Methods and compositions for increasing seed oil without seed yield penalty

Abstract

The invention provides a method for increasing the production of oil in the seed of a plant relative to that in a control plant, without significantly decreasing seed yield, by ectopically expressing an oil-synthesising enzyme and an oil-encapsulating protein in endosperm of the plant, and optionally ectopically expressing an oil-synthesising enzyme and an oil-encapsulating protein in green tissues of the plant. The invention also provides plants and seeds produced or selected by the methods, and methods for processing the seeds into oil and co-products.

Description

[0001] METHODS AND COMPOSITIONS FOR INCREASING SEED OIL WITHOUT SEED YIELD PENALTY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The contents of Australian provisional patent application number 2024900660 filed 13 March 2024, and Australian provisional patent application number 2024900662 filed 13 March 2024, are incorporated herein by reference in their entirety.

[0004] TECHNICAL FIELD

[0005] The invention relates to compositions and methods for increasing oil in the seed of plants without decreasing seed yield.

[0006] BACKGROUND

[0007] Monocotyledonous plants (particularly the grasses) dominate human agriculture. Examples include true grains such as rice, wheat, maize and sorghum, as well as the pasture grasses and sugarcane.

[0008] Nutritionally, true grain seeds tend to be high in carbohydrate and protein but low in fat. This is due to the anatomy of a true grain seed which has smaller embryos when compared to oilseed species such as canola, soybean and camelina. The oil content of true grains typically ranges from: 2%-3% (rice), 3%-4% (wheat bran), 7%-9% (wheat germ), 3%-5% (maize) and 5%-8.2% (sorghum). In contrast oilseeds can have oil content as high as 60% (Sundus et al 2019). Specifically in maize, over 80% of the seed’s volume is endosperm consisting of typically 70-80% starch and 8-10% protein. The embryo makes up only 10-12% of the seed and is typically 25% oil; consequently, bringing the total fat content of a maize seed to 3-5%.

[0009] Among the true grains, maize (Zea mays) is one of the most important global feedstocks for food, feed, and fuels. Approximately 65% of maize grain is used for animal feed and in countries such as the United States it is also the prevalent feedstock for bio-industrial processers that convert maize to renewable fuels such as ethanol, renewable diesel and sustainable aviation fuel. Due to the low level of oil in true grains and the importance of plant oils as a feedstock, research and innovation to increase oil content in true grains has been an important focus for the agricultural industry. For example, high-oil maize shows a greater feed efficiency than normal-oil maize in animal feed trials because the caloric content of oil is 2.25 times greater than that of starch on a weight basis.

[0010] In the industrial processing of maize (wet or dry milling), oil is the most valuable coproduct when compared with ethanol and com distillers grain (Shen et al 2010). Unlike ethanol, which has lower energy density than its fossil fuels counterpart and where carbon dioxide is a byproduct of the fermentation process, maize oil provides a direct one for one conversion into a renewable fuel.

[0011] Maize varieties ranging from 6%-20% oil have been reported (Luo et al 2023). One method to produce high oil hybrids has been to plant a high oil pollinator variety intermixed with conventional hybrids that have normal levels of oil (Lambert et al 1998). The increase in oil levels in these hybrids (relative to the normal oil line) was the result of a small increase in germ weight, an increase in oil concentration in the germ, and a reduction in the percentage of endosperm (Lambert et al 1998).

[0012] However, the increased oil comes with a negative impact on grain yields (aggregate measure driven by average seed weight and seed number per plant) that limits the commercial viability of these strategies as grower revenues are tied to grain yields. The main reasons for the yield reduction are (i) the high metabolic cost of oil synthesis, as 2.25-fold as much energy is needed to produce oil in the kernel as compared with starch (Li et al 2023) and (ii) the limited size of the embryo when compared to oilseeds makes increasing oil content in such a small organ challenging for the plant.

[0013] Newer biotechnology approaches have also been investigated including: increasing carbohydrate availability for oil biosynthesis; overexpression of genes that cause a carbon flux to the fatty acid (FA) biosynthesis pathway; promoting and improving the stability of triacyglycerol (TAG) biosynthesis, enhancing the export of fatty acids from the plastids; and increasing the embryo size to enlarge the volume for oil storage. Many of these are reviewed by Li et al (2023). However, from the literature as discussed above, while there are several methods for improving oil composition in the seed of true grains, those skilled in the art would expect that there will be a decrease in seed (grain) yield if oil content is increased.

[0014] New approaches that can increase seed oil while balancing the plant’s capacity to maintain its seed / grain yield levels are therefore required.

[0015] It is an object of the invention to provide methods and / or for production and / or selection of plants that overcome one or more of the limitations of the prior art and / or at least provide the public with a useful choice.

[0016] SUMMARY OF THE INVENTION

[0017] The present applicants have now surprisingly demonstrated increased oil accumulation in the seed without significantly penalising seed yield, by controlled expression of an oil synthesising enzyme and an oil encapsulating protein in the endosperm.

[0018] Certain embodiments also utilise simultaneous controlled expression of an oil synthesising enzyme and an oil encapsulating protein in the vegetative tissues of the plant.

[0019] These results are surprising for the following reasons:

[0020] • The applicants have increased oil accumulation in the endosperm without the ectopic expression of a transcription factor, such as WRINKLED 1 (WRH -a transcription factor vital for the transcriptional control of plant oil biosynthetic pathways (Kong et al 2019). Prior art indicates that a transcription factor such as WRH is a necessary component to provide the endosperm with oil biosynthesis capabilities. In contrast the applicants have only heterologously expressed a TAG synthesising enzyme and an oil encapsulating protein to achieve increased oil content in the endosperm.

[0021] • Seed oil accumulation was increased significantly while seed yield was at least maintained, which contrasts with the widely accepted inverse relationship between total seed oil and seed yield.

[0022] • Using this simpler genetic approach, the applicants have also selected combinations of proteins and promoters that have temporal and spatial expression profiles typically not used when targeting manipulation of oil in the seed. Methods

[0023] In the first aspect the invention provides a method for increasing the content of oil in the seeds of a plant, relative to that in a control plant, without significantly reducing the seed yield of the plant, wherein the method comprises the step of ectopically expressing at least one oil-synthesising enzyme and at least one oil-encapsulating protein in the endosperm of the plant.

[0024] In one embodiment the method comprises the step of ectopically expressing at least two oilsynthesising enzymes and at least one oil -encapsulating protein in the endosperm of the plant.

[0025] In a further embodiment the method further comprises the step of ectopically expressing at least one additional oil-synthesising enzyme and at least one additional oil-encapsulating protein in the green tissues of the plant.

[0026] In one embodiment the plant does not ectopically express a transcription factor that regulates fatty acid biosynthesis.

[0027] In one embodiment the plant does not ectopically express the transcription factor WRI1.

[0028] In one embodiment the plant does not ectopically express a transcription factor that regulates lipid biosytnesis, in the endosperm of the plant.

[0029] In one embodiment the plant does not ectopically express the transcription factor WRI1, in the endosperm of the plant.

[0030] In one embodiment the increased content of oil in the seeds of a plant is a result of increased content of oil in the endosperm.

[0031] In a further aspect the invention provides a method for producing seed with increased oil content, relative to that in seed of a control plant, the method comprising growing a plant of the invention, or a plant produced by a method of the invention, and collecting seed from the plant. Method including the step of measuring the content of oil in the seed

[0032] In a further embodiment the method includes the step of measuring the content of oil in the seed of the plant.

[0033] Method including the step of selecting the plant based on measuring the content of oil in the seed

[0034] In a further embodiment the method includes the step of selecting the plant based on measuring an increase in the content of oil in the seed.

[0035] Method including the step of measuring the seed yield

[0036] In a further embodiment the method includes the step of measuring the seed yield of the plant.

[0037] Method including the step of selecting the plant based on measuring the seed yield

[0038] In a further embodiment the method includes the step of selecting the plant based on measuring no significant decrease in the seed yield.

[0039] Inceased seed oil content

[0040] In one embodiment content of oil in the seeds of the plant is increased by at least 0.1%, preferably at least 0.2%, more preferably at least 0.3%, more preferably at least 0.4%, more preferably at least 0.5%, more preferably at least 0.6%, more preferably at least 0.7%, more preferably at least 0.8%, more preferably at least 0.9%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more preferably at least 15%, more preferably at least 16%, more preferably at least 17%, more preferably at least 18%, more preferably at least 19%, more preferably at least 20%, more preferably at least 21%, more preferably at least 22%, more preferably at least 23%, more preferably at least 24%, more preferably at least 25%, more preferably at least 26%, more preferably at least 27%, more preferably at least 28%, more preferably at least 29%, more preferably at least 30%, more preferably at least 31%, more preferably at least 32%, more preferably at least 33%, more preferably at least 34%, more preferably at least 35%, more preferably at least 36%, more preferably at least 37%, more preferably at least 38%, more preferably at least 39%, more preferably at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, relative to that in a control plant.

[0041] In one embodiment there is a significant increase in the oil content of the seeds of the plant, relative to that in the control plant.

[0042] In one embodiment the increase in the oil content of the seeds is assessed using near infra red spectroscopy, NIR, (Zhu, Z, Chen, S, Wu X, Xing C, 2018, Food Sci Nutr. 6(4): 1109-1118. Determination of soybean routine quality parameters using near - infrared spectroscopy.) or by gas chromatography (GC) of fatty acid methyl esters (FAMES) (Shantha NC and Napolitano GE, 1992, Gas chromatography of fatty acids, Journal of Chromatography A. 624, 1-2:37-51). Analysis of total fatty acids (crude) AO AC Official Method 996.06 and OACS Official Method Ca 5b-71. Fatty Acid Profile, AOAC Official Methods 996-06 [Analysis of methyl esters by Capillary GLC], AOAC Official Methods Ce 2-66 [Preparation of Methyl Esters of Fatty Acids], AOAC Official Methods 965.49 [Preparation of Methyl Esters of Fatty Acids], AOAC Official Methods 969.33 [Oils and fat, Boron Trifluoride method]. Folch Extraction for Total Lipids from Animal Tissues (Folch et al. 1957, J. Biol. Chem 226:497). It would also be understood by those skilled in the art that a determination of oil content of the seeds could be made by quantifying the oil following industrial processing, including (but not limited to): solvent extraction, crushing, critical point extraction, milling, and fermentation. In one embodiment the solvent extraction is ether extraction. In one embodiment the ether extraction is AOAC Official Method 920:39 (A). In one embodiment the significance of the increase is at the less than 20% probability level, preferably at the less than 15% probability level, more preferably at the less than 10% probability level, more preferably at the less than 5% probability level, more preferably at the less than 1% probability level.

[0043] In one embodiment the significance of the increase in the oil content of the seeds is assessed using AN OVA (SAS Institute, 2016). Preferably the means are separated using Fisher’s Protected LSD at P=0. 1.

[0044] In a further embodiment the significance of the increase in the oil content of the seeds is assessed using Student’s T-test (Microsoft Excel V2108). Preferably the means separated by Fishers Least Significant Difference Test at P=0.05.

[0045] Oil

[0046] In one embodiment the oil is triacylglycerol (TAG).

[0047] Altered oil composition

[0048] In one embodiment the fatty acid profile of the seed of the plant is altered relative to that in the control plant.

[0049] In one embodiment there is an increase in Cl 8:0 fatty acid.

[0050] In a further embodimentt there is an increase in Cl 8: 1 fatty acid.

[0051] In a further embodiment there is an increase in both Cl 8:0 and C18: 1 fatty acids.

[0052] In one embodiment there is a decrease in Cl 8:2 fatty acid.

[0053] In a further embodiment there is a decrease in C18:3 fatty acid.

[0054] In a further embodiment there is a decrease in both C18:2 and C18:3 fatty acid. In a further embodiment there is an increase in both Cl 8:0 and C18: 1, and a decrease in both C18:2 and C18:3 fatty acids.

[0055] Those skilled in the art will know that fatty acid profile with an increase in the proportions of C18:0 and Cl 8: 1 fatty acids and decrease in the proportions of Cl 8:2 and Cl 8:3 fatty acid is characteristic of the activity of an oil synthsising enzyme, such as DGAT1.

[0056] In one embodiment the altered fatty acid profile of the seed is a consequence of the increase in oil as described herein.

[0057] No significant decrease in seed yield

[0058] In one embodiment the there is no significant decrease in seed yield of the plant in which the ectopic expression takes place, relative to that in a control plant.

[0059] Methods for assessing seed yield are known in the art.

[0060] In one embodiment seed yield is assessed by weight of seed produced.

[0061] In a further embodiment seed yield is assessed by number of seed produced.

[0062] In a further embodiment seed yield is for a population of plants in a given area that can also be expressed as total seed weight harvested for the area such as bushels per acre (BU / Acre)

[0063] In a further embodiment any decrease in seed yield relative to that in the control plant is less than 5%, preferably less than 4.9%, more preferably 4.8%, more preferably 4.7%, more preferably 4.6%, more preferably 4.5%, more preferably 4.4%, more preferably 4.3%, more preferably 4.2%, more preferably 4.1%, more preferably 4.0%, more preferably 3.9%, more preferably 3.8%, more preferably 3.7%, more preferably 3.6%, more preferably 3.5%, more preferably 3.4%, more preferably 3.3%, more preferably 3.2%, more preferably 3.1%, more preferably 3.0%, more preferably 2.9%, more preferably 2.8%, more preferably 2.7%, more preferably 2.6%, more preferably 2.5%, more preferably 2.4%, more preferably 2.3%, more preferably 2.2%, more preferably 2.1%, more preferably 2.0%, more preferably 1.9%, more preferably 1.8%, more preferably 1.7%, more preferably 1.6%, more preferably 1.5%, more preferably 1.4%, more preferably 1.3%, more preferably 1.2%, more preferably 1.1%, more preferably 1.0%, more preferably 0.9%, more preferably 0.8%, more preferably 0.7%, more preferably 0.6%, more preferably 0.5%, more preferably 0.4%, more preferably 0.3%, more preferably 0.2%, more preferably 0.1%, more preferably 1.0%, relative to that in a control plant.

[0064] In one embodiment any decrease in seed yield is at the more than 10% probability level, preferably the more than 20% probability level, more preferably the more than 30% probability level.

[0065] In one embodiment the significance of any decrease in seed yield is assessed using ANOVA (SAS Institute, 2016). Preferably the means are separated using Fisher’s Protected LSD at =0.I.

