enhanced canola meal: as used herein, the term "enhanced canola meal" means a canola meal having an enhanced composition resulting from processing of canola seeds having increased protein content and reduced levels of at least some anti-nutritional components. The enhanced canola meal of the present invention may variously be referred to herein as "ECM", "black seed canola ECM", "BSC ECM", or "DAS BSCECM". However, the present invention is not intended to be limited to the ECM germplasm of brassica rapa that knot black seeds only.

Substantive (essentiaily) derived: in some embodiments, manipulation of a plant, seed, or part thereof may result in the creation of a substantially derived variety. As used herein, the term "substantially derived" follows the convention set forth in International Union for the Protection of New Varieties of Plants (UPOV):

[A] a variant should be considered to be substantially derived from another variant ("the original variant"),

(i) it is derived predominantly from an initial variant, or from a variant which itself is derived predominantly from an initial variant, while retaining the expression of a substantial characteristic of the genotype or combination of genotypes derived from the initial variant;

(ii) it is clearly distinguishable from the original variety; and

(iii) it is consistent with the initial variant in expression of substantial characteristics of the genotype or combination of genotypes from the initial variant, in addition to differences arising from the effects of origin.

UPOV, six Meeting with International organization, Geneva, Oct.30,1992 (a document prepared by the Federation office).

Plant commodity: as used herein, the term "plant commodity" refers to a commodity produced from a particular plant or plant part (e.g., a plant comprising a germplasm of the invention, and a plant part obtained from a plant comprising a germplasm of the invention). The merchandise may be, for example but not limited to: a cereal; coarse powder; forage grass; a protein; an isolated protein; flour; an oil; crushed or whole grain or seed; any food product comprising any meal, oil, or ground or whole grain; or silage.

Plant line: as used herein, a "line" refers to a group of plants that exhibit little (e.g., no) genetic variation among individuals in at least one trait. Inbred lines can be created by self-pollination and selection over several generations or alternatively by vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the terms "cultivar", "variety" and "type" are synonymous, and these terms refer to lines used in commercial production.

Plant material: as used herein, the term "plant material" refers to any processed or unprocessed material derived, in whole or in part, from a plant. For example, but not limited to, the plant material can be a plant part, seed, fruit, leaf, root, plant tissue culture, plant explant, or plant cell.

Stability: as used herein, the term "stability" or "stable" refers to a given plant component or trait that can be inherited and maintained at substantially the same level for only multiple seed generations. For example, the stable component may be maintained at substantially the same level for at least three generations. In this context, the term "substantially the same" may refer in some embodiments to being maintained within 25% between two different generations; within 20%; within 15%; within 10%; within 5 percent; within 3 percent; within 2 percent; and/or components within 1%, and components maintained entirely between two different generations. In some embodiments, the stable plant component may be, for example, but not limited to, an oil component; a protein component; a fiber component; a pigment component; a glucosinolate component; and a lignin component. The stability of the components may be affected by one or more environmental factors. For example, the stability of the oil component can be affected by, for example, but not limited to, temperature; a location; stress; and planting time effects. Subsequent generations of plants with stable components under field conditions are expected to produce plant components in a similar manner, for example as listed above.

Trait or phenotype: the terms "trait" and "phenotype" are used interchangeably herein.

Variety or cultivar: the term "variety" or "cultivar" refers herein to a plant line that is unique in its characteristics, stable, and consistent when propagated, for commercial production. In the case of hybrid varieties or cultivars, the parent lines are unique, stable, and consistent in their characteristics.

As used herein, the terms "a" and "an" mean at least one, unless otherwise indicated.

Canola germplasm providing desirable seed component traits in a seed color independent manner

In a preferred embodiment, the present invention provides canola germplasm that can be used to obtain canola plants with desirable seed component traits in a seed color independent manner. Specific exemplary canola inbred lines and hybrids comprising such germplasm are also provided.

In general, canola oil is considered to be a very healthy oil for both human and animal consumption. However, the meal component of canola seeds, which is left over after extraction of the oil component, is inferior to soybean meal due to its high fiber content and reduced nutritional value. In some embodiments, canola plants comprising germplasm of the invention may alleviate or overcome these deficiencies, and may provide canola meal as a highly nutritious and economical source of animal feed. Canola meal is a by-product of canola oil production, and thus canola meal provided by the present invention conserves valuable resources by allowing this by-product to compete with other meals.

Yellow canola seed color was previously considered to be of interest in itself as it was believed to correspond to the improved nutritional profile of the meal component obtained after oil extraction. Some embodiments may provide for the first time a dark seed (e.g., dark, black, and variegated seed) low fiber canola germplasm that also provides excellent high oleic acid and low linolenic acid, which also provides canola meal with improved nutritional characteristics (e.g., improved seed components). In some embodiments, plants comprising the germplasm of the invention may, surprisingly, further provide these traits in combination with other valuable traits, such as, but not limited to, excellent yield, high protein content, high oil content, and high oil quality. The dark skinned seeds in particular embodiments may have considerably thinner seed coats than seeds produced by the brassica variety of standard dark skinned seeds. A thinner seed coat may result in a reduced fiber content in the meal, and an increase in the seed oil and protein content compared to the oil and protein levels in standard darkened seed varieties. Thus, dark seeds produced by plants comprising the germplasm of the invention may have higher oil and protein concentrations in their seeds than observed in seeds produced by standard dark seed brassica plants.

In embodiments, plants comprising the germplasm of the invention exhibit no substantial agronomic and/or seed limitations. For example, such plants may exhibit at least as favorable agronomic and/or seed quality (e.g., germination; early season vigor; effect of seed treatment; seed harvest and storability) as the characteristics exhibited by standard brassica varieties. In particular embodiments, plants comprising the germplasm of the invention may also comprise one or more additional advantageous traits exhibited by pre-existing canola inbreds, such as, but not limited to, an advantageous fatty acid profile.

In embodiments, a plant comprising a germplasm of the invention may produce seed comprising at least one of several nutritional characteristics. In particular embodiments, the seed produced by such a canola plant may comprise at least one nutritional characteristic selected from the group consisting of: a favorable oil profile; high protein content; low fiber content (e.g., ADF and NDF (including low polyphenol content)); (low fiber and high protein impart higher metabolic energy); high phosphorus content; and low erucate (SAE) content. In certain embodiments, "high" or "low" component content refers to a comparison between seed produced from a reference plant comprising the germplasm of the invention and seed produced from a standard canola variety. As such, plants that produce seeds with "low" fiber content can produce seeds with lower fiber content than observed in seeds produced from standard brassica varieties. Also, plants that produce seeds with "high" protein content can produce seeds with higher protein content than is observed in seeds produced from standard brassica varieties.

In some embodiments, a substantially uniform agglomeration of rapeseed produced by a canola plant comprising at least one nutritional characteristic selected from the group described above may be produced. Such seeds can be used to produce a substantially uniform field of canola plants. Particular embodiments provide canola seeds comprising a combination of the foregoing features identified. For example, the combined total oil and protein content of a seed can be a useful measure and unique characteristic of a seed.

Some embodiments provide a canola (e.g., dark seed canola) comprising germplasm of the invention, which is capable of having a nareon-type oil profile or an "Omega-9" oil profile of the canola oil. An "NATRON-type", "NATRON-like", or "Omega-9" oil profile can represent an oleic acid content ranging, for example, from 68-80%, 70-78%, 71-77%, and 72-75%, with an alpha linolenic acid content of less than, for example, 3%. In particular embodiments, seeds obtained from canola plants comprising germplasm of the invention may produce oil with more than 68%, more than 70%, more than 71%, more than 71.5%, and/or more than 72% (e.g., 72.4% or 72.7%) oleic acid, while having a linolenic acid content of less than 3%, less than 2.4%, less than 2%, less than 1.9%, and/or less than 1.8% (e.g., 1.7%). In other embodiments, however, canola comprising germplasm of the invention may produce oil with, for example, an oleic acid content of greater than 80%. In certain embodiments, canola oil produced from canola comprising germplasm of the invention may be naturally stable (e.g., without artificial hydrogenation). The fatty acid content of canola oil can be readily and routinely determined according to known methods.

As such, some embodiments provide canola seeds (e.g., dark canola seeds) comprising an oil fraction and a meal fraction, wherein the oil fraction may have an alpha-linolenic acid content of, for example, 3% or less (relative to the total fatty acid content of the seed) and an oleic acid content of, for example, 68% or more (relative to the total fatty acid content of the seed). The erucic acid (C22:1) content of such seeds may also be less than 2% (by weight) (as compared to the total fatty acid content of the seeds), by definition. In a particular example, the oil content of the canola seed may comprise 48% to 50% by weight of the seed.

The term "high oleic acid" refers to mustard or other brassica species having a higher oleic acid content than the wild type or other reference variety or line, as the context may dictate, and more generally, it dictates a fatty acid composition comprising at least 68.0% (by weight) oleic acid.

"Total saturates" refers to the combined percentages of palmitic (C16:0), stearic (C18:0), arachidic (C20:0), behenic (C22:0) and lignoceric (C24:0) fatty acids. The fatty acid concentrations discussed herein can be determined according to standard procedures well known to those skilled in the art. A specific protocol is set forth in the examples. The fatty acid concentration is expressed as a weight percentage of the total fatty acid content.

As used herein, the term "stability" or "stabilized" with respect to a given genetically controlled fatty acid composition means that the fatty acid component is maintained at substantially the same level, e.g., preferably ± 5%, for at least two generations, and preferably for at least three generations, between generations. The method of the invention enables the creation of a canola line with an improved fatty acid component which is stable up to + -5% between generations. Those skilled in the art understand that the above-mentioned stability can be affected by temperature, location, stress and planting time. Thus, comparison of fatty acid profiles between canola lines should be performed using seeds produced under similar growth conditions.

When the term "canola plant" is used in the context of the present invention, this also includes any single gene transition of the set. As used herein, the term "single-gene transformed plant" refers to those brassica plants developed by a plant breeding technique known as backcrossing, in which substantially all of the desired morphological and physiological characteristics of a variety are restored in addition to the single gene transferred to the variety via the backcrossing technique. Backcrossing methods may be used with the present invention to improve or introduce features into the varieties. As used herein, the term "backcross" refers to a hybrid progeny that returns repeated crosses, i.e., backcrosses, one or more times to the recurrent parent (identified as "BC 1," "BC 2," etc.). The parent brassica plants contributing the desired signature are referred to as "non-recurrent" or "donor parents". This term refers to the fact that the non-recurrent parent is used once in the backcrossing scheme and therefore no longer occurs. A parent Brassica plant that receives one or more gene transfers from a non-recurrent parent is called the recurrent parent because it uses several rounds in the backcrossing scheme (Poehiman & Sleper,1994; Fehr, 1987). In a typical backcrossing scheme, an initial variety of interest (the recurrent parent) is crossed to a second variety (the non-recurrent parent) that carries the single gene of interest to be transferred. Progeny resulting from this cross are then crossed again with the recurrent parent, and the process is repeated until a canola plant is obtained in which, in addition to a single transgene from the non-recurrent parent, substantially all of the desired morphological and physiological characteristics of the recurrent parent are restored in the transformed plant, as determined at a 5% significance level when cultured under the same environmental conditions. In the present application, the term "Brassica" (Brassica) may encompass any or all of the species contained in the genus Brassica, including Brassica napus, Brassica juncea, Brassica nigra (Brassica nigra), Brassica carinata (Brassica carinata), Brassica oleracea (Brassica oleracea), and Brassica rapa (Brassica rapa).

As used herein, brassica juncea refers to mustard that produces seeds having oil and meal qualities that meet the commercial designation requirements for "brassica" oil or meal, respectively (i.e., brassica species plants having less than 2% erucic acid (Δ 1322:1) in the seed oil (by weight) and less than 30 micromoles of glucosinolates per gram of oil-free meal).

