Phytochemical compositions, methods and uses in ruminant animal farming

Abstract

Described herein are phytochemical compositions, methods and uses in ruminant animal farming, along with other methods and uses beyond ruminant farming. Phytochemical compositions are described derived from herb, legume, or grass pasture plant species. The pasture plant species may be characterised by elevated secondary metabolic pathways relative to a conventional pasture plant species. The elevated secondary metabolite compounds, e.g., phenolic concentrations may be a result of an upregulated gene pathway. The elevated secondary metabolite compound concentrations may result in a colouring of the pasture plant or a part thereof. Benefits in ruminant animal farming along with other applications are described such as enhanced animal health and growth; more efficient nitrogen use; lower methane and nitrous emissions from ruminant animals administered with the pasture plant or part thereof; and / or products derived from the pasture plant or part thereof.

Description

[0001] PHYTOCHEMICAL COMPOSITIONS, METHODS AND USES IN RUMINANT ANIMAL FARMING

[0002] TECHNICAL FIELD

[0003] Described herein are phytochemical compositions, methods and uses in ruminant animal farming, along with other methods and uses beyond ruminant farming. More specifically, phytochemical compositions are described derived from herb, legume, or grass pasture plant species. The pasture plant species may be characterised by elevated secondary metabolic pathways, e.g., phenolic concentration in the herb legume or grass pasture plant or part thereof relative to a conventional example of herb, legume, or grass pasture species. The elevated secondary metabolite compounds, e.g., phenolic concentrations may be a result of an upregulated gene pathway. The elevated secondary metabolite compound concentrations may result in a colouring (e.g. purple colouring) of the plant or a part thereof. Benefits in ruminant animal farming along with other applications are described such as enhanced animal health and growth; more efficient nitrogen use; lower methane and nitrous emissions from ruminant animals administered with the pasture plant or part thereof; and / or products derived from the pasture plant or part thereof.

[0004] BACKGROUND

[0005] Pasture plants that ruminant animals graze in pastoral farming convert environmental nitrogen and nitrogen from fertiliser into nitrogen-rich pasture plant materials. The pasture plant materials may be consumed by grazing livestock in the pastoral farm which convert the pasture into nitrogen-rich animal products such as milk, meat, and fibres via the ruminant animals.

[0006] Nitrogen is a key limiting nutrient for pasture plant growth. Sufficient nitrogen is required in soil to match plant growth demands and in turn, to match grazing livestock demands.

[0007] Herb, legume, or grass pasture plants may be useful for supply of dietary nitrogen and carbon for ruminant animals. Use of these plants however can have negative animal health effects or environmental effects, an example being rumen bloating from excess and rapid carbon dioxide, and methane production during fermentation. Typically, the commercial pasture use of varieties of these plant pasture species have relatively low concentrations of secondary metabolite compounds, compared to ornamental or wild relatives (presumably breeding for biomass production efficiency, and palatability, and rapid digestion has lowered the level of such secondary products). The loss of such secondary metabolite compound levels may contribute to rumen and livestock processing of herbage in such a way that animal health or environmental effects are compromised (see examples above). A typical legume pasture plant species such as clover species, may be used in pasture farming as nitrogen fixing plants. Typically, clover leaves A are green in colour B, perhaps with some white variegation C, an example image of which is shown in Figure 1. This is shown in contrast to a legume clover species in which a secondary metabolite pathway is elevated to produce anthocyanins, amongst other compounds, which manifests as a purple-coloured clover Figure 3. Such a contrast is an example of a legume with raised secondary metabolite levels not currently used in pasture that, as demonstrated below, has great potential application.

[0008] Pasture plants that produce greater levels of secondary metabolite e.g., phenolic compounds are of interest. Anthocyanins may for example, produce a plant with red / purple or blue pigmentation although a colour change may not be an inevitable outcome of increased phenolic compound concentration. Where the pigmentation exists in the flowers or fruits of a plant, the colouration may be a cue to pollinators. Colouration in the vegetation of plants may provide benefits to the plant in terms of protection from U\Z light for example. Other types of phenolic compounds may not produce visual colour changes but may provide bioactivity useful for other attributes e.g., livestock pasture digestion, animal health, hence, reference to anthocyanins should not be seen as limiting.

[0009] In a pasture farming context, elevated concentrations of plant secondary metabolite compounds, (e.g. phenolic compounds and in particular, plant derived polyphenolic secondary metabolite (PPSM) compounds in pasture plants), may sequester dietary proteins in ruminant animals by binding to proteins in the rumen during digestion by the ruminant animal of the pasture plants. Sequestered dietary proteins in turn may lead to:

[0010] Reducing bloating, which is a digestive disorder due to excessive rumen gas;

[0011] Reduced methane emissions and nitrogen excretions, which are damaging environmental pollutants; and

[0012] Improved metabolic efficiency by efficient conversion of feeds into valuable animal products including milk, meat, and fibres;

[0013] Improved milk and meat quality growth-promoting, production-enhancing, immunomodulatory, antibacterial, and antioxidant effects on animals.

[0014] Conventional feeds consumed by a ruminant animal contain levels of secondary metabolite compounds that may be too low to reduce bloat, reduce methane emissions and nitrogen excretions, or improve metabolic efficiency. This may lead to undesirable animal health outcomes, higher production costs and polluting environmental waste.

[0015] Phenolic-rich plants do exist but not typically in a pasture context, largely due to issues with ruminant animal palatability, the high cost of supplement manufacture, and the high cost of administration of these plants or compositions derived from these plants. One example of this may be ornamental clover phenotypes that arise in nature as sports. These ornamental clover phenotypes are however only available as clonal plants and not as commercial seed. Other purple-coloured herbs, legumes or grasses may exist but only as ornamental or vegetable varieties and not in a pasture context. Reasons for the lack of pasture applications of ornamental or vegetable varieties may be due to performance issues as noted above. Another example of a purple-coloured variety of clover Z is shown in Figure 2. In this prior art example, the leaves Y have predominantly green leaves X with purple coloured variegation W. The variety shown in Figure 2 was developed through extensive research into genetically modifying Trifolium repens species to enhance purple colour in the leaves and hence, increase PPSM content. Whilst this approach had some success, the variety was never released commercially due to regulatory hurdles, cost of production, lack of stability and, only a slight increase in leaf PPSM content and specifically condensed tannin increase only, and not a wider variety of phenolic compounds. To the inventor's knowledge, despite interest in developing clover plants with increased PPSM compounds, the above examples are all that exists in the pasture plant area.

[0016] It may be useful to provide compositions, pasture plants, pastures and related methods and uses that have elevated plant phenolic concentrations. Ideally, these compositions, pasture plants, pastures are palatable to an animal. The compositions, pasture plants, pastures and related methods may allow for greater efficiency in grazing livestock conversion to nitrogen-rich animal products, along with higher quality products from the animals with potentially longer shelf-life products. The compositions, pasture plants, pastures and methods may avoid or minimise inefficiencies in nitrogen utilisation. The compositions, pasture plants, pastures and related methods and uses may at least provide the public with a choice.

[0017] Further aspects and advantages of the phytochemical compositions, methods and uses in ruminant animal farming will become apparent from the ensuing description that is given by way of example only.

[0018] SUMMARY

[0019] Described herein are phytochemical compositions derived from herb, legume or grass pasture plants. The herb, legume or grass pasture plant may be characterised by generally elevated secondary metabolite levels, e.g. anthocyanin, proanthocyanin and general phenolic concentration in the herb, legume or grass pasture plant vegetation. The herb, legume or grass pasture plant may be characterised by upregulation in genes that encode enzymes within secondary metabolite pathways e.g., the flavonoid biosynthesis pathway of pasture plants. The herb, legume or grass pasture plant may be characterised by purple coloured vegetation. The herb, legume or grass pasture plant, when used as a ruminant animal feed composition, may provide benefits in pastoral farming such as enhanced animal health and growth and more efficient nitrogen use leading to lower methane and nitrous emissions from the ruminant animals consuming the phenolic compositions. The inventors have identified specific lines of herb, legume or grass pasture plants, that are palatable to ruminant animals and which have unexpectedly high levels of secondary metabolite levels compared to traditional pasture plant varieties or cultivars used in pastoral farming. The specific lines identified or created may have a number of attributes not observed in the art, and which differ to genetically modified attempts to produce pasture plants with raised secondary metabolite levels. One method of producing the specific lines or cultivars may be to cross ornamental plants related to pasture species into elite pasture lines varieties or cultivars to produce the described pasture plants herein.

[0020] In a first aspect, there is provided a pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant or part thereof comprises elevated secondary metabolite compound concentrations relative to a conventional herb, legume, or grass pasture plant or part thereof.

[0021] In a second aspect, there is provided a pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant or part thereof comprises upregulated genes that encode enzymes within the flavonoid biosynthesis (ec00941) pathway.

[0022] In a third aspect, there is provided a pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant comprises at least substantially 10% by area purple colouring of leaves of the pasture plant.

[0023] In a fourth aspect, there is provided a composition formulated for ruminant animal administration comprising: the pasture plant or part thereof substantially as described above; or an extract of the pasture plant or part thereof substantially as described above.

[0024] In a fifth aspect, there is provided pasture comprising the pasture plant or part thereof substantially as described above.

[0025] In a sixth aspect, there is provided a method of increasing sequestration of dietary protein in the rumen of a ruminant animal by: selecting the pasture plant or a part thereof substantially as described above; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the sequestration of dietary protein in the rumen of the ruminant animal post administration is increased relative to a ruminant animal administered a conventional pasture plant or a part thereof.

[0026] In a seventh aspect, there is provided a method of decreasing bloating in a ruminant animal by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the amount of bloating that the ruminant animal experiences post administration is reduced relative to the amount of bloating that a ruminant animal would experience if administered a conventional pasture plant or a part thereof.

[0027] In an eighth aspect, there is provided a method of reducing nitrogen excretion from a ruminant animal by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration is reduced, or partitioned differently between urine, faeces and gaseous emissions relative to the amount of nitrogen, or nitrogen partitioning, excreted from a ruminant animal administered a conventional pasture plant or a part thereof.