[0066] In a further embodiment the significance of of any desrease in the seed yield is assessed using Student’s T-test (Microsoft Excel V2108). Preferably the means separated by Fishers Least Significant Difference Test at P=0.05.

[0067] Increase in seed yield

[0068] In a further embodiment there is an increase in seed relative to that in the control plant.

[0069] In one embodiment seed yield is increased by at least 0.1%, preferably at least 0.2%, more preferably at least 0.3%, more preferably at least 0.4%, more preferably at least 0.5%, more preferably at least 0.6%, more preferably at least 0.7%, more preferably at least 0.8%, more preferably at least 0.9%, more preferably at least 1%, more preferably at least 1.1%, more preferably at least 1.2%, more preferably at least 1.3%, more preferably at least 1.4%, more preferably at least 1.5%, more preferably at least 1.6%, more preferably at least 1.7%, more preferably at least 1.8%, more preferably at least 1.9%, more preferably at least 2%, more preferably at least 2.2%, preferably at least 2.4%, preferably at least 2.6%, preferably at least 2.8%, more preferably at least 3%, more preferably at least 3.5%, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more preferably at least 15%, more preferably at least 16%, more preferably at least 17%, more preferably at least 18%, more preferably at least 19%, more preferably at least 20%, relative to that in a control plant.

[0070] In one embodiment there is a significant increase in seed yield, relative to that in a control plant.

[0071] In one embodiment the significance of the increase is at the less than 20% probability level, preferably at the less than 15% probability level, more preferably at the less than 10% probability level, more preferably at the less than 5% probability level, more preferably at the less than 1% probability level.

[0072] In one embodiment the significance of the increase in seed yield is assessed using ANOVA (SAS Institute, 2016). Preferably the means are separated using Fisher’s Protected LSD at =0.I.

[0073] In a further embodiment the significance of the increase in seed yield is assessed using Student’s T-test (Microsoft Excel V2108). Preferably the means separated by Fishers Least Significant Difference Test at / '=0.05.

[0074] Expression in the endosperm

[0075] Oil synthesising enzyme expressed in the endosperm

[0076] Numerous oil synthesising enzymes are known to those skilled in the art, and may be conveniently selected for endosperm expression in accordance with the invention.

[0077] In one embodiment the oil synthesising enzyme is a triacylglycerol (TAG) synthesising enzyme.

[0078] In one embodiment the TAG synthesising enzyme is acyl CoA: diacylglycerol acyltransferase 1 (DGAT1). In a further embodiment the TAG synthesising enzyme is phosphatidylcholine-sterol O- acyltransferase (PDAT).

[0079] In one embodiment both a DGAT1 and a PDAT are expressed

[0080] In one embodiment a modified oil synthesising enzyme is expressed.

[0081] In one embodiment a modified TAG synthesising enzyme is expressed.

[0082] In one embodiment a modified DGAT1 is expressed

[0083] In one embodiment a modified PDAT is expressed

[0084] Oil encapsulating proteins expressed in the endosperm

[0085] Numerous oil-encapsulating proteins are known to those skilled in the art, and may be conveniently selected for use in the invention.

[0086] In one embodiment the oil -encapsulating protein is an oleosin.

[0087] In one embodiment a modified oil -encapsulating protein is expressed.

[0088] In one embodiment a modified oleosin is expressed.

[0089] Seed-preferred expression

[0090] In one embodiment expression is seed-preferred.

[0091] Seed-specific expression

[0092] In one embodiment expression is seed-specific.

[0093] Endosperm-preferred expression In one embodiment expression is endosperm-preferred.

[0094] Endosperm-specific expression

[0095] In one embodiment expression is endosperm-specific.

[0096] Expression in the green tissues

[0097] Oil synthesising enzyme expressed in the green tissue

[0098] Numerous oil synthesising enzymes are known to those skilled in the art, and may be conveniently selected for green tissue expression in accordance with the invention.

[0099] In one embodiment the oil synthesising enzyme is a triacylglycerol (TAG) synthesising enzyme.

[0100] In one embodiment the TAG synthesising enzyme is acyl CoA: diacylglycerol acyltransferase 1 (DGAT1).

[0101] In one embodiment a modified oil synthesising enzyme is expressed.

[0102] In one embodiment a modified TAG synthesising enzyme is expressed.

[0103] In one embodiment a modified DGAT1 is expressed

[0104] Oil encapsulating proteins expressed in the green tissues

[0105] Numerous oil-encapsulating proteins are known to those skilled in the art, and may be conveniently selected for use in the invention.

[0106] In one embodiment the oil -encapsulating protein is an oleosin.

[0107] In one embodiment a modified oil -encapsulating protein is expressed. In one embodiment a modified oleosin is expressed.

[0108] Green-tissue preferred expression

[0109] In one embodiment expression of the oil synthesising enzyme is green-tissue preferred expression.

[0110] In one embodiment expression of the oil-encapsulating protein is green-tissue preferred expression.

[0111] In one embodiment expression of both of the oil synthesising enzyme and the oilencapsulating protein is green-tissue preferred expression.

[0112] Light-induced expression

[0113] In one embodiment expression of the oil synthesising enzyme is light-induced expression.

[0114] In one embodiment expression of the oil-encapsulating protein is light-induced expression.

[0115] In one embodiment expression of both the oil synthesising enzyme and the oil-encapsulating protein is light-induced expression.

[0116] Methods with polynucleotides, constructs and transformation

[0117] Expression in endosperm

[0118] Expression of oil-synthesising enzyme in endosperm

[0119] In one embodiment expression of the oil synthesising enzyme is from a polynucleotide encoding the oil synthesising enzyme.

[0120] In one embodiment the polynucleotide is heterologous with respect to the plant. In a further embodiment polynucleotide is part of a construct comprising a promoter operably linked to the polynucleotide.

[0121] In one embodiment the promoter is heterologous with respect to the polynucleotide.

[0122] In a further embodiment the plant is transformed with the polynucleotide or construct.

[0123] In a further embodiment the method includes the step of transforming the plant with the polynucleotide or construct.

[0124] Those skilled in the art will understand that polynucleotides and constructs for expressing polypeptide s / proteins in cells, plants and other organisms can include various other modifications including restriction sites, recombination / excision sites, codon optimisation, tags to facilitate protein purification, etc. Those skilled in the art will understand how to utilise such modifications, some of which may influence transgene expression, stability and translation. However, an art skilled worker would also understand that these modifications are not essential, and do not limit the scope of the invention.

[0125] Expression of oil-encapsulating protein in endosperm

[0126] In one embodiment expression of the oil-encapsulating protein is from a polynuclotide encoding the oil -encapsulating protein.

[0127] In one embodiment the polynucleotide is heterologous with respect to the plant.

[0128] In one embodiment polynucleotide is part of a construct comprising a promoter operably linked to the polynucleotide.

[0129] In one embodiment the promoter is heterologous with respect to the polynucleotide.

[0130] In a further embodiment the plant is transformed with the polynucleotide or construct.

[0131] In a further embodiment the method includes the step of transforming the plant with the polynucleotide or construct. Those skilled in the art will understand that polynucleotides and constructs for expressing polypeptide s / proteins in cells, plants and other organisms can include various other modifications including restriction sites, recombination / excision sites, codon optimisation, tags to facilitate protein purification, etc. Those skilled in the art will understand how to utilise such modifications, some of which may influence transgene expression, stability and translation. However, an art skilled worker would also understand that these modifications are not essential, and do not limit the scope of the invention.

[0132] Method including the step of measuring the content of oil in the seed

[0133] In a further embodiment the method includes the step of measuring the content of oil in the seed of the plant.

[0134] Method including the step of selecting the plant based on measuring the content of oil in the seed

[0135] In a further embodiment the method includes the step of selecting the plant based on measuring an increase of the content of oil in the seed.

[0136] Method including the step of measuring seed yield

[0137] In a further embodiment the method includes the step of measuring seed yield of the plant.

[0138] Method including the step of selecting the plant based on measuring the seed yield in the seed

[0139] In a further embodiment the method includes the step of selecting the plant based on measuring no significant decrease in seed yield of the plant.

[0140] Promoters

[0141] Those skilled in the art will appreciate that a promoter is used to control expression of an operably linked polynucleotide / s, such as those referred to above. In various embodiments of the invention, the promoter operably linked to the polynucleotide encoding the protein to be expressed may be the same as, or different from, the promoter operably linked to the polynucleotide encoding the oil-encapsulating protein.

[0142] Seed-preferred promoters

[0143] In one embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oil-encapsulating protein is a seed-preferred promoter.

[0144] In a further embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oilencapsulating protein is a seed-specific promoter.

[0145] The term seed-preferred promoter also encompasses seed-specific promoters.

[0146] In one embodiment the seed preferrred promoter is a seed-specific promoter.

[0147] Numerous seed preferrred promoters are known to those skilled in the art.

[0148] Promoters for endosperm expression

[0149] In one embodiment the seed preferrred promoter is an endosperm gene promoter.

[0150] Zein storage promoters

[0151] In one embodiment the seed preferrred promoter is a zein storage gene promoter.

[0152] Promoters for green-tissue expression Green tissue-preferred promoters

[0153] In one embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil-synthesising enzyme and the oil-encapsulating protein is a green tissue preferred promoter.

[0154] In a further embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oilencapsulating protein is a green tissue promoter.

[0155] The term green tissue -preferred promoter also encompasses green tissue-specific promoters.

[0156] In one embodiment the green tissue perferrred promoter is a green tissue-specific promoter.

[0157] Numerous green tissue-preferred promoters are known to those skilled in the art.

[0158] Cab promoters

[0159] In one embodiment the green tissue preferred promoter is a chlorophyll a / b (Cab) binding protein promoter, also known as a cab promoter.

[0160] RbcS promoters

[0161] In a further embodiment the green tissue preferred promoter is a promoter from a small subunit of ribulose-bisphosphate carboxylase (Rubisco) promoter, also known as an rbcS promoter.

[0162] GSE promoters

[0163] In a further embodiment the green tissue-preferred promoter is a promoter from a green special express (GSE) gene, also known as a GSE promoter (Xue M et al., 2018, Int. J. Mol. Sci. 2018). C4 PEPC promoters

[0164] In a further embodiment the green tissue-preferred promoter is a promoter from a phosphoenol pyruvate carboxylase (C4 PEPC) gene, also known as an C4 PEPC promoter.

[0165] C4 PPDK promoters

[0166] In a further embodiment the green tissue-preferred promoter is a promoter from a pyruvate phosphate dikinase (C4 PPDK) gene, also known as an C4 PPDK promoter.

[0167] The term green tissue-preferred also encompassed green tissue-specific promoters.

[0168] In one embodiment the green tissue-preferred promoter is a green tissue-specific promoter.

[0169] Light-induced promoters

[0170] In one embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oil-encapsulating protein is a light-induced promoter.

[0171] Light-induced promoters are known to those skilled in the art and include but are not limited to the green-tissue-preferred promoters described above.

[0172] PLANTS, PLANT PARTS AND SEEDS

[0173] Plants and plants produced by methods of the invention

[0174] In a further aspect the invention provides a plant produced by a method of the invention.

[0175] In a further aspect the invention provides a plant with increased oil content in its seeds, relative to that in a control plant, without decreased seed yield, wherein the plant ectopically expresses at least one oil-synthesising enzyme and at least one oil -encapsulating protein in the endosperm of the plant. In one embodiment the plant ectopically expressing at least two oil-synthesising enzymes and at least one oil -encapsulating protein in the endosperm of the plant.

[0176] In a further embodiment the plant further ectopically expresses at least one oil-synthesising enzyme and at least one oil-encapsulating protein in the green tissues of the plant.

[0177] In one embodiment the plant does not ectopically express a transcription factor that regulates fatty acid biosynthesis.

[0178] In one embodiment the plant does not ectopically express the transcription factor WRI1.

[0179] In one embodiment the plant does not ectopically express a transcription factor that regulates lipid biosytnesis, in the endosperm of the plant.

[0180] In one embodiment the plant does not ectopically express the transcription factor WRI1, in the endosperm of the plant.

[0181] In one embodiment the increased content of oil in the seeds of a plant is a result of increased content of oil in the endosperm.

[0182] Methods for producing plants and seeds produced by crossing

[0183] In a further aspect the invention provides a method for producing a plant with increased oil content in its seed relative to that in a control plant, without significantly decreased seed yield relative to that in the control plant, the method comprising crossing a plant of any preceding claim with another plant, to produce the plant with increased oil content in its seed relative to that in a control plant.

[0184] In a further aspect the invention provides a method for producing a seed with increased oil content relative to that in a control plant, without significantly decreased seed yield relative to that in the control plant, the method comprising: a) crossing a plant of any preceding claim with another plant. b) harvesting the seed produced. Plant selected based on measuring the content of oil in the seed

[0185] In a further embodiment the plant has been selected based on measuring the content of oil in the seed of the plant.

[0186] Plant selected based on measuring the composition of oil in the seed

[0187] In a further embodiment the plant has been selected based on measuring the composition of oil in the seed of the plant.

[0188] Plant selected based on seed yield

[0189] In a further embodiment the plant has been selected based on measuring seed yield.

[0190] In a preferred embodiment the plant has been selected based on measuring an increased oil content and no significant decrease in seed yield.

[0191] Inceased seed oil content

[0192] In one embodiment content of oil in the seeds of the plant is increased by at least 0.1%, preferably at least 0.2%, more preferably at least 0.3%, more preferably at least 0.4%, more preferably at least 0.5%, more preferably at least 0.6%, more preferably at least 0.7%, more preferably at least 0.8%, more preferably at least 0.9%, more preferably at least 1%, preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more preferably at least 15%, more preferably at least 16%, more preferably at least 17%, more preferably at least 18%, more preferably at least 19%, more preferably at least 20%, more preferably at least 21%, more preferably at least 22%, more preferably at least 23%, more preferably at least 24%, more preferably at least 25%, more preferably at least 26%, more preferably at least 27%, more preferably at least 28%, more preferably at least 29%, more preferably at least 30%, more preferably at least 31%, more preferably at least 32%, more preferably at least 33%, more preferably at least 34%, more preferably at least 35%, more preferably at least 36%, more preferably at least 37%, more preferably at least 38%, more preferably at least 39%, more preferably at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, relative to that in a control plant.

[0193] In one embodiment there is a significant increase in the oil content of the seeds of the plant, relative to that in a control plant.

