NZ627107B2

NZ627107B2 - Processes for producing lipids

Info

Publication number: NZ627107B2
Application number: NZ627107A
Authority: NZ
Inventors: Tahchy Anna El; Qing Liu; James Robertson Petrie; Surinder Pal Singh; Thomas Vanhercke
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 2011-12-27
Filing date: 2012-12-21
Publication date: 2017-03-24

Abstract

plant or algae which comprises exogenous polynucleotides and an increased level of non-polar lipids compared to a plant which lacks said exogenous polynucleotides, wherein the first polynucleotide encodes a fatty acid acyltransferase and subsequent polynucleotides encode one or more of i) an RNA molecule which inhibits expression of a gene encoding a polypeptide that is involved in the degradation of lipid or which reduces lipid content such as a lipase, or ii) a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in the plant; and wherein the vegetative part of the plant has a total non-polar lipid content of at least 7%, and wherein if the transcription factor polypeptide of (ii) is present, the plant comprises the RNA molecule of (i). olecule which inhibits expression of a gene encoding a polypeptide that is involved in the degradation of lipid or which reduces lipid content such as a lipase, or ii) a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in the plant; and wherein the vegetative part of the plant has a total non-polar lipid content of at least 7%, and wherein if the transcription factor polypeptide of (ii) is present, the plant comprises the RNA molecule of (i).

Description

PROCESSES FOR PRODUCING LIPIDS FIELD OF THE INVENTION The present invention relates to methods of producing lipids. In particular, the present invention relates to methods of increasing the level of one or more non-polar .lipids and/or the total non-polar lipid content in a transgenic organism or part thereof.

In one particular embodiment, the present invention relates to any ation of one 'or more monoacylglycerol ansferases' (MGATs), diacylglycerol acyltranstransferases (DGATs), glycerolphosphate acyltransferases (GPATs), oil 10 bOdy prOteins and/or transcription factors regulating lipid biosynthesis while silencing key enzymatic steps in the starch biosynthesis and fatty acid desaturation pathways to increase the level of one or more non-polar lipids and/Or the total non-polar lipid content and/or mono-unsaturated fatty acid content in plants or any part thereof including plant seed and/or leaves, algae and fungi. 15 BACKGROUND OF INVENTION The majority of the world's energy, particularly for transportation, is supplied by'petroleum derived fuels, which have a ﬁnite supply. Alternative sources which are renewable are needed, such as from biologically produced oils. 20 Triacylglycerol hesis yglycerols (TAG) constitute the major form of lipids in seeds and consist of three acyl chains esteri'ﬁed to a glycerol backbone. The fatty acids are synthesized . ' in the plastid as acyl-acyl r protein (ACP) intermediates where they can undergo 25 a 'ﬁrst desaturation catalyzed. This reaction is catalyzed by the stearoyl-ACP desaturase and yields oleic acid (C1821A9). uently, the acyl chains are transported to the cytosol and asmic reticulum (ER) as acyl-Coenzyme (CoA) esters. Prior to entering the major TAG biosynthesis' pathway, also known as' the Kennedy or olphosphate (GSP) pathway, the acyl chains are typically 30 integrated into phospholipids of the ER membrane where they can undergo further ration. Two key s in the production of polyunsaturated fatty acids are the membrane-bound FAD2 and FAD3 desaturases which produce linoleic (C18;2A9'12) and a-linolenic acid (Cl 8:3A9'12‘15) respectively.

TAG thesis via the Kennedy pathway consists of a series of Subsequent 35 acylations, each using oA esters as the acyl-donor. The ﬁrst acylation step typically occurs at the snI-position of the G3P backbone and is catalyzed by the olphosphate acyltransferaSe (snI-GPAT). The t, snI-lysophosphatidic acid (snI-LPA) serves as a substrate for the lysophosphatidic acid acyltransferase Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 2'- ) which couples a second acyl chain at the an-p‘osition to. form phosphatidic acid. PA is ﬂirther'dephosphorylated to diacylglycerol (DAG) by the phosphatidic acid phosphatase (PAP) thereby ing the substrate for the ﬁnal acylation step.

Finally, a third acyl chain is esteriﬁed to the sn3-position of DAG in a reaction catalyzed by the glycerol acyltransferase (DGAT) to form TAG which accumulates in oil bodies. A second enzymatic reaction, phosphatidyl glycerol acyltransferase (PDAT), also results in the conversion of DAG to TAG. This reaction ' is unrelated to DGAT and uses phospholipids as the acyl-donors.

To maximise yields for the cial production of lipids, there is a need for .10 r means to increase the levels of , particularly non-polar lipids such as 'DAGs and TAGS, in transgenic organisms or parts thereof such as plants, seeds, leaves, algae and fungi. Attempts at sing neutral lipid yields ‘in plants have mainly focused on individual critical enzymatic steps involved in fatty acid biosynthesis or TAG assembly. These strategies, however, have resulted in modest. 15 increases in seed or leaf oil t. Recent metabolic engineering work in the oleaginous yeast Yarrowia IipOIytica has demonstrated that a combined approach of increasing glycerol-S-phosphate production and preventingvTAG breakdown via B- oxidation resulted in cumulative increases in the total lipid content (Dulermo et a1., I 2011). 20 Plant lipids such as seedoil triaclyglycerols (TAGS) have many uses, for example, culinary uses (shortening, texture, , industrial uses (in soaps, candles, perﬁimes, cosmetics, suitable as drying agents, insulators, lubricants) and provide nutritional value. There is also growing interest in using plant lipids for the . production uel. 25 To maximise yields for the commercial biological production of lipids, there is a need for further means to se the levels of lipids, particularly non-polar lipids such as DAGs and TAGS, in transgenic organisms or parts thereof such as plants, ' seeds, leaves, algae and fungi. 30 SUMMARY OF THE INVENTION _ The present inventors have demonstrated signiﬁcant increases in the lipid content of organisms, particularly in the tive parts and seed 'of plants, by manipulation of both fatty acid biosynthesis and lipid assembly pathways. Various combinations of genes were used to achieve ntial increases in oil content, which 35 is of great signiﬁcance for production of bioﬁiels and other industrial products derived from oil.

D Substitute Sheet (Rule 26) RO/AU In a ﬁrst aspect, the invention provides a process for producing an industrial product from a vegetative plant part or non-human organism or part thereof comprising high levels of lar lipid.

In an embodiment, the invention provides a process for producing an rial product, the process sing the steps of: i) obtaining a vegetative plant part having a total non-polar lipid content of at least about 3%, ably at least about 5% or at least about 7% (w/w dry weight), ii) converting at least some of the lipid in Sim in the vegetative plant part to the rial product by heat, chemical, or tic means, or any combination thereof, 10 and iii) ring the industrial product, thereby producing the industrial product.

In another embodiment, the process for producing an industrial product ses the steps of: 15 i) obtaining a vegetative plant part having a total non-polar lipid content of at least about 3%, preferably at least about 5% or at least about 7% (W/W dry weight), ii) physically processing the vegetative plant part of step i), iii) converting at least some of the lipid in the processed vegetative plant part to the industrial t by applying heat, chemical, or enzymatic means, or any 20 combination thereof, to the lipid in the processed tive plant part, and iv) recovering the industrial product, thereby producing the industrial product. , In another embodiment, the process for producing an industrial product comprises the steps of: 25 i) ing a non—human organism or a part thereof comprising one or more exogenous polynucleotide(s), wherein each of the one or more exogenous polynucleotide(s) is operably linked to a promoter which is e of directing sion of the polynucleotide in a non—human sm or a part thereof, and wherein the non-human organism or part thereof has an increased level of one or more 30 non-polar lipids relative to a corresponding non-human organism or a part thereof lacking the one or more exogenous polynucleotide(s), and ii) converting at least some of the lipid in Situ in the non-human organism or part thereof to the industrial product by heat, chemical, or enzymatic means, or any combination thereof, and 35 iii) recovering the industrial product, thereby producing the industrial product.

In a second aspect, the invention provides a process for producing an industrial product, the process comprising the steps of: (i) obtaining a plant or part thereof, or alga, of the invention, and (ii) optionally, physically processing the plant or part thereof, or alga, of step (i), and (iii) converting at least some of the lipid in the plant or part thereof, or alga of step (i), or in the processed plant or part thereof, or alga, obtained by step (ii), to the industrial product by applying heat, al, or enzymatic means, or any ation thereof, to the lipid, in situ in the plant or part thereof, or alga, of step (i), or in the processed plant or part thereof, or alga, obtained by step (ii), and (iv) recovering the industrial product, 10 thereby producing the industrial product.

In a further embodiment of the above aspects, the process for ing an industrial product comprises the steps of: i) obtaining a non—human organism or a part thereof sing one or more exogenous polynucleotides, wherein the non—human organism or part thereof has an 15 increased level of one or more non—polar lipids relative to a corresponding non—human organism or a part thereof lacking the one or more ous polynucleotides, ii) physically processing the non-human organism or part thereof of step i), iii) converting at least some of the lipid in the processed non—human organism or part thereof to the rial t by applying heat, chemical, or enzymatic 20 means, or any combination thereof, to the lipid in the processed man organism or part thereof, and iv) recovering the industrial product, thereby producing the industrial t.

In each of the above embodiments, it would be understood by a person skilled 25 in the art that the converting step could be done simultaneously with or subsequent to the physical processing step.

In each of the above embodiments, the total non—polar lipid content of the vegetative plant part, or non-human organism or part thereof, preferably a plant leaf or part f, stem or tuber, is at least about 3%, more preferably at least about 5%, 30 preferably at least about 7%, more preferably at least about 10%, more ably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, or more ably at least about 15% (W/w dry weight). In a further preferred embodiment, the total non—polar lipid content is between 5% and 25%, between 7% and 25%, between 10% and 25%, between 12% 35 and 25%, between 15% and 25%, between 7% and 20%, between 10% and 20%, between 10% and 15%, between 15% and 20%, between 20% and 25%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a tage of dry weight. In a particularly preferred ment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred embodiment, the vegetative plant part is a leaf portion having a surface area of at least 1 cm2. rmore, in each of the above embodiments, the total TAG content of the vegetative plant part, or non-human organism or part thereof, preferably a plant leaf or part thereof, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, more preferably at least about 15%, or more 10 preferably at least about 17% (w/w dry weight). In a further preferred embodiment, the total TAG content is between 5% and 30%, between 7% and 30%, between 10% and 30%, between 12% and 30%, between 15% and 30%, between 7% and 30%, between 10% and 30%, between 20% and 28%, between 18% and 25%, between 22% and 30%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, 15 about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more red embodiment, the vegetative plant part is a leaf portion having a e area of at least 1 cm2.

Furthermore, in each of the above embodiments, the total lipid content of the 20 vegetative plant part, or non-human sm or part thereof, preferably a plant leaf or part f, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more ably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, more preferably at least about 15%, more 25 preferably at least about 17% (W/W dry weight), more preferably at least about 20%, more preferably at least about 25%. In a further preferred embodiment, the total lipid content is between 5% and 35%, between 7% and 35%, between 10% and 35%, between 12% and 35%, between 15% and 35%, between 7% and 35%, n 10% and 20%, between 18% and 28%, between 20% and 28%, between 22% and 28%, 30 about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, about 22%, or about 25%, each as a percentage of dry weight. Typically, the total lipid t of the vegetative plant part, or non- human organism or part thereof is about 2-3% higher than the non-polar lipid content.

In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or 35 a portion thereof. In a more preferred embodiment, the vegetative plant part is a leaf portion having a surface area of at least 1 cm2.

The industrial product may be a hydrocarbon t such as fatty acid esters, ably fatty acid methyl esters and/or a fatty acid ethyl esters, an alkane such as e, ethane or a longer-chain alkane, a e of longer chain alkanes, an alkene, a biofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon monoxide, en and biochar. The industrial product may be a mixture of any of these components, such as a mixture of alkanes, or alkanes and alkenes, preferably a mixture which is predominantly (>50%) C4-C8 alkanes, or predominantly C6 to C10 alkanes, or predominantly C6 to C8 alkanes. The industrial product is not carbon dioxide and not water, although these molecules may be produced in combination with the industrial product. The industrial product may be a gas at atmospheric pressure/room 10 temperature, or preferably, a , or a solid such as biochar, or the process may produce a combination of a gas component, a liquid component and a solid component such as carbon monoxide, hydrogen gas, alkanes and biochar, which may subsequently be separated. In an embodiment of the above aspects, the hydrocarbon product is predominantly fatty acid methyl esters. In an alternative embodiment of I5 the above aspects, the hydrocarbon product is a product other than fatty acid methyl esters.

The industrial product may be an intermediate product, for example, a product comprising fatty acids, which can subsequently be converted to, for example, bioﬁlel by, for example, trans-esterification to fatty acid esters. 20 Heat may be applied in the process, such as by pyrolysis, combustion, ation, or together with enzymatic ion (including anaerobic digestion, ting, fermentation). Lower temperature gasification takes place at, for e, between about 700°C to about 1000°C. Higher ature gasification takes place at, for example, n about 1200°C to about 1600°C. Lower 25 temperature pyrolysis (slower pyrolysis), takes place at, for example, about 400°C, whereas higher temperature pyrolysis takes place at, for example, about 500°C.

Mesophilic digestion takes place between, for example, about 20°C and about 40°C.

Thermophilic digestion takes place from, for example, about 50°C to about 65°C.

Chemical means include, but are not limited to, catalytic cracking, anaerobic 30 digestion, fermentation, composting and transesterification. In an embodiment of the above s, a chemical means uses a catalyst or mixture of catalysts, which may be d together with heat. The process may use a neous catalyst, a heterogeneous st and/or an enzymatic catalyst. In an embodiment of the above aspects, the catalyst is a transition metal catalyst, a molecular sieve type catalyst, an 35 activated alumina catalyst or sodium carbonate. Catalysts include acid catalysts such as ric acid, or alkali catalysts such as potassium or sodium ide or other hydroxides. The al means may se transesteriﬁcation of fatty acids in the lipid, which process may use a homogeneous catalyst, a heterogeneous catalyst and/or an enzymatic catalyst. The conversion may comprise pyrolysis, which applies heat and may apply chemical means, and may use a transition metal catalyst, a molecular sieve type catalyst, an activated alumina catalyst and/or sodium carbonate.