[0028] In a ninth aspect, there is provided a method of reducing the risk of nitrogen leaching from pasture by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration is reduced, or partitioned differently between urine, faeces and gaseous emissions, relative to the amount of nitrogen, or nitrogen partitioning, excreted from a ruminant animal administered conventional pasture plant or a part thereof, the reduced nitrogen excreted resulting in a reduced risk of nitrogen leaching from the ruminant animal.

[0029] In a tenth aspect, there is provided a method of reducing the risk of greenhouse gas emissions from pasture by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the amount of methane and / or carbon dioxide excreted from the ruminant animal post administration is reduced relative to a ruminant animal administered conventional pasture plant or a part thereof and, as a result, the amount of greenhouse gas emissions as methane and / or carbon dioxide gas from the ruminant animal is also reduced.

[0030] In an eleventh aspect, there is provided a method of increasing the production of nitrogen-rich ruminant animal products by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the production of nitrogen-rich ruminant animal products from the ruminant animal post administration is increased relative to the production of nitrogen-rich ruminant animal products from a ruminant animal administered conventional pasture plant or a part thereof.

[0031] In a twelfth aspect, there is provided a supplement formulated for oral or topical administration, the supplement comprising: the pasture plant, or part thereof, substantially as described above; or an extract from the pasture plant, or part thereof, substantially as described above.

[0032] In a thirteenth aspect, there is provided a supplement formulated for oral or topical administration, the supplement derived from or comprising flowers, nectar, or flowers and nectar produced from the pasture plant substantially as described above.

[0033] In a fourteenth aspect, there is provided a colourant, the colourant derived from or comprising: the pasture plant or part thereof substantially as described above; or an extract from the pasture plant or part thereof substantially as described above.

[0034] In a fifteenth aspect, there is provided a method of measuring a content of pasture plant with elevated secondary metabolite concentrations in a pasture, the pasture comprising a combination of: pasture plants or parts thereof substantially as described above that undergo a colour change due to the elevated secondary metabolite concentration; and surrounding conventional pasture plants; wherein the extent of the pasture plants or parts thereof substantially as described above in the pasture is assessed by measuring the extent of colour difference between the pasture plants and the surrounding conventional pasture plants.

[0035] In a sixteenth aspect, there is provided seed produced from the pasture plant or part thereof substantially as hereinbefore described.

[0036] In a seventeenth aspect, there is provided a method of production of seed produced from the pasture plant or part thereof substantially as hereinbefore described by: crossing of pasture plants identified as coloured or containing elevated secondary metabolite compounds into elite pasture lines; backcrossing to produce elite line material with the desired trait; and multiplication of the elite line material.

[0037] In an eighteenth aspect, there is provided a method of production of pasture plants or parts thereof substantially as described above by: by gene modification, editing, modulation or mutagenesis of the pasture plant, to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof.

[0038] In a nineteenth aspect, there is provided a method of production of pasture plants or parts thereof substantially as described above by: gene modification, editing, or mutation of the pasture plant, to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof but prevent purple colouration by down regulation or modulation of the UFGT pathway.

[0039] As noted above, the pasture plant, compositions, pasture and various methods may allow use of pasture plants with lower animal health risk issues such as bloating. The pasture plant or a part thereof, compositions, pasture and various methods may also allow for great nitrogen utilisation and nitrogen uptake and reduced risk of carbon dioxide or methane or nitrous emissions or nitrogen leaching.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Further aspects of the phytochemical compositions, methods and uses in ruminant animal farming will become apparent from the following description that is given by way of example only and with reference to the accompanying drawings in which:

[0042] Figure 1 is an image of a prior art Trifolium repens leaflet;

[0043] Figure 2 is an image of a prior art genetically modified Trifolium repens leaflet;

[0044] Figure 3 is an image of clover plants and leaflets of the pasture plants described herein comprising purple coloured leaves / leaflets;

[0045] Figure 4 is an image of a clover plant spade leaf of the pasture plants described herein comprising purple colouring;

[0046] Figure 5 is an image of a pasture comprising the above clover pasture plants growing among other pasture plants;

[0047] Figure 6 is a diagram showing phenolic compound classifications and where the PPSM compounds of the clover plant are understood to largely be located;

[0048] Figure 7 is a graph comparing total monomeric anthocyanins measured for the clover pasture plant described herein (labelled 'purple') compared to other clover plants (labelled 'control' = white clover no purple colour, 'greener' = 20-30% purple leaf colour clover, 'bulk' = 40-60% purple leaf clover);

[0049] Figure 8 is a graph showing total phenolic levels as measured from the clover pasture plant described herein compared to other clover plant samples (labelled 'purple' = 80% purple coloured leaved clover, 'control' = white clover no purple colour, 'greener' = 20-30% purple leaf colour clover, 'bulk' = 40-60% purple leaf clover;

[0050] Figure 9 is a table comparing phenolic compound content in ppm units of two specific phenolic monomer compounds in a control clover pasture plant versus the clover pasture plant described herein where 'purple' = 80% purple coloured leaved clover, 'control' = white clover no purple colour;

[0051] Figure 10 shows a flavonoid biosynthesis pathway diagram illustrating a typical biochemical pathway in gene regulation of flavonoids in plants with the pathways understood to be upregulated highlighted;

[0052] Figure 11 shows side by side comparison images of white clover and grass (B or lower images) contrasted to the upper or A purple clover and pasture images and below in C is the image processing information used to assess the colour difference seen in the right-hand images A and B;

[0053] Figure 12 shows a photograph showing a purple coloured chicory plant with elevated PPSM concentration and related upregulated genes;

[0054] Figure 13 shows a photograph showing a purple coloured plantain / plantago sp. plant with elevated PPSM concentration and related upregulated genes;

[0055] Figure 14 shows a photograph showing a purple coloured grass species plants with elevated PPSM concentration and related upregulated genes;

[0056] Figure 15 shows a photograph showing a purple coloured subterranean clover plants with elevated PPSM concentration and related upregulated genes;

[0057] Figure 16 shows two photographs of images drawn using colourant extracted from clover plant leaves with purple colouring;

[0058] Figure 17 shows a photograph of purple clover plants in flower for seed production;

[0059] Figure 18 shows a photograph of a tray of purple clover being propagated;

[0060] Figure 19 shows a graph illustrating the differences in methane and carbon dioxide emissions from trial sheep as scaled data to the group mean;

[0061] Figure 20 shows a graph illustrating the differences in methane and carbon dioxide emissions from trial sheep as unsealed data;

[0062] Figure 21 shows images of representations of the of Trifolium repens foliar pigmentation patterning associated with the R and V loci, found segregating in the F2 population. Where image (A) No pigmentation; image (B) Red-fleck (Rf); image (C) Red midrib (Rm); image (D) Red leaf (Rl); image (E) Red- . Images B, C and D show pigment phenotypes linked to the T. repens R locus located on chromosome eight of the T. occidentale subgenome. Panel E shows the pigment phenotype linked to the T. repens V locus, located on chromosome five of both the T. occidentale and T. pallescens subgenomes; Figure 22 shows a phylogenetic tree of flavonoid-regulating MYB genes and linkage map locations of the R and V loci in T. repens taken from Albert et al (2015) also used to prepare the F2 plants shown in Figure 21;

[0063] Figure 23 shows a volcano plot illustrating genes that are up regulated (green dots) and genes that are down regulated (red dots) by the purple clover vs the white clover;

[0064] Figure 24 shows a heatmap of genes over expressed in the purple clover compared to white clover where a higher Z score indicates a higher expression and a lower Z score indicates a lower expression and where H1-H5 are purple clover examples and N1-N5 are conventional white clover examples trialled;

[0065] Figure 25 shows a table illustrating the results of BLASTn used to search results of the differentially expressed transcripts from the Trifolium repens F2 population segregating for foliar pigmentation phenotype;

[0066] Figure 26 demonstrates a phenotypically segregating single nucleotide polymorphism downstream of differentially expressed MYB transcription factor indicating allelic segregation;

[0067] Figure 27 shows how primers preferentially amplify the purple version of the TrRED LEAF (samples H2- H9) and can easily be used to discriminate purple from white clover (samples N1-N10);

[0068] Figure 28 shows (A) a photograph of N. benthamiana leaf infiltrated with infiltration buffer containing GV3101 Agrobacterium transformed with the p35S:TrRED LEAF expression vector and, (B) shows a photograph of N. benthamiana leaf infiltrated with infiltration buffer only;

[0069] Figure 29 shows a generalised schematic of the flavonoid biosynthetic pathway. The first eight steps of the pathway can be activated by TrRED LEAF and TrMYB14 transcription factors. Anthocyanin synthesis can be mediated by TrRED LEAF. Proanthocyanidin synthesis can be mediated by TrMYB14 but not TrRED LEAF;

[0070] Figure 30 shows a predicted reduction in pigment accumulation when p35S:TrRED LEAF is co-infiltrated with an RNAi construct targeting the Nicotiana benthamiana UDP-glucose: flavonoid 3-0- glucosyltransferase (UFGT) gene. PAL, Phenylalanine ammonia lyase; C4H, cinnamate 4 hydroxylase; 4CL, 4 coumarate CoA ligase; CHS, chaicone synthase; CHI, chaicone isomerase; F3H, flavanone 3 hydroxylase; F3'5'H, flavonoid 3'5' hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; and

[0071] Figure 31 shows how animal grazing on purple clover phenolic extracts 1-3 had greater phenolic reduction than conventional green clover extracts at increasing concentrations of protein (Bovine Serum Albumin, BSA) concentrations. DETAILED DESCRIPTION

[0072] As noted above, described herein are phytochemical compositions derived from pasture plants. The pasture plant may be characterised by generally elevated secondary metabolite levels, e.g., anthocyanin, proanthocyanin and general phenolic concentration in the pasture plant vegetation. The pasture plant may be characterised by upregulation in genes that encode enzymes within the flavonoid biosynthesis pathway. The pasture plant may be characterised by purple coloured vegetation. The pasture plant, when used as a ruminant animal feed composition, may provide benefits in pastoral farming such as enhanced animal health and growth and more efficient nitrogen use leading to lower methane and nitrous emissions from the ruminant animals consuming the phenolic compositions. The inventors have identified specific lines of pasture plant from herb, legume or grass plants, that are palatable to ruminant animals and which have unexpectedly high levels of phenolic compounds compared to conventional pasture plant varieties used in pastoral farming. The specific lines identified, or created may have a number of attributes not observed in the art, and which differ to genetically modified attempts to produce increased tannin clover plants. One method of producing the specific lines or cultivars may be to cross ornamental lines into elite pasture lines to produce the described pasture plants herein. Another would be to identify one or more regulatory genes in the secondary metabolite pathway and introduce it (or modify existing versions) by way of gene editing or genetic modification.