In one aspect, the invention provides canola plants, such as mustard plants, capable of producing seeds having an endogenous fatty acid content comprising a high percentage of oleic acid and a low percentage of linolenic acid (by weight). In particular embodiments, oleic acid may constitute more than about 68.0%,69.0%,70.0%,71.0%,72.0%,73.0%,74.0%,75.0%,76.0%,77.0%,78.0%,79.0%,80.0%,81.0%,82.0%,83.0%,84.0%, or 85.0%, including all integers and fractions thereof or any integer having a value greater than 85% oleic acid. In particular embodiments, the linolenic acid content of a fatty acid can be less than about 5%,4%,3%,2.5%,2.0%,1.5%,1.0%,0.5%, or 0%, and includes all integers and fractions thereof. In an exemplary embodiment, the plant is mustard, the seeds of which have an endogenous fatty acid content comprising at least 68% oleic acid (by weight) and less than 3% linolenic acid (by weight). In other embodiments, the plant is a mustard plant, the seeds of which have an endogenous fatty acid content comprising at least 68.0% oleic acid (by weight) and no more than about 5% linolenic acid (by weight).

In one aspect, the invention provides canola plants, such as mustard plants, capable of producing seeds having an endogenous fatty acid content of low or high total saturated fatty acids that comprise a high percentage of oleic acid and a low percentage of linolenic acid (by weight) and that may comprise less than about 5.5% total saturated fatty acids or >10% total saturated fatty acids, respectively.

The composition of oil from mustard seed is known to differ from the composition of rape seed oil in terms of two fatty acid components (e.g. higher erucic acid content), essential oils (e.g. allyl isothiocyanate), and minor ingredients (e.g. tocopherols, metals, tannins, phenols, phospholipids, colour bodies, etc.). It has been found that oil (including extract oil) in seeds from mustard is higher in oxidative stability than oil from brassica napus, even though oil from mustard generally has a higher C18:3 level. (C.Wijejunder et al, "" Canola Quality Indian Mustard oil (Brassica juncea) "isMore Stable to Oxidation th an environmental Canola oil (Brassica napus)," J.am.oil chem.Soc. (2008)85: 693-.

In an alternative aspect, the invention provides methods for increasing the oleic acid content and reducing the linolenic acid content of a canola plant. Such methods may involve: (a) inducing mutagenesis in at least some cells from a canola line having an oleic acid content of greater than 55% and a linolenic acid content of less than 14%; (b) regenerating a plant from at least one of said mutagenized cells, and selecting a regenerated plant having a fatty acid content comprising at least 68% oleic acid (or an alternative threshold concentration of oleic acid, as listed above) and less than 3% linolenic acid (or an alternative threshold concentration of linolenic acid, as listed above); and (c) deriving from the regenerated plant a further generation of a plant, individual plants of the further generation of the plant having a fatty acid content comprising at least 68% oleic acid (or alternative threshold concentration of oleic acid) and less than 3% linolenic acid (or alternative threshold concentration of oleic acid). In some embodiments, the brassica can be a mustard. The terms "high oleic acid content" and "low linolenic acid content" encompass the full range of possible values described above. In alternative embodiments, the methods of the invention may further comprise selecting a reduced linolenic acid content, such as the range of possible values described above, for one or more lines, regenerated plants, and further generations of plants. In other embodiments, step (c) may involve selecting and culturing seeds from the regenerated plant of step (b). In other embodiments, the methods of the present invention can include repetition of the prescribed steps until the desired oleic acid content, linolenic acid content, or both are achieved.

In an alternative embodiment, there is provided a method for screening individual seeds for increased oleic acid content and decreased linolenic acid content comprising: determining one or more of: oleic acid content of fatty acids that are part of the seed germinate; or linolenic acid content; or oleic acid content and linolenic acid content; comparing the one or more amounts to a reference value; and extrapolating the likely relative oleic acid, linolenic acid, or oleic and linolenic acid content of the seeds. In particular embodiments, the plant part used for analysis may be a part or whole of a leaf, cotyledon, stem, petiole, stem, or any other tissue or tissue fragment, such as a tissue having a composition that is indicative of reliable correlation with seed composition. In a series of embodiments, the portion of the sprout can be a portion of a leaf. In certain embodiments, the step of inferring the fatty acid composition of the seed may comprise assuming that a significant level of change in a given acid in the leaves reflects a similar relative change in the level of the acid in the seed. In a particular embodiment of the invention, a method for screening canola plants for individual plant lines whose seeds have an endogenous fatty acid content comprising at least 68% oleic acid and less than 3% linolenic acid (by weight) by analyzing leaf tissue. In addition, leaf tissue may be analyzed for fatty acid composition by gas liquid chromatography, wherein extraction of fatty acids may occur by methods such as bulk seed analysis (bulk seed analysis) or half seed analysis (half seed analysis).

In an alternative embodiment, the invention provides a canola plant comprising the previously described alleles of a gene from the mustard line, which may be a mustard plant. In certain embodiments, the plant may be homozygous at fad2-a and fad3-a expressed as mutant alleles. In other embodiments, the brassica juncea plant, plant cell, or portion thereof contains a genetic allele having a nucleic acid sequence from the previously described sequences disclosed herein.

In some embodiments, the invention may involve distinguishing the HOLL Brassica quality mustard of the invention (. gtoreq.68% oleic acid and. ltoreq.5% linolenic acid) from low oleic/high linolenic mustard (about 45% oleic acid and about 14% linolenic acid) by examining the presence or absence of the BJfad2b gene (see, for reference, U.S. patent publication No.20030221217, Yao et al.). This distinction may involve the confirmation that the BJfad2a gene is the only functional oleate fatty acid desaturase gene in the brassica quality mustard line, as is known in the art.

In one embodiment, the brassica juncea line contains the fad2 and fad3 genes as disclosed in international publication No. us2006/0248611a1, exemplified in figures 1 and 3 thereof. The fad2 and fad3 genes are exemplified herein by SEQ ID NOs 1-4. The resulting allele encodes a delta-12 fatty acid desaturase protein, which is exemplified in figure 2 of international publication No. us2006/0248611a 1. In other embodiments, the mustard line may contain mutations at the fad2-a and fad3-a gene loci, and the resulting mutant alleles may encode one or more mutations in the sequences that predict the BJFAD2-a and BJFAD3-a proteins. Representative examples of fad2-a and fad3-a mutant genes and proteins suitable for use in the present invention also include, but are not limited to, those disclosed in: international publication No. WO2006/079567A2 (e.g., FIGS. 1 and 2) such as SEQ ID NOS: 8 and 9, International publication No. WO2007/107590A2 such as SEQ ID NOS: 10-21, U.S. Pat. No.6,967,243B2 (e.g., FIGS. 2 and 3) such as SEQ ID NOS: 22-27; and European publication No.1862551A1 (e.g., FIGS. 1 to 10), such as SEQ ID NOs: 28-39.

In selected embodiments, the invention provides isolated DNA sequences comprising the complete Open Reading Frame (ORF) and/or 5' upstream region of the previously disclosed mutant fad2 and fad3 genes. Thus, the invention also provides polypeptide sequences that are predictive of mutant proteins, which contain mutations from the previously described mutant alleles. Membrane-bound desaturases such as FAD2 are known to have a conserved histidine box. These amino acid residue changes outside the histidine box can also affect FAD2 enzymatic activity (et al.,Molecular Breeding4:543550,1998)。

In one aspect of the invention, the mutant alleles described herein can be used in plant breeding. In particular, high oleic brassica species, such as brassica juncea, brassica napus, brassica rapa, brassica nigra and brassica carinata, can be bred using the alleles of the invention. The present invention provides molecular markers for distinguishing mutant alleles from alternative sequences. Thus, the present invention provides methods for the isolation and selection analysis of genetic crosses involving plants having the alleles of the present invention. Thus, the present invention provides methods for the isolation and selection analysis of progeny derived from genetic crosses involving plants having the alleles of the present invention.

In alternative embodiments, the present invention provides methods for identifying canola plants, such as mustard plants, having a desired fatty acid composition or a desired genetic characteristic. For example, the methods of the invention can involve determining the presence of a particular FAD2 and/or FAD3 allele, such as the invention allele or wild-type J96D-4830/BJfad2a allele, in the genome. In particular embodiments, the method can include identifying the presence of a nucleic acid polymorphism associated with one of the identified alleles or an antigenic determinant associated with one of the alleles of the invention. Such assays can be accomplished, for example, using a range of techniques, such as PCR amplification of relevant DNA fragments, DNA fingerprinting, RNA fingerprinting, gel blotting and RFLP analysis, nuclease protection assays, sequencing of relevant nucleic acid fragments, generation of antibodies (monoclonal or polyclonal), or alternative methods suitable for distinguishing between proteins produced from related alleles from other variants or wild-type forms of the proteins. The invention also provides methods for identifying mustard plants whose seeds have an endogenous fatty acid content comprising at least 68% oleic acid (by weight) by determining the presence of a mutant allele of the invention.

In an alternative embodiment, the present invention provides a canola plant comprising FAD2 and FAD3 coding sequences encoding mutant FAD2 and FAD3 proteins. Such mutant FAD2/FAD3 proteins can contain only one amino acid change compared to wild-type FAD2 protein. In representative embodiments, the plurality of brassica juncea lines contain the previously described mutant FAD2 protein encoded by the previously described alleles. Such alleles can be selected to be effective in conferring increased oleic acid content and decreased linolenic acid content to the plants of the invention. In particular embodiments, the desired allele can be introduced into a plant by breeding techniques. In alternative embodiments, the alleles of the invention can be introduced by molecular biological techniques, including plant transformation. In such embodiments, the plants of the invention can produce seeds having an endogenous fatty acid content comprising: at least about 68% oleic acid (by weight) and less than about 3% linolenic acid (by weight), or any other oleic and linolenic acid content thresholds as listed above. The plants of the present invention may also contain from about 68% to about 85% (by weight) oleic acid, from about 70% to about 78% oleic acid, and from about 0.1% to about 3% linolenic acid, wherein the oil component is genetically derived from a parental line. The plants of the invention may also have a total fatty acid content of less than 7.1% to less than about 6.2% (by weight). In one embodiment, the plant produces seeds having an endogenous fatty acid content comprising at least about 68% oleic acid and less than 3% linolenic acid, wherein the oil composition is genetically derived from a parental line.

In selected embodiments, the present invention provides canola seeds, which may be mustard seeds, having an endogenous oil content having a fatty acid composition as set forth for one or more of the above embodiments, and wherein the genetic determinant of endogenous oil content is derived from a mutant allele of the invention. For example, such seeds may be obtained by self-pollination of each mutant allele line of the invention. Alternatively, such seeds may be obtained, for example, by crossing the mutant allele line with a second parent, followed by selection, wherein the second parent may be any other brassica line such as a brassica line, is brassica quality brassica juncea or non-brassica quality brassica juncea, or any other brassica species such as brassica napus, brassica rapa, brassica nigra, and brassica carinata. Such breeding techniques are well known to those skilled in the art.

In an alternative embodiment, the present invention provides genetically stable plants of the genus brassica, such as brassica juncea plants that form mature seeds having one or more compositions disclosed in the above embodiments. Such plants may be derived from a mustard line with the mutant alleles of the invention. The oil composition of such plants may be genetically derived from a parental line.