[0194] In one embodiment the increase in the oil content of the seeds is assessed using near infra red spectroscopy, NIR, (Zhu, Z, Chen, S, Wu X, Xing C, 2018, Food Sci Nutr. 6(4): 1109-1118. Determination of soybean routine quality parameters using near - infrared spectroscopy.) or by gas chromatography (GC) of fatty acid methyl esters (FAMES) (Shantha NC and Napolitano GE, 1992, Gas chromatography of fatty acids, Journal of Chromatography A. 624, 1-2:37-51). Analysis of total fatty acids (crude) AOAC Official Method 996.06 and OACS Official Method Ca 5b-71. Faty Acid Profile, AOAC Official Methods 996-06 [Analysis of methyl esters by Capillary GLC], AOAC Official Methods Ce 2-66 [Preparation of Methyl Esters of Fatty Acids], AOAC Official Methods 965.49 [Preparation of Methyl Esters of Fatty Acids], AOAC Official Methods 969.33 [Oils and fat, Boron Trifluoride method]. Folch Extraction for Total Lipids from Animal Tissues (Folch et al., 1957, J. Biol. Chem 226:497). It would also be understood by those skilled in the art that a determination of oil content of the seeds could be made by quantifying the oil following industrial processing, including (but not limited to): solvent extraction, crushing, and critical point extraction. In one embodiment the solvent extraction is ether extraction. In one embodiment the ether extraction is AOAC Official Method 920:39 (A).

[0195] In one embodiment the significance of the increase is at the less than 20% probability level, preferably at the less than 15% probability level, more preferably at the less than 10% probability level, more preferably at the less than 5% probability level, more preferably at the less than 1% probability level.

[0196] In one embodiment the significance of the increase in the oil content of the seeds is assessed using AN OVA (SAS Institute, 2016). Preferably the means are separated using Fisher’s Protected LSD at P=0. 1.

[0197] In a further embodiment the significance of the increase in the oil content of the seeds is assessed using Student’s T-test (Microsoft Excel V2108). Preferably the means separated by Fishers Least Significant Difference Test at P=0.05.

[0198] Oil

[0199] In one embodiment the oil is triacylglycerol (TAG).

[0200] Altered oil composition

[0201] In one embodiment the fatty acid profile of the seed of the plant is altered relative to that in the control plant.

[0202] In one embodiment there is an increase in Cl 8:0 fatty acid.

[0203] In a further embodimentt there is an increase in Cl 8: 1 fatty acid.

[0204] In a further embodiment there is an increase in both Cl 8:0 and C18: 1 fatty acids.

[0205] In one embodiment there is a decrease in Cl 8:2 fatty acid.

[0206] In a further embodiment there is a decrease in C18:3 fatty acid.

[0207] In a further embodiment there is a decrease in both C18:2 and C18:3 fatty acid.

[0208] In a further embodiment these is an increase in both Cl 8:0 and Cl 8: 1, and a decrease in both C18:2 and C18:3 fatty acids. Those skilled in the art will know that fatty acid profile with an increase in the proportions of C18:0 and Cl 8: 1 fatty acids and decrease in the proportions of Cl 8:2 and Cl 8:3 fatty acid is characteristic of the activity of an oil synthsising enzyme, such as DGAT1.

[0209] No significant decrease in seed yield

[0210] In one embodiment the there is no significant decrease in seed yield of the plant in which the ectopic expression takes, relative to that in a control plant.

[0211] Methods for assessing seed yield are known in the art.

[0212] In one embodiment seed yield is assessed by weight of seed produced.

[0213] In a further embodiment seed yield is assessed by number of seed produced.

[0214] In a further embodiment seed yield is for a population of plants in a given area that can also be expressed as total seed weight harvested for the area such as bushels per acre (BU / Acre)

[0215] In a further embodiment any decrease in seed yield relative to that in the control plant is less than 5%, preferably less than 4.9%, more preferably 4.8%, more preferably 4.7%, more preferably 4.6%, more preferably 4.5%, more preferably 4.4%, more preferably 4.3%, more preferably 4.2%, more preferably 4.1%, more preferably 4.0%, more preferably 3.9%, more preferably 3.8%, more preferably 3.7%, more preferably 3.6%, more preferably 3.5%, more preferably 3.4%, more preferably 3.3%, more preferably 3.2%, more preferably 3.1%, more preferably 3.0%, more preferably 2.9%, more preferably 2.8%, more preferably 2.7%, more preferably 2.6%, more preferably 2.5%, more preferably 2.4%, more preferably 2.3%, more preferably 2.2%, more preferably 2.1%, more preferably 2.0%, more preferably 1.9%, more preferably 1.8%, more preferably 1.7%, more preferably 1.6%, more preferably 1.5%, more preferably 1.4%, more preferably 1.3%, more preferably 1.2%, more preferably 1.1%, more preferably 1.0%, more preferably 0.9%, more preferably 0.8%, more preferably 0.7%, more preferably 0.6%, more preferably 0.5%, more preferably 0.4%, more preferably 0.3%, more preferably 0.2%, more preferably 0.1%, more preferably 1.0%, relative to that in a control plant. In one embodiment any decrease in seed yield is at the more than 10% probability level, preferably the more than 20% probability level, more preferably the more than 30% probability level.

[0216] In one embodiment the significance of any decrease in seed yield is assessed using ANOVA (SAS Institute, 2016). Preferably the means are separated using Fisher’s Protected LSD at =0.I.

[0217] In a further embodiment the significance of of any desrease in the seed yield is assessed using Student’s T-test (Microsoft Excel V2108). Preferably the means separated by Fishers Least Significant Difference Test at P=0.05.

[0218] Increase in seed yield

[0219] In a further embodiment there is an increase in seed relative to that in the control plant.

[0220] In one embodiment seed yield is increased by at least 0.1%, preferably at least 0.2%, more preferably at least 0.3%, more preferably at least 0.4%, more preferably at least 0.5%, more preferably at least 0.6%, more preferably at least 0.7%, more preferably at least 0.8%, more preferably at least 0.9%, more preferably at least 1%, more preferably at least 1.1%, more preferably at least 1.2%, more preferably at least 1.3%, more preferably at least 1.4%, more preferably at least 1.5%, more preferably at least 1.6%, more preferably at least 1.7%, more preferably at least 1.8%, more preferably at least 1.9%, more preferably at least 2%, more preferably at least 2.2%, preferably at least 2.4%, preferably at least 2.6%, preferably at least 2.8%, more preferably at least 3%, more preferably at least 3.5%, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more preferably at least 15%, more preferably at least 16%, more preferably at least 17%, more preferably at least 18%, more preferably at least 19%, more preferably at least 20%, relative to that in a control plant.

[0221] In one embodiment there is a significant increase in seed yield, relative to that in a control plant. In one embodiment the significance of the increase is at the less than 20% probability level, preferably at the less than 15% probability level, more preferably at the less than 10% probability level, more preferably at the less than 5% probability level, more preferably at the less than 1% probability level.

[0222] In one embodiment the significance of the increase in seed yield is assessed using ANOVA (SAS Institute, 2016). Preferably the means are separated using Fisher’s Protected LSD at =0.I.

[0223] In a further embodiment the significance of the increase in seed yield is assessed using Student’s T-test (Microsoft Excel V2108). Preferably the means separated by Fishers Least Significant Difference Test at P=0.05.

[0224] Endosperm expression

[0225] Oil synthesising enzyme expressed in the endosperm

[0226] Numerous oil synthesising enzymes are known to those skilled in the art, and may be conveniently selected for use endosperm expression in accordance with the invention.

[0227] In one embodiment the oil synthesising enzyme is a triacylglycerol (TAG) synthesising enzyme.

[0228] In one embodiment the TAG synthesising enzyme is acyl CoA: diacylglycerol acyltransferase 1 (DGAT1).

[0229] In a further embodiment the TAG synthesising enzyme is phosphatidylcholine-sterol O- acyltransferase (PDAT).

[0230] In one embodiment both a DGAT1 and a PDAT are expressed

[0231] In one embodiment a modified oil synthesising enzyme is expressed.

[0232] In one embodiment a modified TAG synthesising enzyme is expressed. In one embodiment a modified DGAT1 is expressed

[0233] In one embodiment a modified PDAT is expressed

[0234] Oil encapsulating proteins expressed in the endosperm

[0235] Numerous oil-encapsulating proteins are known to those skilled in the art, and may be conveniently selected for use in the invention.

[0236] In one embodiment the oil -encapsulating protein is an oleosin.

[0237] In one embodiment a modified oil -encapsulating protein is expressed.

[0238] In one embodiment a modified oleosin is expressed.

[0239] Seed-preferred expression

[0240] In one embodiment expression is seed-preferred.

[0241] Seed-specific expression

[0242] In one embodiment expression is seed-specific.

[0243] Endosperm-preferred expression

[0244] In one embodiment expression is endosperm-preferred.

[0245] Endosperm-specific expression

[0246] In one embodiment expression is endosperm-specific.

[0247] Expression in the green tissues

[0248] Oil synthesising enzyme expressed in the green tissue Numerous oil synthesising enzymes are known to those skilled in the art, and may be conveniently selected for use green tissue expression in accordance with the invention.

[0249] In one embodiment the oil synthesising enzyme is a triacylglycerol (TAG) synthesising enzyme.

[0250] In one embodiment the TAG synthesising enzyme is acyl CoA: diacylglycerol acyltransferase 1 (DGAT1).

[0251] In one embodiment a modified oil synthesising enzyme is expressed.

[0252] In one embodiment a modified TAG synthesising enzyme is expressed.

[0253] In one embodiment a modified DGAT1 is expressed

[0254] Oil-encapsulating proteins expressed in the green tissue

[0255] Numerous oil-encapsulating proteins are known to those skilled in the art, and may be conveniently selected for use in the invention.

[0256] In one embodiment the oil -encapsulating protein is an oleosin.

[0257] In one embodiment a modified oil -encapsulating protein is expressed.

[0258] In one embodiment a modified oleosin is expressed.

[0259] Green-tissue preferred expression

[0260] In one embodiment expression of the oil synthesising enzyme is green-tissue preferred expression.

[0261] In one embodiment expression of the oil-encapsulating protein is green-tissue preferred expression. In one embodiment expression of both of the oil synthesising enzyme and the oilencapsulating protein is green-tissue preferred expression.

[0262] Light-induced expression

[0263] In one embodiment expression of the oil synthesising enzyme is light-induced expression.

[0264] In one embodiment expression of the oil-encapsulating protein is light-induced expression.

[0265] In one embodiment expression of both of the oil synthesising enzyme and the oilencapsulating protein is light-induced expression.

[0266] Polynucleotides and constructs

[0267] In one embodiment expression of the oil-synthesising enzyme is from a polynucleotide encoding the oil synthesising enzyme.

[0268] In one embodiment the polynucleotide is heterologous with respect to the plant.

[0269] In a further embodiment polynucleotide is part of a construct comprising a promoter operably linked to the polynucleotide.

[0270] In one embodiment the promoter is heterologous with respect to the polynucleotide.

[0271] In a further embodiment the plant is transformed with the polynucleotide or construct.

[0272] In one embodiment expression of the oil-encapsulating protein is from a polynuclotide encoding the oil -encapsulating protein.

[0273] In one embodiment the polynucleotide is heterologous with respect to the plant.

[0274] In one embodiment polynucleotide is part of a construct comprising a promoter operably linked to the polynucleotide. In one embodiment the promoter is heterologous with respect to the polynucleotide.

[0275] In a further embodiment the plant is transformed with the polynucleotide or construct.

[0276] Promoters

[0277] Those skilled in the art will appreciate that a promoter is used to control expression of an operably linked polynucleotide / s, such as those referred to above.

[0278] In various embodiments of the invention, the promoter operably linked to the polynucleotide encoding the protein to be expressed may be the same as, or different from, the promoter operably linked to the polynucleotide encoding the oil-encapsulating protein.

[0279] Seed-preferred promoters

[0280] In one embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oil-encapsulating protein is a seed-preferred promoter.

[0281] Seed-specific promoter

[0282] In a further embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oilencapsulating protein is a seed-specific promoter.

[0283] The term seed-preferred promoter also encompasses seed-specific promoters.

[0284] In one embodiment the seed preferrred promoter is a seed-specific promoter.

[0285] Numerous seed preferrred promoters are known to those skilled in the art.

[0286] Promoters for endosperm expression In one embodiment the seed preferrred promoter is an endosperm-specific promoter.

[0287] In one embodiment the seed preferrred promoter is an endosperm-preferred promoter.

[0288] Zein storage promoters

[0289] In one embodiment the seed preferrred promoter is a zein storage gene promoter.

[0290] Promoters for green-tissue expression

[0291] Green tissue preferred promoters

[0292] In one embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oil-encapsulating protein is a green tissue preferred promoter.

[0293] In a further embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oilencapsulating protein is a green tissue promoter.

[0294] The term green tissue -preferred promoter also encompasses green tissue -specific promoters.

[0295] In one embodiment the green tissue perferrred promoter is a green tissue -specific promoter.

[0296] Numerous green tissue preferred promoters are known to those skilled in the art.

[0297] Cab promoters

[0298] In one embodiment the green tissue preferred promoter is a chlorophyll a / b (Cab) binding protein promoter, also known as a cab promoter.

[0299] RbcS promoters In a further embodiment the green tissue preferred promoter is a promoter from a small subunit of ribulose-bisphosphate carboxylase (Rubisco) promoter, also known as an rbcS promoter.

[0300] GSE promoters

[0301] In a further embodiment the green tissue-preferred promoter is a promoter from a green special express (GSE) gene, also known as a GSE promoter (Xue M et al., 2018, Int. J. Mol. Sci. 2018).

[0302] C4 PEPC promoters

[0303] In a further embodiment the green tissue-preferred promoter is a promoter from a phosphoenol pyruvate carboxylase (C4 PEPC) gene, also known as an C4 PEPC promoter.

[0304] C4 PPDK promoters

[0305] In a further embodiment the green tissue-preferred promoter is a promoter from a pyruvate phosphate dikinase (C4 PPDK) gene, also known as an C4 PPDK promoter.

[0306] The term green tissue-preferred also encompassed green tissue-specific promoters.

[0307] In one embodiment the green tissue-preferred promoter is a green tissue-specific promoter.

[0308] Light-induced promoters

[0309] In one embodiment the promoter operably linked to the polynucleotide, or in the construct, to drive expression of either, or both, of the oil synthesising enzyme and the oil-encapsulating protein is a light-induced promoter.

[0310] Light-induced promoters are known to those skilled in the art and include but are not limited to the green-tissue-preferred promoters described above. Plant parts, propagule or progeny

[0311] In a further aspect the invention provides a part, propagule or progeny of a plant of the invention.