[0073] For the purposes of this specification, the terms 'about', or 'approximately', or 'substantially' and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% to a reference quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0074] The term 'comprise1and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

[0075] The term 'pastoral farming' and grammatical variations thereof as used herein refers to farming of ruminant animals that occurs predominantly or exclusively by grazing of ruminant animals on pasture plants.

[0076] The term 'pasture plant' and grammatical variations thereof as used herein refers to plant matter used in farming of ruminant animals that ruminant animals graze on.

[0077] The term 'pasture plant or part thereof' or grammatical variations thereof refers to parts of the pasture plant including the aerial parts such as the plant leaves and stolons and optionally, below ground parts such as the plant roots. Reference will generally be made to the plant leaves in the description below. This should not be seen as limiting as other aerial parts of the plant e.g. stolons, may also encompassed under the definition of the clover plant or part thereof. The term 'conventional pasture plant or part thereof' or grammatical variations thereof refers to a pasture plant without an elevated secondary metabolite concentration. In the context of clover legumes, a conventional pasture plant may be termed white clover or a pasture Trifolium repens species. Conventional pasture plants may have a generally green coloured leaf and a PPSM content less than 0.4% dry weight of cut pasture.

[0078] The term 'analogue' or grammatical variations thereof refers to greater than 70, or 75, or 80, or 85, or 90, or 95% homology.

[0079] The term 'ortholog' or grammatical variations thereof refers to greater than 70, or 75, or 80, or 85, or 90, or 95% gene separation by speciation.

[0080] A Pasture Plant

[0081] In a first aspect, there is provided a pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant or part thereof comprises elevated secondary metabolite compound concentrations relative to a conventional herb, legume, or grass pasture plant or part thereof.

[0082] Secondary Metabolite Compounds

[0083] The pasture plant or part thereof may comprise plant polyphenol secondary metabolite (PPSM) compounds.

[0084] The PPSM compounds may comprise flavonoids and / or condensed tannins. The PPSM compounds may comprise a mixture of flavonoids and condensed tannins. The condensed tannins may also be termed proanthocyanins or proanthcyanidins.

[0085] The PPSM compounds may comprise: flavone compounds, flavanol compounds, anthocyanin compounds, and mixtures thereof.

[0086] Example condensed tannin compounds may comprise: procyanidin B2, proanthocyanidin Ai, proanthocyanidin A2 and proanthocyanidin Ci.

[0087] Example flavone compounds may comprise: apigenin, luteolin, chrisin, and tangeretin.

[0088] Example flavanol compounds may comprise: quercetin, kaempferol, rutin, and myricetin.

[0089] Example anthocyanin compounds may comprise: cyanidin, petunidin, malvidin, delphinidin, and pelargonidin.

[0090] The secondary metabolite compound concentration may be at an effective concentration. An effective concentration may refer to at least one of: the total phenolic content of leaves of the pasture plant being at least 20mg / g Gallic Acid Equivalent (GA EQ); and / or the anthocyanin content of leaves of the pasture plant being greater than l.Omg / g; and / or the secondary metabolite compound concentration of leaves of the pasture plant being greater than 0.5% dry weight of cut pasture.

[0091] The secondary metabolite compounds may be PPSM compounds as noted above, and the PPSM concentration of leaves of the pasture plant may be greater than 0.5% dry weight of cut pasture.

[0092] The total phenolic content in leaves of the pasture plant may be at least 20, or 25, or 30, or 35, or 40mg / g GA EQ. The total phenolic content in leaves of the pasture plant may be from 20-40, or 25-35, or 20-30, or 30-40mg / g GA EQ.

[0093] The anthocyanin content in leaves of pasture plant may be greater than 1, or 1.5, or 2, or 2.5, or 3, or 3.5, or 4, or 4.5, or 5, or 5.5, or 6, or 6.5, or 7 mg / g. The anthocyanin content in leaves of the pasture plant may be from 1-7, or 2-6, or 3-5, or 2-4, or 1-3, or 4-7, or 5-7 mg / g.

[0094] The PPSM compound concentration in leaves of the pasture plant may be greater than about 0.5, or 0.6, or 0.7, or 0.8, or 0.9. or 1.0, or 1.1, or 1.2, or 1.3, or 1.4, or 1.5, or 1.6, or 1.7, or 1.8, or 1.9, or 2.0% dry weight of cut pasture. The PPSM compound concentration in leaves of the pasture plant may be from 0.5-2.0, or 0.7 to 1.8, or 1.0 to 1.5, or 0.5 to 1.0, or 1.0 to 2.0, or 1.5 to 2.0% dry weight.

[0095] Conventional pasture crops may have generally low levels of PPSM compounds, typically below 0.4% of the dry weight of cut pasture. For example, white clover only produces PPSM compounds in flowers and trichomes and at levels far too low (substantially below 0.4% PPSM dry weight) to have any effect on nitrogen utilisation by a ruminant animal.

[0096] Gene Upregulation

[0097] In a second aspect, there is provided a pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant or part thereof comprises upregulated genes that encode enzymes within the flavonoid biosynthesis (ec00941) pathway.

[0098] The upregulated genes may be activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof.

[0099] In one embodiment, the upregulated gene may be TrRED LEAF, or analogues, or orthologs thereof.

[0100] Regarding the nomenclature for TrRED LEAF, this comes from the R2R3 family of MYB genes where 'Tr' refers to trifolium repens. ‘TrRED LEAF' refers to the Trifolium repens version of an R2R3 MYB located at the R locus of chromosome 8. For the purposes of this specification, the term ‘TrRED LEAF' is used when referring to Trifolium genus plants and 'TrRED LEAF, or analogues, or orthologs thereof' in respect of non-Trifolium genus plants. In addition, it should be appreciated that analogues or orthologs of the stated genes may also be used and omission of reference to an analogue or an ortholog should not be seen as limiting.

[0101] Upregulation as used herein may refer to an upregulation of genes relative to a conventional pasture plant. The extent of upregulation may be sufficient to increase the presence of secondary metabolites in the pasture plant by at least 5, or 10, or 15, or 20, or 25, or 30% higher than that in a conventional pasture plant or part thereof.

[0102] It is understood by the inventors that TrRED LEAF, or analogues, or orthologs thereof may upregulate the whole flavonoid biosynthesis pathway.

[0103] As will be described further below, it may be possible to knock out / down regulate the enzyme (UFGT discussed further below) that leads to purple colour to produce a non-purple pasture plant in which the anthocyanin production has been curtailed and re-directed to produce more of the other non-coloured PPSM compounds.

[0104] A Pasture Plant With Purple Colouring

[0105] In a third aspect, there is provided a pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant comprises at least substantially 5% by area purple colouring of leaves of the pasture plant.

[0106] Pasture Plant Colour

[0107] As noted above, the pasture plant may be characterised by having purple coloured leaves. Purple colouration refers to the colour of the pasture plant vegetation being a purple colour.

[0108] At least substantially 5, or 10, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95% by area of leaves of the pasture plant may be purple coloured. 5-95, or 10-95, or 20-95, or 30-95, or 40-95, or 50-95, or 60-95, or 70-95, or 80-95% by area of leaves of the pasture plant may be purple coloured.

[0109] The purple colour may be substantially solid across the leaves of the pasture plant.

[0110] The purple colour may not be segregated or patterned across the leaves of the pasture plant.

[0111] The pasture plant may have green coloured edging on edges of the leaves of the pasture plant.

[0112] The purple colour of the leaves may have a wavelength from 380-475nm, or 380-450nm, or 380-435nm or substantially around 420nm. The change in plant colour from conventional green leafed herb, legume, or grass is understood to be associated with changes in plant PPSM content in the herb, legume, or grass plant or part thereof and in particular, a specific final pathway.

[0113] The pasture plant described herein may also be termed a purple leaf pasture plant.

[0114] Pasture Plant Type

[0115] The pasture plant may be derived from the genus Trifolium.

[0116] The pasture plant may be derived from Trifolium repens.

[0117] The pasture plant may be derived from: Trifolium repens purpurascens; Trifolium repens atropurpeum; Trifolium repens 'Pentaphyllum'; Trifolium repens 'Purpurascens Quadrifolium; Black-Leaved Clover; Chocolate Clover; and combinations thereof.

[0118] Note that the clover is not specifically or exclusively a clover of the species Trifolium purpureum (red clover), or Trifolium pratense. The pasture plant may be a pasture clover plant. The pasture clover plant type may be an ecotype naturally found in seed collections of 7. repens or other species noted above.

[0119] Reference may be made hereafter to the herb, legume, or grass plant being a clover plant. This should not be seen as limiting as other pasture plant types may be used e.g., chicory, plantain, or grasses.

[0120] Plant Flowers

[0121] The pasture plant may produce purple-coloured flowers. The pasture plant may not produce purplecoloured flowers.

[0122] The purple colouring of the pasture plant flowers, if they are purple coloured, may have a wavelength from 380-475nm, or 380-450nm, or 380-435nm or substantially around 420nm.

[0123] The pasture plant flower colour is understood to also be associated with changes in plant PPSM content in the pasture plant flowers, flower parts or pollen.

[0124] Alternatively, the pasture plant flowers may have an elevated PPSM compound concentration but no substantial colour difference.

[0125] Pasture Plant Traits

[0126] The increase in secondary metabolite compound concentration and / or upregulation may be a heritable dominant trait. This feature results from the finding that F2 hybrids of the pasture plant express green plants (at least 25%) leading to the conclusion that the purple colour and / or elevated PPSM compounds may be a heritable dominant trait or traits. There may be substantially no seasonal variation. The colour and phenolic or PPSM concentrations in the pasture plant or part thereof, may alter through seasons, but remain substantially greater than a conventional pasture plant.