Enzymatic means e, but are not limited to, digestion by microorganisms in, for example, anaerobic digestion, fermentation or composting, or by recombinant enzymatic proteins.

The lipid that is converted to an industrial product in this aspect of the invention may be some, or all, of the non—polar lipid in the vegetative plant part or non—human organism or part thereof, or preferably the conversion is of at least some 10 of the non—polar lipid and at least some of the polar lipid, and more ably essentially all of the lipid (both polar and non—polar) in the vegetative plant part or non—human organism or part f is converted to the industrial product(s).

In an embodiment of the above aspects, the conversion of the lipid to the industrial product occurs in situ without physical disruption of the vegetative plant 15 part or non—human organism or part thereof. In this embodiment of the above aspects, the vegetative plant part or non-human organism or part thereof may ﬁrst be dried, for example by the application of heat, or the vegetative plant part or non-human organism or part thereof may be used essentially as ted, without drying. In an alternative embodiment of the above aspects, the process comprises a step of 20 physically sing the tive plant part, or the non-human organism or part thereof. The physical sing may compriseone or more of rolling, pressing such as ﬂaking, crushing or grinding the vegetative plant part, non human organism or part thereof, which may be combined with drying of the tive plant part, or the non- human organism or part f. For example, the vegetative plant part, or non- 25 human organism or part f may ﬁrst be substantially dried and then ground to a ﬁner material, for ease of subsequent processing.

In an embodiment of the above aspects, the weight of the vegetative plant part, or the non—human organism or part thereof used in the s is at least 1 kg or preferably at least 1 tonne (dry weight) of pooled vegetative plant parts, or the non- 30 human organisms or parts thereof. The processes may further comprise a ﬁrst step of harvesting vegetative plant parts, for example from at least 100 or 1000 plants grown in a ﬁeld, to provide a collection of at least 1000 such vegetative plant parts, i.e., which are essentially cal. Preferably, the vegetative plant parts are harvested at a time when the yield of non—polar lipids are at their highest. In one embodiment of 35 the above aspects, the vegetative plant parts are harvested about at the time of ng. In another embodiment of the above aspects, the vegetative plant parts are harvested from about at the time of ﬂowering to about the beginning of senescence.

In another embodiment of the above aspects, the vegetative plant parts are harvested when the plants are at least about 1 month of age.

The process may or may not further comprise extracting some of the lar lipid content of the vegetative plant part, or the non-human organism or part thereof prior to the conversion step. In an embodiment of the above aspects, the process further comprises steps of: (a) extracting at least some of the non—polar lipid content of the vegetative plant part or the non—human organism or part thereof as non—polar lipid, and (b) recovering the extracted non-polar lipid, 10 wherein steps (a) and (b) are performed prior to the step of converting at least some of the lipid in the vegetative plant part, or the non—human organism or part thereof to the industrial product. The proportion of non—polar lipid that is ﬁrst extracted may be less than 50%, or more than 50%, or preferably at least 75% of the total non-polar lipid in the tive plant part, or non—human organism or part thereof. In this embodiment, 15 the extracted non—polar lipid comprises triacylglycerols, wherein the triacylglycerols comprise at least 90%, preferably at least 95% of the extracted lipid. The extracted lipid may itself be converted to an rial t other than the lipid itself, for example by trans-esteriﬁcation to fatty acid .

In a third aspect, the invention provides a process for producing extracted lipid 20 from a non-human organism or a part thereof.

In an embodiment, the invention es a process for ing extracted lipid, the s comprising the steps of: p i) obtaining a non—human organism or a part thereof sing one or more exogenous polynucleotide(s) and an increased level of one or more non—polar lipid(s) 25 relative to a corresponding non-human organism or a part thereof, respectively, lacking the one or more exogenous cleotide(s), ii) extracting lipid from the non—human organism or part thereof, and iii) recovering the extracted lipid, thereby producing the ted lipid, n each of the one or more exogenous 30 polynucleotides is operably linked to a promoter which is capable of directing expression of the polynucleotide in a non-human organism or part thereof, and wherein one or more or all of the following features apply: (a) the one or more exogenous polynucleotide(s) comprise a ﬁrst exogenous cleotide which encodes an RNA or transcription factor polypeptide that 35 increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a man organism or a part thereof, and a second exogenous cleotide which encodes an RNA or polypeptide involved in biosynthesis of one or more non—polar lipids, (b) if the non-human organism is a plant, a vegetative part of the plant has a total non-polar lipid content of at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more ably at least about 14%, or more preferably at least about 15% (w/w dry ), (0) the non-human organism is an alga selected from the group ting of diatoms (bacillariophytes), green algae (chlorophytes), blue—green algae (cyanophytes), golden-brown algae (chrysophytes), haptophytes, brown algae and 10 heterokont algae, (d) the one or more non-polar lipid(s) comprise a fatty acid which comprises a yl group, an epoxy group, a cyclopropane group, a double carbon—carbon bond, a triple carbon—carbon bond, conjugated double bonds, a branched chain such as a methylated or hydroxylated branched chain, or a combination of two or more thereof, 15 or any of two, three, four, ﬁve or six of the aforementioned , bonds or ed chains, (6) the total fatty acid content in the non—polar lipid(s) comprises at least 2% more oleic acid and/or at least 2% less palmitic acid than the non-polar lipid(s) in the corresponding non-human organism or part thereof lacking the one or more 20 exogenous polynucleotides, (f) the non-polar lipid(s) comprise a modiﬁed level of total sterols, preferably free (non-esteriﬁed) sterols, steroyl esters, steroyl ides, relative to the non-polar lipid(s) in the corresponding non—human organism or part thereof lacking the one or more exogenous polynucleotides, 25 (g) the non-polar lipid(s) comprise waxes and/or wax , (h) the non-human organism or part thereof is one member of a pooled population or collection of at least 1000 such non—human organisms or parts thereof, respectively, from which the lipid is extracted.

In an embodiment of (b) above, the total non—polar lipid content is between 30 5% and 25%, between 7% and 25%, between 10% and 25%, n 12% and 25%, between 15% and 25%, between 7% and 20%, between 10% and 20%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight.

In an embodiment, the man organism is an alga, or an organism suitable 35 for fermentation such as a fungus, or preferably a plant. The part of the non-human organism may be a seed, fruit, or a vegetative part of a plant. In a red embodiment, the plant part is a leaf n having a surface area of at least 1 cm2. In another preferred embodiment, the non-human organism is a plant, the part is a plant 10 seed and the extracted lipid is seedoil. In a more preferred embodiment, the plant is from an d species, which is used commercially or could be used commercially for oil production. The s may be selected from a group consisting of a Acrocomia aculeata (macauba palm), Arabidopsis na, AraciniS hypogaea 5 (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare (tucuma), Attalea geraenSiS (Indaia-rateiro), Attalea humilis (American oil palm), Attalea oleifera é), a phalerata (uricuri), Attalea Speciosa (babassu), Avena saziva (oats), Beta vulgaris (sugar beet), BraSSica Sp. such as Brassica carinata, BraSSica juncea, Brassica napobraSSica, BraSSica napuS (canola), na sativa (false ﬂax), lO Cannabis sativa , Carthamus tinctorius (safﬂower), Caryocar brasiliense (pequi), Cocos nucifera (Coconut), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon), ElaeiS guineenSiS (African palm), Glycine max (soybean), Gossypium um (cotton), Helianthus Sp. such as Helianthus annuus (sunﬂower), Hordeum vulgare (barley), Jatropha curcaS (physio nut), JoanneSia princepS (arara nut-tree), 15 Lemna Sp. eed) such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swollen duckweed), Lemna ca, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna Zrisulca, Lemna turionifera, Lemna valdiviana, Lemna yungensis, Licania rigida (oiticica), Linum usitatissimum (ﬂax), Lupinus angustifolius (lupin), 20 Mauritia ﬂexuosa i palm), Maximiliana marz'pa (inaja palm), Miscanthus Sp. such as Miscanthus x giganteus and Miscanz‘hus SinenSiS, Nicotiana Sp. (tabacco) such as Nicotiana tabacum or ana benthamiana, Oenocarpus bacaba (bacaba— do—azeite), Oenocarpus bataua (pataua), Oenocarpus distichus (bacaba-de-leque), Orjyza Sp. (rice) such as 00226: sativa and Oryza glaberrima, m virgatum 25 hgrass), Paraqueiba paraenSiS (mari), Persea amencana (avocado), Pongamia pinnata (Indian beech), Populus trichocarpa, Ricinus communiS (castor), Saccharum Sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum Sp. such as Sorghum r, Sorghum vulgare, Theobroma grandiforum (cupuassu), Trifolium Sp., Trithrinax brasz'liensis (Brazilian needle palm), Triticum Sp. (wheat) 30 such as Triticum aestivum and Zea mays (corn). In an embodiment, the ca napuS plant is of the variety Westar. In an alternative ment, if the plant is ca napus, it is of a variety or cultivar other than Westar. In an embodiment, the plant is of a species other than ArabidopSiS thaliana. In another embodiment, the plant is of a species other than Nicotiana tabacum. In another embodiment, the plant 35 is of a species other than Nicotiana benthamiana. In one embodiment, the plant is a ial, for example, a switchgrass. Each of the features described for the plant of the third aspect can be applied mutatis mutandis to the vegetative plant part of the first and/0r second aspects. 11 In an ment, the non-human organism is an oleaginous fungus such as an oleaginous yeast.

In a preferred embodiment, the lipid is extracted without drying the non-human organism or part thereof prior to the extraction. The extracted lipid may subsequently be dried or fractionated to reduce its moisture content.

In further embodiments of this aspect, the invention provides a process for producing extracted lipid from speciﬁc oilseed . In an embodiment, the invention provides a process for producing extracted canola oil, the process comprising the steps of: 10 i) obtaining canola seed comprising at least 45% seedoil on a weight basis, ii) ting oil from the canola seed, and iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) triacylglycerols (TAG), thereby producing the canola oil. In a red embodiment, the canola seed has an 15 oil t on a weight basis of at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55% or at least 56%. The oil content is determinable by measuring the amount of oil that is extracted from the seed, which is threshed ‘seed as commonly harvested, and calculated as a percentage of the seed weight, i.e., % (w/W). re t of the 20 canola seed is between 5% and 15%, and is preferably about 8.5%. In an embodiment, the oleic acid t is between about 58% and 62% of the total fatty acid in the canola oil, preferably at least 63%, and the palmitic acid content is about 4% to about 6% of the total fatty acids in the canola oil. Preferred canola oil has an iodine value of 110-120 and a chlorophyll level of less than 30ppm. 25 In another embodiment, the invention provides a process for producing extracted cornseed oil, the process comprising the steps of: i) obtaining corn seed comprising at least 5% seedoil on a weight basis, ii) extracting oil from the corn seed, and iii) recovering the oil, wherein the recovered oil comprises at least 80%, 30 preferably at least 85% or at least 90% (w/w) triacylglycerols (TAG), thereby producing the cornseed oil. In a red embodiment, the corn seed has an oil content on a seed weight basis (w/w) of at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12% or at least 13%. The re content of the cornseed is about 13% to about 17%, preferably about 15%. Preferred 35 corn oil comprises about 0.1% tocopherols.

In another embodiment, the invention provides a s for producing extracted soybean oil, the process comprising the steps of: i) obtaining soybean seed comprising at least 20% seedoil on a weight basis, 12 ii) extracting oil from the soybean seed, and iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) triacylglycerols (TAG), thereby producing the n oil. In a red embodiment, the soybean seed has an oil t on a seed weight basis (w/w) of at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 31%. In an embodiment, the oleic acid content is between about 20% and about 25% of the total fatty acid in the soybean oil, preferably at least 30%, the linoleic acid content is n about 45% and about 57%, preferably less than 10 45%, and the palmitic acid content is about 10% to about 15% of the total fatty acids in the soybean oil, preferably less than 10%. Preferably the soybean seed has a protein content of about 40% on a dry weight basis, and the moisture t of the n seed is about 10% to about 16%, preferably about 13%.

In r embodiment, the invention provides a process for producing 15 extracted lupinseed oil, the process comprising the steps of: i) obtaining lupin seed comprising at least 10% seedoil on a weight basis, ii) extracting oil from the lupin seed, and iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) triacylglycerols (TAG), 20 thereby producing the lupinseed oil. In a preferred embodiment, the lupin seed has an oil content on a seed weight basis (w/w) of at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least 16%.

In another embodiment, the invention provides a process for producing extracted peanut oil, the process comprising the steps of: 25 i) obtaining peanuts comprising at least 50% seedoil on a weight basis, ii) extracting oil from the peanuts, and iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) triacylglycerols (TAG), thereby producing the peanut oil. In a preferred embodiment, the peanut seed 30 ts) have an oil content on a seed weight basis (w/w) of at least 51%, at least 52%, at least 53%, at least 54%, at least 55% or at least 56%. In an embodiment, the oleic acid content is between about 38% and 59% of the total fatty acid in the peanut oil, preferably at least 60%, and the palmitic acid content is about 9% to about 13% of the total fatty acids in the peanut oil, preferably less than 9%. 35 In r embodiment, the invention provides a process for producing extracted sunﬂower oil, the process comprising the steps of: i) obtaining sunﬂower seed comprising at least 50% l on a weight basis, ii) extracting oil from the sunflower seed, and 13 iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) triacylglycerols (TAG), thereby ing the sunﬂower oil. In a preferred embodiment, the sunﬂower seed have an oil content on a seed weight basis (w/w) of at least 51%, at least 52%, at least 53%, at least 54%, or at least 55%.