[0312] In one embodiment the part, propagule or progeny comprises at least one of the polynucleotides, promoters and constructs as herein described.

[0313] In a further embodiment the part, propagule or progeny is transgenic for at least one polynucleotide or construct encoding at least one an oil synthesising enzyme as herein described.

[0314] In a further embodiment the part, propagule or progeny is transgenic for at least one polynucleotide or construct encoding at least one oil encapsulating protein as herein described.

[0315] In a further embodiment the part, propagule or progeny is transgenic for at least one polynucleotide or construct encoding at least one oil synthesising enzyme as herein described, and at least one polynucleotide or construct encoding at least one oil encapsulating protein as herein described.

[0316] In one embodiment the progeny has increased oil content in its seeds, relative to that in a control plant, without a significant reduction in seed yield, and ectopically expresses at least one oil-synthesising enzyme, and at least one oil -encapsulating protein, as herein described.

[0317] In one embodiment the part, propagule or progeny does not ectopically express a transcription factor that regulates fatty acid biosynthesis.

[0318] In one embodiment the part, propagule or progeny does not ectopically express the transcription factor WRI1.

[0319] In one embodiment the part, propagule or progeny does not ectopically express a transcription factor that regulates lipid biosytnesis, in the endosperm. In one embodiment the part, propagule or progeny does not ectopically express the transcription factor WRI1, in the endosperm.

[0320] Seed

[0321] In one embodiment the plant part is a seed with increased oil content.

[0322] In one embodiment the seed does not ectopically express a transcription factor that regulates fatty acid biosynthesis.

[0323] In one embodiment the seed does not ectopically express the transcription factor WRI1.

[0324] In one embodiment the seed does not ectopically express a transcription factor that regulates lipid biosytnesis, in the endosperm.

[0325] In one embodiment the seed does not ectopically express the transcription factor WRI1, in the endosperm.

[0326] In one embodiment the increased content of oil in the seed is a result of increased content of oil in the endosperm.

[0327] In one embodiment the increased oil content is as herein described.

[0328] Method of testing the oil content of the seed, and selecting seed

[0329] In a further aspect the invention provides a method for testing the oil content of the seed of the invention, or the seed of a plant of the invention.

[0330] In a further aspect the invention provides a method selecting seed based in measuring the oil content of the seed of the invention, or the seed of a plant of the invention, or the seed of a plant produced by a method of the invention.

[0331] In one embodiment the oil content is as herein described. Method of testing the seed yield, and selecting plant

[0332] In a further aspect the invention provides a method for testing the seed yield a plant of the invention.

[0333] In a further aspect the invention provides a method selecting the plant based in measuring the seed yield of a plant of the invention.

[0334] In one embodiment the seed yield is as herein described.

[0335] Method of testing the oil content and seed yield and selecting plants and seed

[0336] In a further aspect the invention provides a method for testing oil content of the seed of a plant of the invention, or the seed of a plant of the invention, or the seed of a plant produced by a method of the invention.

[0337] In a further aspect the invention provides a method for testing the seed of a plant of the invention, or the seed of a plant produced by a method of the invention.

[0338] In a further aspect the invention provides a method selecting seed based in measuring the oil content of the seed of a plant of the invention, or the seed of a plant of the invention, or the seed of a plant produced by a method of the invention.

[0339] In a further aspect the invention provides a method selecting seed, the method comprising the steps: a) measuring the oil content of the seed of a plant of the invention, or the seed of a plant of the invention, or the seed of a plant produced by a method of the invention, b) measuring the seed yield of the plant in a) c) selecting a plant based on the measurement in both a) and b) and d) collecting seed from the plant in c).

[0340] In one embodiment the oil content is as herein described.

[0341] In one embodiment the seed yield is as herein described. In a further aspect the invention provides a method for producing oil, the method comprising extracting oil from the seeds of a plant of the invention, or a seed of the invention.

[0342] Method for producing oil

[0343] In a further aspect the invention provides a method for producing oil the method comprising producing or selecting a plant or seed according to the invention, extracting oil from the seeds of the plant, or the seed.

[0344] In one embodiment the oil extraction is by at least one of: a) solvent extraction, b) crushing c) milling d) critical point extraction e) separation during a fermentation process f) wet milling or dry milling

[0345] In a further embodiment the oil is processed into at least one of: a) a fuel, b) an oleochemical, c) a nutritional oil, d) a cosmetic oil, e) a polyunsaturated fatty acid (PUFA), and f) a combination of any of a) to e).

[0346] Methods with reduced carbon dioxide production

[0347] In a further aspect the invention provides a method for producing oil from seed of the invention, or seed produced by a method of the invention, in a fermentation process, that reduces the production of carbon dioxide as a byproduct per seed input unit relative to that when control seed is used.

[0348] In one embodiment the method comprises: a) providing seed of the invention, or seed produced by a method of the invention, or a mash produced from the seed, b) subjecting the seed or mash to fermentation c) extracting oil from the fermenting or fermented seed or mash.

[0349] In a further aspect the invention provides a method for producing oil from seed of the invention, or seed produced by a method of the invention, in a fermentation process, that reduces the production of carbon dioxide as a byproduct per unit oil output relative to that when control seed is used.

[0350] In one embodiment the method comprises: a) providing seed of the invention, or seed produced by a method of the invention, or a mash produced from the seed, b) subjecting the seed or mash to fermentation c) extracting oil from the fermenting or fermented seed or mash.

[0351] In one embodiment the reduction in carbon dioxide is a result of reduced carbohydrate content in the seed of the invention, or seed produced by a method of the invention.

[0352] In a further embodiment the method produces more oil than when control seed is used.

[0353] In one embodiment the method produces at least 0.1%, preferably at least 0.2%, more preferably at least 0.3%, more preferably at least 0.4%, more preferably at least 0.5%, more preferably at least 0.6%, more preferably at least 0.7%, more preferably at least 0.8%, more preferably at least 0.9%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, more preferably at least 4%, more preferably at least 5%, more preferably at least 6%, more preferably at least 7%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, more preferably at least 15%, more preferably at least 16%, more preferably at least 17%, more preferably at least 18%, more preferably at least 19%, more preferably at least 20%, more preferably at least 21%, more preferably at least 22%, more preferably at least 23%, more preferably at least 24%, more preferably at least 25%, more preferably at least 26%, more preferably at least 27%, more preferably at least 28%, more preferably at least 29%, more preferably at least 30%, more preferably at least 31%, more preferably at least 32%, more preferably at least 33%, more preferably at least 34%, more preferably at least 35%, more preferably at least 36%, more preferably at least 37%, more preferably at least 38%, more preferably at least 39%, more preferably at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60% more oil than when control seed is used.

[0354] In one embodiment the oil is TAG.

[0355] DETAILED DESCRIPTION OF THE INVENTION

[0356] The applicant’s invention involves increasing the production of oil in the seeds of a plant without the usual decrease in seed yield seen when oil content is increased. This is surprisingly achieved by ectopically expressing at least one oil-synthesising enzyme and at least one oil-encapsulating protein in the endosperm of the plant.

[0357] In some embodiments the method comprises the step of ectopically expressing at least two oil-synthesising enzymes and at least one oil-encapsulating protein in the endosperm of the plant.

[0358] In some embodiments the method further comprises the step of ectopically expressing at least one oil-synthesising enzyme and at least one oil-encapsulating protein in the green tissues of the plant.

[0359] Oil

[0360] In one embodiment the oil is triacylglycerol (TAG)

[0361] Oil synthesising enzyme Oil synthesising enzymes for use in the invention are well-known to those skilled in the art and include for example DGAT1 (Liu Q, Siloto RM, Lehner R, Stone SJ, Weselake RJ 2012. Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog. Lipid Res. 51:350-377), DGAT2 (Liu Q, Siloto RM, Lehner R, Stone SJ, Weselake RJ 2012. Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog. Lipid Res. 51:350-377), DGAT3 (Chi X, Hu R, Zhang X, Chen M, Chen N, Pan L, Want T, Wang M, Yang Z, Want Q, Yu S 2014. Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L.) Pios One https: / / doi.org / 10.1371 / joumal.pone.0105834), PDAT (Lager I, Jeppson S, Gippert A-L, Feussner l, Stymne S, Mannon S 2020. Acyltransferases regulate oil quality in Camelina sativa through both acyl donor and acyl acceptor specificities. Front.

[0362] Plant Sci. https: / / d0i.0rg / 10.3389 / fpls.2020.01144), MGAT (Liu Q, Siloto RM, Lehner R, Stone SJ, Weselake RJ 2012. Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog. Lipid Res. 51:350-377), and triacylglycerol (TAG) synthesising enzymes.

[0363] In one preferred embodiment the oil synthesising enzymes is a TAG synthesising enzyme.

[0364] Tri acylglycerol (TAG) biosynthesis

[0365] The only committed step in TAG biosynthesis is the last one, i.e. the addition of a third fatty acid to an existing diacylglycerol, thus generating TAG. In plants this step is predominantly (but not exclusively) performed by one of five (predominantly ER localised) TAG synthesising enzymes including: acyl CoA: diacylglycerol acyltransferase (DGAT1); an unrelated acyl CoA: diacylglycerol acyl transferase (DGAT2); a soluble DGAT (DGAT3) which has less than 10% identity with DGAT1 or DGAT2 (Saha et al., 2006); phosphatidylcholine-sterol O-acyltransferase (PDAT); and a wax synthase (WSDl, Li et al., 2008). The DGAT1 and DGAT2 proteins are eoncoded by two distinct gene families, with DGAT1 containing approximately 500 amino acids and 10 predicted transmembrane domains and DGAT2 has only 320 amino acids and two transmembrane domains (Shockey et al., 2006). The term “triacylglycerol synthesising enzyme” or “TAG synthesising enzyme” as used herein means an enzyme capable of catalysing the addition of a third fatty acid to an existing diacylglycerol, thus generating TAG.

[0366] Preferred TAG synthesising enzymes include but are not limited to: acyl CoA: diacylglycerol acyltransferase 1 (DGAT1); diacylglycerol acyl transferase2 (DGAT2); phosphatidylcholinesterol O-acyltransferase (PDAT) and cytosolic soluble form of DGAT (soluble DGAT or DGAT3).

[0367] The invention also contemplates use of modified TAG synthesizing enzymes, that are modified (for example in their sequence by substitutions, insertions or additions and the like) to alter their specificity and or activity.

[0368] DGAT1

[0369] In one embodiment the TAG synthesizing enzymes is DGAT1.

[0370] The term "DGAT1" as used herein means acyl CoA: diacylglycerol acyltransferase (EC 2.3.1.20)

[0371] DGAT1 is typically the major TAG synthesising enzyme in both the seed and senescing leaf (Kaup etal., 2002, Plant Physiol. 129(4): 1616-26; for reviews see Lung and Weselake 2006, Lipids. Dec 2006;41(12): 1073-88; Cahoon et al., 2007, Current Opinion in Plant Biology. 10:236-244; and Li et al., 2010, Lipids. 45: 145-157).

[0372] DGAT1 contains approximately 500 amino acids and has been reported to have up to 10 predicted transmembrane domains whereas DGAT2 has only 320 amino acids and is predicted to contain only two transmembrane domains; both proteins were also predicted to have their N- and C-termini located in the cytoplasm (Shockey et al., 2006, Plant Cell 18:2294-2313). Both DGAT1 and DGAT2 have orthologues in animals and fungi and are transmembrane proteins located in the ER. In most dicotyledonous plants DGAT1 & DGAT2 appear to be single copy genes whereas there are typically two versions of each in the grasses which presumably arose during the duplication of the grass genome (Salse et al., 2008, Plant Cell, 20: 11-24). Examples of these DGAT1 sequences, suitable for use in the methods and compositions of the invention, from members of several plant species are provided in Table 1 below. The sequences (both polynucleotide and polypeptide are provided in the Sequence Listing).

[0373] Table 1 - DGAT1 sequences from multiple species In one embodiment the DGAT1 has the amino acid sequence of any one of SEQ ID NO: 30 to 58 (Table 1), or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 30 to 58. In a further embodiment the DGAT1 has the amino acid sequence of any one of SEQ ID NO: 30 to 58.

[0374] In one embodiment the DGAT1 is encoded by the polynucleotide sequence of any one of SEQ ID NO: 1 to 29, or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 1 to 29. In a further embodiment the DGAT1 is encoded by the polynucleotide sequence of any one of SEQ ID NO: 1 to 29.

[0375] Chimeric DGAT1

[0376] Chimeric DGAT1 sequences are know to those skilled in the art and are described, for example, in WO2014068439.

[0377] Non-limiting examples of chimeric DGAT1 sequences, suitable for use in the methods and compositions of the invention, from members of several plant species are provided in Table 2 below. The sequences are provided in the Sequence Listing.

[0378] Table 2. Examples of chimeric DGAT1 polypeptide sequences from a variety of plants.

[0379] In one embodiment the chimeric DGAT1 has the amino acid sequence of any one of SEQ ID NO: 59 to 94 (Table 2), or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 59 to 94. In a further embodiment the DGAT1 has the amino acid sequence of any one of SEQ ID NO: 59 to 94.

[0380] PDAT

[0381] In one embodiment the TAG synthesizing enzymes is PDAT.

[0382] The term "PDAT" as used herein means phosphatidylcholine-sterol O-acyltransferase.

[0383] Examples of these PDAT sequences, suitable for use in the methods and compositions of the invention, from members of several plant species are provided in Table 3 below. The sequences (both polynucleotide and polypeptide are provided in the Sequence Listing). Table 3. Examples of PDAT sequences from from multiple species.

[0384] In one embodiment the PDAT has the amino acid sequence of any one of SEQ ID NO: 97 to 103 (Table 3), or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 97 to 103. In a further embodiment the DGAT1 has the amino acid sequence of any one of SEQ ID NO: 97 to 103.

[0385] In one embodiment the PDAT is encoded by the polynucleotide sequence of any one of SEQ ID NO: 95 to 96; 187 to 191 or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 95 to 96 and 187 to 191. In a further embodiment the PDAT is encoded by the polynucleotide sequence of any one of SEQ ID NO: 95 to 96 and 187 to 191.