In an alternative embodiment, the present invention provides a method of producing a genetically stable canola plant, such as a mustard plant, that produces mature seeds having an endogenous fatty acid content comprising one or more of the compositions specified in the above embodiments. The method of the invention may involve the following steps: the Omega9 gene from Brassica napus (e.g., fad2a and fad3a) is crossed with other Brassica plants, such as Brassica juncea, to form F₁The progeny. Propagation of F, for example, by means which may include self-pollination or the formation of doubled haploid plants₁The progeny. By combining mutant FAD2 alleles and mutant FAD3 alleles, plants with dual mutant gene alleles (FAD2 and FAD3) can have oil fatty acid profiles superior to any single mutant plant. The resulting progeny may be selected for genetically stable plants that produce seeds having the composition disclosed in one or more of the preceding embodiments. For example, such seeds may have a stabilized fatty acid profile comprising a total saturates content of about 7.1% to about 6.5% in the total extractable oil. In certain variants, the progeny may themselves produce seeds or oils having the compositions set forth above for the alternative embodiments. Having an oleic acid content of greater than about 68% (by weight) and a linolenic acid content of less than about 3% (by weight).

In one aspect, the present invention provides plants having a stable, heritable high oleic and low linolenic acid phenotype. For example, the high oleic and low linolenic acid phenotypes derived from the mutant alleles of the invention are genetically heritable to the M2, M3, and M4 generations.

In alternative embodiments, the invention provides brassica juncea plants in which the activity of a fatty acid desaturase is altered, the oleic acid content is altered, or the linolenic acid content is altered relative to wild-type brassica juncea used in mutagenesis experiments. Fatty acid desaturase ("FAD") means that proteins exhibit the activity of introducing double bonds in the biosynthesis of fatty acids. For example, the FAD2/FAD3 enzyme can be characterized by the activity of introducing a second double bond in the biosynthesis of linolenic acid from oleic acid. Altered desaturase activity can comprise an increase, decrease, or elimination of desaturase activity as compared to a reference plant, cell, or sample.

In other aspects, reduction of desaturase activity can comprise abolishing expression of a nucleic acid sequence encoding a desaturase, such as a nucleic acid sequence of the invention. By abolishing expression is meant herein that the functional amino acid sequence encoded by the nucleic acid sequence is not produced at detectable levels. Decreasing desaturase activity can comprise ablating transcription of a nucleic acid sequence encoding a desaturase, such as a sequence of the present invention encoding a FAD2 enzyme or a FAD3 enzyme. Abolishing transcription means herein that the mRNA sequence encoded by the nucleic acid sequence is not transcribed at a detectable level. Reduced desaturase activity can also include the generation of truncated amino acid sequences from a nucleic acid sequence encoding a desaturase. Generating a truncated amino acid sequence means herein that the amino acid sequence encoded by the nucleic acid sequence lacks one or more amino acids of the functional amino acid sequence encoded by the wild-type nucleic acid sequence. Additionally, the decreased desaturase activity can comprise generating a variant desaturase amino acid sequence. Generating a variant amino acid sequence means herein that the amino acid sequence has one or more amino acids that are different from the amino acid sequence encoded by the wild-type nucleic acid sequence. As discussed in more detail herein, the present invention discloses that the mutation system of the present invention produces FAD2 and FAD3 enzymes with variant amino acids compared to the wild type line J96D-4830. To reduce desaturase activity, various types of mutations can be introduced into nucleic acid sequences, such as frameshift mutations, substitutions, and deletions.

In some embodiments, the present invention provides novel FAD2/FAD3 polypeptide sequences, which can be modified according to alternative embodiments of the present invention. It is well known in the art that certain modifications and variations can be made to the structure of a polypeptide without substantially altering the biological function of the peptide to obtain a biologically equivalent polypeptide. As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid for another at a given position in a peptide, where the substitution can be made to obtain a biologically equivalent polypeptide without any significant loss of function or gain. In making such changes, for example, substitutions of amino acid residues can be made based on the relative similarity of the side-chain substituents, e.g., their size, charge, hydrophobicity, hydrophilicity, etc., and the effect of such substitutions on peptide function can be determined by routine testing. In contrast, as used herein, the term "non-conservative amino acid substitution" refers to the substitution of one amino acid for another at a given position in a peptide, wherein the substitution results in a significant loss of function or gain of function of the peptide, to obtain a polypeptide that is not a biological equivalent.

Fibers are a component of plant cell walls and include carbohydrate polymers (e.g., cellulose (linear glucose polymeric chains)); hemicelluloses (e.g. branching of heterogeneous copolymers of galactose, xylose, arabinose, rhamnose, to which phenolic molecules are attached); and pectin (water soluble polymers of galacturonic acid, xylose, arabinose, with varying degrees of methylation). Cellulose also contains polyphenol polymerases (e.g., lignin-like polymers and condensed tannins). In theory, ADF fibers consist of cellulose and lignin. Condensed tannins are generally contained in the ADF fraction, but the condensed tannin content does not vary depending on the ADF. In contrast, TDF is a coarse powder from which proteins, solubles and starch have been removed, and is composed of insoluble cell wall components (e.g., cellulose, hemicellulose, polyphenols and lignin).

In particular embodiments, seeds of a brassica plant (e.g., a dark seed brassica plant) comprising the germplasm of the invention may have reduced ADF as compared to a brassica variety. In particular examples, the fiber content of canola meal (whole seed, excluding oil, on a dry weight basis) may include, for example and without limitation: less than about 18% ADF (e.g., about 18% ADF, about 17% ADF, about 16% ADF, about 15% ADF, about 14% ADF, about 13% ADF, about 12% ADF, about 11% ADF, and about 10% ADF, and/or less than about 22% NDF (e.g., about 22.0% NDF, about 21% NDF, about 20% NDF, about 19% NDF, about 18% NDF, and about 17% NDF).

In particular embodiments, seeds of a canola plant comprising the germplasm of the invention may have increased protein content as compared to a standard dark-colored seed canola variety. In a specific example, the protein content of canola meal (whole seed, oil removed, on a dry weight basis) may include, for example, but not limited to, greater than about 45% (e.g., about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, and about 58%) crude protein. Different brassica varieties are characterized by specific protein content. The protein content (% nitrogen x6.25) can be determined using a variety of well known and conventional analytical techniques such as NIR and Kjeldahl.

Phosphorus content can also be used to define seeds, plants, and lines of brassica varieties. Such canola varieties may, in some embodiments, produce canola meal (whole seed, oil removed, on a dry weight basis) having increased phosphorus content when compared to meal produced from standard canola varieties. For example, canola meal of the invention may have a phosphorus content of more than 1.2%, more than 1.3%, more than 1.4%, more than 1.5%, more than 1.6%, more than 1.7%, and/or more than 1.8%.

Various combinations of the above traits may also be identified in, and exemplified by, the inbred canola lines and hybrids provided in several examples. These line illustrations various novel combinations of a wide variety of advantageous canola characteristics and/or traits may be provided and obtained using the germplasm of the invention. For example, an inbred canola line comprising a germplasm of the invention may be crossed with another canola line comprising the desired characteristics and/or traits to result in a desired seed component characteristic of the inbred line comprising a germplasm of the invention. Calculations of seed components (e.g., fiber content, glucosinolate content, oil content, etc.) and other plant traits may be obtained using techniques known in the art and recognized in the industry. By selecting and propagating progeny plants from crosses that comprise the desired characteristics and/or traits of the parent varieties, new varieties can be created that comprise the desired combinations of characteristics and/or traits.

Canola meal with improved nutritional characteristics

Some embodiments provide a meal comprising a canola seed, wherein the canola seed has oil and meal characteristics, as discussed above. For example, some embodiments include hexane-extracted, air-dried canola meal (white flakes or WF) comprising a novel combination of features (e.g., seed components) as discussed above. Particular embodiments include meals comprising canola seeds produced from plants comprising the germplasm of the invention, and meals comprising seeds of progeny of plants comprising the germplasm of the invention.

In some embodiments, canola inbred lines and hybrids comprising the germplasm of the invention may provide nutritionally enhanced meal characteristics when utilized directly as feed or food ingredients and/or when utilized as feedstock for processing protein isolates and concentrates. For example, such canola inbred lines and hybrids can provide animal feed that is superior to standard canola meal. In some embodiments, the canola meal components (and animal feeds containing them) may be utilized to provide good nutrition for monogastric animals (e.g., pigs and poultry).

In some embodiments, the canola meal components (and animal feeds containing them) may be further utilized to provide good nutrition for ruminants (e.g., bovine animals, sheep, goats, and other animals of the suborder ruminants (ruminants)). Feeding ruminants presents special problems and special opportunities. A particular opportunity arises from the ability of ruminants to utilize insoluble cellulose fibers that can be broken down by certain microorganisms in the rumen of these animals, and are generally indigestible by monogastric mammals such as pigs. Particular problems arise from the tendency of certain feeds to inhibit fiber digestion in the rumen, and from the tendency of the rumen to limit the utilization of certain components of certain feeds, such as fat and protein.

Oil-extractable canola seeds are a potential source of high quality protein for use in animal feed. After oil extraction, commercial canola meal comprises about 37% protein, as compared to about 44-48% in soybean meal (which is currently widely preferred for feed and food purposes). The proteins contained in canola are rich in methionine and contain sufficient amounts of lysine, both of which are the limiting amino acids in most cereal and oilseed proteins. However, the use of canola meal as a protein source is somewhat limited in certain animal feeds because it contains undesirable constituents such as fiber, glucosinolates, and phenols (phenolics).

One nutritional aspect of canola derived rapeseed is its high (30-55 μmol/g) level of glucosinolates, a sulfur-based compound. Upon crushing canola leaves or seeds, isothiocyanates are produced by the action of myrosinase on glucosinolates. These products inhibit thyroxine synthesis by the thyroid gland and have other antimetabolic effects. Paul et al (1986) the or. appl. Genet.72: 706-9. As such, for human food use, for example, the glucosinolate content of the protein derived from rapeseed meal should be reduced or eliminated to provide product safety.

Improved canola seeds having, for example, favorable oil profiles and levels in the seed and low glucosinolate levels, will significantly reduce the need for hydrogenation. For example, the higher oleic acid and lower alpha-linolenic acid content of such oils can impart increased oxidative stability, thus reducing the need for hydrogenation and the production of trans fatty acids. The reduction in seed glucosinolates significantly reduces the residual sulfur content of the oil. Sulfur damages the nickel catalysts that are commonly used for hydrogenation. Koseoglu et al, Chapter8, in Canola and Rapeded: Production, Chemistry, Nutrition, and Processing Technology, Ed. Shahidi, Van nonstandard Reinhold, N.Y.,1990, pp.123-48. In addition, oils from brassica varieties with low seed glucosinolates can be less expensive to hydrogenate.

The phenolic compounds in the canola meal impart a bitter taste and are believed to be necessarily related to dark colour in the final protein product. The seed hulls (which are present in large quantities in standard canola meal) are indigestible in humans and other monogastric animals, and also provide an unsightly heterogeneous product.

The meal component of a seed produced by a canola plant comprising the germplasm of the invention may have, for example and without limitation: high protein; low fiber; higher phosphorus; and/or low SAE. Insoluble fibers and polyphenols are anti-nutritional and impair protein and amino acid digestion. As such, canola meal and animal feeds comprising canola meal having at least one seed component characteristic selected from the group consisting of: reduced fiber content, increased protein content, reduced polyphenol content, and increased phosphorus content.

In a particular example, canola meal (oil-free, on a dry matter basis) may comprise at least about 45% (e.g., about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, abo about 53%, about 54%, about 55%, about 56%, about 57%, and about 58%) protein content.

Canola varieties comprising the germplasm of the invention may have good yields and produce seeds with much lower Acid Detergent Fiber (ADF) than the search for canola lines. In some embodiments, any empirical value determined for the composition of seeds produced from a plant variety comprising the germplasm of the invention may be used to define the plant, seed, and oil of the plant variety. In some such instances, specific numbers may be used as endpoints to define ranges above, below, or between any of the measured values. Exemplary ranges for oil characteristics and other seed components are listed above. The lines and seeds of the plants thereof may also be defined in combinations of such ranges. For example, the oil characteristics discussed above, along with characteristic fiber levels, polyphenol levels, glucosinolate levels, protein levels, and phosphorus levels, for example, may be used to define a particular line and its seeds.