In another embodiment, the invention provides a process for producing extracted cottonseed oil, the process comprising the steps of: i) obtaining cottonseed comprising at least 41% seedoil on a weight basis, ii) extracting oil from the cottonseed, and 10 iii) recovering the oil, wherein the red oil comprises at least 90% (w/w) triacylglycerols (TAG), thereby producing the cottonseed oil. In a prefrred embodiment, the cotton seed have an oil content on a seed weight basis (w/w) of at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50%. In 15 an embodiment, the oleic acid content is between about 15% and 22% of the total fatty acid in the cotton oil, preferably at least 22%, the linoleic acid content is between about 45% and about 57%, preferably less than 45%, and the ic acid content is about 20% to about 26% of the total fatty acids in the cottonseed oil, preferably less than 18%. In an embodiment, the cottonseed oil also contains cyclopropanated fatty 20 acids such as sterculic and malvalic acids, and may n small amounts of ol.

In another embodiment, the invention provides a process for producing extracted safﬂower oil, the process comprising the steps of: i) obtaining safﬂower seed comprising at least 35% seedoil on a weight basis, 25 ii) extracting oil from the safﬂower seed, and iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) lglycerols (TAG), thereby producing the safﬂower oil. In a preferred embodiment, the er seed have an oil content on a seed weight basis (w/w) of at least 36%, at least 37%, at least 30 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, or at least 45%.

In another embodiment, the invention es a process for producing extracted ﬂaxseed oil, the process comprising the steps of: i) obtaining ﬂax seed comprising at least 36% seedoil on a weight basis, 35 ii) ting oil from the ﬂax seed, and iii) ring the oil, wherein the recovered oil comprises at least 90% (w/w) triacylglycerols (TAG), 14 thereby producing the flaxseed oil. In a preferred embodiment, the ﬂax seed have an oil content on a seed weight basis (w/w) of at least 37%, at least 38%, at least 39%, or at least 40%.

In another embodiment, the invention provides a s for producing extracted Camelina oil, the process comprising the steps of: i) obtaining Camelina sativa seed comprising at least 36% seedoil on a weight basis, ii) extracting oil from the Camelina sativa seed, and iii) recovering the oil, wherein the recovered oil comprises at least 90% (w/w) 10 triacylglycerols (TAG), thereby producing the Camelina oil. In a preferred ment, the na sativa seed have an oil content on a seed weight basis (w/w) of at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, or at least 45%. 15 The s of the third aspect may also comprise measuring the oil and/or protein content of the seed by near-infrared ance spectroscopy as described in Horn et a1. (2007).

In an embodiment, the process of the third aspect of the ion comprises partially or completely drying the vegetative plant part, or the man organism, 20 or part thereof, or the seed, and/or one or more of rolling, pressing such as ﬂaking, crushing or grinding the tive plant part, or the non—human organism or part thereof, or the seed, or any combination of these methods, in the extraction process.

‘ The process may use an organic solvent (e. g., hexane such as n—hexane or a combination of n-hexane with isohexane, or butane alone or in combination with 25 ) in the extraction process to extract the lipid or oil or to increase the efﬁciency of the extraction process, particularly in combination with a prior drying process to reduce the moisture content.

In an embodiment, the process comprises recovering the extracted lipid or oil by collecting it in a container, and/or purifying the extracted lipid or seedoil, such as, 30 for example, by degumming, deodorising, decolourising, drying and/or fractionating the extracted lipid or oil, and/or removing at least some, preferably substantially all, waxes and/or wax esters from the extracted lipid or oil. The process may se analysing the fatty acid composition of the extracted lipid or oil, such as, for example, by converting the fatty acids in the extracted lipid or oil to fatty acid methyl esters and 35 analysing these using GC to determine the fatty acid composition. The fatty acid composition of the lipid or oil is determined prior to any fractionation of the lipid or oil that alters its fatty acid ition. The extracted lipid or oil may comprise a 15 mixture of lipid types and/or one or more derivatives of the lipids, such as free fatty acids.

In an embodiment, the process of the third aspect of the invention results in substantial quantities of extracted lipid or oil. In an embodiment, the volume of the extracted lipid or oil is at least 1 litre, preferably at least 10 litres. In a preferred embodiment, the extracted lipid or oil is packaged ready for transportation or sale.

In an embodiment, the ted lipid or oil comprises at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% TAG on a weight basis.

The extracted lipid or oil may comprise phospholipid as a minor component, up to 10 about 8% by weight, preferably less than 5% by weight, and more preferably less than 3% by weight.

In an embodiment, the process results in extracted lipid or oil wherein one or more or all of the following features apply: (i) triacylglyeerols comprise at least 90%, preferably at least 95% or 96%, of 15 the extracted lipid or oil, (ii) the extracted lipid or oil comprises free sterols, steroyl esters, steroyl glycosides, waxes or wax esters, or any combination thereof, and (iii) the total sterol t and/or composition in the extracted lipid or oil is signiﬁcantly different to the sterol content and/or composition in the extracted lipid or 20 oil produced from a corresponding man organism or part f, or seed.

In an embodiment, the process further comprises converting the extracted lipid or oil to an industrial product. That is, the extracted lipid or oil is converted post— extraction to another al form which is an industrial product. Preferably, the industrial product is a arbon product such as fatty acid esters, preferably fatty 25 acid methyl esters and/or fatty acid ethyl esters, an alkane such as methane, ethane or a -chain alkane, a mixture of longer chain alkanes, an alkene, a biofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such as ethanol, ol, or butanol, biochar, or a ation of carbon monoxide, hydrogen and biochar.

In the s of either the ﬁrst, second or third aspects of the invention, the 3O vegetative plant part, or the part of the non-human organism may be an aerial plant part or a green plant part such as a plant leaf or stem, a woody part such as a stern, branch or trunk, or a root or tuber. Preferably, the plants are grown in a ﬁeld and the parts such as seed harvested from the plants in the ﬁeld.

In an embodiment, the process further comprises a step of ting the 35 vegetative plant part, non-human organism or part thereof, preferably with a ical harvester.

Preferably, the vegetative plant parts are harvested at a time when the yield of non—polar lipids are at their highest. In one embodiment, the vegetative plant parts are 16 harvested about at the time of ﬂowering. In another ment, the vegetative plant parts are harvested from about at the time of ﬂowering to about the beginning of senescence. In r embodiment, the vegetative plant parts are harvested when the plants are at least about 1 month of age.

If the organism is an algal or fungal organism, the cells may be grown in an enclosed container or in an open-air system such as a pond. The ant organisms comprising the non—polar lipid may be ted, such as, for example, by a process comprising ﬁltration, centrifugation, sedimentation, ﬂotation or ﬂocculation of algal or fungal organisms such as by adjusting pH of the medium. Sedimentation is less 10 red.

In the process of the third aspect of the invention, the total non—polar lipid content of the non-human organism or part thereof, such a vegetative plant part or seed, is increased relative to a corresponding tive plant part, non—human organism or part thereof, or seed. 15 In an embodiment, the vegetative plant part, or non—human organism or part thereof, or seed of the ﬁrst, second or third aspects of the invention is further deﬁned by three es, namely e (i), Feature (ii) and e (iii), singly or in combination: Feature (i) quantiﬁes the extent of the increased level of the one or more non- 20 polar lipids or the total non-polar lipid content of the tive plant part, or non- human organism or part thereof, or seed which may be expressed as the extent of increase on a weight basis (dry weight basis or seed weight basis), or as the relative se compared to the level in the corresponding vegetative plant part, or non— human organism or part thereof, or seed. Feature (ii) speciﬁes the plant genus or 25 species, or the fungal or algal species, or other cell type, and Feature (iii) speciﬁes one or more speciﬁc lipids that are increased in the non—polar lipid content.

For Feature (i), in an embodiment, the extent of the increase of the one or more non-polar lipids is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, 30 at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25% or at least 26% greater on a dry weight or seed weight basis than the corresponding vegetative plant part, or non-human organism or part thereof.

Also for Feature (i), in a preferred embodiment, the total non-polar lipid 35 content of the vegetative plant part, or non-human organism or part thereof, or seed is increased when ed to the corresponding vegetative plant part, or non-human organism or part thereof, or seed. In an embodiment, the total non—polar lipid content is increased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 17 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25% or at least 26% greater on a dry weight or seed weight basis than the ponding vegetative plant part, or non-human organism or part thereof,or seed.

Further, for Feature (i), in an embodiment, the level of the one or more non— polar lipids and/or the total non—polar lipid content is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, 10 at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater on a relative basis than the corresponding vegetative plant part, or non— human organism or part f, or seed. 15 Also for Feature (i), the extent of increase in the level of the one or more non— polar lipids and/or the total non—polar lipid content may be at least 2-fold, at least 3- fold, at least , at least 5-fold, at least 6-fold, at least 7—fold, at least 8—fold, at least 9-fold, at least lO-fold, or at least 12-fold, preferably at least about 13-fold or at least about 15-fold greater on a relative basis than the ponding vegetative plant 20 part, or non-human organism or part f, or seed.

As a result of the increase in the level of the one or more non-polar lipids and/or the total non—polar lipid content as deﬁned in Feature (i), the total non—polar lipid content of the vegetative plant part, or non—human organism or part f, or seed is preferably between 5% and 25%, between 7% and 25%, between 10% and 25 25%, between 12% and 25%, between 15% and 25%, between 7% and 20%, n 10% and 20%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight or seed .

For Feature (ii), in an embodiment, the non-human organism is a plant, alga, or 30 an organism suitable for fermentation such as a yeast or other fungus, preferably an oleaginous fungus such as an oleaginous yeast. The plant may be, or the tive plant part may be from, for example, a plant which is Acrocomia aculeata (macauba palm), Arabidopsis thaliana, Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru), Astrocaryum vulgare (tucuma), Attalea geraensis (Indaia-rateiro), 35 Attalea humilis (American oil palm), Attalea olez‘fera (andaia), Attalea phaleraz‘a (uricuri), Attalea sa (babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica Sp. such as Brassica carinata, Brassica juncea, ca napobrassica, Brassica napus (canola), Camelina sativa (false ﬂax), Cannabis sativa (hemp), 18 Carthamus zinctorius (safﬂower), Caryocar brasilz'emse (pequi), Cocos mucifera (Coconut), Crambe mica (Abyssinian kale), Cucumis melo ), Elaez's guimeemsis (African palm), Glycine max (soybean), Gossypiam hirsutum (cotton), Heliamthus Sp. such as Heliamthus ammuuS (sunﬂower), Hordeum vulgare (barley), Jatropha cureas (physic nut), Joammesz'a primeeps (arara ee), Lemma Sp. (duckweed) such as Lemma aequimoclialis, Lemma disperma, Lemma ecuaa’oriemSiS, Lemma gibba (swollen duckweed), Lemma japomica, Lemma minor, Lemma mimuta, Lemma obscura, Lemma paucicostata, Lemma perpusilla, Lemma , Lemma trisulca, Lemna turiomifera, Lemma valdiviama, Lemma yumgemsis, Licamia rigida 10 (oiticica), Limum usitatissimum (ﬂax), LupimuS ifolius (lupin), Mauritia ﬂexuosa (buriti palm), Maximiliana maripa (inaja palm), Miscamthus Sp. such as Miscamthus x gigamteus and thus SimemSiS, Nicotiama Sp. (tabacco) such as Nicotiama tabacum or Nicotiama bemthamiama, Oemocarpus bacaba a—do— azeite), Oemocarpus bataua (pataua), Oemocarpus distichus (bacaba—de—leque), Oryza 15 Sp. (rice) such as Oryza sativa and Oryza glaberrima, Pamicum virgatum (switchgrass), Paraqueiba paraemsz's (mari), Persea amemcama (avocado), Pomgamia pimmata (Indian beech), Populus trichocarpa, Ricimus commumis (castor), Saccharum Sp. (sugarcane), Sesamum imdicum (sesame), Solamum tuberosum (potato), Sorghum Sp. such as Sorghum r, Sorghum vulgare, Theobroma gramdiforum (cupuassu), 2O ium Sp., Trithrimax brasiliemsz's (Brazilian needle palm), Triticum Sp. (Wheat) such as um aestivum and Zea mayS (corn). In an embodiment, the Brassica mapus plant is of the variety Westar. In an ative embodiment, if the plant is BraSSica mapas, it is of a variety or ar other than Westar. In an embodiment, the plant is of a species other than ArabidopSiS thaliama. In another embodiment, the 25 plant is of a species other than Nicotiama tabacum. In r embodiment, the plant is of a species other than Nicotiama bemthamiama. In one embodiment, the plant is a perennial, for example, a switchgrass. Each of the features described for the plant of the third aspect can be applied mutatiS mutamdis to the tive plant part of the ﬁrst and/0r second aspect. 30 For Feature (iii), TAG, DAG, TAG and DAG, MAG, total saturated fatty acid (PUFA), or a speciﬁc PUFA such as eicosadienoic acid (EDA), arachidonic acid (ARA), alpha linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), tetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or a fatty acid which comprises a yl 35 group, an epoxy group, a cyclopropane group, a double carbon-carbon bond, a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a methylated or hydroxylated branched chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or branched chains, 19 is/are increased or sed. The extent of the increase of TAG, DAG, TAG and DAG, MAG, PUFA, speciﬁc PUFA, or fatty acid, is as deﬁned in Feature (i) above.

In a red embodiment, the MAG is 2-MAG. Preferably, DAG and/or TAG, more preferably the total of DAG and TAG, or MAG and TAG, are increased. In an embodiment, TAG levels are increased without sing the MAG and/0r DAG content.

Also for Feature (iii), in an embodiment, the total fatty acid content and/or TAG content of the total non-polar lipid content comprises (a) at least 2% more, preferably at least 5% more, more preferably at least 7% more, most ably at 10 least 10% more, at least 15% more, at least 20% more, at least 25% more oleic acid, or at least 30% more relative to the non-polar lipid(s) in the corresponding vegetative plant part, or man organism or part thereof, or seed lacking the one or more exogenous polynucleotides. In an embodiment, the total fatty acid content in the non- polar lipid(s) comprises (b) at least 2% less, ably at least 4% less, more 15 preferably at least 7% less, at least 10% less, at least 15% less, or at least 20% less palmitic acid relative to the non—polar lipid(s) in the corresponding vegetative plant part, or non—human organism or part thereof, or seed lacking the one or more exogenous polynucleotides. In an embodiment, the total fatty acid content of the total non-polar lipid content comprises (c) at least 2% less, preferably at least 4% less, 20 more preferably at least 7% less, at least 10% less, or at least 15% less ALA relative to the non-polar lipid(s) in the corresponding vegetative plant part, or non-human organism or part thereof, or seed g the one or more ous polynucleotides.