[0386] Oil encapsulating proteins

[0387] Oil encapsulating proteins for use in the invention are well-known to those skilled in the art and include for example ol eosins (Shao et al., 2019, New insights into the role of seed oil body proteins in metabolism and plant development, Front. Plant Sci., https: / / doi.org / 10.3389 / fpls.2019.01568), steroleosins (Lin et al., 2002, Steroleosin, a sterol- binding dehydrogenase in seed oil bodies. Plant Physiol. 128: 1200-1211), caoleosins (Hsieh and Huan, 2004, Endoplasmic reticulum, oleosins, and oil seeds in tapetum cells. Plant Physiology, 136:3427-3434), polyoleosins, (W02007045019 ), oleosin including at least one artificially introduced cysteine (WO2011 / 053169), oleosin where the lysine residues in the amphipathic arms have been replaced with arginine (US02021 / 0261632 Al). In one embodiment the oil encapsulating protein is an oleosin.

[0388] Oleosins

[0389] Oleosins are comparatively small (15 to 24 kDa) proteins which allow the OBs to become tightly packed discrete organelles without coalescing as the cells desiccate or undergo freezing conditions (Leprince et al., 1998; Siloto et al., 2006; Slack et al., 1980; Shimada et a / .2008).

[0390] Oleosins have three functional domains consisting of an amphipathic N-terminal arm, a highly conserved central hydrophobic core (~72 residues) and a C-terminal amphipathic arm. The accepted topological model is one in which the N- and C-terminal amphipathic arms are located on the outside of the OBs and the central hydrophobic core is located inside the OB (Huang, 1992; Loer and Herman, 1993; Murphy 1993). The negatively charged residues of the N- and C-terminal amphipathic arms are exposed to the aqueous exterior whereas the positively charged residues are exposed to the OB interior and face the negatively charged lipids. Thus, the amphipathic arms with their outward facing negative charge are responsible for maintaining the OBs as individual entities via steric hinderance and electrostatic repulsion both in vivo and in isolated preparation (Tzen et al., 1992). The N-terminal amphipathic arm is highly variable and as such no specific secondary structure can describe all examples. In comparison the C-terminal arm contains a a-helical domain of 30-40 residues (Tzen et al. , 2003). The central core is highly conserved and thought to be the longest hydrophobic region known to occur in nature; at the centre is a conserved 12 residue proline knot motif which includes three spaced proline residues (for reviews see Frandsen et al., 2001; Tzen et al., 2003). The secondary, tertiary and quaternary structure of the central domain is still unclear. Modelling, Fourier Transformation-Infra Red (FT-IR) and Circular Dichromism (CD) evidence exists for a number of different arrangements (for review see Roberts et al., 2008).

[0391] The properties of the major oleosins is relatively conserved between plants and is characterised by the following:

[0392] • 15-25kDa protein corresponding to approximately 140-230 amino acid residues.

[0393] • The protein sequence can be divided almost equally along its length into 4 parts which correspond to a N-terminal hydrophilic region, two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region. • The topology of oleosin is attributed to its physical properties which includes a folded hydrophobic core flanked by hydrophilic domains. This arrangement confers an amphipathic nature to oleosin resulting in the hydrophobic domain being embedded in the phospholipid monolayer (Tzen etal., 1992) while the flanking hydrophilic domains are exposed to the aqueous environment of the cytoplasm.

[0394] • Typically oleosins do not contain cysteines.

[0395] Preferred oleosins for use in the invention are those which contain a central domain of approximately 70 non-polar amino acid residues (including a proline knot) uninterrupted by any charged residues, flanked by two hydrophilic arms. Examples of oleosin sequences suitable for use in the invention in their native form, or suitable to be modified for use in the invention, and modified oleosins, are shown in Table 4 below. The sequences (both polynucleotide and polypeptide are provided in the Sequence Listing).

[0396] In a further embodiment the oil encapsulating protein is a steroleosin. In a further embodiment the oil encapsulating protein is a caloleosin.

[0397] Table 4. Examples of oleosin, steroleosin, caloleosin and cysteine oleosin ORFs and peptide sequences from from multiple species.

[0398] In one embodiment the oleosin has the amino acid sequence of any one of SEQ ID NO: 125 to 134 and 144 to 145 (Table 4), or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 125 to 134 and 144 to 145. In a further embodiment the oleosin has the amino acid sequence of any one of SEQ ID NO: 125 to 134 and 144 to 145.

[0399] In one embodiment the oleosin is encoded by the polynucleotide sequence of any one of SEQ ID NO: 104 to 113 and 123 to 124 (Table 4) or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 104 to 113 and 123 to 124. In a further embodiment the oleosin is encoded by the polynucleotide sequence of any one of SEQ ID NO: 104 to 113 and 123 to 124.

[0400] In one embodiment the steroleosin has the amino acid sequence of any one of SEQ ID NO: 135 to 138 (Table 4), or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 135 to 138. In a further embodiment the oleosin has the amino acid sequence of any one of SEQ ID NO: 135 to 138.

[0401] In one embodiment the steroleosin is encoded by the polynucleotide sequence of any one of SEQ ID NO: 114 to 117 (Table 4) or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 114 to 117. In a further embodiment the oleosin is encoded by the polynucleotide sequence of any one of SEQ ID NO: 114 to 117.

[0402] In one embodiment the caloleosin has the amino acid sequence of any one of SEQ ID NO: 139 to 143 (Table 4), or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 139 to 143. In a further embodiment the oleosin has the amino acid sequence of any one of SEQ ID NO: 139 to 143.

[0403] In one embodiment the caloleosin is encoded by the polynucleotide sequence of any one of SEQ ID NO: 118 to 122 (Table 4) or a variant thereof. Preferably the variant has at least 70% identity to any one of SEQ ID NO: 118 to 122. In a further embodiment the oleosin is encoded by the polynucleotide sequence of any one of SEQ ID NO: 118 to 122.

[0404] Oleosin, steroleosins, caloleosins are well known to those skilled in the art. Further sequences from many different species can be readily identified by methods well-known to those skilled in the art. For example, further sequences can be easily identified by an NCBI Entrez Cross-Database Search (available at ncbi.nlm.nih.gov / sites / gquery) using oleosin as a search term.

[0405] Modified oleosins

[0406] The invention also contemplates the use of modified oleosins including polyoleosins (WO / 2007 / 045019), cysteine oleosins (WO / 2011 / 053169) and lysine modified oleosins (US02021 / 0261632 Al).

[0407] Cysteine oleosins

[0408] Cysteine oleosins for use in the methods of the invention, are modified to contain at least one artificially introduced cysteine residue. Preferably the engineered oleosins contain at least two cysteines.

[0409] Various methods well-known to those skilled in the art may be used in production of the modified oleosins with artificially introduced cysteines. Such methods include site directed mutagenesis (US 6,448,048) in which the polynucleotide encoding an oleosin is modified to introduce a cysteine into the encoded oleosin protein.

[0410] Alternatively, the polynucleotide encoding the modified oleosins, may be synthesised in its entirety.

[0411] Further methodology for producing modified oleosins and for use in the methods of the invention are described in WO / 2011 / 053169, US 8,987,551, and WO / 2013 / 022353.

[0412] The introduced cysteine may be an additional amino acid (i.e. an insertion) or may replace an existing amino acid (i.e. a replacement). Preferably the introduced cysteine replaces an existing amino acid. In a preferred embodiment the replaced amino acid is a charged residue. Preferably the charged residue is predicted to be in the hydrophilic domains and therefore likely to be located on the surface of the oil body.

[0413] The hydrophilic, and hydrophobic regions / arms of the oleosin can be easily identified by those skilled in the art using standard methodology (for example: Kyte and Doolitle (1982).

[0414] The modified oleosins for use in the methods of the invention are preferably range in molecular weight from 5 to 50 kDa, more preferably, 10 to 40kDa, more preferably 15 to 25 kDa.

[0415] The modified oleosins for use in the methods of the invention are preferably in the size range 100 to 300 amino acids, more preferably 110 to 260 amino acids, more preferably 120 to 250 amino acids, more preferably 130 to 240 amino acids, more preferably 140 to 230 amino acids.

[0416] Preferably the modified oleosins comprise an N-terminal hydrophilic region, two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region.

[0417] Preferably the modified oleosins can be divided almost equally their length into four parts which correspond to the N-terminal hydrophilic region (or arm), the two centre hydrophobic regions (joined by a proline knot or knob) and a C-terminal hydrophilic region (or arm).

[0418] Preferably the topology of modified oleosin is attributed to its physical properties which include a folded hydrophobic core flanked by hydrophilic domains. Preferably the modified oleosins can be formed into oil bodies when combined with triacylglycerol (TAG) and phospholipid.

[0419] Preferably topology confers an amphipathic nature to modified oleosin resulting in the hydrophobic domain being embedded in the phospholipid monolayer of the oil body while the flanking hydrophilic domains are exposed to the aqueous environment outside the oil body, such as in the cytoplasm.

[0420] Preferably the modified oleosin includes at least one artificially introduced cysteine, wherein the cysteine is introduced into at least one of: a) in the N-terminal hydrophilic region of the oleosin, and b) in the C-terminal hydrophilic region of the oleosin.

[0421] In one embodiment the modified oleosin for use in the method of the invention, comprises a sequence with at least 70% identity to the hydrophobic domain of any of the oleosin protein sequences referred to in Table 4 above.

[0422] In one embodiment the modified oleosin has the amino acid sequence with 70% identity to any one of SEQ ID NO: 125 to 134, 144 to 145 and 186 (Table 4).

[0423] In a further embodiment the modified oleosin has the amino acid sequence of SEQ ID NO: 186.

[0424] In one embodiment the modified oleosin is encoded by the polynucleotide sequence with 70% identity to of any one of SEQ ID NO: 104 to 113, 123 to 124 and 185 (Table 4).

[0425] In a further embodiment the modified oleosin is encoded by the polynucleotide sequence of SEQ ID NO: 185.

[0426] In one embodiment the modified oleosin for use in the method of the invention, comprises a sequence with at least 70% identity to the hydrophobic domain of any of the amino acid sequences of SEQ ID NO: 125 to 134, 144 to 145 and 186. In one embodiment the modified oleosin for use in the method of the invention, comprises a sequence with the hydrophobic domain of any of the amino acid sequences of SEQ ID NO: 125 to 134, 144 to 145 and 186.

[0427] In further embodiment the modified oleosin is essentially the same as any of the unmodied oleosins referred above, apart from the additional artificially introduced cysteine or cysteines.

[0428] Ectopic expression

[0429] Ther term "ectopic expression", and grammatical equivalents thereof, as used here in means expression of a polynucleotide, gene or protein in a cell type, tissue type, or developmental stage, or an expression level, in / at which the polynucleotide, gene or protein is not usually endogenously expressed. Ectopic expression as used herein also encompassed transgenic expression, including over-expression.

[0430] Seed-preferred expression

[0431] The term "seed-preferred expression", and grammatical equivalents thereof, as used herein means expression predominantly in the seed of a plant relative to other tissues and parts of the plant. This term does not exclude some, albeit relatively low, expression in the seed of the plant.

[0432] The term "seed-preferred expression", and grammatical equivalents thereof, as used herein means also encompasses "seed-specific expression", and grammatical equivalents thereof.

[0433] In one embodiment "seed-preferred expression" is "seed-specific expression".

[0434] Seed-specific expression

[0435] The term "seed-specific expression", and grammatical equivalents thereof, as used herein means expression exclusively in the seed of a plant, and not in other tissues or parts of the plant. Endosperm-preferred expression

[0436] The term "endosperm-preferred expression", and grammatical equivalents thereof, as used herein means expression predominantly in endosperm of the seed of a plant relative to other tissues and parts of the plant. This term does not exclude some, albeit relatively low, expression in the endosperm of the seed of the plant.

[0437] The term "endosperm-preferred expression", and grammatical equivalents thereof, as used herein means also encompasses "endosperm-specific expression", and grammatical equivalents thereof.

[0438] In one embodiment "endosperm-preferred expression" is " endosperm-specific expression".

[0439] Endosperm-specific expression

[0440] The term "endosperm-specific expression", and grammatical equivalents thereof, as used herein means expression exclusively in the endosperm of a plant, and not in other tissues or parts of the plant.

[0441] Green tissue-preferred

[0442] The term "green tissue-preferred expression", and grammatical equivalents thereof, as used herein means expression predominantly in the green tissues of a plant relative to other tissues of the plant. This term does not exclude some, albeit relatively low, expression in non-green tissues of the plant.

[0443] The term "green tissue-preferred expression", and grammatical equivalents thereof, as used herein also encompassed "green tissue-specific expression", and grammatical equivalents thereof.

[0444] In one embodiment "green tissue-preferred expression" is "green tissue-specific expression".

[0445] Green tissue-specific The term "green tissue-specific expression", and grammatical equivalents thereof, as used herein means expression exclusively in the green tissues of a plant, and not in other nongreen tissues of the plant.

[0446] Light-induced expression

[0447] The term " Light-induced expression" , and grammatical equivalents thereof, as used herein means expression induced by light, in the plant.

[0448] Promoters

[0449] Seed-preferred promoters

[0450] Seed-preferred promoters drive expression of operably linked polynucleotides predominantly in the seeds of plants. This term does not exclude some, albeit relatively low, expression in other non-seed tissues of the plant.

[0451] Numerous examples of seed-preferred promoters are known by those skilled in the art and include, by way of example, but are not limited to: seed-preferred promoters found in US 6,342,657; and US 7,081,565; and US 7,405,345; and US 7,642,346; and US 7,371,928, and napin promoters, legumin B4, 7S globulin, and 11 S globulin (Zakharov et al., 2004, J. Exp. Bot., 55: 1463-1471), dlec2, Arc5-1, lectin, and usp (Stoger et al., 2005, Current Opinion in Biotechnology, 16: 167-173).

[0452] The term "seed-preferred promoter", and grammatical equivalents thereof, as used herein also encompasses "seed-specific promoter", and grammatical equivalents thereof.

[0453] In one embodiment "seed-preferred promoter" is a "seed-specific promoter".

[0454] Seed specific promoters

[0455] Seed-specific promoters drive expression of operably linked polynucleotides exclusively in the seeds of plants. Endosperm preferred promoters

[0456] Endosperm-preferred promoters drive expression of operably linked polynucleotides predominantly in the endosperm of seeds of plants. This term does not exclude some, albeit relatively low, expression in other non-endosperm tissues of the plant.

[0457] The term "endosperm-preferred promoter", and grammatical equivalents thereof, as used herein also encompasses " endosperm -specific promoter", and grammatical equivalents thereof.

[0458] In one embodiment "endosperm-preferred promoter" is an " endosperm-specific promoter".