Not all of the foregoing features (e.g., seed component features) are required to define lines and seeds of some embodiments, but other features may be used to define such lines and seeds (e.g., without limitation, metabolic energy, digestive energy, bioenergy, and net energy).

Plants comprising germplasm that confers desirable seed component traits in a manner independent of seed color

The desired traits of particular canola inbred lines and hybrids comprising the germplasm of the invention may be transferred to other types of canola (via conventional breeding and the like), such as turnip and mustard, and the resulting plants produce seeds having desired characteristics (e.g., seed component characteristics) that are independent of seed color expression. Thus, a brassica variety that has received one or more desired trait transfers for a particular brassica inbred line or hybrid comprising germplasm of the invention can produce seed with desired characteristics having yellow-colored or dark-colored seeds. The meal and seeds of such new or modified brassica varieties may have reduced seed fiber levels, increased protein levels, increased phosphorus levels, and/or reduced polyphenol levels.

Some embodiments include not only yellow and dark seeds of brassica comprising germplasm as described and exemplified herein, but also plants planted or otherwise produced from such seeds, and tissue cultures of regenerable cells of the subject brassica plants. Exemplary lines and hybrids were obtained without genetic engineering and without mutagenesis, thereby demonstrating the utility of the germplasm in generating new and modified brassica varieties.

In some particular embodiments, particular exemplary canola inbred lines and hybrids are provided. As part of this disclosure, at least 2500 seeds of each of CL065620, CL044864, CL121460H, CL166102H, and CL121466H have been deposited with the American Type Culture Collection (ATCC), Rockville, md.20852, and are publicly available, subject to patent rights, but are not otherwise limited (except for those limitations expressly allowed by 37c.f.r. § 1.808 (b)). The deposits are designated as ATCC deposits No. PTA-11697, PTA-11696, PTA-11698, PTA-12570, PTA-11699, respectively, with a deposit date of 2011 at 22 months (for PTA11696 to PTA11699) and 2012 at 21 months (for PTA-12570). As set forth above, the deposit will be maintained in the ATCC deposit center (which is the public deposit center) for a period of 30 years, or 5 years after the most recent request, or for the life of the patent, whichever is longer, and will be replaced when it becomes non-viable during that period.

Some embodiments include seeds of any of the brassica varieties disclosed herein. Some embodiments also include canola plants produced from such seeds, and tissue cultures of regenerable cells of such plants. Also included are canola plants regenerated from such tissue cultures. In particular embodiments, such plants may be capable of expressing all of the morphological and physiological properties of the exemplified varieties. The canola plants of particular embodiments may have been identified for physiological and/or morphological characteristics from plants grown from the deposited seed.

Also provided are methods of crossing in at least one parent of a progeny of the seeds described above using germplasm of the invention (e.g., as found in the exemplary canola inbred lines and hybrids provided herein). For example, some embodiments include F1 hybrid brassica napus plants having any of the plants exemplified herein as one or both parents. Other embodiments include those through such F₁The hybrid produces a brassica napus hybrid. In particular embodiments, for generating F₁A method of hybridizing brassica napus seeds includes crossing an exemplary plant with a different inbred plant brassica plant and harvesting the resulting hybrid seed. The canola plants (e.g., parent canola plants, and methods of making F₁A brassica plant produced by such methods of crossing) can be a female or male plant.

In some embodiments, the characteristics (e.g., oil and protein levels and/or profiles) of a canola plant may be further modified and/or improved by crossing a plant of the invention with another line having a modified characteristic (e.g., high oil and protein levels). Likewise, other characteristics may be improved by taking care of the parent plant. A canola line comprising the germplasm of the invention may be beneficial for crossing its desired seed component characteristics into other canola or canola lines in a seed color independent manner. The germplasm of the invention allows for the transfer of these traits to other plants within the same species by conventional plant breeding techniques, including cross pollination and selection of progeny. In some embodiments, conventional plant breeding techniques involving pollen transfer and selection may be used to transfer desired traits between species. See, for example, Brassica cropping and wire alloys biology and weaving, Eds.Tsunada et al, Japan Scientific Press, Tokyo (1980); Physiological stresses for Yield Improvement of Annual Oil and protein clones, Eds.Dipenbrock and Becker, Blackwell Wissenschafts-VerlagBerlin, Vinnna (1995); Canola and Rapeeed, Ed.Shahidi, Van nonstrand Reinhold, N.Y. (1990); and Breeding Oilseed Brassica, Eds.Labana et al, Narosa publishing House, New Dehl (1993).

In some embodiments, a method for transferring at least one desired seed component characteristic in a seed color independent manner comprises self-pollinating members of the F1 generation to generate F2 seed after interspecific crossing. Backcrossing can then be performed to obtain lines that exhibit the desired characteristics of the seed components. In addition, protoplast fusion and nuclear transfer methods can be used to transfer traits from one species to another. See, for example, Ruesink, "Fusion of high Plant Protoplasts," Methods in enzymology, Vol.LVIII, eds. Jakoby and Pastan, Academic Press, Inc., New York, N.Y. (1979), and the relaying cis treated thermoin, and Carlson et al (1972) Proc.Natl.Acad. Sci.USA69: 2292.

Exemplary canola lines obtained and generated comprising germplasm of the invention can now be readily transferred to other canola species by conventional plant breeding techniques as set forth above, along with the desired seed component characteristics. For example, it is now possible to easily transfer the dark seed coat color to commercial turnip varieties such as, but not limited to, Tobin, Horizon and Colt, along with the desired seed component characteristics. It should be understood that the dark seed color need not be transferred with other characteristics of the seed.

Given an exemplary variation as a starting point, a person skilled in the art of double-stranded technology can manipulate the specific benefits provided by the variation in a variety of ways without departing from the scope of the invention. For example, the seed oil profile present in the exemplary variety can be transferred to an agronomically desirable brassica napus variety by conventional plant breeding techniques involving cross-pollination and selection of progeny, e.g., wherein the germplasm of the exemplary variety is incorporated into other agronomically desirable varieties.

Particular embodiments may include exemplified varieties of brassica napus, as well as substantially derived varieties that have been substantially derived from at least one of the exemplified varieties. Additionally, embodiments of the invention may include plants of at least one of the exemplified variants, plants of such substantially derived variants, and/or canola plants regenerated from plants or tissues (including pollen, seeds, and cells) produced therefrom.

Plant material capable of regeneration may be selected, for example, seeds, microspores, ovules, pollen, vegetative parts, and microspores. In general, such plant cells may be selected from any brassica variety, including those having desirable agronomic traits.

Regeneration techniques are known in the art. Cells capable of regeneration (e.g., seeds, microspores, ovules, pollen, and vegetative parts) may be initially selected from a selected plant or variety. Optionally, these cells may be subjected to mutagenesis. Plants can then be formed from the cells using regeneration, pollination, and/or growth techniques based on the cell type (and whether they are mutagenized or not). Manipulation of the plant or seed or portion thereof can result in the creation of a substantially derived variant.

In some embodiments, the desired seed component characteristics exhibited by plants comprising germplasm of the invention may be introduced into plants comprising a plurality of individual desired traits in a manner that is independent of seed color to produce plants having both the desired seed component characteristics and the plurality of desired traits. The method of introducing desired seed fraction characteristics into plants comprising one or more desired traits in a manner independent of seed color is referred to as "stacking" of these traits. In some instances, the superposition of the desired seed component characteristics with the plurality of desired traits may result in further improvements in the seed component characteristics. In some examples, the superposition of a desired seed component characteristic with a plurality of desired traits may result in a canola plant having the desired seed component characteristic in addition to one or more (e.g., all) of the plurality of desired traits.

Examples of traits that may be desired for combination with desired seed component characteristics include, for example and without limitation: plant disease resistance genes (see, e.g., Jones et al (1994) Science266:789 (tomato Cf-9 gene for resistance to Phyllomycete (Cladospora fulvum)), Martin et al (1993) Science262:1432 (tomato Pto gene for resistance to Pseudomonas syringae), and Mindrinos et al (1994) Cell78:1089 (RSP 2 gene for resistance to Pseudomonas syringae)); a gene conferring resistance to a pest; bacillus thuringiensis (Bacillus thuringiensis) proteins, derivatives thereof, or synthetic polypeptides modeled thereon (see, e.g., Geiser et al (1986) Gene48:109 (Bt. delta. -endotoxin Gene; DNA molecules encoding. delta. -endotoxin Gene are available, e.g., as ATCC accession No. 40098;67136;31995; and 31998 from the American type culture Collection (Manassas, Va))); lectins (see, e.g., Van Damme et al (1994) Plant Molec. biol.24:25 (Clivia miniata) mannose-binding lectin gene)); vitamin binding proteins, such as avidin (see international PCT publication US93/06487 (using avidin and avidin homologs as larvicides against insect pests)); an enzyme inhibitor; protease or proteolytic enzyme inhibitors (see, e.g., Abe et al (1987) J. biol. chem.262:16793 (rice cysteine protease inhibitor); Huub et al (1993) Plant Molec. biol.21:985 (Nicotiana protease inhibitor I; and U.S. Pat. No.5,494,813); amylase inhibitors (see Sumitoani et al (1993) biosci. Biotech. biochem.57:1243 (Streptomyces nitrosperus) alpha-amylase inhibitor)), insect-specific hormones or pheromones, e.g., ecdysteroids (ecdysteroids) or juvenile hormones, variants thereof, mimetics based thereon, or antagonists or agonists thereof (see, e.g., Hammock et al (124) Nature344:458 (juvenile hormone inactivating agent)); insect-specific peptides or neuropeptides that destroy affected pests (see, e.g., Regan insect-specific peptide (see, e.317) J. Biotin. chem.269: 1990 (Pacific) receptor for neuroparal insect-specific peptides (1985. pacific hormone; pacific receptor for insects) (pacific insect-specific neuropeptide) and pacific peptide (pacific receptor) (1989. pacific urinary tract) receptor); insect-specific venom produced in nature by snakes, wasps, or other organisms (see, e.g., Pang et al (1992) Gene116:165 (scorpion-insect-toxic peptide)); an enzyme responsible for the hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-proteinaceous molecule with pesticidal activity; enzymes involved in modification (including post-translational modification) of biologically active molecules, such as glycolytic enzymes; a proteolytic enzyme; a lipolytic enzyme; a nuclease; a cyclase; a transaminase; an esterase; a hydrolase; a phosphatase enzyme; a kinase; a phosphorylase enzyme; a polymerase; an elastase; chitinase; or dextranase, whether natural or synthetic (see International PCT publication WO93/02197 (cellobiase (callase) gene), DNA molecules containing chitinase coding sequences (e.g., from ATCC under accession Nos.39637 and 67152), Kramer et al (1993) Insect biochem. molecular. biol.23:691 (Helicoverpa virens larva) chitinase), and Kawawack et al (1993) Plant molecular. biol.21:673 (parsubi 4-2 polyubiquitin gene), molecules that stimulate signal transduction (see, e.g., Botella et al (1994) Plant molecular. biol.24:757 (calmodulin), and Griess et al (1994) Plant physiol.104:1467 (maize calmodulin), hydrophobic peptides (hydrophthalide) peptides (hydrophyllides) (see, e.g., International PCT publication WO 95/18855) for the synthesis of fungal cell membrane resistance inhibitors of fungal pathogens; see, PCT/3655, and derivatives of fungal genes for fungal infections), Channel formers, or channel blockers (see, e.g., Jaynes et al (1993) Plant Sci 89:43 (cecropin- β lytic peptide analogue which makes transgenic plants resistant to Pseudomonas solanacearum); viral entry proteins or complex toxins derived therefrom (see, e.g., Beach et al (1990) Ann. rev. Phytopathohol.28: 451 (for coat protein mediated resistance of alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potexvirus, potyvirus, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus); insect-specific antibodies or immunotoxins derived therefrom (see, e.g., Taylor et al, Abstract #497, Seven' l Symposiumon-Microbe (Edinbuild) (see, for example, Nature et al; for the production of recombinant antibodies for enzymatic challenge of strains of viruses via Taynes et al (1993); production of antibodies for protecting strains of plants (see, e.g., Nature 469 (1993) Antibody genes)); development-arresting proteins (developmental-arrestive proteins) produced by pathogens or parasites in nature (see, e.g., Lamb et al (1992) Bio/Technology10:1436 (fungal. endo-. alpha. -l, 4-D-polygalacturonase promotes fungal colonization and Plant nutrient release by solubilizing the Plant cell wall with-. alpha. -1, 4-D-galacturonase (galacturonase); Toubart et al (1992) Plant J.2:367 (endopolygalacturonase inhibitor))), and development-arresting proteins produced by plants in nature (see, e.g., Logemann et al (1992) Bio/Technology10:305 (inactivated ribosomal genes of barley, which provide increased resistance to fungal disease)).