[0127] The purple colour expression may begin already at an early stage of pasture plant growth e.g. the spade leaf stage of pasture plant growth.

[0128] The pasture plant may be a perennial plant. The pasture plant may be annual or hybrid.

[0129] Stability

[0130] The pasture plant or part thereof may be stable. Stability in this context refers to the concentration of secondary metabolite compounds / PPSM compounds in the pasture plant or part thereof, and being relative to conventional pasture plants. Stability also may refer to the pasture plant stability between generations with the trait being heritable and expressed between generations.

[0131] The inventors have found that the secondary metabolites / PPSM compound concentration and / or purple colour trait(s) is / are substantially stable across seasons relative to what may be observed for a conventional pasture plant i.e. is not necessarily induced by cold or warm temperatures or changes in U\Z light concentration, but such variables may affect absolute levels of secondary metabolite / PPSM production.

[0132] Breeding and Isolation

[0133] The pasture plant or part thereof may be bred via traditional selection and cultivar enhancement methods. This approach may be of benefit to reduce the extent of regulation needed to produce a commercial product. The pasture plant may not be produced by genetic modification. Artificial breeding may be via traditional selection and cultivar enhancement methods.

[0134] In the inventor's experience, traditional methods of selection and breeding are a simple and effective means to produce the described pasture plant populations. Traditional methods of breeding by the inventors produced a superior variety to published art genetically modified pasture plants, at least in terms of PPSM content and related gene upregulation.

[0135] Alternatively, it may also be possible to produce the pasture plant via genetically engineered techniques. The pasture plant may be produced by genetic modification, or gene editing, or directed mutation. This may be completed to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof. The pasture plant may be artificially bred to enhance phenolic expression, upregulate genes encoding phenolic expression and / or purple colour. The pasture plant described may be isolated from a wider population of pasture plants. Isolation may be completed by analysis of phenolic concentration, gene upregulation in favour of phenolic content, purple colouration content, protein content, the noted activator MYB components, or other characterising traits described herein.

[0136] One method of producing the specific lines or cultivars may be to cross ornamental clover into elite pasture clover to produce the described pasture plants herein.

[0137] As may be appreciated, purple vegetation pasture plants may occur at random in nature. However, purple colouration to the extent identified is rare or not observed in natural pasture plant populations and seldom reaches populations in a pasture that would have any substantial impact on phenolic levels in a pasture as a whole. Producing a pasture plant or pasture comprising the pasture plant with any substantial effects on phenolic concentration in the leaves, particularly in a pastoral farm system is therefore an artificial creation. Due to the dominance found by the inventors of the purple trait of the above described pasture plant, multiplication and growth of more plant mass may spread quickly once initial pasture plants are established.

[0138] If genetic modification were to be used, the method may utilise editing or genetic modification to up regulate the transcription factor to create the pasture plant with the above described characteristics.

[0139] Purple Colour and Secondary Metabolite Compound Relationship

[0140] The purple colour of the leaves and the secondary metabolite compound concentration may be linked. The inventors have found that there may be a relationship between greater purple colour and greater secondary metabolite compound concentration. The relationship may be non-linear.

[0141] The pasture plant or part thereof identified may have a substantially higher ((e.g. 50%, or 100%, or 150%, or 200%, 250% more) total monomeric anthocyanin measure than standard green coloured leaf herb, legume, or grass.

[0142] The herb, legume, or grass pasture plant or part thereof identified may have a substantially higher total phenolic content (e.g. 50%, or 100%, or 150%, or 200%, 250% more) than conventional green coloured leaf herb, legume, or grass.

[0143] Phenolic monomers may be substantially higher in the pasture plant or part thereof than in conventional pasture plant. For example, quercetin-3-glucoside may be 42 times more concentrated in the clover plant leaves described than in the leaves of conventional clover. Delphinidin-3-rutinoside may be present in the clover plant leaves described whilst not being detectable at all in the leaves of conventional clover.

[0144] A Composition In a fourth aspect, there is provided a composition formulated for ruminant animal administration comprising: the pasture plant or part thereof substantially as described above; or an extract of the pasture plant or part thereof substantially as described above.

[0145] The compositions described, when administered to ruminant animals, may result in increased secondary metabolites compound consumption by the ruminant animal.

[0146] The composition may be a feed.

[0147] The composition may be a feed supplement.

[0148] The composition may be in a dry form. The composition may be in a solid form. For example, the composition may be in a powder form, or a shape form (e.g. bar, ball, pellet, clusters, tablet, or bolus).

[0149] The composition may be in a liquid or slurry form (e.g. a drench, tonic, or drink).

[0150] The composition may be in a fresh form (e.g. cut pasture, uncut pasture).

[0151] The composition may be in a preserved form (e.g. dried, hay, silage).

[0152] The composition may be in a form configured to be suitable for administration to a ruminant animal.

[0153] The composition may be self-administered by a ruminant animal. For example, the composition may be self-administered by the ruminant animal as a component of fresh or cut pasture, as a dried hay or, in wet silage form.

[0154] The composition may be prepared as a supplement for administration to a ruminant animal by harvesting the herb, legume, or grass plant when the plant leaves have PPSM levels greater than 0.5% of the dry weight, and subsequently prepared for administration.

[0155] The composition may be made via a forming step, wherein the herb, legume, or grass plant may be harvested, processed and then moulded, pressed, spray dried or otherwise formed into a shape (e.g. bar, ball, pellet, clusters, tablet). Shape forming may be with dimensions and / or textures suitable for consumption by the ruminant animal.

[0156] The composition may comprise a housing for the composition such as in a specific delivery device such as a syringe.

[0157] The composition may be prepared for self-administration by a ruminant animal by permitting the animal to graze a grown pasture that comprises the herb, legume, or grass plant.

[0158] The composition may be prepared as for self-administration by a ruminant animal by cutting a grown pasture that comprises the herb, legume, or grass pasture plant and drying the cut pasture for hay or for manufacturing silage. Pasture

[0159] In a fifth aspect, there is provided pasture comprising the pasture plant or part thereof substantially as described above.

[0160] The pasture comprising the pasture plant or part thereof may be selected from or mixed into: field; fodder; grass; herb, legume, hay; paddock; silage; sward, turf; ryegrass (Lolium sp.); Plantain (Plantago sp.); Chicory (Chichorium intybus); Clover (Trifolium sp.); and combinations thereof.

[0161] The pasture plants described above may be grown or overplanted to form the pasture.

[0162] The term 'growing' or grammatical variations as used herein refers to: cultivating, planting, seeding, broadcast seeding, drill seeding, dispersing mechanically, dispersing by animals, transplanting, and combination thereof and a subsequent step of subsequent plant growth.

[0163] The pasture may be formed by growing the pasture plants on bare land.

[0164] The pasture may be formed by overplanting existing pasture with the pasture plants.

[0165] Method of Increasing Sequestration

[0166] In a sixth aspect, there is provided a method of increasing sequestration of dietary protein in the rumen of a ruminant animal by: selecting the pasture plant or a part thereof substantially as described above; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the sequestration of dietary protein in the rumen of the ruminant animal post administration is increased relative to a ruminant animal administered a conventional pasture plant or a part thereof.

[0167] Method of Decreasing Bloating

[0168] In a seventh aspect, there is provided a method of decreasing bloating in a ruminant animal by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the amount of bloating that the ruminant animal experiences post administration is reduced relative to the amount of bloating that a ruminant animal would experience if administered a conventional pasture plant or a part thereof.

[0169] Method of Reducing Nitrogen Excretion In an eighth aspect, there is provided a method of reducing nitrogen excretion from a ruminant animal by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration is reduced, or partitioned differently between urine, faeces and gaseous emissions relative to the amount of nitrogen, or nitrogen partitioning, excreted from a ruminant animal administered a conventional pasture plant or a part thereof.

[0170] Method of Reducing the Risk of Nitrogen Leaching

[0171] In a ninth aspect, there is provided a method of reducing the risk of nitrogen leaching from pasture by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration is reduced, or partitioned differently between urine, faeces and gaseous emissions, relative to the amount of nitrogen, or nitrogen partitioning, excreted from a ruminant animal administered conventional pasture plant or a part thereof, the reduced nitrogen excreted resulting in a reduced risk of nitrogen leaching from the ruminant animal.

[0172] Method of Reducing the Risk of Greenhouse Gas Emissions

[0173] In a tenth aspect, there is provided a method of reducing the risk of greenhouse gas emissions from pasture by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the amount of methane and / or carbon dioxide excreted from the ruminant animal post administration is reduced relative to a ruminant animal administered conventional pasture plant or a part thereof and, as a result, the amount of greenhouse gas emissions as methane and / or carbon dioxide gas from the ruminant animal is also reduced.

[0174] Method of Increasing the Production of Nitrogen-Rich Ruminant Animal Products

[0175] In a eleventh aspect, there is provided a method of increasing the production of nitrogen-rich ruminant animal products by: selecting the pasture plant or part thereof substantially as described above; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the production of nitrogen-rich ruminant animal products from the ruminant animal post administration is increased relative to the production of nitrogen-rich ruminant animal products from a ruminant animal administered conventional pasture plant or a part thereof.

[0176] Nitrogen Rich Animal Products

[0177] Nitrogen-rich animal products that may result from ruminant animal grazing of the pasture plant or part thereof described above may be tissue-based commodities. E.g. meat, offal, fibres, extracts e.g. BSA.

[0178] Another example of valuable nitrogen-rich animal products that may result from ruminant animal grazing of the pasture plant or part thereof described above may be secretion-based commodities and products of those commodities. E.g. milk, whole milk, milk powder, cream, ice-cream, cheese, and yoghurt.

[0179] Another example of valuable nitrogen-rich animal products may be fibre-based commodities. E.g. wool, horn, and antler.

[0180] The nitrogen rich animal products may also comprise increased concentrations of secondary metabolites / PPSM compounds or derivatives thereof relative to animals fed on a diet comprising conventional pasture plant.