[0459] Endosperm specific promoters

[0460] Endosperm -specific promoters drive expression of operably linked polynucleotides exclusively in the endosperm of seeds of plants.

[0461] Zein storage protein promoter

[0462] In a further embodiment the seed-preferred or seed-specific promoter is a Zein storage promoter.

[0463] Zein storage proteins, genes and promoters are known to those skilled in the art.

[0464] In one embodiment the promoter is from a zein storge gene selected from is selected from: beta, 18 delta zein, gamma, PMS2, A30, 19A2, PMS1, 191B, A20, 19C2, PZ22.3, Z1C1-7, PZ22.1, ZA / M1, B49, Z1D-4, and ZD1-2 (Chen et al, 2014).

[0465] In a further embodiment the promoter is from a zein storge gene selected from is selected from: beta, A20, PMS1, DI -2 and gamma.

[0466] In one embodiment the promoter is from Zea mays. In one embodiment the zein storge gene promoter has at least 70% identity to the polynucleotdies sequence of any one of SEQ ID NO: 146, 150, 152, 154, 155, 156, 158, 160, and 162 to 165.

[0467] In a further embodiment the zein storge gene promoter has the polynucleotide sequence of any one of SEQ ID NO: 146, 150, 152, 154, 155, 156, 158, 160, and 162 to 165.

[0468] Green tissue-preferred and green tissue-specific promoters

[0469] Cab promoters

[0470] In a further embodiment the green tissue-preferred or green tissue-specific promoter is a chlorophyll a / b (Cab) binding promoter.

[0471] In one embodiment the cab promoter has at least 70% identity to the polynucleotdies sequence of any one of SEQ ID NO: 158 and 160.

[0472] In a further embodiment the cab promoter has the polynucleotide sequence of any one of SEQ ID NO: 158 and 160.

[0473] Rbcs promoters

[0474] In a further embodiment the green tissue-preferred or green tissue-specific promoter is a promoter from a small subunit of ribulose-bisphosphate carboxylase (Rubisco) gene, also know as an rbcS promoter.

[0475] In one embodiment the rbcS promoter has at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 162 and 163.

[0476] In a further embodiment the rbcS promoter has the polynucleotide sequence of any one of SEQ ID NO: 162 and 163.

[0477] PEPC promoters In a further embodiment the green tissue-preferred or green tissue-specific promoter is a promoter from a phosphoenol-pyruvate carboxylase (PEPC) gene, also know as a PEPC promoter.

[0478] In one embodiment the PEPC promoter has at least 70% identity to the polynucleotide sequence of any one of SEQ ID NO: 164 and 165.

[0479] In a further embodiment the PEPC promoter has the polynucleotide sequence of any one of SEQ ID NO: 164 and 165.

[0480] Source of plant sequences and plants used in the invention.

[0481] The plant-derived oil synthesising enzymes, the oil encapsulating proteins, promoters and plants used in the invention, may be from any plant species.

[0482] In one embodiment the plant is derived from a gymnosperm plant species.

[0483] In a further embodiment the plant is derived from an angiosperm plant species.

[0484] In a further embodiment the plant is derived from a from dicotyledonous plant species.

[0485] In a further embodiment the plant is derived from a monocotyledonous plant species.

[0486] In a further embodiment the plant is derived from the botanical family Poaceae.

[0487] Preferred plants include true cereal grains, as herein defined.

[0488] Preferred true cereal grains are from the botanical family Poaceae.

[0489] Preferred plants are true cereal grains include wheat, oats, rice, com (maize), barley, sorghum, rye, and millet.

[0490] Preferred wheat cereal grains also include farro, freekeh, emmer and spelt.

[0491] A preferred true cereal grain genus is Eragrostis.

[0492] A preferred Eragrostis species is Eragrostis tef. A futher preferred true cereal grain genus is Triticum

[0493] A preferred Triticum species is Triticum aestivum.

[0494] A futher preferred true cereal grain genus is Avena.

[0495] A preferred Avena species is Avena sativa.

[0496] A further preferred true cereal grain genus is Oryza.

[0497] A preferred Oryza species is Oryza sativa.

[0498] A further preferred true cereal grain genus is Hordeum.

[0499] A preferred Hordeum species is Hordeum vulgare.

[0500] A further preferred true cereal grain genus is Sorghum.

[0501] A preferred Sorghum species is Sorghum hicolor.

[0502] A further preferred true cereal grain genus is Secale.

[0503] A preferred Secale species is Secale cereale.

[0504] A further preferred true cereal grain genus is Pennisetum.

[0505] A preferred Pennisetum species is Pennisetum glaucum.

[0506] A further preferred true cereal grain genus is Triticale.

[0507] A paticularly preferred true cereal grain genus is Zea.

[0508] A paticularly preferred Zea species is Zea mays.

[0509] Other preferred plants are forage plant species from a group comprising but not limited to the following genera: Zea, Sorghum, Lolium, Hordium, Miscanthus, Saccharum, Festuca, Dactylis, Bromus, Thinopyrum, Trifolium, Medicago, Pheleum, Phalaris, Holcus, Glycine, Lotus, Plantago and Cichorium.

[0510] Other preferred plants are leguminous plants. The leguminous plant or part thereof may encompass any plant in the plant family Leguminosae or Fabaceae. For example, the plants may be selected from forage legumes including, alfalfa, clover; leucaena; grain legumes including, beans, lentils, lupins, peas, peanuts, soybean; bloom legumes including lupin, pharmaceutical or industrial legumes; and fallow or green manure legume species.

[0511] A further contemplated genus is Trifolium. Preferred Trifolium species include Trifolium repens,' Trifolium arvense,' Trifolium affine,' and Trifolium occidentale. A particularly preferred Trifolium species is Trifolium repens.

[0512] Another preferred genus is Medicago. Preferred Medicago species include Medicago sativa and Medicago truncatula. A particularly preferred Medicago species is Medicago sativa, commonly known as alfalfa.

[0513] Another preferred genus is Glycine. Preferred Glycine species include Glycine max, Glycine wightii (also known as Neonotonia wightii) and Glycine soja. A particularly preferred Glycine species is Glycine max, commonly known as soybean. A particularly preferred Glycine species is Glycine wightii, commonly known as perennial soybean.

[0514] Another preferred genus is Vigna. A particularly preferred Vigna species is Vigna unguiculata commonly known as cowpea.

[0515] Another preferred genus is Mucana. Preferred Mucana species include Mucana pruniens. A particularly preferred Mucana species is Mucana pruniens commonly known as velvet bean.

[0516] Another preferred genus is Arachis. A particularly preferred Arachis species is Arachis glahrata commonly known as perennial peanut.

[0517] Another preferred genus is Pisum. A preferred Pisum species is Pisum sativum commonly known as pea.

[0518] Another preferred genus is Lotus. Preferred Lotus species include Lotus corniculatus , Lotus pedunculatus , Lotus glahar, Lotus tenuis and Lotus uliginosus. A preferred Lotus species is Lotus corniculatus commonly known as Birdsfoot Trefoil. Another preferred Lotus species is Lotus glahar commonly known as Narrow -leaf Birdsfoot Trefoil. Another preferred preferred Lotus species is Lotus pedunculatus commonly known as Big trefoil. Another preferred Lotus species is Lotus tenuis commonly known as Slender trefoil. Another preferred genus is Brassica. A preferred Brassica species is Brassica oleracea, commonly known as forage kale and cabbage.

[0519] Other preferred species are oil seed crops.

[0520] Preferably oil seed crops include but are not limited to the following genera: Brassica, Carthumus, Helianthus, Zea and Sesamum.

[0521] A preferred oil seed genera is Glycine. Preferred Glycine species include Glycine max and Glycine soja. Preferred Glycine species is Glycine max.

[0522] A preferred oil seed genera is Brassica. A preferred oil seed species is Brassica napus.

[0523] A preferred oil seed genera is Brassica. A preferred oil seed species is Brassica oleraceae.

[0524] A preferred oil seed genera is Carthamus. A preferred oil seed species is Carthamus tinctorius.

[0525] A preferred oil seed genera is Helianthus. A preferred oil seed species is Helianthus annuus.

[0526] A preferred oil seed genera is Zea. A preferred oil seed species is Zea mays.

[0527] A preferred oil seed genera is Sorghum. A preferred oil seed species is Sorghum hicolor.

[0528] A preferred oil seed genera is Sesamum. A preferred oil seed species is Sesamum indicum.

[0529] A preferred silage genera is Zea. A preferred silage species is Zea mays.

[0530] A preferred silage genera is Sorghum. A preferred silage species is Sorghum hicolor.

[0531] A preferred silage genera is Brachiaria. A preferred silage species is Zea mays.

[0532] A preferred grain producing genera is Hordeum. A preferred grain producing species is Hordeum vulgare.

[0533] A preferred grain producing genera is Sorghum. A preferred grain producing species is Sorghum hicolor.

[0534] A preferred grain producing genera is Zea. A preferred grain producing species is Zea mays.

[0535] A preferred grazing genera is Lolium. A preferred grazing species is Lolium perenne. A preferred grazing genera is Lolium. A preferred grazing species is Lolium arundinaceum .

[0536] A preferred grazing genera is Trifolium. A preferred grazing species is Trifolium repens.

[0537] A preferred grazing genera is Hordeum. A preferred grazing species is Hordeum vulgare.

[0538] A preferred grazing genera is Brachicirici. A preferred grazing species is Brachicirici sp.

[0539] Preferred plants also include forage, or animal feedstock plants. Such plants include but are not limited to the following genera: Miscanthus, Sciccharum, Pcmicum.

[0540] A preferred biofuel genera is Miscanthus . A preferred biofuel species is Miscanthus giganteus .

[0541] A preferred biofuel genera is Saccharum. A preferred biofuel species is Saccharum officinarum.

[0542] A preferred biofuel genera is Panicum. A preferred biofuel speices is Panicum virgatum.

[0543] A preferred biofuel genera is Zea. A preferred biofuel speices is Zea mays.

[0544] A preferred biofuel genera is Sorghum. A preferred biofuel speices is Sorghum hicolor.

[0545] A particularly preferred genus of plant in which to increase seed oil content, without decreasing seed protein content, in accordance with the invnention, is Glycine. A particularly preferred Glycine species in Glycine max.

[0546] Particularly preferred genera as sources of the oil synthesizing enzyme for use in the invention are Tropaeolum, Glycine, Zea, Sorghum. A particularly preferred Tropaeolum species is Tropaeolum majus. A particularly preferred Glycine species in Glycine max. A particularly preferred Zea species in Zea mays. A particularly preferred Sorghum species in Sorghum hicolor.

[0547] Particularly preferred genera as sources of the oil encapsulating for use in the invention are Sesamum, Glycine, Zea, Sorghum. A particularly preferred Sesamum species is Sesamum indicum. A particularly preferred Glycine species in Glycine max. A particularly preferred Zea species in Zea mays. A particularly preferred Sorghum species in Sorghum hicolor.

[0548] Particularly preferred genera as sources of the constitutive promoters for use in the invention are Arahidopsis , Glycine, Oryza, Zea, Sorghum. A particularly preferred Arahidops is species is Arabidopsis thaliana. A particularly preferred Glycine species in Glycine max. A particularly preferred Oryza species in Oryza sativa. A particularly preferred Zea species in Zea mays. A particularly preferred Sorghum species in Sorghum bicolor.

[0549] Particularly preferred genera as sources of the endosperm-preferred and endosperm-specific promoters for use in the invention are Oryza, Zea, Sorghum. A particularly preferred Oryza species in Oryza sativa. A particularly preferred Zea species in Zea mays. A particularly preferred Sorghum species in Sorghum bicolor.

[0550] Particularly preferred genera as sources of the green tissue-preferred and green tissue specific promoters for use in the invention are Pisum, Glycine, Oryza, Zea, Sorghum. A particularly preferred Pisum species is Pisum sativa. A particularly preferred Glycine species in Glycine max. A particularly preferred Oryza species in Oryza sativa. A particularly preferred Zea species in Zea mays. A particularly preferred Sorghum species in Sorghum bicolor.

[0551] Plant parts, propagues and progeny

[0552] The term “plant” is intended to include a whole plant, any part of a plant, a seed, a fruit, propagules and progeny of a plant.

[0553] The term ‘propagule’ means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.

[0554] The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting progeny, comprising the polynucleotides or constructs of the invention, also form a part of the present invention.

[0555] Preferably the plants, plant parts, propagules and progeny comprise a polynucleotide or construct according to the invention, and / or express a sequence according to the invention.

[0556] Control plant

[0557] Those skilled in the art will know how to choose a suitable control plant.

[0558] In one embodiment the control plant is of the same type, and age or developmental stage, but does not ectopically express the oil synthesising enzyme in accordance with the invention. In one embodiment the control plant is of the same type, and age or developmental stage, but does not ectopically express the oil encapsulating protein in accordance with the invention.

[0559] In a further embodiment the control plant is of the same type, and age or developmental stage, but does not ectopically express the oil synthesising enzyme in accordance with the invention, or the oil encapsulating protein in accordance with the invention.

[0560] In a further embodiment the control plant is not transformed with the polynucleotide, or construct, encoding the oil synthesising enzyme in accordance with the invention.

[0561] In a further embodiment the control plant is not transformed with the polynucleotide, or construct, encoding the oil encapsulating protein in accordance with the invention.

[0562] In a further embodiment the control plant is not transformed with the polynucleotide, or construct, encoding the oil synthesising enzyme in accordance with the invention, or with the polynucleotide, or construct, encoding the oil encapsulating protein in accordance with the invention.

[0563] In one embodiment the control plant is an untransformed plant.

[0564] In a further embodiment the control plant is transformed with a control construct. In one embodiment the control construct is an "empty vector" construct.

[0565] In a further embodiment the control plant is a null segregant.

[0566] In a further embodiment the control plant is a plant that has not been modified, by a geneediting technique to express the protein according to the invention.

[0567] Preferably the control part, propagule or progeny is from a control plant as described above.

[0568] Plant parts

[0569] In one embodiment the part is from a reproductive tissue. In a further embodiment the part is a seed. Animal feedstock

[0570] In a further aspect the invention provides an animal feedstock comprising a plant, plant part, seed, or co-product of the invention, or produced by a method of the invention.

[0571] Food ingredient

[0572] In a further aspect the invention provides a food ingredient comprising a plant, plant part, seed, or co-product of the invention, or produced by a method of the invention.

[0573] Method for producing oil

[0574] In a further aspect the invention provides a method for producing oil, the method comprising extracting lipid from at least one of a plant, plant part, propagule and progeny of the invention, or produced by a method of the invention.