Other examples of traits that may be desired for combination with desired seed component characteristics include, for example and without limitation: genes conferring resistance to herbicides (Lee et al (1988) EMBO J.7:1241 (mutant ALS enzyme); Miki et al (1990) the or. appl. Genet.80:449 (mutant AHAS enzyme); U.S. Pat. Nos. 4,940,835 and 6,248,876 (mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) gene providing glyphosate resistance); U.S. Pat. No.4,769,061 and ATCC accession No. 39256(aroA gene); Glyphosate acetyltransferase genes (glyphosate resistance); other phosphono compounds from Streptomyces species (Streptomyces species), including Streptomyces hygroscopicus and Streptomyces viridochromogenes), such as those described in European application No. 0246 and DeGreef et al (1989) Biotechnology (PAT gene 7) providing glyphosate resistance; pyridyloxy or phenoxypropionic acid and cyclohexanone (glyphosate resistant); european patent application No.0333033 and U.S. Pat. No.4,975,374 (glutamine synthetase genes that provide resistance to herbicides such as L-phosphinothricin); marshall et al (1992) the or. appl. Genet.83:435(Acc1-S1, Acc1-S2, and Acc1-S3 genes, which provide resistance to phenoxy propionic acid and cyclohexanone, such as sethoxydim and haloxyfop); WO2005012515 (GAT gene providing glyphosate resistance); WO2005107437 (gene conferring resistance to 2,4-D, fop and pyridyloxy auxin herbicides); and herbicides that inhibit photosynthesis, such as triazines (psbA and gs + genes) or benzonitrile (nitrilase gene) (see, e.g., Przibila et al (1991) Plant Cell3:169 (mutant psbA gene); nucleotide sequences of nitrilase genes are disclosed in U.S. Pat. No.4,810,648, and DNA molecules containing these genes are available under ATCC accession numbers 53435,67441, and 67442; and Hayes et al (1992) biochem. J.285:173 (glutathione S-transferase)).

Other examples of traits that may be desired for combination with desired seed component characteristics include, for example and without limitation: genes conferring or contributing to a value-added trait, e.g., modified fatty acid metabolism (see, e.g., Knultzon et al (1992) Proc.Natl.Acad.Sci.U.S.A.89:2624 (antisense gene to stearoyl-ACP desaturase that increases stearic acid content of plants)); reduced phytate content (see, e.g., Van Hartingsveldt et al (1993) Gene127:87 (Aspergillus niger phytase Gene enhances the breakdown of phytate, adding more free phosphate to the transformed plant), and Raboy et al (1990) Maydica35:383 (cloning and reintroduction of DNA associated with alleles of maize mutants responsible for having low levels of phytate)); and modified carbohydrate composition, for example, by transforming plants with genes encoding enzymes that alter the branching pattern of starch (see, e.g., Shiroza et al (1988) J.Bacteol.170:810 (Streptococcus) mutant fructosyltransferase genes); Steinmetz et al (1985) mol.Gen.Genet.20:220 (levansucrase) genes); Pen et al (1992) Bio/Technology10:292 (alpha-amylase); Elliot et al (1993) Plant mol.biol.21: 515 (tomato invertase genes); Sogaard et al (1993) J.biol. chem.268:22480 (barley alpha-amylase genes); and Fisher et al (1993) Plant 102: Physiol.102:1045 (corn starch branching enzyme II)).

The following examples are provided to illustrate certain specific features and/or aspects of the claimed invention. The embodiments should not be construed as limiting the disclosure to the specific features or aspects described.

Examples

Example 1: enhanced Canola Meal (ECM) and conventional canola meal mean nutrient composition and values.

Several analytical and functional studies were conducted between 2009 and 2012 to evaluate the nutrient composition and value of the ECM lines and hybrids of the invention. Whole unprocessed seeds, partially processed meal and fully processed meal were tested to account for possible processing effects on nutrient composition and values. Samples were analyzed at the university of illinois, missouri, georgia, and manitoba. This compositional information was used to evaluate the energy value of enhanced canola meal versus conventional canola meal using standard predictive equations. Biological assessment of poultry energy and amino acid digestibility was done at the university in illinois and georgia on samples. Biological evaluation of samples for pig energy and amino acid digestibility was performed at the university of illinois. Summary nutrient composition differences between ECM lines (range or mean) and conventional canola meal are shown in table 1. Details of relevant procedures and studies are summarized in the examples that follow.

Table 1: average nutrient composition of ECM and regular canola meal.

Numbers in parentheses are mean values

Prediction from nutrient composition

The ECM line shows several unique improvements in nutrient composition that provide animal feeding value. As shown in table 1, the ECM was about 7% more dotted in the protein than the conventional canola meal. In addition, the balance of essential amino acids (as a percentage of protein) is maintained at higher protein levels. Poultry and swine digestibility of amino acids in ECM is at least as good as in conventional canola meal, and the key amino acid lysine appears to have a slightly higher digestibility, the ECM line showing lower levels of fiber components found in cell walls and hulls, specifically, levels of lignin/polyphenols are about 2% lower points, cellulose levels are 1% lower points, ADF residues are 3% lower points (3% points), and ADF levels are 5% lower points.

Higher protein levels and lower fiber component levels were associated with about 10% increased bioenergy in ECM lines. These lines also show higher levels of phosphorus, which is an expensive nutrient added to animal feed. Higher protein (amino acids), energy and phosphorus are associated with an increase in canola meal value ($/t) of about 20-32% in swine and poultry feeds, as reflected in an increase in the opportunistic price of chicks and swine growing feeds. Table 1.

Example 2: POS White Flake (WF), LT and HT coarse powder method

ECM seeds and conventional canola seeds were processed at the POS test Plant (Pilot Plant) of the sas Cartoon (CA) according to the following protocol:

material

Canola seeds were received at the POS on day 2/8/2011 on about 1.5MT ECM test line (CL 44864). Approximately 3.0MT commodity control brassica seed was received at the POS on day 3/8/2011. The main material sources are as follows. Hexane/isohexane: univar, saskaton, SK.

Hyflo Super-cel filter aided: manville Products corp., Denver, CO.

Nitrogen gas: air Liquide, saskaton, SK.

Filter cloth, monofilament Porrits and Spensor, Point Claire, PQ.

Filter paper, 55lb tanned (tan style)1138-55 Porrits and Spensor, Point Claire, PQ.

The method comprises the following steps: processing in a laboratory

Between each canola variety, all equipment of the "primary" processing plant is evacuated or cleaned. Flammable, without shutting down the extractor between trials. However, the extractor chain Schnecken and solvent recovery system remained running to empty the equipment between brassica varieties. The vacuum was not turned off, so all vapors were drawn to the condenser, concentrated, and released into the solvent work tank. This prevents water from concentrating in Schnecken and plugging the conveyor. Canola samples were pressed/extracted in the following order:

1. control HT

2. Control LT

ECM test line (CL44864) LT

Flaking (Flaking)

Flaking is performed to rupture the oil cells and to prepare flakes with a large surface area for cooking/pre-pressing by passing the seeds through a set of glossing rollers. The sheet thickness and moisture are adjusted to minimize the amount of fines generated. High fines levels produce pressed cakes with poor solvent percolation characteristics.

Canola seeds are embryonated using a minimum roll gap setting. The flake thickness range for each batch was as follows:

1. controls HT 0.21-0.23 mm

2. Control LT 0.19-0.23 mm

ECM test line (CL44864) LT 0.21-0.23 mm

The feed rate was controlled by the press rate and was about 133-150 kg/hr.

Slicing machine: 14 "diameter x 28" width Lauhoff Flakmaster Mill model S-28, series No.7801, manufactured by Lauhoff Corporation.

Cooking (Conditioning)

Cooking is done to further break the oil cells, make the sheet pliable, and by this reducing the viscosity of the contained oil, increase the efficiency of the screw press (expeller). Cooking is also done to inactivate enzymes in the seeds. The digester was preheated before each run was developed. Steam pressure is adjusted during operation to maintain the desired flake temperature.

The temperatures in the trays used for the control HT batches were as follows:

60 + -5 deg.C for the top plate

Bottom plate 97 + -3 deg.C

The temperatures in the disks used for the control LT lot plus ECM test line (CL44864) LT lot were as follows:

60 + -5 deg.C for the top plate

Bottom plate 93 + -2 deg.C

A cooking pan: two pan Simon-Rosedown steamers were used. Each compartment was 36cm high (21cm working height) and 91cm in diameter and was supplied with a sweeping arm for material agitation. Steam is used on the jacket for dry heating, and direct steam may be added to the vessel contents. A digester was installed on the screw extruder (screw) for direct feeding.

Squeezing machine

Pressing takes about 2/3 of the oil and produces a pressed cake (presscake) suitable for solvent extraction. Press cakes require crush resistance (to lift in the extractor) and porosity (for good mass transfer and drainage). The padded and cooked seeds were pressed using a Simon-Rosedown Prepress (pre-press).

The crude press oil was collected in a tank.

A pre-squeezer: Simon-Rosedowns 9.5cm diameter x94cm long screw press. An operating screw speed of 17rpm was used.

Solvent extraction and desolvation

Solvent extraction is by contacting the pressed cake with hexane to measure oil from the cake mass. Two mechanisms are in operation: the oil is immersed in the solvent and the press residue (hexane-solid) is washed with increasingly weaker solvent mixed oil (miscella) (hexane-oil). The extraction is usually a continuous countercurrent process.

The canola control HT press cake was extracted with isohexane/hexane using a total residence time (loop in) to loop out) of about 90 minutes, a solvent to solids ratio of about 2.5:1(w: w), and a solvent mixed oil temperature of 52 ± 5 ℃. (the canola press cake feed rate is about 90kg/hr at 90 minute residence time, and the solvent flow rate is 220 ± 10 kg/hr.).

Samples of commercial white canola flake (WF) were removed and air dried prior to desolvation.

The crude oil was desolvated in a rising film evaporator and a steam stripper.

Desolvation of the press cake (hexane-solids) was accomplished in a steam-jacketed Schnecken screw and 2-pan desolvator-oven. The jet was added to the top DT disc. The target temperatures in the disks were as follows:

schnecken outlet: <60 deg.C

Desolvated disk 102 + -3 deg.C

Baking plate at 102 + -3 deg.C

Canola control LT and ECM test line (CL44864) LT batch presscakes were extracted with isohexane/hexane using a total residence time of about 90 minutes (ring to ring out), a solvent to solids ratio of about 2.5:1(w: w), and a solvent mixed oil temperature of 52 ± 5 ℃. (the canola press cake feed rate is about 80kg/hr at 110 minutes residence time, and the solvent flow rate is 220 ± 10 kg/hr.).