In an embodiment, the total fatty acid content of the total non-polar lipid content comprises ((1) at least 2% more, ably at least 5% more, more preferably at least 25 7% more, most preferably at least 10% more, or at least 15% more, LA, relative to the non—polar lipid(s) in the corresponding vegetative plant part, or man organism or part thereof, or seed lacking the one or more exogenous polynucleotides. Most preferably, the total fatty acid and/or TAG content of the total non~polar lipid content has an increased oleic acid level according to a ﬁgure deﬁned in (a) and a sed 3O palmitic acid content according to a ﬁgure deﬁned in (b). In an embodiment, the total sterol content is increased by at least 10% relative to seedoil from a corresponding seed. In an embodiment, the extracted lipid or oil comprises at least 10ppm chlorophyll, preferably at least 30ppm chlorophyll. The chlorophyll may subsequently be removed by de-colourising the extracted lipid or oil. 35 In red embodiments, the one or more non-polar lipids and/or the total non-polar lipid content is deﬁned by the combination of Features (i), (ii) and (iii), or Features (i) and (ii), or Features (i) and (iii), or Features (ii) and (iii). 20 The process of the third aspect of the invention provides, in an embodiment, that one or more or all of the following features apply: (i) the level of one or more lar lipids in the vegetative plant part, or non- human organism or part f, or seed is at least 0.5% greater on a weight basis than the level in a corresponding vegetative plant part, non-human organism or part thereof, or seed, tively, lacking the one or more exogenous polynucleotide(s), or preferably as further deﬁned in Feature (i), (ii) the level of one or more non-polar lipids in the vegetative plant part, non- human organism or part thereof, or seed is at least 1% greater on a relative basis than 10 in a corresponding vegetative plant part, non-human organism or part thereof, or seed, respectively, lacking the one or more exogenous cleotide(s), or preferably as further deﬁned in Feature (i), (iii) the total non-polar lipid content in the vegetative plant part, non-human organism or part thereof, or seed is at least 0.5% greater on a weight basis than the 15 level in a corresponding vegetative plant part, non—human sm or part f, or seed, respectively, g the one or more exogenous polynucleotide(s), or preferably as further deﬁned in Feature (i), (iv) the total non-polar lipid content in the vegetative plant part, non-human organism or part thereof, or seed is at least 1% greater on a ve basis than in a 20 corresponding vegetative plant part, non-human organism or part f, or seed, respectively, lacking the one or more exogenous polynucleotide(s), or preferably as further deﬁned in Feature (i), (v) the level of one or more non—polar lipids and/or the total non-polar lipid content of the vegetative plant part, non—human organism or part thereof, or seed, is at 25 least 0.5% greater on a weight basis and/or at least 1% greater on a relative basis than a corresponding vegetative plant part, non—human organism or a part thereof, or seed, respectively, which is lacking the one or more exogenous polynucleotides and which comprises an exogenous polynucleotide encoding an Arabidopsis thaliana DGATl, or preferably as further deﬁned in Feature (i), 30 (vi) the TAG, DAG, TAG and DAG, or MAG content in the lipid in the vegetative plant part, non-human organism or part thereof, or seed, and/or in the extracted lipid therefrom, is at least 10% greater on a ve basis than the TAG, DAG, TAG and DAG, or MAG content in the lipid in a ponding vegetative plant part, non-human organism or a part thereof, or seed lacking the one or more 35 exogenous polynucleotide(s), or a corresponding extracted lipid therefrom, respectively, or preferably as further deﬁned in Feature (i), and (vii) the total polyunsaturated fatty acid (PUFA) content in the lipid in the tive plant part, non—human organism or part thereof, or seed and/or in the 21 extracted lipid therefrom, is increased (e.g., in the presence of a MGAT) or decreased (e.g., in the absence of a MGAT) relative to the total PUFA content in the lipid in a ponding vegetative plant part, non-human organism or part thereof, or seed lacking the one or more exogenous polynucleotide(s), or a corresponding extracted lipid therefrom, respectively, or preferably as r deﬁned in Feature (i) or Feature (iii).

In an embodiment, the level of a PUFA in the tive plant part, non-human organism or part thereof, or seed and/or the extracted lipid rom, is increased relative to the level of the PUFA in a corresponding vegetative plant part, non-human 10 organism or part thereof, or seed, or a corresponding extracted lipid therefrom, respectively, wherein the polyunsaturated fatty acid is eicosadienoic acid, arachidonic acid (ARA), alpha nic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or a combination of two of more thereof. 15 Preferably, the extent of the increase is as defined in Feature (i).

In an embodiment of the third aspect, the corresponding vegetative plant part, or non—human organism or part thereof, or seed is a non-transgenic vegetative plant part, or non—human organism or part thereof, or seed, respectively. In a preferred embodiment, the corresponding vegetative plant part, or non-human sm or part 20 thereof, or seed is of the same ar, strain or variety but lacking the one or more exogenous polynucleotides. In a further preferred ment, the corresponding vegetative plant part, or non-human organism or part thereof, or seed is at the same pmental stage, for example, ﬂowering, as the vegetative plant part, or non- human sm or part thereof, or seed. In another embodiment, the vegetative plant 25 parts are harvested from about at the time of ﬂowering to about the beginning of senescence. In another embodiment, the seed is ted when the plants are at least about 1 month of age.

In an embodiment, part of the non-human organism is seed and the total oil content, or the total fatty acid content, of the seed is at least 0.5% to 25%, or at least 30 1.0% to 24%, greater on a weight basis than a corresponding seed lacking the one or more exogenous polynucleotides.

In an embodiment, the relative DAG content of the seedoil is at least 10%, at least 10.5%, at least 11%, at least 11.5%, at least 12%, at least 12.5%, at least 13%, at least 13.5%, at least 14%, at least 14.5%, at least 15%, at least 15.5%, at least 16%, at 35 least 16.5%, at least 17%, at least 17.5%, at least 18%, at least 18.5%, at least 19%, at least 19.5%, at least 20% greater on a relative basis than of seedoil from a ponding seed. In an embodiment, the DAG content of the seed is increased by 22 an amount as deﬁned in e (i) and the seed is from a genus and/or species as deﬁned in Feature (ii).

In an embodiment, the ve TAG content of the seed is at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, at least 9.5%, at least 10%, or at least 11% greater on an absolute basis relative to a corresponding seed. In an embodiment, the TAG content of the seed is increased by an amount as deﬁned in Feature (i) and the seed is from a genus and/or species as deﬁned in Feature (ii).

In another embodiment, the part of the non-human organism is a vegetative 10 plant part and the TAG, DAG, TAG and DAG, or MAG content of the vegetative plant part is at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30% at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at 15 least 90%, or at least 100% greater on a relative basis than the TAG, DAG, TAG and DAG, or MAG content of a corresponding vegetative plant part lacking the one or more exogenous polynucleotides. In a preferred embodiment, the MAG is 2—MAG.

In an ment, the TAG, DAG, TAG and DAG, or MAG content of the vegetative plant part is determined from the amount of these lipid ents in the 20 extractable lipid of the vegetative plant part. In a further embodiment, the TAG, DAG, TAG and DAG, or MAG content of the enic vegetative plant part is increased by an amount as deﬁned in Feature (i).

‘ In an embodiment, at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%) or at least 60% (mol%), preferably 25 at least 65% (mol%), more preferably at least 66% (mol%), at least 67% (mol%), at least 68% , at least 69% (mol%) or at least 70% (mol%) of the fatty acid content of the total non-polar lipid content of the vegetative plant part, non-human sm or part thereof, or seed, or of the lipid or oil ted therefrom, preferably of the TAG fraction, is oleic acid. Such high oleic contents are preferred for use in 30 biodiesel applications.

In r embodiment, the PUFA content of the vegetative plant part, or non- human organism or part thereof, or seed is increased (e.g., in the presence of a MGAT) or sed (e.g., in the absence of a MGAT) When compared to the corresponding vegetative plant part, or non-human organism or part thereof, or seed. 35 In this context, the PUFA content includes both esteriﬁed PUFA (including TAG, DAG, etc.) and non-esteriﬁed PUFA. In an embodiment, the PUFA content of the vegetative plant part, or non—human organism or part thereof, or seed is preferably determined from the amount of PUFA in the extractable lipid of the vegetative plant 23 part, or non-human organism or part thereof, or seed. The extent of the increase in PUFA content may be as deﬁned in Feature (i). The PUFA content may comprise EDA, ARA, ALA, SDA, ETE, ETA, EPA, DPA, DHA, or a combination of two of more thereof.

In another embodiment, the level of a PUFA in the vegetative plant part, non- human organism or part thereof, or seed, or the lipid or oil extracted therefrom is increased or decreased when compared to the corresponding vegetative plant part, man organism or part thereof, or seed, or the lipid or oil extracted therefrom.

The PUFA may be EDA, ARA, ALA, SDA, ETE, ETA, EPA, DPA, DHA, or a 10 combination of two of more thereof. The extent of the increase in the PUFA may be as deﬁned in Feature (i).

In another embodiment, the level of a fatty acid in the extracted lipid or oil is increased when ed to the lipid extracted from the corresponding vegetative plant part, or non-human organism or part thereof, or seed and wherein the fatty acid 15 comprises a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon- carbon bond, a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a methylated or ylated ed chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or ed chains. The extent of the increase in the fatty acid may be as 20 deﬁned in e (i).

In an embodiment, the level of the one or more non—polar lipids (such as TAG, DAG, TAG and DAG, MAG, PUFA, or a speciﬁc PUFA, or a speciﬁc fatty acid) and/or the total non-polar lipid content is determinable by analysis by using gas chromatography of fatty acid methyl esters obtained from the extracted lipid. 25 Alternate methods for determining any of these contents are known in the art, and include s which do not require extraction of lipid from the organism or part thereof, for example, analysis by near ed (NIR) or nuclear magnetic resonance (NMR).

In a further embodiment, the level of the one or more non—polar lipids and/or 3O the total non-polar lipid content of the tive plant part, or non—human sm or part thereof, or seed is at least 0.5% greater on a dry weight or seed weight basis and/or at least 1% greater on a relative basis, preferably at least 1% or 2% greater on a dry weight or seed weight basis, than a corresponding vegetative plant part, or non- human organism or a part thereof, or seed lacking the one or more exogenous 35 polynucleotides but comprising an exogenous polynucleotide ng an Arabidopsis thaliana DGATl (SEQ ID N0183).

In yet a further ment, the vegetative plant part or the non-human organism or part thereof, or seed further comprises (i) one or more introduced 24 mutations, and/or (ii) an exogenous polynucleotide which down-regulate the production and/or activity of an endogenous enzyme of the vegetative plant part or the non-human organism or part thereof, the endogenous enzyme being selected from a fatty acid acyltransferase such as DGAT, sn-l glycerol-3—phosphate acyltransferase (sn-l GPAT), l-acyl-glycerol-3—phosphate acyltransferase (LPAAT), acyl- CoA:lysophosphatidylcholine acyltransferase (LPCAT), phosphatidic acid phosphatase (PAP), an enzyme involved in starch biosynthesis such as (ADP)-glucose pyrophosphorylase (AGPase), a fatty acid rase such as a A12 fatty acid desaturase (FAD2), a ptide ed in the degradation of lipid and/0r which 10 reduces lipid content such as a lipase such as CGi58 polypeptide or SUGAR- DEPENDENTl triacylglycerol lipase, or a combination of two or more thereof. In an alternative embodiment, the vegetative plant part or the non-human organism or part thereof does not comprise (i) above, or does not comprise (ii) above, or does not comprise (i) above and does not comprise (ii) above. In an embodiment, the 15 exogenous polynucleotide which down—regulates the tion of AGPase is not the polynucleotide disclosed in Sanjaya et al. (2011). In an embodiment, the ous polynucleotides in the vegetative plant part or the non-human organism or part thereof, or seed does not consist of an exogenous polynucleotide encoding a WRIl and an exogenous polynucleotide encoding an RNA molecule which inhibits 20 sion of a gene encoding an AGPase.

In the process of either the first, second or third aspects, the vegetative plant part, or non—human organism or part f, or seed, or the extracted lipid or oil, is further deﬁned in red embodiments. Therefore, in an embodiment one or more or all of the following features apply 25 (i) oleic acid comprises at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%), preferably at least 65% (mol%) or at least 66% (mol%) of the total fatty acid content in the non-polar lipid or oil in the vegetative plant part, non—human sm or part thereof, or seed, 30 ii) oleic acid comprises at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% , or at least 60% (mol%), preferably at least 65% (mol%) or at least 66% (mol%) of the total fatty acid content in the ted lipid or oil, (iii) the non-polar lipid or oil in the vegetative plant part, non-human organism 35 or part thereof, or seed ses a fatty acid which ses a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon-carbon bond, a triple carbon— carbon bond, conjugated double bonds, a branched chain such as a methylated or 25 hydroxylated branched chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the entioned groups, bonds or branched chains, and (iv) the extracted lipid or oil comprises a fatty acid which comprises a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon—carbon bond, a triple carbon—carbon bond, conjugated double bonds, a ed chain such as a methylated or hydroxylated branched chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or branched chains. The fatty acid composition in this embodiment is measured prior to any ation of the fatty acid composition, such as, for example, by fractionating the 10 extracted lipid or oil to alter the fatty acid composition. In preferred embodiments, the extent of the increase is as deﬁned in Feature (i).

In an embodiment, the level of a lipid in the tive plant part, man organism or part thereof, or seed and/or in the ted lipid or oil is determinable by analysis by using gas chromatography of fatty acid methyl esters prepared from the 15 extracted lipid or oil. The method of analysis is preferably as described in Example 1 herein.