[0575] In a preferred embodiment the plant part is a seed.

[0576] In one embodiment the method includes the step of extracting lipid via crushing.

[0577] In one embodiment the crushing is expeller crushing.

[0578] In a further embodiment the method includes the step of extracting lipid via solvent extraction.

[0579] In one embodiment the solvent is hexane.

[0580] In a further embodiment the solvent is ether.

[0581] In one embodiment the ether extraction is AOAC Official Method 920:39 (A).

[0582] In a further embodiment the method includes the step of extracting lipid via critical point extraction. In a further embodiment the oil is separated out as part of a fermentation process

[0583] In a further embodiment the oil extraction is part of a wet milling or dry milling process

[0584] In a further embodiment the lipid is processed into at least one of: a) a fuel, b) an oleochemical, c) a nutritional oil, d) a cosmetic oil, e) a polyunsaturated fatty acid (PUFA), and f) a combination of any of a) to e).

[0585] Lipid production

[0586] In certain embodiments the cell, tissues, plants and plant parts of the invention produces more lipid than control cells, tissues, plants and plant parts.

[0587] Those skilled in the art are well aware of methods for measuring lipid production. This may typically be done by quantitative fatty acid methyl ester gas chromatography mass spectral analysis (FAMES GC-MS). Suitable methods are also described in the examples section of this specification.

[0588] Polynucleotides and fragments

[0589] The term “polynucleotide(s),” as used herein, means a single or double -stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre- mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

[0590] A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides. Proteins / Polypeptides and fragments

[0591] The term “polypeptide”, as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides or proteins of the present invention, or used in the methods of the invention, may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The modified DGAT1 proteins may also be expressed fom endogenous polynucleotides that have been modified using gene editing approaches.

[0592] A “fragment” of a polypeptide is a subsequence of the polypeptide that preferably performs a function of and / or provides three-dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.

[0593] The term “isolated” as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.

[0594] The term “recombinant” refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and / or is recombined with sequences that are not present in its natural context.

[0595] A “recombinant” polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.

[0596] The term “derived from” with respect to polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.

[0597] Variants As used herein, the term “variant” refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides. The term “variant” with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.

[0598] Polynucleotide variants

[0599] Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention. Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from the NCBI website on the World Wide Web at ftp: / / ftp.ncbi.nih.gov / blast / . The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

[0600] The identity of polynucleotide sequences may be examined using the following unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn

[0601] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line “Identities = “.

[0602] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,!. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from the world wide web at hgmp.mrc.ac.uk / Software / EMBOSS / . The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at ebi.ac.uk / emboss / align / .

[0603] Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235. A preferred method for calculating polynucleotide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.).

[0604] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the World Wide Web at ftp : / / ftp .ncbi .nih .gov / blast / .

[0605] The similarity of polynucleotide sequences may be examined using the following unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx

[0606] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

[0607] Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 -6 more preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x 10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than 1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 - 70, more preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably less than 1 x 10-100 when compared with any one of the specifically identified sequences. Alternatively, variant polynucleotides of the present invention, or used in the methods of the invention, hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.

[0608] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

[0609] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook etal., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0. 1% SDS at 65°C.

[0610] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500 / oligonucleotide length) °C.

[0611] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm. Variant polynucleotides of the present invention, or used in the methods of the invention, also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a “silent variation”. Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

[0612] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0613] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the World Wide Web at ftp: / / ftp.ncbi.nih.gov / blast / via the tblastx algorithm as previously described.

[0614] Polypeptide variants

[0615] The term variant with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.

[0616] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from the NCBI website on the World Wide Web at ftp: / / ftp.ncbi.nih.gov / blast / . The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[0617] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0618] A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.).

[0619] Polypeptide variants of the present invention, or used in the methods of the invention, also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from the NCBI website on the World Wide Web at ftp: / / ftp.ncbi.nih.gov / blast / . The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp

[0620] Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 -6 more preferably less than 1 x 10 -9, more preferably less than 1 x 10 -12, more preferably less than 1 x 10 -15, more preferably less than 1 x 10 -18, more preferably less than 1 x 10 -21, more preferably less than 1 x 10 -30, more preferably less than 1 x 10 -40, more preferably less than 1 x 10 -50, more preferably less than 1 x 10 -60, more preferably less than 1 x 10 -70, more preferably less than 1 x 10 -80, more preferably less than 1 x 10 -90 and most preferably 1x10-100 when compared with any one of the specifically identified sequences.

[0621] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.

[0622] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0623] Constructs, vectors and components thereof

[0624] The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and / or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector. The term “vector” refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.

[0625] The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5’ to 3’ direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and c) a terminator functional in the host cell into which the construct will be transformed.

[0626] The term “coding region” or “open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and / or a polypeptide under the control of appropriate regulatory sequences. The coding sequence may, in some cases, identified by the presence of a 5’ translation start codon and a 3’ translation stop codon. When inserted into a genetic construct, a “coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.

[0627] “Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.

[0628] The term “noncoding region” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5 ’ UTR and the 3 ’ UTR. These regions include elements required for transcription initiation and termination, mRNA stability, and for regulation of translation efficiency.

[0629] Terminators are sequences, which terminate transcription, and are found in the 3 ’ untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions. The term “promoter” refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors. Introns within coding sequences can also regulate transcription and influence post-transcriptional processing (including splicing, capping and polyadenylation) .

[0630] A promoter may be homologous with respect to the polynucleotide to be expressed. This means that the promoter and polynucleotide are found operably linked in nature.

[0631] Alternatively, the promoter may be heterologous with respect to the polynucleotide to be expressed. This means that the promoter and the polynucleotide are not found operably linked in nature.

[0632] In certain embodiments the polynucleotides / polypeptides of the invention may be advantageously expessed under the contol of selected promoter sequences as described below.

[0633] Vegetative tissue specific promoters

[0634] An example of a vegetative specific promoter is found in US 6,229,067; and US 7,629,454; and US 7,153,953; and US 6,228,643.

[0635] Photosynthetic tissue preferred promoters

[0636] Photosynthetic tissue preferred promoters include those that are preferrentially expressed in photosynthetic tissues of the plants. Photosynthetic tissues of the plant include leaves, stems, shoots and above ground parts of the plant. Photosynthetic tissue preferred promoters include light regulated promoters.

[0637] Light regulated promoters

[0638] Numerous light regulated promoters are known to those skilled in the art and include for example chlorophyll a / b (Cab) binding protein promoters and Rubisco Small Subunit (SSU) promoters. An example of a light regulated promoter is found in US 5,750,385. Uight regulated in this context means light inducible or light induced. A “transgene” is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.

[0639] Transgenic plant

[0640] A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.

[0641] Methods for isolating or producing polynucleotides

[0642] The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference.

[0643] The polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.

[0644] Further methods for isolating polynucleotides of the invention include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65°C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardfs solution; washing (three washes of twenty minutes each at 55°C) in 1. 0 X SSC, 1% (w / v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5 X SSC, 1% (w / v) sodium dodecyl sulfate, at 60°C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1 X SSC, 1% (w / v) sodium dodecyl sulfate, at 60°C.

[0645] The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.

[0646] A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5’RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridizationbased method, computer / database -based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).

[0647] It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species.

[0648] Variants (including orthologues) may be identified by the methods described.

[0649] Methods for identifying variants

[0650] Physical methods

[0651] Variant polypeptides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variants of polynucleotide molecules of the invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.

[0652] Alternatively, library screening methods, well known to those skilled in the art, may be employed (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe sequence, hybridization and / or wash stringency will typically be reduced relatively to when exact sequence matches are sought. Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.

[0653] Computer based methods

[0654] The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.

[0655] An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp: / / ftp.ncbi.nih.gov / blast / ) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.

[0656] The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997. The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

[0657] The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce “Expect” values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.

[0658] Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, igbmc.u-strasbg.fr / BioInfo / ClustalW / Top.html) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).

[0659] Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego. PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (expasy.org / prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.

[0660] Methods for isolating polypeptides

[0661] The polypeptides of the invention, or used in the methods of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.

[0662] The polypeptides and variant polypeptides of the invention, or used in the methods of the invention, may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification,).

[0663] Alternatively the polypeptides and variant polypeptides of the invention, or used in the methods of the invention, may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.

[0664] Methods for producing constructs and vectors

[0665] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and / or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined. Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987.

[0666] Methods for producing host cells comprising polynucleotides, constructs or vectors

[0667] The invention provides a host cell which comprises a genetic construct or vector of the invention.

[0668] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel etal., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

[0669] Methods for producing plant cells and plants comprising constructs and vectors

[0670] The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention, or used in the methods of the invention. Plants comprising such cells also form an aspect of the invention.

[0671] Methods for transforming plant cells, plants and portions thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory ManuaL Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London. Methods for genetic manipulation of plants

[0672] A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297; Hellens et al., 2000, Plant Mol Biol 42: 819-32; Hellens et al., Plant Meth 1: 13). For example, strategies may be designed to increase expression of a polynucleotide / polypeptide in a plant cell, organ and / or at a particular developmental stage where / when it is normally expressed or to ectopically express a polynucleotide / polypeptide in a cell, tissue, organ and / or at a particular developmental stage which / when it is not normally expressed. The expressed polynucleotide / polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

[0673] Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.

[0674] The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35 S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02 / 00894 and WO2011 / 053169, which is herein incorporated by reference.

[0675] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP -glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.

[0676] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance.

[0677] Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and / or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.

[0678] Numerous binary vectors that replicate in Agrobacterium and can be used for delivery of constructs into plant cells are known to those skilled in the art and include for example: pBIN19 (Bevan M, 1984, Nucleic Acids Research. 12 (22): 8711-21), pGreenOOOO (Hellens etal., Plant Molecular Biology. 42 (6): 819-32.) and pLX-B2 (Pasin et al., 2017, ACS Synthetic Biology. 6 (10): 1962-1968). Binary vectors for Ochrobactrum -mediated transformation are also well- known in the art as described for example in EP3341483B1, US20180216123A1, PCT / US2016 / 049135, and Cho et al., 2022, Plant Biotechnol J., https: / / doi.org / 10. l l l l / pbi.13777.

[0679] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial No. 5, 159, 135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent Nos. 5, 187, 073 and 6. 020, 539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci.104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(l):45-51); Prunus (Ramesh et al., 2006 Plant Cell Rep. 25(8):821-8; Song and Sink 2005 Plant Cell Rep. 2006; 25(2): 117-23;

[0680] Gonzalez Padilla et al., 2003 Plant Cell Rep.22(l):38-45); strawberry (Oosumi etal., 2006 Planta. 223(6): 1219-30; Folta et a / ., 2006 Planta Apr 14; PMID: 16614818), rose (Ui et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995;44: 129-33), tomato (Dan et al., 2006, Plant Cell Reports V25: 432-441), apple (Y ao et al., 1995, Plant Cell Rep. 14, 407- 412), Canola (Brassica napus U.). (Cardoza and Stewart, 2006 Methods Mol Biol. 343:257- 66), safflower (Orlikowska et al, 1995, Plant Cell Tissue and Organ Culture 40:85-91), ryegrass (Altpeter et al., 2004 Developments in Plant Breeding 11(7):255-250), rice (Christou et al., 1991 Nature Biotech. 9:957-962), maize (Wang et al., 2009 In: Handbook ofMaize pp. 609-639) and Actinidia eriantha (Wang etal., 2006, Plant Cell Rep. 25,5: 425-31).

[0681] Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.

[0682] Modification of endogenous genomes

[0683] Targeted genome editing using engineered nucleases such as clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, is an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of cell types and in organisms that have traditionally been challenging to manipulate genetically. A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells (Nature Biotechnology 32, 347- 355 (2014). The system is applicable to plants, and can be used to regulate expression of target genes. (Bortesi and Fischer, Biotechnology Advances Volume 33, Issue 1, January-February 2015, Pages 41-52). Use of CRISPR technology in plants is also reviewed in Zhang et al., 2019, Nature Plants, Volume 5, pages778-794. EXAMPLES

[0684] The invention will now be described with reference to the following non-limiting examples.

[0685] Example 1: Endosperm Preferred Expression Promoter Selection and Construct Design.

[0686] In maize, the endosperm makes up approximately 83% of the kernel’s dry weight; this contains 70-80% starch and 8-10% protein. Endosperm cells do not contain chloroplasts but they do contain plastids and particularly amyloplasts (which mainly function in starch production and storage) and as well as endoplasmic reticulum. Plastids are the progenitors of chloroplasts and amyloplasts. The applicants postulated that the biochemistry for producing lipids will still exist at sufficient levels in the plastids and amyloplasts given the cells contain the sub-cellular organelles which are all encapsulated by lipid membranes. As such, although this goes against prevalent thinking by those skilled in the art, the applicants postulated that transcription factors such as WRI1 are not required to be heterologously expressed in the endosperm to obtain higher oil content in the seed.

[0687] The applicants postulate that to bring about a change in composition without impacting field yields (aggregate of total seed number and weight), the DGAT, the oleosin and the PDAT (if included) can be expressed in the endosperm. To achieve this, the applicants have used Zein storage protein promoters. The Zein promoters are endosperm preferred (seed specific), they consist of four families (alpha, beta, gamma and delta). The complete list of zeins includes beta, 18 delta zein, gamma, PMS2, A30, 19A2, PMS1, 191B, A20, 19C2, PZ22.3, Z1C1-7, PZ22.1, ZA / M1, B49, Z1D-4, and ZD1-2 (Chen et al, 2014).

[0688] Design of constructs to co-express DGAT1 and oleosin in Zea mays endosperm.

[0689] Two combinations of promoters were selected for endosperm preferred expression of DGAT1 and either native Zea mays oleosin (ZmOle) or Zea mays cysteine oleosin (ZmCysOle). Combined, these were used to generate three constructs Ml, M2 and M3. These constructs are described briefly below.

[0690] Ml. Includes ZmUbil promoter +5’UTR + ZmUbil intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein A20 promoter driving ZmCysOle ORF and Zm-Zein A20 terminator, as well as Zm-beta Zein promoter driving TmZmDGATl ORF and ZmOle terminator facing the RB. See Tables 5, and 12.