Samples of ECM test line White Flakes (WF) were removed and air dried prior to desolvation.

The crude oil was desolvated in a rising film evaporator and a steam stripper.

Desolvation of the press cake (hexane-solids) was accomplished in a steam-jacketed Schnecken screw and 2-pan desolvator-oven. The jet was added to the top DT disc. The target temperatures in the disks were as follows:

schnecken export <60 DEG C

93 + -2 deg.C for desolvated disks

Baking plate at 93 + -2 deg.C

An extractor: all stainless Crown Iron Works Loop Extractors (type II). The extraction bed was 20.3cm wide by 12.7cm deep by 680cm long. In addition, the unit included solvent mixed oil desolvation using a climbing film evaporator and a steam stripper and press residue (solid plus solvent) desolvation using a steam jacketed Schnecken screw and a 2-pan desolvator-oven. The recovered solvent is collected and recycled.

Vacuum drying

Vacuum drying was done to dry the defatted LT canola meal to <12% moisture.

The defatted canola meal batches that require drying are only control LT batches. About 225kg of defatted meal was loaded into a Littleford reactor dryer. The meal was then heated to 75 + -2 deg.C under 10-15' HG vacuum. Meal sampling was started at about 60 ℃ for moisture analysis and occurred every 15 minutes until moisture < 12%. The meal was then placed in bulk sacks. The above procedure was repeated until all the meal was dried.

A vacuum drier: a600 liter Littleford reactor of the FKM600-D (2Z) type, sequence #5132, LittlefordDay, Florence, KY.

Hammer mill (Hammer milling)

Hammer milling was performed to produce consistent particle sizes.

The dried meal was hammer milled using an 8/64 "screen. The hammer mill was vacuum swept between each batch of meal. The meal is packaged into fiber drums and stored at ambient temperature until shipment.

The order of hammer milling the canola meal is as follows:

1. control HT.

ECM test line (CL44864) LT.

3. Control LT.

A hammer mill: prater Industries, G5HFSI type, series #5075, Chicago, IL

Example 3: indianapolis white flake process

The canola seeds of the invention may be processed to produce canola white flakes using the procedures originally described in Bailey's Industrial Oil & Fat Products (1996),5th ed., Chapter2, wiley inter science Publication, New York.

To extract oil from canola seeds, canola seeds are first plumped, i.e., coffee ground, and heat treated in an oven to 85 ℃ ± 10 ℃ for at least 20 minutes. After heat treatment, a Taby press was usedMachine 20A type press (Skeppsta,Sweden) pressed the ground seeds. The resulting pressed cake from the Taby press was subjected to solvent extraction to remove any remaining residual oil.

The pressed cake from the oil seed pressing step is then subjected to solvent extraction to remove and collect any remaining residual oil. The press cake was placed into a stainless steel sleeve (thimbles) which was placed into a custom Soxhlet^TMIn an extractor (from lasale Glassware (Guelph, ON)). Hexane can be used as extraction solvent and is tolerated by Soxhlet^TMThe extractor system was run for 9-10 hours. The solvent extracted pressed cake was then removed from the sleeve and spread throughout the pan to a cake thickness of less than 1 inch. The solvent extracted cake was allowed to air desolvate for 24 hours before milling. The solvated white flakes are then milled away using, for example, RobotCoupe R2N Ultra B (Jackson, MS).

Example 4: sample analysis

Chemical and nutrient analysis of ECM and conventional canola samples can be performed differently using methods as outlined below. Canola meal samples were analyzed for dry matter (method 930.15; AOAC international.2007.official methods of AOAC int.18th. rev.2.w. hortwitzand g.w.latimer jr., eds. asoc. of. anal. chem. Int., gaithersburg.md. (hereinafter "AOAC Int., 2007")), ash (method 942.05; AOAC Int.), and GE (via bomb calorimeter (model 6300, Parr Instruments, Moline, IL)). AOAC International (2007) office Methods of Analysis of AOAC int, 18th ed.Rev.2, Hortwitz and Latimer, eds. Assoc. off.anal. chem.int., Gaithersburg. MD. Acid hydrolyzed ether extracts (AEE) were determined by acid hydrolysis using 3N HCl (Sanderson), followed by crude fat extraction with petroleum ether on a Soxtec2050 automated analyzer (FOSS North America, Eden Prairie, MN) (method 954.02; AOAC Int.). Sanderson (1986), "A new method of analysis of feeding decisions for the determination of crop oils and falls," Pages77-81, in Recent Advances in Animal Nutrition, Haresign and Cole, eds. Crude protein was measured by combustion (method 990.03; AOAC Int.) on an Elementar rapid N-cube (cube) protein/nitrogen apparatus (Elementar americas inc., mt. laurel, NJ); amino acids [ AOAC Int. ], were determined according to method 982.30E (a, B and C); crude fiber was determined according to method 978.10(AOAC Int.); ADF and lignin were measured according to method 973.18(AOAC Int.); and NDF was determined according to Holst (Holst, D.O.1973.Holst filtration apparatus for VanSoest reagent analysis.J.AOAC.56: 1352-. The sugar profiles (glucose, fructose, sucrose, lactose, maltose) follow the Churms (Churms,1982, Carbohydrates in Handbook of chromatography. Zweight and Sherma, eds. CRC Press, Boca Raton, FL.), and Kakehi and Honda (1989.Silyl ethers of Carbohydrates. Page43-85in analytes of Carbohydrates by GLC and MS.C.J.biermann and G.D.McGinnis, eds. CRC Press, Boca Raton, FL). Oligosaccharides (raffinose, stachyose, verbascose) were analyzed according to Churms; minerals (Ca, P, Fe, Mg, Mn, Cu, Na, K, S, Mo, Zn, Se, Co, Cr) were determined by inductively Coupled Plasma Optical emission spectroscopy (ICP-OES) [ method 985.01(A, B, and C); AOAC Int ], while phytates were determined according to Ellis et al (1977.Quantitative determination of phytate in the presence of the precursor of high organic phosphorus. Anal. biochem.77: 536) in a single-step reaction.

Example 5: baseline analysis of ECM Indianapolis white flake samples and regular canola meal

Nutrient composition of the roasted ECM and regular canola meal prepared at the pilot plant. Several ECM lines (44864,121460,121466 and 65620) were processed in indianapolis' Dow agro sciences laboratory using a process similar to commercial canola meal processing, but without the final step of desolventizer/toasting after solvent extraction of oil from the seeds. This method and the resulting samples are referred to as "indianapolis white flakes". The processing parameters are outlined in example 3. These ECM indianapolis white flake samples were tested at university in illinois and mississippi and the results are shown in tables 2a,2b and 2 c. Canola meal control was toasted, commercially prepared canola meal. Values are expressed on a dry matter basis (but including oil).

Table 2 a: nutrient composition of ECM indianapolis white flake canola meal samples compared to conventional canola meal.

The results of the analysis of samples of ECM indianapolis white flakes from the university of illinois and mississippi were similar to the results from the mannich university for whole seeds. Oligosaccharides were lower in samples 44864(2010) than in other ECM samples, including 2011 planted 44864, while monosaccharides were higher. It appears that for the 2010 sample, the growing plants will catalyze the formation of monosaccharides with some sucrose and oligosaccharides near harvest.

Using the indianapolis white flake protocol, higher protein, lower ADF, and lower lignin and polyphenols were seen in the ECM line compared to the conventional canola meal, which is similar to the results seen with whole seeds. The 33% NDF value of the commercial meal is at the upper end of the typical range.

Table 2 b: amino acid composition (% crude protein) of ECM indianapolis white flake samples compared to regular canola meal.

Essential amino acids considered as major limitations in poultry and pig feed

As with the whole seed, the results in table 2b show that the amino acid composition (as a percentage of crude protein) is similar for the ECM indianapolis white flake sample and the commercial canola meal. This indicates that important amino acids are increased proportionally due to the increase of proteins in the ECM line.

Table 2 c: mineral composition of indianapolis ECM white flake samples compared to regular canola meal.

The mineral content of the ECM indianapolis white flake sample was similar to that of conventional canola meal, with two exceptions being phosphorus and sodium. As was the case with the mannich toba university results for whole seeds, phosphorus in the ECM line did appear consistently higher than conventional canola meal. The additional sodium in the conventional canola meal is undoubtedly due to sodium added during conventional canola processing.

Example 6: ECM processing at POS test plant of Sass cartoon, Canada to simulate commercial processing

In animal feeding evaluations to prepare ECM, it was determined that canola meal samples should be prepared under commercial processing conditions in view of the impact of processing on nutritional value. Subsequently, the samples were processed at the POS test plant of the sas cartoon. Two processing conditions were used: regular temperatures (HT) and Lower Temperatures (LT) in desolventizer/oven to ensure that processing conditions do not impose more than an all over impact on nutritional value. The processing conditions used for the POS are outlined in example 2.

Table 3: nutrient composition of ECM and conventional canola meal prepared at POS test plant of the sass cartoon, canada under simulated commercial processing conditions. (analyses were performed at university in Illinois and Missippi).

The test processed meal showed similar composition to the whole seed and indianapolis white flake samples, and the differences between the ECM sample and the conventional canola were consistent with the analyses described in tables 2a and 2 b: protein 7% higher, ADF 5% lower, lignin and polyphenol 4% lower, and phosphorus 0.35% lower.

Example 7: complete analysis of unprocessed ECM and conventional canola seeds

Nutrient composition of unprocessed canola seeds. 5 whole seed samples from ECM lines produced in 2010 and 2011 were analyzed at the university of manitoba. These were compared to the official Canadian Grain Commission (CGC) composite seed samples produced in 2011, which by definition is the average quality of current commercial brassica varieties cultivated in the west canada during the season. The nutrient composition results are expressed on an oil-free dry matter basis and are shown in tables 4a and 4 b.

Table 4 a: nutrient composition of ECM seed samples compared to conventional canola seed

The results show that the greatest difference between ECM and conventional canola is higher protein content. ECM was 7.2% point higher on an oil-free dry matter basis (51.1% vs. 43.9%) and 6.1% point higher on a 3% oil, 88% dry matter basis (typical specification basis for commercial canola meal) (43.5% vs. 37.4%) in protein content. See tables 4a,4 b. Higher protein seems to be explained by a 2% lower lignin and polyphenol in ECM and a 3% lower ADF residual (ADF-lignin/polyphenol-cellulose). ADF residues may be a combination of glycoprotein and hemicellulose components. The fiber components are mainly present in the cell walls and the shell. ECM has a phosphorus content that is almost 30% higher than in conventional canola and it appears to be evenly distributed between phytic acid and non-phytic acid forms. Phosphorus is a valuable nutrient in animal feed and even if phytate-bound phosphorus is incompletely digested by poultry and swine, the usual use of phytase in animal feed makes this phosphorus available to the animal. Table 4b provides a similar comparison of amino acid composition in whole plant seeds.

Table 4 b: amino acid composition of ECM seed samples (% crude protein) compared to conventional canola seed.

Essential amino acids considered as major limiting in poultry and pig feed

The results in table 4b show that the amino acid composition (as a percentage of crude protein) is similar between ECM and commercial canola meal. This indicates that as proteins are increased in the ECM line, important amino acids are also increased.

Example 8: TME and amino acid digestibility in poultry

True Metabolic Energy (TME) and True Available Amino Acid (TAAA) assays were developed by doctor Ian sibvald of Agriculture Canada in ottawa in 1976 and 1981, respectively. Due to its direct and non-destructive nature, the assay has become the method of choice in many countries of the world, including the united states, for determining the energy and amino acid availability in poultry feed ingredients.