[0181] Administration

[0182] Administration may comprise growing the pasture plant to form pasture with at least a substantial portion of the pasture comprising the pasture plant and, grazing the ruminant animal on the pasture.

[0183] Growing may occur in settings selected from: a laboratory, a greenhouse, a field, pasture, mixed pasture, and combinations thereof.

[0184] Alternatively, administration may comprise feeding out, to the ruminant animal, a feed, or a feed supplement, the feed or the feed supplement comprising the pasture plant or a part thereof and, grazing the ruminant animal on the feed or the feed supplement.

[0185] Ruminant Animal

[0186] The ruminant animal may comprise ruminant animals selected from the suborders Ruminantia and Tylopoda.

[0187] The ruminant animal may be selected from: cows, cattle, goats, deer, sheep, and combinations thereof Secondary Metabolite Value to Pasture Farming

[0188] Secondary metabolite compounds may be desirable in ruminant feeds because they sequester dietary protein by binding to proteins from feed in the digestive tract of ruminant animals. The binding of dietary protein by secondary metabolite compounds may reduce the fermentation of plant protein by ruminal microbiota and its conversion into carbon dioxide and / or methane, which in excess, causes bloating in ruminants. This also may reduce carbon dioxide and / or methane emissions and may reduce urinary nitrogen excretion by increasing nitrogen utilization. Some PPSMs are also known to be antimethanogens acting directly on methanogenic microbiota.

[0189] When secondary metabolite compounds are administered to ruminant animals as feed or as feed supplements, there may be a reduction in the emission of carbon dioxide and / or methane, which may additionally reduce the excretion of urinary nitrogen, and may increase the production of valuable animal products.

[0190] The inventors understand that administration of the herb, legume, or grass pasture plant (or parts thereof), or compositions comprising the herb, legume, or grass pasture plant or parts thereof to ruminant animals may provide unexpected benefits. Examples of these benefits may comprise one or more of the following effects:

[0191] ® Increased sequestration of dietary protein by binding of secondary metabolite compounds to plant feed proteins in the rumen;

[0192] * The administration of anti-methanogen secondary metabolite compounds;

[0193] ® Reducing the population of methanogens in the rumen;

[0194] ® Reducing excessive fermentation in the rumen;

[0195] ® Reducing the production of methane gas in the rumen;

[0196] ® Reducing the production of methane which is exhaled as a potent greenhouse gas;

[0197] * Reducing the production of urinary nitrogen which is excreted;

[0198] ® Redirecting metabolic energy to animal growth and the production of valuable animal products; and / or

[0199] ® Production of higher quality / stable animal products.

[0200] Without being bound by theory, the herb, legume, or grass pasture plant or part thereof and compositions described above are understood to cause changes to ruminant animal digestion by increasing plant protein sequestration through binding of plant proteins to secondary metabolite compounds thereby preventing their utilisation by methanogenic microorganisms in the rumen. Additionally, secondary metabolite compounds inhibit methanogenic microorganisms or otherwise deplete the methanogen microorganisms in the animal. Ruminant methane is a by-product of ruminant fermentation and is produced by a complex community of microorganisms, mainly in the rumen (87% - 90%) and, to a lesser extent (13% - 10%), in the large intestine, by the process called methanogenesis. Cattle produce about 7 and 9 times as much methane as sheep and goats, respectively.

[0201] Carbon dioxide and / or methane production by methanogens in the rumen due to fermentation of excessive dietary plant protein may cause bloat. As a result, high nitrogen forage plants such as clover or other herb, legume, or grass plants may require the additional burden of careful grazing management to prevent or otherwise limit the incidence of bloat.

[0202] Carbon dioxide and / or methane production by methanogens in the rumen may be emitted into the atmosphere by exhaling the gases mainly through the mouth and nostrils of the ruminant animal.

[0203] Carbon dioxide and / or methane are greenhouse gases. Methane has a global warming potential (GWPioo) 28 times that of carbon dioxide. Methane production represents a loss of energy, from 2 to 12% of gross energy intake from feed. Inhibiting methanogens may reduce methane production and / or may redirect otherwise wasted metabolic energy into useful anabolic growth processes.

[0204] Anabolic processes may also utilise nitrogen-rich materials from feed, which reduces excreted urinary nitrogen, and reduces the nitrogen deposited in urine patches and faecal deposits on pastures. When excess excreted nitrogen is deposited on pastures at a rate greater than required for optimal pasture plant efficiency, excess nitrogen may be lost via nitrate (NO3-) leaching, and ammonia (NH3), nitrous oxide (N2O) and nitrogen (N2) volatilisation. Nitrous oxide is particularly damaging to the atmosphere as a greenhouse gas with a global warming potential 298 times that of carbon dioxide. Nitrogen loss to ground water can cause uncontrolled growth of aquatic biota thereby damaging ecosystems, causing toxic algal blooms, and the eutrophication of water bodies.

[0205] Given all of the above potential issues, herb, legume, or grass pasture plants or parts thereof, compositions and methods that may result in more efficient sequestration of dietary nitrogen may be of significant benefit and value, at least in pastoral farming.

[0206] A Supplement

[0207] In a twelfth aspect, there is provided a supplement formulated for oral or topical administration, the supplement comprising: the pasture plant, or part thereof, substantially as described above; or an extract from the pasture plant, or part thereof, substantially as described above.

[0208] A Supplement from Flowers and / or Nectar In a thirteenth aspect, there is provided a supplement formulated for oral or topical administration, the supplement derived from or comprising flowers, nectar, or flowers and nectar produced from the pasture plant substantially as described above.

[0209] In the above aspects, the pasture plant described may be used for human or other animal nutrition supplementation. The pasture plant or part thereof as noted above may have an elevated secondary metabolite compound concentration. Secondary metabolite compounds are known and used as nutritional supplements for a variety of conditions e.g. as an anti-oxidant.

[0210] Typically, secondary metabolite compounds are found and consumed directly or extracted from products such as wine, grape seed extracts, honey, tea, tea extracts and so on.

[0211] In one example, the pasture plant leaves, flowers, or leaves and flowers, may be used directly or extracted to concentrate the respective PPSM content in the production of a nutritional product.

[0212] In another example, nectar produced by the pasture plant flowers may be used by bees to make honey and create a unique floral based honey product that may be used as a honey or extracted to enrich the secondary metabolite compound content of the honey.

[0213] A Colourant

[0214] In a fourteenth aspect, there is provided a colourant, the colourant derived from or comprising: the pasture plant or part thereof substantially as described above; or an extract from the pasture plant or part thereof substantially as described above.

[0215] In this aspect, the colourant may be used for printing, for paints, or for food industries. The colouring may be a naturally derived colourant and hence of use where alternative naturally based colourants are of value.

[0216] A Pasture Management Tool

[0217] In a fifteenth aspect, there is provided a method of measuring a content of pasture plant with elevated secondary metabolite concentrations in a pasture, the pasture comprising a combination of: pasture plants or parts thereof substantially as described above that undergo a colour change due to the elevated secondary metabolite concentration; and surrounding conventional pasture plants; wherein the extent of the pasture plants substantially as described above in the pasture is assessed by measuring the extent of colour difference between the pasture plants and the surrounding conventional pasture plants.

[0218] As may be appreciated from other aspects herein, the secondary metabolite compounds described may cause a change in colour to the plants in which the PPSM compounds are produced. This change in colour makes these plants stand out from a pasture of conventional green pasture and as a result, it is very easy to visually estimate the herb, legume, or grass content of a pasture. This may be useful to aid assessment of the pasture composition or diversity. This may apply to any components of the pasture being different to any other components such that they can easily be discerned by the human eye, or image capture devices and software (e.g., camera's and image software).

[0219] Seed and A Method of Seed Production

[0220] In a sixteenth aspect, there is provided seed produced from the pasture plant or part thereof substantially as hereinbefore described.

[0221] In a seventeenth aspect, there is provided a method of production of seed produced from the pasture plant or part thereof substantially as hereinbefore described by: crossing of pasture plants identified as coloured or containing elevated secondary metabolite compounds into elite pasture lines; backcrossing to produce elite line material with the desired trait; and multiplication of the elite line material.

[0222] In an eighteenth aspect, there is provided a method of production of pasture plants or parts thereof substantially as described above by: gene modification, editing, modulation or mutagenesis of the pasture plant, to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof.

[0223] In a nineteenth aspect, there is provided a method of production of pasture plants or parts thereof substantially as described above by: gene modification, editing, or mutation of the pasture plant, to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof; and further preventing purple colouration by down regulation or modulation of the UFGT pathway. The first steps of the secondary metabolite pathway can be activated by TrRED LEAF and TrMYB14 transcription factors (or by analogues thereof). Anthocyanin synthesis can be mediated by TrRED LEAF or analogues, or orthologs but is understood to be dependent upon a functional UFGT gene. Thus by down regulating, silencing, or mutation of the UFGT to be non-functional, it may be possible to upregulate proanthocyanidin synthesis, mediated by TrMYB14, but not TrRED LEAF i.e. producing a green leaf plant with upregulated proanthocyanidin synthesis and concentration of the resulting compounds.

[0224] This would reduce or stop the production of purple colouration and raise precursor levels, increasing the proanthocyanidin (tannin) levels. It may be that the version of the TrMYB14 requires some modification to upregulate the LAR and ANR genes but this can be achieved by gene editing or site directed mutagenesis.

[0225] Advantages

[0226] As noted above, the pasture plant, compositions, pasture and various methods may allow use of pasture plants with lower animal health risk issues such as bloating. The pasture plant or a part thereof, compositions, pasture and various methods may also allow for great nitrogen utilisation and nitrogen uptake and reduced risk of carbon dioxide or methane or nitrous emissions or nitrogen leaching.

[0227] The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features.