[0691] M2. Includes ZmUbil promoter + 5’UTR + ZmUbil intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein PMS2 promoter driving ZmCysOle ORF and Zm-Zein A20 terminator, as well as Zm- Zein ZD 1-2 promoter driving TmZmDGATl ORF and ZmOle terminator facing the RB. See Tables 6, and 12. M3. Includes ZmUbil promoter +5’UTR + ZmUbil intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein A20 promoter driving ZmOle ORF and Zm-Zein A20 terminator, as well as Zm-beta Zein promoter driving TmZmDGATl ORF and ZmOle terminator facing the RB. See Tables 7, and 12.

[0692] Table 5. Component list and position for construct Ml Table 6. Component list and position for construct M2

[0693] Table 7. Component list and position for construct M3 Construct to co-express DGAT1, PDAT and oleosin in Zea mays endosperm.

[0694] One combination of promoters was selected for endosperm preffered expression of DGAT, PDAT and Zea mays cysteine oleosin (ZmCysOle). These were used to generate the construct M4 which is described briefly below.

[0695] M4. Includes ZmUbil promoter +5’UTR + ZmUbil intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein A20 promoter driving ZmCysOle ORF and Zm-Zein A20 terminator, as well as Zm-beta Zein promoter driving TmZmDGATl ORF and ZmOle terminator, and Zm Zein-gamma promoter driving AtPDAT and OsOle terminator facing the RB. See Tables 8 and 12.

[0696] Table 8. Component list and position for construct M4 Example 2: Leaf Promoter Selection and Construct Design.

[0697] C4 plants typically have two main cells in the leaf. The mesophyll cells where CO2 is fixed by phosphoenol-pyruvate carboxylase (PEPC) the most abundant protein in mature mesophyll cells (and is also specific to mesophyll cells) of maize, compromising 10-15% of the soluble protein. Within the mesophyll the carboxylation of phosphoenol-pyruvate forms oxaloacetate which is usually converted to malate and diffuses from the mesophyll cells to the bundle sheath cells. Here it is decarboxylated to produce CO2 and pyruvate. In the bundle sheath cells the CO2 is fixed by Ribulose biphosphate carboxylase (RBCS, and is also specific to bundle sheath cells). In comparison to PEPC and RBCS the chlorophyll a / b binding proteins (CAB) are found in both the mesophyll cells and the bundle sheath cells, although predominantly in the mesophyll (Kailash et al 1992, PNAS 89:3654-3658).

[0698] Design of constructs to co-express DGAT1 and cysteine oleosin in green tissue as well as coexpress DGAT1 and oleosin in Zea mays endosperm.

[0699] Three combinations of promoters (Phosphoenolpyruvate carboxylase - PEPC, Ribulose- 1,5- bisphosphate carboxylase / oxygenase - RBCS and chlorophyll a / b-binding protein - CAB) have been used to co-express DGAT1 and cysteine oleosin in green tissue of C4 plants. These have been named M5, M6, and M7 and are described briefly below.

[0700] M5. Incudes ZmUbil promoter +5’UTR + ZmUbil intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein A20 promoter driving ZmCysOle ORF and Zm-Zein A20 terminator, as well as Zm-beta Zein promoter driving TmZmDGATl ORF and ZmOle terminator, and ZmCABl promoter driving ZmCysOle ORF and AtUBQlO terminator and ZmCAB7 promoter driving TmDGATl ORF and AtUBQ14 terminator facing the RB. See Tables 9 and 12.

[0701] M6. Includes ZmUbi 1 promoter +5 ’UTR + ZmUbi 1 intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein A20 promoter driving ZmCysOle ORF and Zm-Zein A20 terminator, as well as Zm-beta Zein promoter driving TmZmDGATl ORF and ZmOle terminator, and ZmRBCS-m3 promoter driving ZmCysOle ORF and AtUBQlO terminator and ZmRBCS-o promoter driving Tm-DGATl ORF and AtUBQ14 terminator facing the RB. See Tables 10 and 12.

[0702] M7. Includes ZmUbi 1 promoter +5 ’UTR + ZmUbi 1 intron driving BAR (with AtACT2 intron) and OsUBQ2t terminator facing the LB and in a back-to-back orientation uses Zm- Zein A20 promoter driving ZmCysOle ORF and Zm-Zein A20 terminator, as well as Zm-beta Zein promoter driving TmZmDGATl ORF and ZmOle terminator, and ZmPEPC promoter driving ZmCysOle ORF and AtUBQlO terminator and SbPEPC promoter driving TmDGATl ORF and AtUBQ14 terminator facing the RB. See Tables 11 and 12. Table 9. Component list and position for construct M5

[0703] Table 10. Component list and position for construct M6

[0704] Table 11. Component list and position for construct M7 Table 12. List of nucleic acid and residue sequences of promoters, introns, terminators, open reading frames, peptides described in this document.

[0705] Example 3: Zea mays Transformation.

[0706] The Zea mays elite inbred LH244 variety was transformed by The Wisconsin Crop Innovation Center (W CIC) using an immature embryo and following a modified protocol of Raji et al, (2018) (Raji, J.A., Frame, B., Little, D., Santoso, T.J., Wang, K. (2018).

[0707] Agrobacterium- and Biolistic-Mediated Transformation of Maize Bl 04 Inbred. In: Lagrimini, L. (eds) Maize. Methods in Molecular Biology, vol 1676. Humana Press, New York, NY. doi.org / 10. 1007 / 978-1-4939-7315-6_2).

[0708] Ti and T2 seed were produced under glasshouse conditions. T2 seed were evaluated by Proximate analysis; lines with elevated fat content were subsequently used in field trial evaluation. Proximate analysis was conducted using AO AC approved methods for measuring crude protein (combustion analysis (LECO) AOAC Official Method 990.03, 2006), crude fat (by ether extraction, AOAC Official Method 920.39 (A)), ash (AOAC Official Method 943.05), moisture (by AOAC Offical Method 934.01, 2006), crude fiber (by AOAC Official Method 978.10, 2006), total carbohydrates crude “by difference” (100%-%(crude protein +Ash +crude fat + moisture) and gross calories by calculation (protein= 4 calories / gram, carbohydrate = 4 calories / gram, fat = 9 calories / gram)

[0709] Example 4 Transgenic Zea mays Field Trial Evaluation.

[0710] T2 Transgenic lines were evaluated in a non-irrigated field trial using a replicated paired plot arrangement. T2 seeds were planted in 76cm (30 inch) wide rows, 2 rows / rep, approximately 42 seed / row (~84 seed / rep), and typically 2-3 replications / line. Seed planting density was maintained at 74,130 seeds / hectare (30,000 seeds / acre). Plant numbers were determined for each row and subsequent yields were adjusted to bushels / acre (bu / ac) at a population of 30,000 per acre.

[0711] Com was harvested with a small plot combine (Wintersteiger Delta, Salt Lake City, UT). Grain yield was adjusted to 150 g kg-1 moisture before analysis, and moisture was determined using GAC 2100 (DICKEY-john Corp., Auburn, IL). Grain quality was analyzed for starch, oil, and protein concentrations (Foss Infratec 1241, Eden Prairie, MN). The NIR machine averages 10 subsamples of the collected grain sample.

[0712] Table 13 shows the population adjusted yields for each line compared to the relevant null;

[0713] Table 14 shows the NIR analysis of seed oil, protein and starch content for each line compared to the relevant null. Combined, the data show several constructs increase oil content by up to 37% (average increase of 19.7% for all events) without reducing population adjusted seed yield by more than 5% compared to the relevant null. Six of 14 events show an improved yield (at least a 10% increase) compared to the relevant null while improving seed oil content by at least 13 %. Eight of 14 events show improved seed oil content (11.5-36.7% increase) compared to the relevant null while maintaining field yields (% difference to relevant null + / -5%).

[0714] The applicants postulate that the amount of carbohydrates (used in ethanol production via fermentation) is reduced in field grown transgenic seed as it is in greenhouse grown seed (~I% reduction in carbohy crates compared to average null) and that this decrease in carbohydrates will reduce the CO2 production in the ethanol fermentation process per unit seed input while increasing oil extracted per unit.

[0715] Proximate analysis can be performed on field produced seed giving crude protein, crude fat, moisture, crude fiber, total carbohydrates and gross calories. Table 13. Construct and line number (transgenic and null), field yields, population adjusted field yields. Table 14. Construct and line number (transgenic and null), NIR determined seed oil, protein and starch contents Endosperm oil content was measured in a subset of events and their relevant controls. See Table 15 below.

[0716] Table 15. Construct and line number (transgenic and null) and oil content in endosperm

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Claims

CLAIMS1 A method for increasing the oil content in the seed of a plant relative to that in a control plant, without significantly decreasing the seed yield of the plant relative to that in the control plant, the method comprising the step of ectopically expressing at least one oil-synthesising enzyme and at least one oil-encapsulating protein in the endosperm of the plant.

2. The method of claim 1 comprising ectopically expressing at least two oil-synthesising enzymes and at least one oil-encapsulating protein in the endosperm of the plant.

3. The method of any preceding claim further comprising the step of ectopically expressing at least one oil-synthesising enzyme and at least one oil-encapsulating protein in the green tissues of the plant.

4. The method of any preceding claim including the step of measuring oil content in the seed of the plant and measuring the seed yield, and selecting the plants on the basis of increased seed oil content without a significant decrease in the seed yield.

5. A method for producing seed with increased oil content, relative to that in seed of a control plant, the method comprising growing a plant produced by the method of any preceding claim, and collecting seed from the plant.

6. The method of any preceding claim wherein the production or content oil content in the seeds of the plant is increased by at least 5%.

7. The method of any preceding claim in which expression of the oil-synthesising enzyme in the endosperm is from a polynucleotide encoding the oil synthesising enzyme, and expression of the polynucleotide is controlled by an endospermpreferred promoter operably linked to the polynucleotide.

8. The method of any preceding claim in which expression of the oil-encapsulating protein in the endosperm is from a polynucleotide encoding the oil-encapsulating protein, and expression of the polynucleotide is controlled by an endosperm-preferred promoter operably linked to the polynucleotide.

9. The method of any preceding claim in which expression of the oil-synthesising enzyme in green tissues is from a polynucleotide encoding the oil synthesising enzyme, and expression of the polynucleotide is controlled by a green tissue-preferred promoter operably linked to the polynucleotide.

10. The method of any preceding claim in which the expression of the oil -encapsulating protein in green tissues is from a polynucleotide encoding the oil -encapsulating protein, and expression of the polynucleotide is controlled by an green tissue preferred promoter operably linked to the polynucleotide.

11. The method of any one of claims 7 to 10 in which the plant is transformed with the polynucleotide and operably linked promoter.

12. A plant or seed produced or selected by the method of any preceding claim.

13. A part, propagule or progeny of the plant of claim 12.

14. The part of claim 13 that is a seed.

15. The plant, plant part, propagule, progeny or seed of any one of claims 12 to 14 in which the plant is transgenic for at least one polynucleotide encoding the oilsynthesising enzyme and operably linked promoter, and at least one polynucleotide encoding the oil -encapsulating protein and operably linked promoter.

16. The seed of claim 14 that has increased oil content relative to that in seed of a control plant, or control seed.

17. A plant with increased seed oil content relative to that in a control plant, without significantly decreased seed yield relative to that in the control plant, wherein the plant ectopically expresses: a) at least one oil-synthesising enzyme in the endosperm of plant, and b) at least one oil-encapsulating protein in the endosperm of the plant.

18. The plant of claim 17 that further ectopically expresses : c) at least one oil-synthesising enzyme in the green tissues of plant, andd) at least one oil-encapsulating protein in the green tissues of the plant.

19. A method for producing a plant with increased production or content of oil in its seed relative to that in a control plant, without significantly decreased seed yield relative to that in the control plant, the method comprising crossing a plant of any preceding claim with another plant.

20. A method for producing a seed with increased oil content relative to that in a control plant, the method comprising: a) growing a plant of any preceding claim, or produced by a method of any preceeding claim, and b) harvesting the seed produced.

21. A method for producing a seed with increased oil production that in a control plant, the method comprising: a) crossing a plant of any preceding claim with another plant. b) harvesting the seed produced.

22. The plant or seed of any preceding claim, or produced by the method of any preceding claim, in which oil content in the seeds of the plant, or in the seed, is increased by at least 5%.

23. A method for producing oil, the method comprising extracting oil from the seeds of a plant of any preceding claim, or a seed of any preceding claim.

24. A method for producing oil the method comprising producing a plant or seed according to any preceeding claim, extracting oil from the seeds of the plant, or the seed.

25. The method of any of claims 23 to 24 in which the oil extraction is by at least one of: a) solvent extraction, b) crushing, c) critical point extraction, d) separation during a fermentation process, and e) a wet milling or dry milling process.

26. The method of any of claims 23 to 25 in which the oil is processed into at least one of: a) a fuel, b) an oleochemical, c) a nutritional oil, d) a cosmetic oil, e) a polyunsaturated fatty acid (PUFA), and f) a combination of any of a) to e).

27. A method for producing a co-product, the method comprising extracting oil from a seed of any preceding claim, and collecting the remaining co-product.

28. A method for producing a co-product, the method comprising providing a plant or seed according to any preceeding claim, extracting oil from the seeds of the plant, or the seed, and collecting the co-product before, at the same time or after extracting the oil.

29. The method of any of claims 27 to 28 in which extraction is by at least one of: a) solvent extraction, b) crushing, c) critical point extraction, d) separation during a fermentation process, and e) wet or dry milling.

30. A co-product produced by the method of any one of claims 27 to 29.

31. An animal feedstock comprising a co-product of claim 30.

32. A food ingredient comprising a co-product of claim 30.

33. A fuel feedstock comprising a co-product of claim 30.

34. A method for producing oil from seed of any preceding claim, or seed produced by a method of any preceding claim, in a fermentation process with reduced production of carbon dioxide as a byproduct per seed input unit relative to that when control seed is used.

35. A method for producing oil from seed of any preceding claim, or seed produced by a method of any preceding claim, in a fermentation process with reduced production of carbon dioxide as a byproduct per unit oil output relative to that when control seed is used.

36. The method of claim 34 in which the method comprises: a) providing seed of any preceding claim, or seed produced by a method of any preceding claim, or a mash produced from the seed, b) subjecting the seed or mash to fermentation, and c) extracting oil from the fermenting or fermented seed or mash.

37. The method of claim 35 in which the method comprises: a) providing seed of any preceding claim, or seed produced by a method of any preceding claim, or a mash produced from the seed, b) subjecting the seed or mash to fermentation, and c) extracting oil from the fermenting or fermented seed or mash.

38. The method of claim 34 to 37 that produces more oil than when control seed is used.