Mature Single Comb White Leghorn (SCWL) young rooster were used as the experimental animals of choice in different studies conducted at illinois university and georgia university. Birds are known to have a rapid intestinal clearance time (gut-clearance time). By removing the feed for a period of 24 hours, it is reliably assumed that the digestive tract of the test subject is emptied of previously consumed food residues.

Each bird (typically 8 individuals per treatment) was precision-fed 35 grams of the test feed, placed directly into the crop via intubation. The space volume is similar, usually with 25 instead of 35 grams of the fiber high ingredient fed. After intubation, the birds were provided with water, but no additional feed, for a period of 40 hours during which the excreta were collected quantitatively. After collection, the excreta are dried in a forced air oven, typically at 80C. Subsequently, it is weighed and ground for determining the total energy (GE) in a TME assay, or to determine the amino acid content. The GE and amino acid composition of the fractions were similarly determined. Once weighed, fecal samples were pooled and homogenized for single GE or amino acid assays. The amount of excreta mass per bird varies much more than the GE or amino acid composition of a particular excreta. This observation and the expense and time delay of GE and amino acid determinations justify the combination.

Digestibility is calculated using methods well known in the art for energy or individually for each amino acid. Estimates of endogenous loss of GE and amino acids were used to correct for experimental artifacts.

Example 9: pig Digestive Energy (DE), Metabolic Energy (ME)

DE and ME. 48 growing castrated pigs (initial BW:20kg) were included in the randomized complete block design study at the university of Illinois. Pigs will be assigned 1 of 6 diets, with 8 replicates per diet. Pigs will be placed in metabolism cages equipped with feeders and nipple suckers (nipple drinker), full slatted floors, screen floors (screen floor), and urine pans. This would allow total, but separate urine and fecal material collection from each pig.

The amount of feed provided per pig per day will be maintained at the minimum pig energy per repeat (i.e. 106kcalME per kg)^0.75NRC,1998) was calculated 3-fold and divided into 2 equal meals. NRC1998, Nutrient requirements of Swine, Tenth reviewed edition, national academy Press, Washington, DC. Water will be available at all times. The experiment will last for 14 days. The first 5 days are considered to be a dietary adaptation period and urine and fecal material are collected during the last 5 days using a marker-to-marker method (marker to marker approach) according to standard protocols (Adeola, O.2001, diagnostic and analysis techniques in pigs, pages903-916in Swine Nutrition.2nd ed.A.J.Lewisand L.L.southern, ed.CRC Press, New York, NY.NRC.1998.NutrientRequirements of ine Swine 10th rev.ed.Natl.Acad.Press, Washington DC.). Urine samples were collected on 50mL hydrochloric acid protectant in a urine bucket. Immediately after collection, the feces samples and 10% of the collected urine were stored at-20 ℃. At the end of the experiment, the urine sample will be thawed and mixed in the animal and diet and the subsamples will be used for chemical analysis.

Stool samples will be dried in a forced air oven and finely ground prior to analysis. Stool, urine, and feed samples were analyzed in duplicate for DM and total energy using a bomb calorimeter (Parr Instruments, Moline, IL). After chemical analysis, total tract digestibility values were calculated for energy in each diet using the previously described protocol (Widmer, m.r., l.m.mcginnis, and h.h.stein.2007.energy, phophorus, and amino acid diagnostic of high-protein variants dried grains and corn fed to growing slices.j.anim.sci.85: 2994-. The amount of energy lost in the feces and urine, respectively, was calculated, and the amount of DE and ME in each of the 24 diets was calculated (Widmer et al, 2007). The DE and ME of corn will be calculated by dividing the DE and ME values of the corn diet by the corn inclusion rate in the diet. These values would then be used to calculate the contribution to DE and ME from corn in the corn-canola meal diet and the corn-soybean meal diet, and then the DE and ME in each source of canola meal and soybean meal sample would be calculated by difference, as previously described (Widmeret al, 2007).

The data were analyzed using the Proc Mixed protocol in SAS (SAS Institute inc., Cary, NC). The data obtained for each diet and each ingredient will be compared using ANOVA. Homogeneity of variance was confirmed using the UNIVARIATE protocol in Proc Mixed. Diet or composition will be a fixed effect, while swine and repeat will be random effects. The least squares means will be calculated using the LSD test and the means will be separated using the pdf statements in procmix. Pigs will be the experimental unit for all calculations and alpha level 0.05 will be used to assess significance between means.

Example 10: pig amino acid digestibility (AID and SID)

Porcine AIDs and SID were analyzed in a study at the university of illinois. 12 growing castrated pigs (initial BW:34.0 + -1.41 kg) were fitted with T-cannulas near the distal ileum and included a repeated 6x6 latin square design, each with 6 diets and 6 sessions. Pigs were individually housed in 1.2x1.5m pens in environmentally controlled rooms. The enclosure has solid side panels, a full slatted floor, and a feeder, and each side panel mounts a nipple sucker.

A 6-part diet was prepared. The 5 diets were based on corn starch, sugar, and SBM or canola meal were the only source of AA in these diets. The last diet was an N-free diet used to assess the basal ileal endogenous loss of CP and AA. Vitamins and minerals are included in all diets to meet or exceed the current needs assessment for growing pigs (NRC, 1998). All diets also contained 0.4% chromium oxide as a non-digestible marker.

The weight of the pigs was recorded at the beginning and end of each session, and the amount of feed supplied per day was also recorded. All pigs were fed at a level of 2.5 times the daily maintenance energy requirement and water was available at all times throughout the experiment. The first 5 days of each period are considered as the dietary adaptation period. Ileal digest (digesta) samples were collected for 8 hours on days 6 and 7 using standard procedures. The plastic bag was attached to the cannula barrel using a cable tie and the digest collected in the bag. The bags are removed at any time they are filled with digesta, or at least 30 minutes, and immediately frozen at-20 ℃ to prevent bacterial degradation of the amino acids in the digesta. After completion of one experimental period, animals were deprived of feed overnight and the next morning and provided a new experimental diet.

At the end of the experiment, ileal samples were thawed, pooled in animals and diets, and subsamples were collected for chemical analysis. Samples were also collected for each diet and each canola meal and SBM sample. The digest samples were lyophilized and finely ground prior to chemical analysis. All samples of diet and digests were analyzed for DM, chromium, crude protein, and AA, and canola meal and SBM for crude protein and AA.

Apparent Ileal Digestibility (AID) values for AA in each diet were calculated using equation [1 ]:

AID,(%)=[1-(AAd/AAf)x(Crf/Crd)]x100, [1]

wherein AID is the apparent ileal digestibility value of AA (%), AAd is the concentration of said AA in ileal digest DM, AAf is the concentration of said AA in feed DM, Crf is the chromium concentration in feed DM, and Crd is the chromium concentration in ileal digest DM. The AID of the CP is also calculated using this equation.

The basal endogenous flow of each AA to the distal ileum was determined based on the flow obtained after feeding the N-free diet using equation [2 ]:

IAA_{end up}=AAd x(Crf/Crd) [2]

Wherein IAA_{End up}Is the basal endogenous loss of AA (mg per kg DMI). The same equation will be used to determine the basal endogenous loss of CP.

By correcting the IAA of each AA for AID_{End up}Using equation [3 ]]Normalized ileal AA digestibility values were calculated:

SID,(%)=AID+[(IAA_{end up}/AAf)x100] [3]

Where SID is the normalized ileal digestibility value (%).

Data were analyzed using the Proc GLM protocol of SAS (SAS inst.inc., Cary, NC). The 5 diets containing canola meal or SBM were compared using ANOVA with canola meal source, pigs, and time periods as the primary effect. The mean was separated using the LSD test. Alpha level 0.05 was used to assess significance between means. The pig individuals were the experimental unit for all analyses.

Example 11: dietary AA digestibility

Amino acid digestibility of ECM will be assessed by incubating ECM meal samples in situ in rumen-intubated animals, such as cows, to assess soluble and degradable protein content, and to determine the degradation rate (Kd) of the degradable fraction.

Cattle will be fed a mixed diet of Total Mixed Ration (TMR) containing 28.1% corn silage, 13.0% alfalfa silage, 7.4% alfalfa hay, 20.4% ground corn, 14.8% wet brewer's spent grain (brewer's), 5.6% whole cottonseed, 3.7% soybean hulls, and 7.0% supplements (protein, minerals, vitamins). Standard polyester in situ bags (R510,5CM x10CM, 50-micron pore size) containing about 6g Dry Matter (DM) soybean meal (SBM), regular Canola Meal (CM), or Enhanced Canola Meal (ECM) were incubated in the rumen for 0,2,4,8,12,16,20,24,32,40,48, and 64 hours. Duplicate bags would be removed at each time point and rinsed in tap water until the effluent was clean. The bags were dried at 55 ℃ for 3 days, then the residue was taken out and weighed to determine the disappearance of the Dry Matter (DM). The residue was analyzed for N content using Leco's combustion method. The samples were not incubated in the rumen for 0 time, but were washed and processed in the same manner as the rumen incubated samples.

Samples of the 0 time residue and residue remaining after 16 hours incubation of the rumen will be analyzed for approximate composition (DM, crude fat, crude fiber, and ash) and Amino Acid (AA) composition (no tryptophan). These parameters can be used to generate estimates of Ruminal Degrading Protein (RDP) and ruminal non-degrading protein (RUP), as used in the National Research Council (2001) guidelines for dairy cow nutrient requirements.

The percentage of the original sample N retained at each time point can be calculated and the replicates for each time point within the cow are averaged. Values from three cows will be fitted toand McDonald (1979). In this method, assuming rumen CP disappearance follows first order kinetics, as defined by the equation, CP disappearance = a + B × (1-e)^-Kd×t) Where A is the soluble CP fraction (% CP), B is the potentially degradable CP fraction (% CP), and Kd is the degradation rate constant (h)^-1) And t is the rumen incubation time (h). Fraction C (non-degradable in the rumen) was calculated as fraction a minus fraction B. The equation would be fitted using SAS's PROC NLIN (9.2 th edition; SAS Institute Inc., Cary, NC), which was performed using the Marquardt calculation method.

The equations for calculating the RDP and RUP values (in percent of CP) are: RDP = a + B [ Kd/(Kd + Kp) ], and RUP = B [ Kp/(Kd + Kp) ] + C, where Kp is the rate of passage from the rumen. Since the transit rate cannot be calculated directly from these data (where the rumen contains matrix and which prevents passage to the next tract), the rate of Kp must be assumed. In this study, a value of 0.07 would be used for Kp, which is similar to the value calculated in NRC (2001) for the equation for high-producing cows consuming a typical lactating diet. Since the purpose of this project was to compare protein sources and ruminal digestibility estimates under the same conditions, the choice of passage rate for determining RDP and RUP was arbitrary.

The final equation for each sample will be generated using samples incubated for 0,2,4,8,16,24 and 48 hours according to the NRC (2001) recommendations. Data from additional incubation time points (i.e., 12,20,32,40, and 64 hours) in this study can be used to confirm the kinetics of the system and ensure that the modified canola meal is consistent with the assumptions in the NRC (2001) specification.

Example 12: poultry TME and TAAA, including true TME versus predicted TME based on analytical results from illinois, missouri, and manitoba university.

Poultry True Metabolic Energy (TME) assessments of ECM samples were conducted at both illinois university and georgia university. The protocol is described in example 8.

Table 5: TME content of ECM and conventional canola meal in the university of illinois and university of georgia studies.

Mean values within columns and groups with different letters are significantly different (p <.05)

**(SE)

(difference of percentage)

In the case of POS prepared ECM and canola meal samples, a suitable comparison was made between the two LT meals to eliminate processing effects. The results were comparable in both the university of illinois and the university of georgia studies. Poultry TME was significantly higher for ecm (LT) than conventional canola meal (LT): the study at university of illinois was 9% higher and that at university of georgia was 14% higher. These results confirm the predictive equation results below. Table 4.