Again with respect to either the ﬁrst, second or third aspects, the invention provides for one or more ous polynucleotides in the vegetative plant part, or non-human sm or part thereof, or seed used in the process. Therefore, in an 20 embodiment, the vegetative plant part, or the non-human organism or part f, or the seed ses a ﬁrst exogenous polynucleotide which encodes an RNA or preferably a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid thetic genes in a vegetative plant part, or a non— human organism or a part thereof, or a seed, respectively, and a second ous 25 polynucleotide which encodes an RNA or a polypeptide involved in biosynthesis of one or more non-polar , wherein the ﬁrst and second exogenous polynucleotides are each operably linked to a promoter which is capable of directing expression of the polynucleotide in a vegetative plant part, or a non-human organism or a part thereof, or a seed, respectively. That is, the ﬁrst and second exogenous polynucleotides 3O encode different factors which together provide for the increase in the non-polar lipid content in the vegetative plant part, or the non-human organism or part thereof, or the seed.

The increase is preferably additive, more preferably synergistic, relative to the presence of either the ﬁrst or second exogenous polynucleotide alone. The factors 35 encoded by the ﬁrst and second cleotides operate by different mechanisms.

Preferably, the transcription factor polypeptide increases the availability of substrates for non-polar lipid synthesis, such as, for example, increasing ol—3—phosphate and/or fatty acids preferably in the form of acyl-CoA, by increasing expression of 26 genes, for example at least 5 or at least 8 genes, involved in glycolysis or fatty acid biosynthesis (such as, but not limited to, one or more of ACCase, e transporters (SuSy, cell wall invertases), ketoacyl synthase (KAS), phosphofructokinase (PFK), pyruvate kinase (PK) (for example, 2920, At3g22960), te dehydrogenase, hexose transporters (for example, GPT2 and PPTl), cytosolic fructokinase, cytosolic phosphoglycerate mutase, enoyl-ACP reductase (At2g05990), and phosphoglycerate mutase (Atlg22170)) preferably more than one gene for each category. In an embodiment, the first exogenous polynucleotide encodes a Wrinkled l (WRIl) transcription factor, a Leafy Cotyledon l (Lecl) transcription factor, a 10 Leafy Cotyledon 2 (LEC2) transcription factor, a Fu53 transcription factor, an ABI3 ription factor, a Dof4 transcription factor, a BABY BOOM (BBM) transcription factor or a Dofll transcription factor. In one embodiment, the LEC2 is not an Arabidopsis LEC2. As part of this embodiment, or separately, the second exogenous cleotide may encode a polypeptide having a fatty acid ansferase activity, 15 for example, monoacylglycerol acyltransferase (MGAT) activity and/or diacylglycerol acyltransferase (DGAT) activity, or glycerol-S-phosphate acyltransferase (GPAT) activity. In one embodiment, the DGAT is not an Arabidopsis DGAT.

In a preferred embodiment, the vegetative plant part, or non-human organism 20 or a part f, or the seed, of the ﬁrst, second or third s of the invention comprises two or more exogenous polynucleotide(s), one of which encodes a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in the vegetative plant part, or non—human sm or a part thereof, or seed such as a Wrinkled l (WRIl) transcription factor, 25 and a second of which encodes a polypeptide involved in biosynthesis of one or more lar lipids such as a DGAT.

In an embodiment, the vegetative plant part, non—human organism or a part thereof, or the seed of the first, second or third aspects of the invention may further comprise a third, or more, exogenous polynucleotide(s). The third, or more, 30 exogenous polynucleotide(s) may encode one or more or any combination of: i) a further RNA or transcription factor polypeptide that increases the sion of one or more glycolytic or fatty acid biosynthetic genes in a non-human organism or a part thereof (for example, if the first exogenous cleotide encodes a Wrinkled l (WRII) transcription factor, the third exogenous polynucleotide may 35 encode a LEC2 or BBM transcription factor (preferably, LEC2 or BBM expression lled by an inducible promoter or a promoter which does not result in high transgene expression levels), 27 ii) a further RNA or polypeptide involved in biosynthesis of one or more non— polar lipids (for example, if the second exogenous polynucleotide encodes a DGAT, the third exogenous polynucleotide may encode a MGAT or GPAT, or two further exogenous polynucleotides may be present encoding an MGAT and a GPAT), iii) a polypeptide that stabilizes the one or more non—polar lipids, preferably an Oleosin, such as a polyoleosin or a caleosin, more preferably a eosin, iv) an RNA molecule which ts expression of a gene encoding a polypeptide involved in starch biosynthesis such as a AGPase polypeptide, V) an RNA molecule which inhibits expression of a gene encoding a 10 polypeptide involved in the degradation of lipid and/or which s lipid content such as a lipase such as CGi58 polypeptide or SUGAR—DEPENDENTI triacylglycerol lipase, or Vi) a silencing suppressor polypeptide, wherein the third, or more, exogenous cleotide(s) is operably linked to a 15 promoter which is capable of directing expression of the polynucleotide(s) in a vegetative plant part, or a man organism or a part thereof, or a seed, respectively.

A number of speciﬁc combinations of genes are shown herein to be effective for increasing non-polar lipid contents. Therefore, regarding the process of either the 20 ﬁrst, second or third aspects of the invention, in an embodiment, the vegetative plant part, or the man organism or part thereof, or the seed comprises one or more exogenous cleotide(s) which encode: i) a Wrinkled l (WRIl) transcription factor and a DGAT, ii) a WRll transcription factor and a DGAT and an Oleosin, 25 iii) a WRll transcription factor, a DGAT, a MGAT and an Oleosin, iv) a monoacylglycerol acyltransferase (MGAT), v) a diacylglycerol acyltransferase 2 (DGATZ), vi) a MGAT and a ol—3-phosphate ansferase , Vii) a MGAT and a DGAT, 30 viii) a MGAT, a GPAT and a DGAT, ix) a WRIl transcription factor and a MGAT, x) a WRll transcription factor, a DGAT and a MGAT, xi) a WRIl transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, xii) a DGAT and an Oleosin, or 35 xiii) a MGAT and an Oleosin, and xiv) optionally, a silencing suppressor polypeptide, wherein each of the one or more exogenous polynucleotide(s) is operably linked to a er which is capable of ing expression of the polynucleotide in 28 a tive plant part, or a non—human organism or part thereof, or seed, respectively.

Preferably the one or more ous polynucleotides are stably ated into the genome of the vegetative plant part, or the non~human organism or part thereof, or the seed, and more preferably are t in a homozygous state. The polynucleotide may encode an enzyme having an amino acid sequence which is the same as a sequence of a naturally ocurring enzyme of, for example, plant, yeast or animal origin. Further, the polynucleotide may encode an enzyme having one or more conservative mutations When compared to the naturaly ocurring enzyme.

In an embodiment, 10 (i) the GPAT also has phosphatase activity to e MAG, such as a ptide having an amino acid sequence ofArabidopsis GPAT4 or GPAT6, and/or (ii) the DGAT is a DGAT] or a DGATZ, and/or (iii) the MGAT is an MGATl or an MGAT2.

In a preferred embodiment, the vegetative plant part, the non-human organism 15 or part thereof, or the seed ses a ﬁrst exogenous polynucleotide encoding a WRII and a second exogenous polynucleotide encoding a DGAT, preferably a DGATl.

In another preferred embodiment, the vegetative plant part, the non—human organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide 20 encoding a WRII, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, and a third ous polynucleotide encoding an oleosin.

In a further embodiment, the vegetative plant part, the non—human organism or part thereof, or the seed ses a ﬁrst exogenous polynucleotide encoding a WRIl , a second exogenous polynucleotide encoding a DGAT, ably a DGATI, a third 25 exogenous polynucleotide encoding an oleosin, and a fourth ous polynucleotide encoding an MGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-human organism or part thereof, or the seed comprises a ﬁrst ous polynucleotide encoding a WRII , a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third 30 exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding LEC2 or BBM.

In a further embodiment, the vegetative plant part, the non-human organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide encoding a WRIl , a second exogenous cleotide encoding a DGAT, preferably a DGAT], a third 35 exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an MGAT, preferably an MGAT2, and a ﬁfth exogenous polynucleotide encoding LEC2 or BBM. 29 In a further embodiment, the vegetative plant part, the non-human organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide encoding a WRIl, a second exogenous polynucleotide encoding a DGAT, ably a DGATl, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as CGi58 polypeptide.

In a further embodiment, the vegetative plant part, the non—human organism or part thereof, or the seed comprises a ﬁrst exogenous cleotide encoding a WRIl , a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third 10 exogenous polynucleotide encoding an oleosin, a fourth exogenous cleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGiSS polypeptide, and a ﬁfth exogenous polynucleotide encoding LEC2 or BBM.

In a further embodiment, the vegetative plant part, the non—human organism or 15 part thereof, or the seed comprises a ﬁrst exogenous cleotide encoding a WRIl , a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an RNA molecule which ts sion of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth exogenous polynucleotide encoding an 20 MGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-human organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide ng a WRIl , a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide 25 encoding an RNA molecule which inhibits sion of a gene encoding a lipase such as a CGi58 polypeptide, a ﬁfth exogenous polynucleotide encoding an MGAT, preferably an MGAT2, and a sixth exogenous polynucleotide encoding LEC2 or BBM.

In an embodiment, the seed comprises a ﬁrst exogenous polynucleotide 30 encoding a WRIl, a second exogenous polynucleotide ng a DGAT, preferably a DGATl, a third exogenous cleotide encoding an n, and a fourth exogenous polynucleotide ng an MGAT, preferably an MGAT2. Preferably, the seed further comprises a ﬁfth exogenous polynucleotide ng a GPAT.

Where relevant, instead of a polynucleotide encoding an RNA molecule which 35 ts expression of a gene ng a lipase such as a CGi58 polypeptide, the vegetative plant part, the non—human organism or part thereof, or the seed has one or more introduced mutations in the lipase gene such as a CGi58 gene which confers 30 reduced levels of the lipase polypeptide when compared to a corresponding vegetative plant part, non-human organism or part thereof, or seed lacking the mutation.

In a preferred embodiment, the exogenous polynucleotides ng the DGAT and oleosin are ly linked to a constitutive promoter, or a promoter active in green tissues of a plant at least before and up until ﬂowering, which is capable of directing expression of the polynucleotides in the vegetative plant part, the non—human sm or part thereof, or the seed. In a further preferred embodiment, the exogenous polynucleotide encoding WRII, and RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, is ly 10 linked to a constitutive promoter, a promoter active in green tissues of a plant at least before and up until ﬂowering, or an inducible promoter, which is capable of directing expression of the polynucleotides in the vegetative plant part, the non-human organism or part f, or the seed. In yet a further preferred ment, the exogenous polynucleotides encoding LEC2, BBM and/or MGATZ are operably linked 15 to an inducible promoter which is capable of directing expression of the polynucleotides in the vegetative plant part, the man organism or part thereof, or the seed.

In each of the above embodiments, the cleotides may be provided as separate molecules or may be provided as a contiguous single molecule, such as on a 20 single T-DNA molecule. In an embodiment, the orientation of transcription of at least one gene on the T—DNA molecule is opposite to the orientation of transcription of at least one other gene on the T—DNA molecule.

In each of the above embodiments, the total non-polar lipid content of the vegetative plant part, or non-human organism or part thereof, or the seed, preferably a 25 plant leaf or part thereof, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, or more preferably at least about 15% (w/w dry weight). In a further preferred embodiment, the total non—polar lipid 30 t is between 5% and 25%, between 7% and 25%, between 10% and 25%, between 12% and 25%, between 15% and 25%, between 7% and 20%, between 10% and 20%, between 10% and 15%, between 15% and 20%, between 20% and 25%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight 35 or seed . In a particularly preferred ment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred embodiment, the tive plant part is a leaf portion having a surface area of at least 1 cm2. 31 Furthermore, in each of the above embodiments, the total TAG content of the vegetative plant part, or non-human organism or part thereof, or the seed, preferably a plant leaf or part thereof, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more ably at least about 13%, more preferably at least about 14%, more preferably at least about 15%, or more preferably at least about 17% (w/w dry weight). In a further preferred embodiment, the total TAG content is n 5% and 30%, between 7% and 30%, between 10% and 30%, between 12% and 30%, between 15% and 30%, between 7% 10 and 30%, between 10% and 30%, between 20% and 28%, between 18% and 25%, between 22% and 30%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight or seed weight. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred 15 embodiment, the vegetative plant part is a leaf portion having a surface area of at least 1 cm2.