[0228] Further, where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relate, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0229] WORKING EXAMPLES

[0230] The above described pasture phytochemical compositions, methods and uses in ruminant animal farming are now described by reference to specific examples and the items and item numbers listed below:

[0231] 1 Purple leaf clover plant

[0232] 2 Leaves

[0233] 3 Purple colouring

[0234] 4 Green edging

[0235] 5 Flower 10 Pasture

[0236] 20 Non-purple leaf clover pasture

[0237] 30 Phenolic compounds categories identified

[0238] Note that clover is used as a representative herb, legume, or grass pasture plant for the purposes of the examples unless otherwise stated. As noted elsewhere, other herb, legume, or grass pasture plants may be used instead of clover such as chicory, plantain, or grasses containing raised levels of secondary metabolites e.g., PPSM compared to conventional pasture cultivars. Reference to clover should not be seen as limiting.

[0239] EXAMPLE 1

[0240] Compositions comprising purple-leafed / higher PPSM compound concentration clover were selected, isolated and grown in test fields. Selection and isolation was completed via traditional selection and cultivar enhancement methods and not via genetic modification. Selection and isolation was completed based on purple colouration and PPSM compound leaf content, the inventors finding a correlation between leaf purple colour and PPSM compound content.

[0241] Figure 3 illustrates a close up photograph of the clover plant 1 described herein. The clover plant 1 shown is a perennial clover plant 1 derived from the Trifolium genus in which 90-95% by area of the leaves 2 of the clover plant 1 are purple coloured 3. As shown in Figure 4, purple colour 3 expression starts at a spade leaf 4 stage (early) in the plant 1 growth cycle.

[0242] The purple colour 3 is substantially solid across the leaves 2 of the clover plant 1 and not segregated or patterned across the leaves 2 of the clover plant 1.

[0243] The clover plant 1 leaves 2 can have some green coloured edging 4 on edges of the leaves 2 of the clover plant 1.

[0244] The clover plant 1 produced conventional white coloured flowers 5. It is envisaged that the flower colour and phenolic content of the flowers could be coloured or have elevated phenolic content and reference to a white flower should not be seen as limiting.

[0245] The leaves 2 of the clover plant 1 identified were formed almost exclusively as three leaflet leaves 2.

[0246] The purple colour 3 and PPSM compound concentration of the leaves 2 was identified to be a heritable dominant trait.

[0247] No substantial seasonal variation in purple colour 3 or PPSM compound content was noted and the identified clover plant 1 remained a perennial plant with substantially purple colouration 3 and substantially stable PPSM compound content throughout the year. It is envisaged that the colour and phenolic / PPSM compound content could vary seasonally and reference to a perennial plant should not be seen as limiting. Figure 5 shows a pasture 10 comprising the clover plants 1 described herein. In this image, the clover plants 1 were overplanted in existing pasture 20 to give 40-60% area coverage by the clover plants 1 on the pasture 10.

[0248] EXAMPLE 2

[0249] The clover plant 1 leaves 2 were collected and samples taken to determine the type and quantity of PPSM content in the leaves 2 of the clover plant 1 identified versus art clover plants.

[0250] Figure 6 shows a diagram of phenolic compounds and the circled categories 30 were the compounds that were dominant in the clover plant 1 samples collected. The range of compounds identified in the samples was unexpectedly greater than the prior art would have indicated with art genetically modified varieties tending to only have phenolic compounds in one category (condensed tannins) and not multiple categories as found for the described clover plant 1.

[0251] Figure 7 is a graph showing total monomeric anthocyanins measured in a control 'conventional' green clover leaf versus that of clover plants 1 of increasing purple leaf colour 3. As shown, there is a relationship between purple colour 3 and anthocyanin content of the leaves 2. The clover plant 1 identified had a significantly higher (250% higher) total monomeric anthocyanin measure than the control or other purple colouration varieties selected.

[0252] Figure 8 is a graph showing total phenolic levels measured in a control 'conventional' green clover leaf versus that of clover plants 1 of increasing purple leaf colour 3. As shown, there is a relationship between purple colour 3 and total phenolic levels of the leaves 2. The clover plant 1 identified had a significantly higher total phenolic content (250% more) than the control conventional white clover varieties compared against.

[0253] Figure 9 is a table comparing two specific measured HPLC monomers tested for comparing the clover plant 1 identified against a control 'conventional' white clover leaf sample. For quercetin-3-glucoside, the clover plant 1 identified had 42 times more monomer present than the control. For delphinidin-3- rutinoside, the clover plant 1 identified had this compound present (89ppm) while the control did not have detectable levels of this monomer at all. Rutin concentration almost doubled as well relative to conventional clover (conventional = 1.60ppm; purple leaf = 2.82). The clover plant identified was unexpectedly different in phenolic content compared to control art clover plants.

[0254] Additional trials were completed to determine the genetic pathways for flavonoid biosynthesis and in doing so, to understand which pathways were upregulated by the clover plants described with the elevated PPSM compound content. The trials completed examined the pathway shown in Figure 10 and the highlighted enzymes were the ones found to be significantly upregulated relative to enzymes in conventional or green clover. Figure 10 only shows enzymes upregulated within the flavonoid biosynthesis (ec00941) pathway. There are other pathways that may be upregulated also within other secondary metabolism pathways. The three activator MBW components (TrRED LEAF, TrANl and TrWRKY 44) were especially active in the clover plants with purple colouring and elevated PPSM compounds described herein.

[0255] EXAMPLE 3

[0256] As noted above, the colour change resulting from the presence of PPSM compounds may lead to methods of better, easier, improved, or new management of pasture through measurement or estimation of the herb, legume, or grass in the pasture via its colour difference, e.g., purple coloured chicory, or clover plants within a principally green paddock, or farm, to aid assessment of the pasture composition or diversity.

[0257] This may apply to any components of the pasture being different to any other components such that they can easily be discerned by the human eye, or image capture devices and software (e.g., camera's and image J software).

[0258] Currently it is extremely difficult to estimate clover content within a pasture;

[0259] For example farmers aim to achieve ~30% clover within the paddock but, with green coloured white clover this is difficult to discern against a green grass background as shown in Figure 11B for example.

[0260] Using PPSM coloured plants of pasture species other than green makes it much easier to discern the proportion of said species within the pasture. From such information / knowledge it is then easy to make decisions about how to better manage the pasture e.g., but not limited to grazing decisions, spraying decisions, irrigation decisions, fertilizer decisions, and re-sowing, or, over-sowing, or stitching in seeding decisions.

[0261] Figure 11 (left A and B) are RGB images of picture of green, white clover in a pasture (bottom) showing visually the ease in which the purple clover (top) can be discerned from the green grass.

[0262] Figure 11 (right A and B) are the same images analysed using ImageJ software and using simple threshold colour discrimination (conditions shown in Figure 11 C) to identify the purple clover from the grass (right A), the same conditions cannot discern the green clover (right B). This is depicted to serve as an example of how such image capture and software can discern pasture composition when different colour parameters are used but is not limiting as to which of the many applications might be used to discern such colour differences.

[0263] EXAMPLE 4

[0264] As noted in the above description, the described aspects may relate to a variety of herb, legume and grass pasture plants and is not limited to just clover. As a demonstration that the composition of colour and compounds are not limited to just clover as a pasture species, trials were completed using chicory (Cichorium intybus), plantain / plantago sp., grass species, and subterranean clover, that could be used as pasture plants with up regulated secondary metabolites compared to conventional herb, legume and grass pasture plants.

[0265] The results are shown as photographs to illustrate the purple colouring from such selection, or from examples of plants with upregulated PPSM compound expression and concentrations are shown in Figure 12 (chicory), Figure 13 (plantain / plantago sp.), Figure 14 (grass species), and Figure 15 (subterranean clover). As demonstrated, all of these plant types may be propagated, crossed into elite pasture material and seed produced and used as integral components of a pasture and the purple colour used to show that the traits are heritable and dominant.

[0266] EXAMPLE 5

[0267] As noted above, the herb, legume, or grass pasture plant or part thereof, or an extract from the herb, legume, or grass pasture plant or part thereof may be used as a colourant e.g., for printing, for paints, or for food industries.

[0268] To demonstrate this, purple-coloured leaves from a clover plant described in earlier examples were simply used in place of a pen or crayon to make two drawings as shown in Figure 16. The fact that the colour transferred easily to the paper medium suggests it would be suitable for easy extraction and commercialisation. The colour appears to sit on the leaf and is easily transferred.

[0269] EXAMPLE 6

[0270] As a demonstration of an example of seed production from coloured pasture species, Figure 17 shows purple clover in flower for seed production and, Figure 18 shows a tray of purple clover being propagated using seeds collected from the flowering purple clover of Figure 17. This shows that it is possible to produce seeds from the herb, legume, or grass pasture plant and propagate these in a further generation of herb, legume, or grass pasture plant.

[0271] EXAMPLE 7

[0272] The above description notes the way that the described herb, legume, or grass pasture plant or an extract thereof may be used to reduce greenhouse gas emissions from animals. To demonstrate this effect, an experiment was undertaken to measure methane output from sheep consuming purple clover (0.5% dry weight of cut pasture or greater PPSM compounds) vs sheep consuming conventional (green) clover (<0.4% dry weight or no PPSM compounds). In the experiment, 40 sheep were first assessed for methane production using portable accumulation chambers and a subset of 20 sheep with similar methane producing outputs were chosen from the wider group of 40 sheep.

[0273] The 20 selected sheep were then blocked into two groups of 10 sheep. 5 sheep from each group were randomly chosen to consume a purple clover and ryegrass diet. The remaining 5 sheep in each group consumed a conventional (green) clover and ryegrass diet.

[0274] The sheep were all pre-fed for 10 days for conditioning, then blocked into pens and fed for 12 days on their respective diets. Over the last 3 days after feeding in the morning and afternoon, the sheep were placed into the portable accumulation chambers and methane and carbon dioxide emissions measured.

[0275] On each measure, the sheep fed purple clover demonstrated a lower methane and carbon dioxide emission than the sheep fed conventional (green) clover. The results found are illustrated in the tables shown in Figure 19 and Figure 20. These tables show the differences in methane and carbon dioxide emissions either as scaled data to the group mean (Figure 19) or as unsealed data (Figure 20).

[0276] EXAMPLE 8

[0277] As noted in the description, the traits around elevated secondary metabolite compounds and upregulation of genes was found to be a heritable trait.