White flake samples of ECM and regular canola meal were also collected at the POS immediately after the solvent extractor stage and prior to the DT stage. These WF meal poultry TMEs were compared in different studies at georgia university and, as with the LT samples, the ECM WF had significantly higher TMEs than the conventional canola meal WF. Table 4.

4 ECM variants were processed independently in the Dow Agrosciences laboratories of Indianapolis using the white flake process described in example 3. These samples were then subjected to poultry TME analysis at two colleges. There was no significant difference in TME between the ECM lines tested, except that 121460 line appeared to have a lower TME than either 121466 or 65620 line.

The observed TME values from these results are consistent with the metabolic energy content predicted below. National Research country nutrients of Poultry (NRC,1984, nutrients of Poultry. ninth viewed edition. National Academy press. washington, DC)) has the prediction equation for ME in canola meal (double zero rapeseed meal): ME kcal/kg = (32.76x CP%) + (64.96x EE%) + (13.24x NFE%)

By calculation, a CP that is 7% higher should be compensated by a NFE that is 7% lower, so the net coefficient of CP should be: 32.76-13.24 = 19.52. This resulted in 137kcal/kg more ME in the ECM than in canola meal (7% x19.52= 137). The problem with this equation is that NFE is a poor estimate of the sugar and starch energy values.

An alternative equation is the EEC prediction equation for poultry ME (adult). (Fisher, C and J.M.McNab.1987.techniques for determining the ME content of a pulmonary feed. in: Haresign and D.J.A.Cole (Eds), Recent Advances in Animal Nutrition-1987. Butterworks, London.P.3-17): ME, kcal/kg = (81.97x EE%) + (37.05x CP%) + (39.87x starch%) + (31.08x sugar%)

The EEC equation is a "positive contribution" equation that gives values to digestible nutrients in canola meal, such as protein, fat, starch, and free sugars. Since the only analytical difference between ECM and canola meal is protein, we can use the coefficient 37.05 to calculate the additional energy: 37.05x7% =259 kcal/kg. The EEC equation is designed for whole feed, which generally has a higher digestibility than canola meal. Therefore, the 37.05 coefficient is too high.

An alternative approach is to use the first principle of protein mass energy values. The approximate estimate was 4 calories total energy per gram of protein x80% protein digestibility x5% loss of nitrogen excretion = about 75% total calories per gram (3 calories of metabolic energy per gram or 30x protein%. this yields a metabolic energy in ECM of 30x7% =210kcal/kg extra ME.

In summary, ECM meal would be expected to have 140-260 kcal/kg more poultry ME than conventional canola meal. The 140kcal/kg value may be heavily underestimated, while 260kcal/kg may be on the higher side. It is possible to increase 200-. This is expressed on an "as is" basis (Table 1), and the commercial ECM may have 2000kcal/kg poultry ME to conventional canola meal of 2200 kcal/kg. This is a 10% energy increase.

Poultry true amino acid digestibility (TAAA) was also measured at both the university of illinois and the university of georgia. In this case, only POS-prepared meal samples were analyzed, as it is believed that the much higher amino acid digestibility of white flakes relative to roasted canola meal is not commercially relevant. Table 6.

Table 6: poultry True Amino Acid Availability (TAAA) of key amino acids of the POS-prepared ECM and conventional canola meal were studied.

There were no statistically significant differences in the availability of true amino acids to poultry between the different canola meal samples. Table 6.

Example 13: porcine amino acid digestibility (AID and SID) and predicted NE

A study of porcine ileal amino acid digestibility was conducted at the university of illinois. The comparison was carried out using the meal prepared by the POS pilot plant.

Table 7: porcine apparent ileal amino acid digestibility (AID) and porcine normalized ileal amino acid digestibility (SID) of proteins and key amino acids in ECM and conventional canola meal prepared at POS in the university of illinois study.

Mean values within rows and groups with different letters are significantly different (p <.05)

Some statistically significant differences in protein and amino acid digestibility were noted between the ECM and canola meal samples. ECM had a higher crude protein AID than canola meal, but the difference in protein SID was not significant. For both AID and SID, lysine is more digestible in the ECM than in conventional canola meal that has undergone the same heat treatment. Table 7.

For pigs, well-established equations for predicting DE, ME, and NE in pigs are the equations for Noblet, as outlined in EvaPig (2008, Version1.0.INRA, AFZ, Ajinomoto Eurosyne) and NRC NutrientRequirements of Swine (NRC,1998, NutrientRevised Requirements of Swine; TenthRevised Edition; National Academy Press, Washington, DC):

equations 1-4: DE, kcal/kg = 4151- (122x Ash%) + (23x CP%) + (38x EE%) - (64x CF%)

Equations 1-14: NE, kcal/kg =2790+ (41.22x EE%) + (8.1x Starch%) - (66.5xAsh%) - (47.2x ADF%)

The Noblet equation is a hybrid of both positive and negative contribution factors: fat, protein and starch have positive coefficients, while ash, CF and ADF have negative coefficients. No protein was used in the Net Energy (NE) equation, but the difference in ADF captured the difference between ECM and canola meal. Since starch and ash are the same in ECM and canola meal, the key difference is ADF. ADF 5% lower point results in 47.2x5% =236kcal/kg more NEs in the ECM. This prediction number is similar to the poultry ME number, so again, a net pig energy increase of 200kcal/kg in ECM is possible on an "as is" basis (table 1). This should result in an energy increase of about 12%.

Example 14: other ECM hybrids

The new canola hybrid CL166102H also exhibits enhanced meal (ECM) properties. Performance and quality traits, including oil, meal protein, ADF, and total glucosinolates (Tgluc), were measured on this hybrid seed (harvested from the 2011 pilot). See table 8.

The results in table 8 clearly indicate that this new DAS ECM line is superior to the commercial variant in terms of coarse meal properties.

Table 8 b: agronomic Performance of the ECM line (C3B03 test)

Claims (42)

1. A canola germplasm conferring on a canola seed the traits of high protein content and low ADF content, wherein the canola plant produces a seed having at least 68% oleic acid (C18:1) and less than 3% linolenic acid (C18: 3).

2. The canola germplasm of claim 1, which confers to a canola seed the additional traits of at least about 45% crude protein content and no more than about 18% acid detergent fiber on an oil-free, dry mass basis.

3. A canola plant comprising the canola germplasm of claim 1.

4. The canola plant of claim 3, wherein the canola plant produces a seed having an average of at least about 45% crude protein content and no more than about 18% acid detergent fiber, as determined on an oil-free, dry mass basis.

5. The canola plant of claim 3, wherein the canola plant produces seeds having an average of less than 2% erucic acid.

6. The canola plant of claim 3, further comprising at least one additional trait selected from the group consisting of: reduced polyphenol content and increased phosphorus content.

7. The canola plant of claim 3, further comprising an average crude protein content of at least about 45%.

8. The canola plant of claim 3, further comprising an average of no more than about 18% acid detergent fiber on an oil-free dry mass basis.

9. The canola plant of claim 3, wherein the seed has less than 11% acid detergent fiber on an oil-free dry mass basis.

10. The canola plant of claim 3, wherein the seed comprises at least 43% oil.

11. The canola plant of claim 3, wherein the seed comprises at least 45% protein.

12. The canola plant of claim 3, wherein the seed comprises at least 43% oil and at least 44% protein on an oil-free, dry matter basis.

13. A field comprising the plants of claim 3, wherein said plants produce an average of at least 1700 kilograms of seed per hectare.

14. The canola plant of claim 3, wherein the plant is selected from the group consisting of: CL065620, CL044864, CL121460H, CL166102H, and CL 121466H.

15. The plant of claim 3, wherein said plant was produced without genetic engineering and without mutagenesis.

16. The plant of claim 3, wherein said seed has a reduced anti-nutritional component.

17. The plant of claim 3, wherein said canola plant produces a seed comprising a phosphorus content of greater than 1.3% on an oil-free, dry matter basis.

18. Seeds produced by the canola plants of claims 3 to 12, and 14 to 17.

19. A progeny plant grown from the seed of claim 18.

20. The progeny plant of claim 19, wherein the progeny plant produces seed having the traits of, on average, at least 68% oleic acid (C18:1) and less than 3% linolenic acid (C18:3), and high protein content and low ADF content.

21. The progeny plant of claim 20, wherein the progeny plant produces seed having at least about 45% crude protein content and no more than about 18% ADF, as determined on an oil-free, dry matter basis.

22. Canola meal is produced from one or more seeds of claim 18.

23. The canola meal of claim 22, wherein said meal has a mean true metabolizable energy of at least 2400 kcal/kg.

24. The canola meal of claim 23, wherein said meal has a favorable amino acid digestibility profile.

25. The canola meal of claim 22, wherein said canola meal comprises an amino acid digestibility of at least about 90% of the amino acid digestibility of soybean meal (10% moisture content).

26. The canola meal of claim 22, wherein said canola meal comprises a digestible or metabolizable energy content of at least about 80% of the digestible or metabolizable energy content of soybean meal.

27. A canola seed which is genetically stable in traits of high protein content and low ADF content, and having an average of at least 68% oleic acid (C18:1) and less than 3% linolenic acid (C18: 3).

28. The canola seed of claim 27, wherein the canola seed is genetically stable having an average of at least about 45% crude protein content, and no more than about 18% acid detergent fiber, as determined on an oil-free, dry matter basis.

29. The canola seed of claim 27, further comprising an average of at least about 45% crude protein content.

30. The canola seed of claim 27, further comprising an average of no more than about 18% acid detergent fiber content.

31. The canola seed of claim 27, wherein the canola seed is genetically stable in terms of comprising at least one additional trait selected from the group consisting of: reduced polyphenol content and increased phosphorus content.

32. Canola meal produced from the seeds of claims 18 and 27 to 31.

33. A method of introducing at least one desired trait selected from the group consisting of: high protein content, low ADF content, at least 68% oleic acid (C18:1), and less than 3% linolenic acid (C18:3), wherein the method comprises:

crossing the plant of claim 2 with a second different canola plant to produce F₁A progeny plant;

selecting one or more progeny plants having the desired trait to produce selected progeny plants;

backcrossing the selected progeny plant with the plant of claim 2 to produce a backcross progeny plant;

selecting a backcross progeny plant having the desired trait and the physiological and morphological characteristics of the second different brassica cultivar to produce selected backcross progeny plants; and are

Repeating the backcross and selection steps three or more times to generate backcross progeny plants of the fourth or higher generation comprising the inbred selection for the desired trait.

34. The method according to claim 33, wherein the desired trait comprises seed having a crude protein content of at least about 45% and no more than about 18% acid detergent fiber, as determined on an oil-free, dry matter basis, and an average of at least 68% oleic acid (C18:1) and less than 3% linolenic acid (C18: 3).

35. A plant produced by the method according to claim 33.

36. A plant commodity obtained from the plant of claims 3 to 12, 14 to 17, or 35.

37. An enhanced canola meal obtainable directly from a canola seed comprising at least about 45% crude protein content and no more than about 18% acid detergent fiber, on average at least 68% oleic acid (C18:1) and less than 3% linolenic acid (C18:3), as determined on an oil-free, dry matter basis.

38. The enhanced canola meal of claim 37, further comprising an average crude protein content of at least about 49%.

39. The enhanced canola meal of claim 37, further comprising an average of no more than about 18% acid detergent fiber content.

40. The enhanced canola meal of claim 37, wherein the canola seed is genetically stable in terms of comprising at least one additional trait selected from the group consisting of: reduced polyphenol content and increased phosphorus content.

41. The enhanced canola meal of claim 37, wherein the canola seed further comprises reduced anti-nutritional factor levels.

42. The enhanced canola meal of claim 37, wherein said enhanced canola meal may replace another, different conventional canola meal or soybean meal as a protein or energy supplement in ruminant, swine, or poultry diets.