Furthermore, in each of the above ments, the total lipid content of the vegetative plant part, or man organism or part thereof, or the seed, preferably a plant leaf or part f, stem or tuber, is at least about 3%, more preferably at least 2O about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, more preferably at least about 15%, more ably at least about 17% (w/w dry weight), more preferably at least about 20%, more preferably at least about 25%. In a further preferred embodiment, 25 the total lipid content is n 5% and 35%, between 7% and 35%, between 10% and 35%, between 12% and 35%, between 15% and 35%, between 7% and 35%, between 10% and 20%, between 18% and 28%, between 20% and 28%, between 22% and 28%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, about 22%, or about 25%, each as a 30 tage of dry weight. Typically, the total lipid t of the vegetative plant part, or non—human organism or part f is about 2—3% higher than the non—polar lipid content. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred embodiment, the vegetative plant part is a leaf portion having a surface area of at least 1 cm2. 35 In an embodiment, the tive plant part, the non-human organism or part thereof, or the seed, preferably the vegetative plant part, comprises a ﬁrst exogenous polynucleotide encoding a WRll, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third ous polynucleotide encoding an MGAT, 32 preferably an MGAT2, and a fourth ous polynucleotide encoding an oleosin, wherein the vegetative plant part, non—human organism or part f, or seed has one or more or all of the following features: i) a total lipid content of at least 8%, at least 10%, at least 12%, at least 14%, or at least 15.5% (% weight), ii) at least a 3 fold, at least a 5 fold, at least a 7 fold, at least an 8 fold, or least a 10 fold, at higher total lipid content in the vegetative plant part or non—human organism relative to a corresponding vegetative plant part or non-human sm lacking the exogenous polynucleotides, 10 iii) a total TAG content of at least 5%, at least 6%, at least 6.5% or at least 7% (% weight of dry weight or seed weight), iv) at least a 40 fold, at least a 50 fold, at least a 60 fold, or at least a 70 fold, or at least a 100 fold, higher total TAG content relative to a corresponding vegetative plant part or non-human organism lacking the exogenous polynucleotides, 15 v) oleic acid ses at least 15%, at least 19% or at least 22% (% weight) of the fatty acids in TAG, vi) at least a 10 fold, at least a 15 fold or at least a 17 fold higher level of oleic acid in TAG relative to a corresponding vegetative plant part or non—human sm lacking the exogenous polynucleotides, 20 vii) palmitic acid comprises at least 20%, at least 25%, at least 30% or at least 33% (% weight) of the fatty acids in TAG, viii) at least a 1.5 fold higher level of palmitic acid in TAG relative to a corresponding vegetative plant part or non-human organism lacking the exogenous polynucleotides, 25 ix) linoleic acid comprises at least 22%, at least 25%, at least 30% or at least 34% (% weight) of the fatty acids in TAG, x) a—linolenic acid comprises less than 20%, less than 15%, less than 11% or less than 8% (% weight) of the fatty acids in TAG, and xi) at least a 5 fold, or at least an 8 fold, lower level of OL—linolenic acid in TAG 30 relative to a corresponding vegetative plant part or non human organism lacking the exogenous polynucleotides. In this embodiment, preferably the vegetative plant part at least has feature(s), i), ii) iii), iv), i) and ii), i) and iii), i) and iv), i) to iii), i), iii) and iv), i) to iv), ii) and iii), ii) and iv), ii) to iv), or iii) and iv). In an embodiment, % dry weight is % leaf dry weight. 35 In a further embodiment, the vegetative plant part, the non-human organism or part thereof, or the seed, preferably the vegetative plant part, ses a ﬁrst exogenous polynucleotide ng a WRIl, a second exogenous polynucleotide ng a DGAT, preferably a DGATl, a third ous polynucleotide encoding 33 an oleosin, wherein the vegetative plant part, non-human organism or part thereof, or seed has one or more or all of the following features: 1) a total TAG content of at least 10%, at least 12.5%, at least 15% or at least 17% (% weight of dry weight or seed weight), ii) at least a 40 fold, at least a 50 fold, at least a 60 fold, or at least a 70 fold, or at least a 100 fold, higher total TAG content in the vegetative plant part or non-human organism relative to a corresponding vegetative plant part or non human sm lacking the exogenous polynucleotides, iii) oleic acid comprises at least 19%, at least 22%, or at least 25% (% weight) 10 of the fatty acids in TAG, iv) at least a 10 fold, at least a 15 fold, at least a 17 fold, or at least a 19 fold, higher level of oleic acid in TAG in the vegetative plant part or non—human organism relative to a corresponding vegetative plant part or non—human organism lacking the exogenous polynucleotides, 15 v) palmitic acid comprises at least 20%, at least 25%, or at least 28% (% ) of the fatty acids in TAG, vi) at least a 1.25 fold higher level of palmitic acid in TAG in the vegetative plant part or non-human organism relative to a corresponding tive plant part or non-human organism lacking the exogenous polynucleotides, 20 vii) linoleic acid ses at least 15%, or at least 20%, (% weight) of the fatty acids in TAG, viii) olenic acid comprises less than 15%, less than 11% or less than 8% (% weight) of the fatty acids in TAG, and ix) at least a 5 fold, or at least an 8 fold, lower level of oc—linolenic acid in TAG 25 in the vegetative plant part or non-human organism ve to a corresponding vegetative plant part or man organism lacking the exogenous polynucleotides.

In this embodiment, preferably the vegetative plant part at least has feature(s), i), ii), or i) and ii). In an embodiment, % dry weight is % leaf dry weight.

Preferably, the deﬁned features for the two above embodiments are as at the 30 ﬂowering stage of the plant.

In an alternate ment, the vegetative plant part, the non—human organism or part thereof, or the seed consists of one or more exogenous polynucleotides encoding a DGATl and a LEC2.

In a preferred embodiment, the exogenous cleotide encoding WRIl 35 comprises one or more of the following: 1) nucleotides whose sequence is set forth as any one of SEQ ID NOs123l to 278, 34 ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOS:279 to 337, or a biologically active fragment thereof, iii) nucleotides whose sequence is at least 30% identical to i) or ii), and iv) tides which hybridize to any one of i) to iii) under stringent conditions.

In a preferred embodiment, the exogenous polynucleotide encoding DGAT comprises one or more of the following: i) nucleotides whose ce is set forth as any one of SEQ ID NOs:204 to 10 211, 338 to 346, ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:83, 212 to 219, 347 to 355, or a biologically active fragment thereof, iii) nucleotides whose sequence is at least 30% identical to i) or ii), and 15 iv) a polynucleotide which hybridizes to any one of i) to iii) under stringent conditions.

In another preferred embodiment, the exogenous cleotide encoding MGAT comprises one or more of the following: i) nucleotides whose sequence is set forth as any one of SEQ ID NOs:1 to 44, 20 ii) nucleotides ng a polypeptide sing amino acids whose ce is set forth as any one of SEQ ID NOsz45 to 82, or a biologically active fragment thereof, iii) nucleotides whose sequence is at least 30% cal to i) or ii), and iv) a cleotide which hybridizes to any one of i) to iii) under stringent 25 ions.

In another preferred embodiment, the exogenous polynucleotide encoding GPAT comprises one or more of the following: i) nucleotides whose sequence is set forth as any one of SEQ ID NOsz84 to 143, 30 ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOszl44 to 203, or a ically active fragment thereof, iii) nucleotides whose sequence is at least 30% identical to i) or ii), and iv) a polynucleotide which hybridizes to any one of i) to iii) under stringent 35 conditions.

In another preferred embodiment, the exogenous polynucleotide encoding DGATZ comprises one or more of the following: 35 i) nucleotides whose sequence is set forth as any one of SEQ ID NOs2204 to 21 1, ii) nucleotides encoding a ptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOStZlZ to 219, or a biologically active fragment thereof, iii) nucleotides whose sequence is at least 30% identical to i) or ii), and iv) a polynucleotide which hybridizes to any one of i) to iii) under stringent conditions.

In another preferred embodiment, the exogenous polynucleotide encoding an 10 oleosin comprises one or more of the following: i) tides whose sequence is set forth as any one of SEQ ID NOs:389 to 408, ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID 2 to 388, or a biologically active fragment I5 thereof, iii) nucleotides whose sequence is at least 30% identical to i) or ii), and iv) a ce of nucleotides which hybridizes to any one of i) to iii) under stringent conditions.

In an embodiment, the CGi58 polypeptide comprises one or more of the 20 following: i) nucleotides whose ce is set forth as any one of SEQ ID NOsz422 to 428, ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOs:429 to 436, or a ically active fragment 25 thereof, iii) nucleotides whose sequence is at least 30% identical to i) or ii), and iv) a sequence of nucleotides which hybridizes to any one of i) to iii) under stringent conditions.

In another embodiment, the exogenous polynucleotide encoding LEC2 30 comprises one or more of the following: i) nucleotides whose sequence is set forth as any one of SEQ ID NOS:437 to 439, ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID 2 to 444, or a biologically active fragment 35 thereof, iii) nucleotides whose ce is at least 30% identical to i) or ii), and iv) a sequence of nucleotides which hybridizes to any one of i) to iii) under stringent conditions. 36 In a further embodiment, the exogenous polynucleotide encoding BBM comprises one or more of the ing: 1) nucleotides whose sequence is set forth as any one of SEQ ID NOsz440 or 441 ii) nucleotides encoding a polypeptide comprising amino acids whose sequence is set forth as any one of SEQ ID NOsz445 or 446, or a biologically active fragment thereof, iii) nucleotides whose ce is at least 30% identical to i) or ii), and iv) a sequence of tides which hybridizes to any one of i) to iii) under 10 stringent conditions. y, sequences preferred in one ment can be combined with sequences preferred in another embodiment and more ageously further combined with a sequence preferred in yet another embodiment.

In one ment, the one or more ous cleotides encode a 15 mutant MGAT and/or DGAT and/or GPAT. For example, the one or more exogenous polynucleotides may encode a MGAT and/or DGAT and/or GPAT having one, or more than one, conservative amino acid substitutions as exempliﬁed in Table 1 relative to a wildtype MGAT and/or DGAT and/or GPAT as deﬁned by a SEQ ID NO herein. Preferably the mutant polypeptide has an equivalent or greater activity 20 relative to the non-mutant polypeptide.

In an embodiment, the vegetative plant part, non-human organism or part thereof, or seed comprises a ﬁrst exogenous polynucleotide that {encodes a MGAT and a second exogenous polynucleotide that encodes a GPAT. The ﬁrst and second polynucleotides may be provided as separate molecules or may be provided as a 25 contiguous single molecule, such as on a single T—DNA molecule. In an embodiment, the orientation of transcription of at least one gene on the T-DNA molecule is opposite to the orientation of transcription of at least one other gene on the T—DNA molecule. In a preferred embodiment, the GPAT is a GPAT having phosphatase activity such as an Arabidopsis GPAT4 or GPAT6. The GPAT having phosphatase 30 activity acts to catalyze the formation of MAG from GP (i.e., acylates G—3-P to form LPA and uently removes a phosphate group to form MAG) in the non- human organism or part thereof. The MGAT then acts to catalyze the formation of DAG in the non—human organism or part thereof by acylating the MAG with an acyl group derived from fatty acyl—CoA. The MGAT such as A. thaliana MGATl may 35 also act to catalyze the formation of TAG in the non-human sm or part thereof if it also has DGAT ty.

The vegetative plant part, non—human organism or part thereof, or seed may comprise a third exogenous polynucleotide encoding, for example, a DGAT. The 37 ﬁrst, second and third polynucleotides may be provided as separate molecules or may be provided as a contiguous single molecule, such as on a single T-DNA molecule.

The DGAT acts to se the formation of TAG in the transgenic vegetative plant part, non-human sm or part thereof, or seed by acylating the DAG (preferably produced by the MGAT pathway) with an acyl group derived from fatty oA. In an embodiment, the orientation of transcription of at least one gene on the T—DNA molecule is opposite to the orientation of transcription of at least one other gene on the T—DNA molecule.

In another embodiment, the vegetative plant part, non-human organism or part 10 f, or seed comprises a ﬁrst exogenous polynucleotide that encodes a MGAT and a second exogenous polynucleotide that encodes a DGAT. The ﬁrst and second polynucleotides may be ed as separate molecules or may be provided as a contiguous single molecule, such as on a single T-DNA molecule. In an embodiment, the orientation of transcription of at least one gene on the T—DNA molecule is 15 opposite to the orientation of transcription of at least one other gene on the T-DNA molecule. The vegetative plant part, man organism or part thereof, or seed may comprise a third exogenous polynucleotide encoding, for example, a GPAT, preferably a GPAT having phosphatase activity such as an Arabidopsis GPAT4 or GPAT6. The ﬁrst, second and third polynucleotides may be provided as separate 20 molecules or may be provided as a uous single le.

Furthermore, an endogenous gene activity in the plant, vegetative plant part, or the non—human organism or part thereof, or the seed may be egulated.

Therefore, in an ment, the vegetative plant part, the non-human organism or part thereof, or the seed comprises one or more of: 25 (i) one or more introduced mutations in a gene which encodes an endogenous enzyme of the plant, vegetative plant part, non-human organism or part thereof, or seed, respectively, or (ii) an exogenous polynucleotide which down—regulates the production and/or activity of an endogenous enzyme of the plant, vegetative plant part, man 30 organism or part thereof, or seed, tively, wherein each endogenous enzyme is selected from the group consisting of a fatty acid ansferase such as DGAT, an sn-l glycerol-3—phosphate acyltransferase (sn—l GPAT), a l-acyl-glycerol-3—phosphate acyltransferase (LPAAT), an acyl- CoA:lysophosphatidylcholine acyltransferase ), a phosphatidic acid 35 phosphatase (PAP), an enzyme involved in starch biosynthesis such as (ADP)-glucose pyrophosphorylase (AGPase), a fatty acid desaturase such as a A12 fatty acid desaturase (FAD2), a polypeptide involved in the degradation of lipid and/or which reduces lipid content such as a lipase such as a CGi58 polypeptide or SUGAR- 38 ENTl triacylglycerol lipase, or a combination of two or more thereof. In an embodiment, the exogenous polynucleotide is selected from the group ting of an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a microRNA, a cleotide which encodes a polypeptide which binds the endogenous enzyme, a double stranded RNA le or a processed RNA molecule derived therefrom. In an ment, the exogenous polynucleotide which down- regulates the production of AGPase is not the polynucleotide disclosed in Sanjaya et a1. (2011). In an embodiment, the exogenous polynucleotides in the vegetative plant part or the non-human organism or part thereof, or seed does not consist of an 10 exogenous polynucleotide encoding a WRIl and an exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding an AGPase.

Increasing the level of lar lipids is ant for applications involving particular fatty acids. ore, in an embodiment, the total non-polar lipid, the extracted lipid or oil comprises: 15 (i) non-polar lipid which is TAG, DAG, TAG and DAG, or MAG, and (ii) a speciﬁc PUFA which is EDA, ARA, SDA, ETE, ETA, EPA, DPA, DHA, the speciﬁc PUFA being at a level of at least 1% of the total fatty acid content in the non-polar lipid, or a combination of two or more of the speciﬁc PUFA, or (iii) a fatty acid which is present at a level of at least 1% of the total fatty acid 20 content in the lar lipid and which comprises a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon—carbon bond, a triple carbon—carbon bond, conjugated double bonds, a branched chain such as a ated or hydroxylated branched chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or branched chains. 25 In a fourth aspect, the invention provides non—human organisms, preferably plants, or parts f such as vegetative plant parts or seed, which are useful in the processes of the ﬁrst, second or third aspects or in further aspects described hereafter.

Each of the features in the embodiments described for the ﬁrst, second or third aspects can be applied mutatis is to the non-human organisms, ably plants, or 3O parts thereof such as vegetative plant parts or seed of the fourth aspect. Particular embodiments are emphasized as follows.