[0278] As a way to demonstrate this heritability, representations of the of Trifolium repens foliar pigmentation patterning associated with the TrRED LEAF (R) loci and TrRED LEAF (V) loci, found in the F2 population were phenotyped, and their presence in subsequent populations established, examples of phenotypes are shown in Figure 21. Figure 22 shows a phylogenetic tree of flavonoid-regulating MYB genes and linkage map locations of the R and V loci in T. repens taken from Albert et al (2015) to demonstrate the heritability of the F2 leaf colouration shown in Figure 21.

[0279] As shown in Figure 21, images of representations of the of Trifolium repens foliar pigmentation patterning associated with the R and loci, found segregating in the F2 population are demonstrated, where image (A) No pigmentation; image (B) Red-fleck (Rf); image (C) Red midrib (Rm); image (D) Red leaf (Rl); image (E) Red-\Z.

[0280] Images B, C and D show pigment phenotypes linked to the 7. repens R locus located on chromosome eight of the 7. occidentale subgenome. Panel E shows the pigment phenotype linked to the 7. repens V locus, located on chromosome five of both the 7. occidentale and 7. pallescens subgenomes.

[0281] This demonstrates the crossing that was performed, the phenotypes seen and heritability of the phenotype and its association with the specific allelic version of TrRED LEAF (R) loci by association with the phylogeny tree of the TrRED LEAF family. This aspect is described further below and focuses on the TrRED LEAF (R) loci. EXAMPLE 9

[0282] To show gene upregulation was controlled by TrRED LEAF (R) loci, analysis was completed to show the number of genes differentially expressed in the purple clover (containing the TrRED LEAF functional R loci - high pigmentation plants, labelled H) compared to the white clover (no visible pigmentation plants, labelled N).

[0283] The results are illustrated in the volcano plot shown in Figure 23 which shows genes that are up regulated (green dots) and genes that are down regulated (red dots) by the purple clover vs the conventional white clover.

[0284] In more detail, Figure 23 shows differentially expressed transcripts determined by Illumina short-read sequencing and RNA-Seq analysis of a Trifolium repens F2 population segregating for foliar pigmentation phenotype. The volcano plot of significantly up- and down-regulated genes in samples with high pigment accumulation plants (H) relative to plants lacking visible pigmentation (N).

[0285] Five biological replicates of H and N were used per RNA-Seq study group. RNA reads were mapped to a de novo transcriptome established using RNA reads from H and N study groups and assembled using Trinity v2.1.1 software. Read counts per transcript were determined using RSEM vl.3.3 software, and the resulting read-count matrix analysed via EdgeR v3.38.0 software, to identify the transcripts that were differentially regulated by the functional TrRED LEAF loci.

[0286] The results found demonstrated that there are over 100 genes up regulated by TrRED LEAF and a smaller number down regulated. Expressed another way, the results showed the there are differences in gene expression profiles associated with the pigmentation phenotype.

[0287] EXAMPLE 10

[0288] A heatmap of genes over expressed in the purple clover (H) compared to white clover (N) is shown in Figure 24.

[0289] This heatmap shows differential gene expression profiles determined by Illumina short-read sequencing and RNA-Seq analysis of a Trifolium repens F2 population segregating for foliar pigmentation phenotype. The heat map also indicates specific genes that are up regulated by the purple clover TrRED LEAF allele.

[0290] Distinct gene expression profiles were shown between samples with high pigment accumulation (H) and samples lacking visible pigmentation (N).

[0291] Five biological replicates were used per RNA-Seq study group.

[0292] RNA reads were mapped to a de novo transcriptome established using RNA reads from H and N study groups and assembled using Trinity v2.1.1 software. The identity of the transcripts is shown on the right of the heat map Read counts per transcript were determined using RSEM vl.3.3 software, and the resulting read-count matrix analysed via DESeq2 vl.38.3 software.

[0293] In a further test, BLASTn was used to search results of the differentially expressed transcripts from the Trifolium repens F2 population segregating for foliar pigmentation phenotype, and identify their putative function. The results for this are shown in the table in Figure 25.

[0294] The results showed enrichment of genes involved in flavonoid and anthocyanin biosynthesis in samples with high pigment accumulation (H) relative to samples lacking visible pigmentation (N) (see for example column 7 of Figure 25). Columns eight and nine of Figure 25 show the average number of RNA reads mapping to each transcript per N sample and H sample, respectively.

[0295] EXAMPLE 11

[0296] An experiment was completed to identify the difference in expression of TrRED LEAF between purple and conventional white clover, and link this to the presence of the functional TrRED LEAF loci, through the presence of a SNP in a closely (42KB) linked gene (PS6L) as in Figure 26 (section labelled B), which allows homozygous or heterozygous individuals carrying the TrRED LEAF gene to be identified e.g. through the application of a CAPS (cleaved amplified polymorphic sequences) as in Figure 26 (section labelled C), or KASP (Kompetitive allele specific PCR).

[0297] In more detail, Figure 26 demonstrates a phenotypically segregating single nucleotide polymorphism (SNP), just over 42Kb downstream, of differentially expressed TrRED LEAF (R) locus MYB transcription factor indicating allelic segregation, where, in Figure 26:

[0298] (A) shows RNA reads from samples with high pigment accumulation (H) and samples lacking visible pigmentation (N) aligned to the UTM_Trep_vl.O_hapl (GeneBank accession:

[0299] GCA 030914245.1) Trifolium repens reference genome. o The left panel shows an R2R3 activator MYB transcription factor, TrRED LEAF, being expressed only in high pigment accumulating samples; o The right panel shows a C > T single nucleotide polymorphism (SNP), co-segregating with the pigment phenotype (and thus the TrRED LEAF allele associated with the purple phenotype), present in the second exon of the proteosome subunit 6-1 i ke (PS6L) gene, 42 kb downstream of TrRED LEAF.

[0300] (B) shows multiple-alignment of five haplotype sequences derived from long-read Oxford Nanopore Technology sequencing and contig assembly using Flye v2.9.2 software. T. occidentale subgenome haplotypes are marked Occl-4 and T. pallescens subgenome are marked Pall-3. o A 31 bp deletion is shown in all four T. occidentale subgenome haplotypes (and none of the T. pallescens subgenome haplotypes); o This variant is 132 bp upstream of a C > T single nucleotide polymorphism (SNP) cosegregating with the pigment phenotype present in the second exon of proteosome subunit 6-1 ike (PS6L) gene, 42 kb downstream of TrRED LEAF; o Among the T. occidentals subgenome haplotypes, this SNP is only present in Occ3; the haplotype which contains the version of the TrRED LEAF gene associated with the purple phenotype; o The C > T SNP forms a Msel restriction enzyme binding site.

[0301] (C) shows gel electrophoresis of a Cleaved Amplified Polymorphic Sequences (CAPS) assay screening for TrRED LEAF zygosity using the PS6L C>T SNP as a marker, i.e., the SNP can be used as a molecular marker to identify the closely associated TrRED LEAF, in its homozygous or hemizygous condition.

[0302] EXAMPLE 12

[0303] In this example, results from a polymerase chain reaction (PCR) amplification are shown (see Figure 27), of TrRED LEAF from a Trifolium repens F2 population segregating for foliar pigmentation phenotype, across an 885bp region of the TrRED LEAF coding sequence.

[0304] Genomic DNA from 10 samples lacking visible pigmentation (N1-N10) and with nine samples with high pigment accumulation (H1-H9) were amplified with polymerase chain reaction (PCR) primers targeting an 885 bp fragment of the TrRED LEAF coding sequence.

[0305] Amplicons from each PCR reaction were run on a 1.5% agarose gel using gel electrophoresis.

[0306] As shown in Figure 27, clear single ~880 bp bands were found to be present in all nine samples exhibiting foliar pigment accumulation. No visible bands were found present in all 10 samples lacking foliar pigmentation. L; 1.5 kb DNA ladder, -ve; dFhO negative control.

[0307] The primers preferentially amplify the purple version of the TrRED LEAF and can easily be used to discriminate purple from white clover

[0308] In Figure 26 of Example 11, the primers of the CAPS assay were targeting the PS6L gene fragment that contains the C > T SNP. In this Example and Figure 27, the primers are targeting an 885 bp fragment TrRED LEAF CDS itself.

[0309] The results in this Example provide further evidence that the TrRED LEAF gene is absent in F2 plants that lack pigment.

[0310] EXAMPLE 13

[0311] As noted elsewhere in this specification, a variety of herbs, legumes and grasses may be used for upregulation of pathways leading to increased concentration of secondary metabolite compounds. To demonstrate this further, Nicotiana benthamiana leaves were agroinfiltrated with the functional TrRED LEAF gene to demonstrate the upregulation of the secondary metabolite compound pathway in other plant species. This represents a deliberate upregulation method as opposed to using naturally occurring upregulated plants crossed into elite lines.

[0312] Figure 28 shows the results where pigment accumulation in Nicotiana benthamiana leaves infiltrated with a p35S:TrRED LEAF expression vector. TrRED LEAF coding sequence was cloned into a GoldenGate Level 2 vector under the control of the cauliflower mosaic virus 35S promoter.

[0313] Specifically, in Figure 28, (A) shows a photograph of N. benthamiana leaf infiltrated with infiltration buffer containing GV3101 Agrobacterium transformed with the p35S:TrRED LEAF expression vector (shown as purple colouration) and, (B) shows a photograph of N. benthamiana leaf infiltrated with infiltration buffer only (no purple colouring).

[0314] Upregulation is therefore possible in a variety of plant species.

[0315] EXAMPLE 14

[0316] In this example, development of a non-purple purple pasture plant is described. It is proposed that the purple pasture plants may be modified to redirect the metabolic flux within the flavonoid biosynthetic pathway.

[0317] Figure 29 shows a generalised schematic of the flavonoid biosynthetic pathway. The first eight steps of the pathway can be activated by TrRED LEAF and TrMYB14 transcription factors. Anthocyanin synthesis can be mediated by TrRED LEAF but is dependent upon a functional UFGT gene. Thus by down regulating, silencing, or mutation of the UFGT to be non-functional, in a TrRED LEAF positive plant, it may be possible to upregulate proanthocyanidin synthesis, mediated by TrMYB14, i.e. producing a green leaf plant with upregulated proanthocyanidin synthesis and concentration of the resulting compounds.