In an embodiment of the fourth aspect, the invention provides a plant comprising a vegetative part, or the vegetative part thereof, wherein the vegetative part has a total non-polar lipid content of at least about 3%, more preferably at least 35 about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, or more preferably at least about 15% (w/w dry weight). In a further preferred embodiment, the total non-polar lipid 39 content is between 5% and 25%, n 7% and 25%, n 10% and 25%, between 12% and 25%, between 15% and 25%, between 7% and 20%, n 10% and 20%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more red embodiment, the vegetative plant part is a leaf portion having a e area of at least 1 cm2. In a further embodiment, the non—polar lipid comprises at least 90% triacylglycerols (TAG). Preferably the plant is fertile, morphologically normal, and/or agronomically useful. Seed of the 10 plant preferably germinates at a rate substantially the same as for a corresponding wild~type plant. Preferably the vegetative part is a leaf or a stem, or a combination of the two, or a root or tuber such as, for example, potato tubers.

In r embodiment, the man organism, preferably plant, or part thereof such as tive plant part or seed comprises one or more exogenous 15 polynucleotides as deﬁned herein and has an increased level of the one or more nonpolar lipids and/or the total non-polar lipid content which is at least 2-fold, at least 3- fold, at least 4-fold, at least 5—fold, at least 6-fold, at least 7—fold, at least 8—fold, at least 9-fold, at least lO-fold, or at least l2-fold, preferably at least about 13—fold or at least about 15-fold greater on a relative basis than a corresponding non-human 20 organism, preferably plant, or part thereof such as vegetative plant part or seed lacking the one or more exogenous polynucleotides.

In an embodiment, the invention provides a canola plant sing canola seed whose oil content is at least 45% on a weight basis. Preferably, the canola plant or its seed have es as described in the ﬁrst, second or third aspects of the 25 invention.

In an ment, the invention provides a corn plant comprising corn seed whose oil content is at least 5% on a weight basis. Preferably, the corn plant or its seed have features as described in the ﬁrst, second or third aspects of the invention.

In an ment, the invention provides a soybean plant comprising soybean 30 seed whose oil content is at least 20% on a weight basis. Preferably, the soybean plant or its seed have features as described in the ﬁrst, second or third aspects of the invention.

In an embodiment, the invention provides a lupin plant comprising lupin seed whose oil content is at least 10% on a weight basis. ably, the lupin plant or its 35 seed have features as described in the ﬁrst, second or third aspects of the invention.

In an embodiment, the invention provides a peanut plant comprising peanuts whose oil content is at least 50% on a weight basis. Preferably, the peanut plant or its seed have features as described in the ﬁrst, second or third aspects of the invention. 40 In an embodiment, the invention es a sunﬂower plant sing sunﬂower seed whose oil content is at least 50% on a weight basis. Preferably, the sunﬂower plant or its seed have features as described in the ﬁrst, second or third aspects of the invention.

In an embodiment, the invention provides a cotton plant comprising cotton seed whose oil content is at least 41% on a weight basis. Preferably, the cotton plant or its seed have features as described in the ﬁrst, second or third aspects of the invention.

In an embodiment, the invention provides a safﬂower plant comprising 10 er seed whose oil content is at least 35% on a weight basis. Preferably, the safﬂower plant or its seed have features as described in the ﬁrst, second or third aspects of the invention.

In an embodiment, the ion provides a ﬂax plant comprising ﬂax seed whose oil content is at least 36% on a weight basis. Preferably, the ﬂax plant or its 15 seed have es as described in the first, second or third s of the invention.

In an embodiment, the ion provides a Camelina sativa plant comprising Camelina sativa seed whose oil content is at least 36% on a weight basis. Preferably, the Camelina sativa plant or its seed have features as described in the ﬁrst, second or third aspects of the invention. 20 In embodiments, the plants may be further deﬁned by Features (i), (ii) and (iii) as described hereinbefore. In a preferred embodiment, the plant or the vegetative part comprises one or more or all of the following features: (i) oleic acid in a vegetative part or seed of the plant, the oleic acid being in an esteriﬁed or non—esteriﬁed form, n at least 20% (mol%), at least 22% (mol%), 25 at least 30% , at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%), preferably at least 65% (mol%) or at least 66% (mol%) of the total fatty acids in the lipid t of the vegetative part or seed is oleic acid, (ii) oleic acid in a vegetative part or seed of the plant, the oleic acid being in an esteriﬁed form in non—polar lipid, wherein at least 20% (mol%), at least 22% (mol%), 30 at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%), preferably at least 65% (mol%) or at least 66% (mol%) of the total fatty acids in the non-polar lipid content of the vegetative part or seed is oleic acid, (iii) a modiﬁed fatty acid in a vegetative part or seed of the plant, the modiﬁed fatty acid being in an ﬁed or non-esteriﬁed form, preferably in an esteriﬁed form 35 in non-polar lipids of the vegetative part or seed, wherein the modiﬁed fatty acid comprises a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon- carbon bond, a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a methylated or hydroxylated branched chain, or a combination of two or 41 more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or branched , and (iv) waxes and/or wax esters in the non—polar lipid of the vegetative part or seed of the plant.

In an embodiment, the plant or the vegetative plant part is a member of a population or collection of at least about 1000 such plants or parts. That is, each plant or plant part in the population or tion has essentially the same properties or se the same ous nucleic acids as the other members of the population or collection. Preferably, the plants are homozygous for the exogenous polynucleotides, 10 which provides a degree of uniformity. Preferably, the plants are growing in a ﬁeld.

The collection of vegetative plants parts have preferably been harvested from plants growing in a ﬁeld. Preferably, the vegetative plant parts have been harvested at a time when the yield of non-polar lipids are at their highest. In one ment, the vegetative plant parts have been harvested about at the time of ﬂowering. In another 15 embodiment, the vegetative plant parts are harvested when the plants are at least about 1 month of age. In another embodiment, the vegetative plant parts are harvested from about at the time of ﬂowering to about the beginning of senescence. In another embodiment, the vegetative plant parts are harvested at least about 1 month after induction of expression of inducible genes. 20 In a further embodiment of the fourth aspect, the invention provides a vegetative plant part, non—human organism or a part f, or seed comprising one or more exogenous polynucleotide(s) and an increased level of one or more non-polar lipid(s) relative to a corresponding vegetative plant part, non-human organism or a part thereof, or seed lacking the one or more exogenous polynucleotide(s), wherein 25 each of the one or more exogenous polynucleotides is operably linked to a promoter which is capable of directing expression of the polynucleotide in a vegetative plant part, non-human organism or part f, or seed and wherein one or more or all of the following features apply: (i) the one or more ous polynucleotide(s) comprise a ﬁrst exogenous 30 polynucleotide which encodes an RNA or transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a vegetative plant part, man organism or a part thereof, or seed and a second exogenous cleotide which encodes an RNA or ptide involved in biosynthesis of one or more non-polar lipids, 35 (ii) if the non-human organism is a plant, a vegetative part of the plant has a total non—polar lipid content of at least about 3%, more ably at least about 5%, ably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 42 13%, more preferably at least about 14%, or more preferably at least about 15% (w/w dry weight), (iii) the non-human organism is an alga selected from the group consisting of diatoms (bacillariophytes), green algae ophytes), blue-green algae phytes), golden-brown algae (chrysophytes), haptophytes, brown algae and heterokont algae, (iv) the non—polar lipid(s) comprise a fatty acid which comprises a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon-carbon bond, a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a methylated 10 or hydroxylated branched chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or branched chains, (V) the vegetative plant part, non-human organism or part f, or seed comprises oleic acid in an esterified or non—esterified form in its lipid, wherein at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 15 50% (mol%), or at least 60% (mol%), preferably at least 65% (mol%) or at least 66% (mol%) of the total fatty acids in the lipid of the vegetative plant part, non-human organism or part thereof, or seed is oleic acid, (vi) the vegetative plant part, non-human organism or part thereof, or seed comprises oleic acid in an esteriﬁed form in its non-polar lipid, wherein at least 20% 20 (mol%), at least 22% , at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), or at least 60% (mol%), preferably at least 65% (mol%) or at least 66% (mol%) of the total fatty acids in the lar lipid of the vegetative plant part, non- human sm or part thereof, or seed is oleic acid, (vii) the total fatty acid content in the lipid of the vegetative plant part, non- 25 human organism or part thereof, or seed comprises at least 2% more oleic acid and/or at least 2% less palmitic acid than the lipid in the corresponding vegetative plant part, non—human organism or part f, or seed lacking the one or more exogenous polynucleotides, and/or (viii) the total fatty acid t in the non—polar lipid of the vegetative plant 30 part, non-human organism or part thereof, or seed ses at least 2% more oleic acid and/or at least 2% less palmitic acid than the non—polar lipid in the corresponding vegetative plant part, non-human organism or part thereof, or seed lacking the one or more ous polynucleotides, (ix) the non-polar lipid(s) comprise a d level of total s, preferably 35 free sterols, steroyl esters and/or steroyl glycosides, (x) the non-polar lipid(s) comprise waxes and/0r wax esters, and (xi) the non-human organism or part thereof is one member of a population or collection of at least about 1000 such non-human organisms or parts thereof. 43 In an ment, the one or more exogenous polynucleotide(s) comprise the ﬁrst exogenous cleotide and the second exogenous polynucleotide, and wherein one or more or all of the features (ii) to (xi) apply.

In an embodiment of (ii) above, the total lar lipid content is between 5% and 25%, between 7% and 25%, between 10% and 25%, between 12% and 25%, between 15% and 25%, between 7% and 20%, between 10% and 20%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight. In a more preferred embodiment, the tive plant part is a leaf portion having a e area 10 of at least 1 cm2.

In preferred embodiments, the non-human organism or part thereof is a plant, an alga or an organism suitable for fermentation such as a fungus. The part of the non-human organism may be a seed, fruit, or a vegetative part of a plant such as an aerial plant part or a green part such as a leaf or stem. In another embodiment, the 15 part is a cell of a multicellular organism. With respect to the part of the man organism, the part comprises at least one cell of the non-human organism. In further red embodiments, the non—human organism or part thereof is further deﬁned by features as deﬁned in any of the embodiments bed in the ﬁrst, second or third aspects of the invention, including but not limited to Features (i), (ii) and (iii), and the 20 exogenous polynucleotides or combinations of ous polynucleotides as defined in any of the ments described in the ﬁrst, second or third aspects of the invention.

. In an embodiment, the plant, vegetative plant part, non-human organism or part thereof, or seed comprises one or more exogenous polynucleotides which encode: 25 i) a Wrinkled l (WRIl) transcription factor and a DGAT, ii) a WRIl transcription factor and a DGAT and an Oleosin, iii) a WRIl transcription factor, a DGAT, a MGAT and an Oleosin, iv) a monoacylglycerol acyltransferase (MGAT), v) a diacylglycerol acyltransferase 2 (DGAT2), 30 Vi) a MGAT and a glycerol—3—phosphate acyltransferase (GPAT), Vii) a MGAT and a DGAT, viii) a MGAT, a GPAT and a DGAT, ix) a WRIl transcription factor and a MGAT, x) a WRIl ription factor, a DGAT and a MGAT, 35 xi) a WRIl transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, xii) a DGAT and an Oleosin, or xiii) a MGAT and an Oleosin, and xiv) optionally, a silencing suppressor polypeptide, 44 wherein each exogenous polynucleotide is operably linked to a er which is capable of directing expression of the polynucleotide in a plant, vegetative plant part, non-human organism or part thereof, or seed, respectively. The one or more exogenous polynucleotides may comprise nucleotides whose sequence is deﬁned herein. Preferably, the plant, vegetative plant part, non—human organism or part thereof, or seed is homozygous for the one or more exogenous polynucleotides.

Preferably, the ous polynucleotides are integrated into the genome of the plant, vegetative plant part, non-human organism or part thereof, or seed. The one or more polynucleotides may be provided as separate molecules or may be provided as a 10 contiguous single molecule. Preferably, the ous polynucleotides are integrated in the genome of the plant or organism at a single c locus or genetically linked loci, more preferably in the homozygous state. More preferably, the integrated exogenous polynucleotides are genetically linked with a selectable marker gene such as an herbicide nce gene. 15 In a preferred ment, the vegetative plant part, the non-human organism or part f, or the seed comprises a ﬁrst exogenous polynucleotide encoding a WRIl and a second exogenous polynucleotide encoding a DGAT, preferably a DGATl.

In another preferred embodiment, the vegetative plant part, the non—human 20 organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide encoding a WRII, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, and a third exogenous polynucleotide encoding an oleosin.

In a further embodiment, the vegetative plant part, the non—human organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide ng a WRIl , 25 a second exogenous polynucleotide encoding a DGAT, preferably a DGATI, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding an MGAT, preferably an MGATZ.

In a further embodiment, the vegetative plant part, the non—human organism or part thereof, or the seed comprises a first exogenous polynucleotide encoding a WRIl, 30 a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous cleotide encoding LEC2 or BBM.

In a r embodiment, the vegetative plant part, the man organism or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide ng a WRIl, 35 a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third ous cleotide encoding an n, a fourth exogenous polynucleotide encoding an MGAT, ably an MGATZ, and a ﬁfth exogenous polynucleotide encoding LECZ or BBM. 45 In a further embodiment, the vegetative plant part, the non—human sm or part thereof, or the seed comprises a ﬁrst exogenous polynucleotide encoding a WRIl , a second exogenous polynucleotide encoding a DGAT, preferably a DGATI, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide ng an RNA molecule which inhibits expression of a gene ng a lipase such as a CGi58 ptide.

In a further embodiment, the vegetative plant part, the non-human sm or part thereof, or the seed comprises a ﬁrst exogenous cleotide encoding a WRIl, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third 10 exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth ous polynucleotide encoding LEC2 or BBM.

In a further embodiment, the vegetative plant part, the non—human organism or 15 part thereof, or the seed comprises a ﬁrst exogenous polynucleotide encoding a WRIl , a second exogenous polynucleotide encoding a DGAT, preferably a DGATI, a third exogenous cleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth exogenous polynucleotide encoding an 20 MGAT, preferably an MGAT2.