[0318] This would reduce or stop the production of purple colouration and raise precursor levels, increasing the proanthocyanidin (tannin) levels. It may be that the version of the TrMYB14 requires some modification to upregulate the LAR and ANR genes but this can be achieved by gene editing or site directed mutagenesis.

[0319] Figure 30 is a diagram to further illustrate the above approach. As demonstrated in Figure 28 the TrRED LEAF can be used to upregulate the pathway in species other than clover, in the case in point, by agroinfiltration of a plasmid containing a functioning TrRED LEAF into a N. benthamiana leaf. To show that the metabolic flux can be altered, the same experiment can be conducted but this time including an additional plasmid, containing a RNAi (gene silencing construct) directed against the N. benthamiana UFGT gene. The resulting agroinfiltration (right hand side of Figure 30 would produce no, or less, purple pigment than the TrRED LEAF construct on its own (left hand side of Figure 30). EXAMPLE 15

[0320] As noted elsewhere, the described herb, legume or grass pasture plants or parts thereof may alter products produced from animals eating the plants or supplemented with the plants.

[0321] To demonstrate this effect, the protein, Bovine Serum Albumin (BSA), produced by animals grazing on varying pastures was tested by incubating with PPSM compounds extracted from purple or conventional (green) clover. Following incubation, and centrifugation to remove precipitated BSA, the level of phenolic extract was remeasured. The greater the reduction in phenolic extract, the greater the binding to BSA. Figure 31 shows the results of this trial. BSA incubated with purple clover phenolic extracts 1-3 shown in Figure 31 had greater phenolic reduction than conventional green clover extracts at increasing concentrations of protein (Bovine Serum Albumin, BSA) concentrations. Reduction was measured relative to initial phenolic amount in each extract. Phenolic reduction is an indication of protein loss from solution due to phenolic-protein precipitation. Such associations have been shown to help transfer protein to the abomasum where it is more efficiently processed by the ruminant animal. Aspects of the above-described phytochemical compositions, methods and uses in ruminant animal farming have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein.

Claims

WHAT IS CLAIMED IS:

1. A pasture plant or part thereof, the pasture plant selected from a herb, legume, or grass pasture plant or part thereof; and wherein the pasture plant or part thereof comprises elevated secondary metabolite concentrations relative to a conventional herb, legume, or grass pasture plant or part thereof.

2. The pasture plant or part thereof as claimed in claim 1 wherein the pasture plant or part thereof comprises plant polyphenol secondary metabolite (PPSM) compounds.

3. The pasture plant or part thereof as claimed in claim 2 wherein the PPSM compounds comprise a mixture of flavonoids and condensed tannins.

4. The pasture plant or part thereof as claimed in claim 2 wherein the PPSM compounds comprise a mixture of flavones, flavanols and anthocyanins.

5. The pasture plant or part thereof as claimed in claim 1 comprising upregulated genes that encode enzymes within the flavonoid biosynthesis (ec00941) pathway.

6. The pasture plant or part thereof as claimed in claim 5 wherein the upregulated genes are activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof.

7. The pasture plant or part thereof as claimed in claim 5 wherein the upregulated gene is TrRED LEAF or analogues, or orthologs thereof.

8. The pasture plant or part thereof as claimed in claim lwherein the pasture plant is bred via traditional selection and cultivar enhancement methods and wherein the pasture plant is not produced by genetic modification.

9. The pasture plant or part thereof as claimed in claim 1 wherein the pasture plant is bred by gene modification, editing, modulation, or mutagenesis, to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof.

10. The pasture plant or part thereof as claimed in claim 1 wherein the total phenolic content in leaves of the pasture plant is at least 20mg / g GA EQ.

11. The pasture plant or part thereof as claimed in claim 1 wherein the anthocyanin content in leaves of the pasture plant is greater than l.Omg / g.

12. The pasture plant or part thereof as claimed in claim 1 wherein leaves of the pasture plant comprise a secondary metabolite compound content greater than 0.5% dry weight.

13. The pasture plant or part thereof as claimed in claim 1 wherein the elevated secondary metabolite concentrations in the pasture plant or part thereof is a heritable dominant trait.

14. The pasture plant or part thereof as claimed in claim 1 wherein the pasture plant comprises at least substantially 5% by area purple colouring of leaves of the pasture plant.

15. The pasture plant or part thereof as claimed in claim 14 wherein the purple colour is substantially solid across the leaves of the pasture plant.

16. The pasture plant or part thereof as claimed in claim 14 wherein the purple colour is not segregated or patterned across the leaves of the pasture plant.

17. The pasture plant or part thereof as claimed in claim 14 wherein the pasture plant has green coloured edging on edges of the leaves of the pasture plant.

18. The pasture plant or part thereof as claimed in claim 14 wherein the purple colour of the leaves of the pasture plant has a wavelength from 380-475nm.

19. The pasture plant as claimed in claim 1 wherein the pasture plant is derived from the Trifolium genus.

20. The pasture plant as claimed in claim 1 wherein the pasture plant is derived from Trifolium repens.

21. A composition formulated for ruminant animal administration comprising: the pasture plant or part thereof as claimed in any one of the above claims; or an extract of the pasture plant or part thereof as claimed in any one of the above claims.

22. Pasture comprising the pasture plant or a part thereof as claimed in any one of claims 1-20.

23. A method of increasing sequestration of dietary protein in the rumen of a ruminant animal by: selecting the pasture plant or part thereof as claimed in any one of claims 1-20; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the sequestration of dietary protein in the rumen of the ruminant animal post administration is increased relative to a ruminant animal administered a conventional pasture plant or a part thereof.

24. A method of decreasing bloating in a ruminant animal by: selecting the pasture plant or part thereof as claimed in any one of claims 1-20; administering the pasture plant, or a part thereof, to the ruminant animal; wherein the amount of bloating that the ruminant animal experiences post administration is reduced relative to the amount of bloating that a ruminant animal would experience if administered a conventional pasture plant or a part thereof.

25. A method of reducing nitrogen excretion from a ruminant animal by: selecting the pasture plant or part thereof as claimed in any one of claims 1-20;administering the pasture plant, or a part thereof, to the ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration is reduced, or partitioned differently between urine, faeces and gaseous emissions, relative to the amount of nitrogen, or nitrogen partitioning, excreted from a ruminant animal administered a conventional pasture plant or a part thereof.

26. A method of reducing the risk of nitrogen leaching from pasture by: selecting the pasture plant or part thereof as claimed in any one of claims 1-20; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration is reduced, or partitioned differently between urine, faeces and gaseous emissions, relative to the amount of nitrogen, or nitrogen partitioning, excreted from a ruminant animal administered conventional pasture plant or a part thereof, the reduced nitrogen, or nitrogen partitioning, excreted resulting in a reduced risk of nitrogen leaching from the ruminant animal.

27. A method of reducing the risk of greenhouse gas emissions from pasture by: selecting the pasture plant or part thereof as claimed in any one of claims 1-20; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the amount of nitrogen excreted from the ruminant animal post administration as carbon dioxide and / or nitrous containing gas is reduced relative to a ruminant animal administered conventional pasture plant or a part thereof and, as a result, the amount of greenhouse gas emissions as carbon dioxide and / or nitrous containing gas from the ruminant animal is also reduced.

28. A method of increasing the production of nitrogen-rich ruminant animal products by: selecting the pasture plant or part thereof as claimed in any one of claims 1-20; administering the pasture plant, or a part thereof, to a ruminant animal; wherein the production of nitrogen-rich ruminant animal products from the ruminant animal post administration is increased relative to the production of nitrogen-rich ruminant animal products from a ruminant animal administered conventional pasture plant or a part thereof.

29. The method as claimed in any one of claims 23 to 28 wherein administration comprises growing the pasture plant or a part thereof to form pasture with at least a substantial portion of the pasture comprising the pasture plant or a part thereof and, grazing the ruminant animal on the pasture.

30. The method as claimed in any one of claims 23 to 28 wherein administration comprises feeding out, to the ruminant animal, a feed, or a feed supplement, the feed or the feed supplement comprising the pasture plant or a part thereof and, grazing the ruminant animal on the feed or the feed supplement.

31. The method as claimed in any one of claims 23 to 30 wherein the ruminant animal is selected from: cows, cattle, goats, deer, sheep, and combinations thereof.

32. A supplement formulated for oral or topical administration, the supplement comprising: the pasture plant or part thereof as claimed in any one of claims 1-20; or an extract from the pasture plant or part thereof as claimed in any one of claims 1-20.

33. A supplement formulated for oral or topical administration, the supplement derived from or comprising flowers, nectar, or flowers and nectar produced from the pasture as claimed in any one of claims 1-20.

34. A colourant, the colourant derived from or comprising: the pasture plant or part thereof as claimed in any one of claims 1-20; or an extract from the pasture plant or part thereof as claimed in any one of claims 1-20.

35. A method of measuring a content of pasture plant with elevated secondary metabolite concentrations in a pasture, the pasture comprising a combination of: pasture plants or parts thereof as claimed in any one of claims 1-20 that undergo a colour change due to the elevated secondary metabolite concentration; and surrounding conventional pasture plants; wherein the extent of the pasture plants with elevated secondary metabolite concentrations in the pasture is assessed by measuring the extent of colour difference between the pasture plants and the surrounding conventional pasture plants.

36. Seed produced from the pasture plant or part thereof as claimed in any one of claims 1-20.

37. A method of production of seed produced from the pasture plant or part thereof as claimed in any one of claims 1-20 by: crossing of pasture plants identified as coloured or containing elevated secondary metabolite compounds into elite pasture lines; backcrossing to produce elite line material with the desired trait; and multiplication of the elite line material.

38. A method of production of pasture plants or parts thereof as claimed in any one of claims 1-20 by:

39. gene modification, editing, modulation or mutagenesis of the pasture plant, to cause upregulation of activator MYB components selected from one or more of: TrRED LEAF, TrANl, TrWRKY 44, or analogues, or orthologs thereof.

40. The method of production of pasture plants or parts thereof as claimed in claim 38 by: further preventing purple colouration by down regulation or modulation of the UFGT pathway.

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