In a further embodiment, the vegetative plant part, the non-human sm or part thereof, or the seed comprises a first exogenous polynucleotide ng a WRIl, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide 25 encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, a ﬁfth exogenous polynucleotide encoding an MGAT, preferably an MGATZ, and a sixth exogenous polynucleotide encoding LEC2 or BBM.

In an embodiment, the seed comprises a ﬁrst exogenous polynucleotide 30 encoding a WRIl, a second exogenous polynucleotide encoding a DGAT, preferably a DGATI, a third exogenous polynucleotide ng an n, and a fourth exogenous polynucleotide encoding an MGAT, preferably an MGAT2. Preferably, the seed r comprises a ﬁfth exogenous polynucleotide ng a GPAT.

Where relevant, instead of a polynucleotide encoding an RNA molecule which 35 inhibits expression of a gene encoding a lipase such as a CG158 polypeptide, the vegetative plant part, the non-human organism or part thereof, or the seed has one or more introduced mutations in the lipase gene such as a CGi58 gene which confers 46 reduced levels of the lipase polypeptide when compared to an isogenic vegetative plant part, non-human organism or part thereof, or seed lacking the mutation.

In a preferred embodiment, the exogenous polynucleotides encoding the DGAT and oleosin are operably linked to a constitutive promoter, or a promoter active in green tissues of a plant at least before and up until ﬂowering, which is capable of directing expression of the polynucleotides in the vegetative plant part, the non—human organism or part thereof, or the seed. In a further preferred embodiment, the exogenous polynucleotide encoding WRIl, and RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, is operably 10 linked to a constitutive promoter, a promoter active in green tissues of a plant at least before and up until ﬂowering, or an inducible promoter, which is capable of directing expression of the polynucleotides in the vegetative plant part, the non-human organism or part thereof, or the seed. In yet a further preferred embodiment, the ous polynucleotides encoding LECZ, BBM and/or MGATZ are operably linked 15 to an inducible er which is capable of directing expression of the polynucleotides in the vegetative plant part, the non—human organism or part thereof, or the seed.

In each of the above ments, the total lar lipid t of the vegetative plant part, or non-human organism or part thereof, or the seed, preferably a 20 plant leaf or part thereof, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, or more preferably at least about 15% (w/w dry weight or seed weight). In a further red embodiment, the total 25 non~polar lipid content is between 5% and 25%, between 7% and 25%, between 10% and 25%, between 12% and 25%, between 15% and 25%, between 7% and 20%, n 10% and 20%, between 10% and 15%, between 15% and 20%, between 20% and 25%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of 3O dry weight or seed weight. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred embodiment, the vegetative plant part is a leaf portion having a surface area of at least 1 cm2.

Furthermore, in each of the above embodiments, the total TAG t of the vegetative plant part, or non—human sm or part thereof, or the seed, preferably a 35 plant leaf or part thereof, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more ably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more ably at least about 14%, more preferably at least about 47 15%, or more preferably at least about 17% (w/w dry weight or seed ). In a further preferred embodiment, the total TAG content is between 5% and 30%, between 7% and 30%, n 10% and 30%, between 12% and 30%, between 15% and 30%, between 7% and 30%, between 10% and 30%, between 20% and 28%, between 18% and 25%, between 22% and 30%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, or about 22%, each as a percentage of dry weight or seed weight. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred embodiment, the vegetative plant part is a leaf portion 10 having a surface area of at least 1 cm2.

Furthermore, in each of the above embodiments, the total lipid content of the vegetative plant part, or non-human sm or part thereof, or the seed, preferably a plant leaf or part thereof, stem or tuber, is at least about 3%, more preferably at least about 5%, preferably at least about 7%, more preferably at least about 10%, more 15 ably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, more preferably at least about 15%, more preferably at least about 17% (w/w dry weight or seed weight), more preferably at least about 20%, more ably at least about 25%. In a further preferred embodiment, the total lipid content is between 5% and 35%, between 7% 20 and 35%, between 10% and 35%, between 12% and 35%, between 15% and 35%, n 7% and 35%, between 10% and 20%, between 18% and 28%, between 20% and 28%, between 22% and 28%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 20%, about 22%, or about 25%, each as a tage of dry weight or seed weight. Typically, the total 25 lipid content of the tive plant part, or man organism or part thereof is about 2-3% higher than the non-polar lipid content. In a particularly preferred embodiment, the vegetative plant part is a leaf (or leaves) or a portion thereof. In a more preferred embodiment, the vegetative plant part is a leaf portion having a surface area of at least 1 cm2. 30 In an embodiment, the vegetative plant part, the non-human organism or part f, or the seed, preferably the vegetative plant part, comprises a ﬁrst exogenous polynucleotide encoding a WRIl, a second exogenous polynucleotide ng a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an MGAT, preferably an MGAT2, and a fourth exogenous polynucleotide encoding an oleosin, 35 wherein the vegetative plant part, non-human organism or part thereof, or seed has one or more or all of the following features: i) a total lipid content of at least 8%, at least 10%, at least 12%, at least 14%, or at least 15.5% (% weight of dry weight or seed weight), 48 ii) at least a 3 fold, at least a 5 fold, at least a 7 fold, at least an 8 fold, or least a 10 fold, at higher total lipid content in the vegetative plant part or non-human organism relative to a corresponding vegetative plant part or non-human organism g the ous polynucleotides, iii) a total TAG content of at least 5%, at least 6%, at least 6.5% or at least 7% (% weight of dry weight or seed weight), iv) at least a 40 fold, at least a 50 fold, at least a 60 fold, or at least a 70 fold, or at least a 100 fold, higher total TAG content relative to a corresponding vegetative plant part or non-human organism lacking the exogenous polynucleotides, 10 v) oleic acid comprises at least 15%, at least 19% or at least 22% (% weight) of the fatty acids in TAG, vi) at least a 10 fold, at least a 15 fold or at least a 17 fold higher level of oleic acid in TAG relative to a corresponding vegetative plant part or non-human organism lacking the exogenous cleotides, 15 vii) ic acid comprises at least 20%, at least 25%, at least 30% or at least 33% (% weight) of the fatty acids in TAG, viii) at least a 1.5 fold higher level of palmitic acid in TAG relative to a corresponding tive plant part on non—human organism g the exogenous polynucleotides, 2O ix) linoleic acid comprises at least 22%, at least 25%, at least 30% or at least 34% (% weight) of the fatty acids in TAG, x) a—linolenicacid ses less than 20%, less than 15%, less than 11% or less than 8% (% weight) of the fatty acids in TAG, and xi) at least a 5 fold, or at least an 8 fold, lower level of OL-linolenic acid in TAG 25 ve to a corresponding vegetative plant part or non-human organism lacking the exogenous polynucleotides. In this embodiment, preferably the vegetative plant part at least has feature(s), i), ii) iii), iv), i) and ii), i) and iii), i) and iv), i) to iii), i), iii) and iv), i) to iv), ii) and iii), ii) and iv), ii) to iv), or iii) and iv). In an embodiment, % dry weight is % leaf dry weight. 30 In a further embodiment, the vegetative plant part, the non-human organism or part thereof, or the seed, preferably the vegetative plant part, comprises a ﬁrst exogenous polynucleotide encoding a WRIl, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, wherein the vegetative plant part, non—human organism or part thereof, or 35 seed has one or more or all of the ing features: i) a total TAG content of at least 10%, at least 12.5%, at least 15% or at least 17% (% weight of dry weight or seed weight), 49 ii) least a 40 fold, at least a 50 fold, at least a 60 fold, or at least a 70 fold, or at least a 100 fold, higher total TAG content in the vegetative plant part or non-human organism relative to a corresponding vegetative plant part or non-human organism lacking the exogenous polynucleotides, iii) oleic acid comprises at least 19%, at least 22%, or at least 25% (% weight) of the fatty acids in TAG, iv) at least a 10 fold, at least a 15 fold, at least a 17 fold, or at least a 19 fold, higher level of oleic acid in TAG in the vegetative plant part or non-human organism relative to a corresponding vegetative plant part or non-human sm lacking the 10 exogenous cleotides, v) palmitic acid comprises at least 20%, at least 25%, or at least 28% (% weight) of the fatty acids in TAG, vi) at least a 1.25 fold higher level of palmitic acid in TAG in the vegetative plant part or man organism ve to a corresponding vegetative plant part or 15 non—human organism lacking the ous polynucleotides, vii) linoleic acid comprises at least 15%, or at least 20%, (% weight) of the fatty acids in TAG, viii) olenic acid comprises less than 15%, less than 11% or less than 8% (% weight) of the fatty acids in TAG, and 2O ix) at least a 5 fold, or at least an 8 fold, lower level of a-linolenic acid in TAG in the vegetative plant part or non-human organism relative to a corresponding tive plant part or non—human sm. lacking the exogenous polynucleotides.

In this embodiment, preferably the vegetative plant part at least has feature(s), i), ii), or i) and ii). In an embodiment, % dry weight is % leaf dry weight. 25 Preferably, the deﬁned features for the two above embodiments are as at the ﬂowering stage of the plant.

In a fifth aspect, the invention provides a plant or part thereof, or alga comprising one or more exogenous polynucleotide(s) and an increased level of one or more non-polar lipid(s) relative to a corresponding plant or a part thereof, or alga 30 lacking the one or more exogenous polynucleotide(s), wherein the one or more exogenous polynucleotide(s) comprise a ﬁrst exogenous polynucleotide which s a ﬁrst fatty acid acyltransferase polypeptide and one or more further exogenous polynucleotide(s) encoding one or more of the following: (i) an RNA molecule which ts expression of a gene encoding a 35 polypeptide involved in the degradation of lipid and/or which reduces lipid content such as a lipase, (ii) a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a plant or part thereof, or alga, 50 (iii) a second fatty acid acyltransferase polypeptide, or (iv) an RNA molecule which inhibits expression of a gene encoding a polypeptide involved in starch biosynthesis such as a AGPase polypeptide, wherein a vegetative part of the plant, has a total lar lipid content of at least about 7% (w/w dry weight), and wherein, if the transcription factor polypeptide is present, expression of the first fatty acid acyltransferase polypeptide and the transcription factor polypeptide in the plant or part thereof, or alga has an effect on non-polar lipid accumulation in the plant or part thereof, or alga that is larger than an additive effect of the individual effects of each polypeptide alone.

IO In a sixth aspect, the invention es a plant or part thereof, or alga, comprising two or more exogenous cleotides and an increased level of one or more non—polar lipid(s) relative to a corresponding plant or a part thereof, or alga, lacking the two or more exogenous polynucleotides, wherein the two or more exogenous polynucleotides comprise a ﬁrst exogenous polynucleotide which encodes a 15 ﬁrst fatty acid acyltransferase polypeptide and one or more further exogenous polynucleotide(s) encoding one or both of the following: (i) an RNA molecule which inhibits expression of a gene encoding a polypeptide ed in the ation of lipid and/or which reduces lipid content such as a lipase, or 20 (ii) a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a plant or part thereof, or alga, wherein a vegetative part of the plant has a total non—polar lipid content of at least about 7% (w/w dry weight), and wherein, if the transcription factor polypeptide of (ii) is t, the plant or part thereof, or alga, comprises the RNA 25 le of (i).

In a seventh aspect, the invention provides a plant seed capable of growing into a plant of the invention,‘or obtained from a plant of the invention, for example a non— human organism of the invention which is a plant. In an embodiment, the seed comprises one or more exogenous polynucleotides as defined herein. 30 In an eighth aspect, the ion provides a process for ing a cell with enhanced y to produce one or more non-polar , the process comprising the steps of: a) introducing into a cell one or more exogenous polynucleotides, b) expressing the one or more exogenous polynucleotides in the cell or a 35 y cell thereof, c) ing the lipid content of the cell or progeny cell, and 51 d) selecting a cell or progeny cell having an increased level of one or more non— polar lipids relative to a corresponding cell or progeny cell lacking the exogenous polynucleotides, wherein the one or more exogenous polynucleotides encode i) a Wrinkled l (WRIl) transcription factor and a DGAT, ii) a WRIl transcription factor and a DGAT and an Oleosin, iii) a WRIl transcription factor, a DGAT, a MGAT and an Oleosin, iv) a monoacylglycerol acyltransferase (MGAT), v) a diacylglycerol acyltransferase 2 (DGAT2), 10 vi) a MGAT and a glycerol—3-phosphate acyltransferase , vii) a MGAT and a DGAT, viii) a MGAT, a GPAT and a DGAT, ix) a WRIl transcription factor and a MGAT, x) a WRIl transcription factor, a DGAT and a MGAT, 15 xi) a WRIl transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, xii) a DGAT and an Oleosin, or xiii) a MGAT and an Oleosin, and xiv) optionally, a silencing suppressor polypeptide, wherein each exogenous polynucleotide is operably linked to a promoter that is 20 capable of directing expression of the exogenous polynucleotide in the cell or progeny cell.

In a ninth aspect, the invention es a process for obtaining the cell of the invention, the process comprising the steps of: i) introducing into a cell two or more exogenous cleotides, 25 ii) expressing the two or more exogenous polynucleotides in the cell or a progeny cell thereof, iii) ing the lipid content of the cell or progeny cell, and iv) ing a cell or progeny cell having an increased level of one or more non- polar lipids relative to a corresponding cell or progeny cell lacking the 30 exogenous cleotides, n the two or more ous cleotides encode i) a Wrinkled l (WRll) transcription factor and a DGAT, ii) a WRIl transcription factor and a DGAT and an Oleosin, iii) a WRll transcription factor, a DGAT, a MGAT and an Oleosin, 35 iv) a MGAT and a glycerolphosphate acyltransferase (GPAT), v) a MGAT and a DGAT, 52 vi) a MGAT, a GPAT and a DGAT, vii) a WRIl ription factor and a MGAT, viii) a WRll transcription factor, a DGAT and a MGAT, ix) a WRll transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, and x) optionally, a ing suppressor polypeptide, wherein each exogenous polynucleotide is operably linked to a promoter that is capable of directing expression of the exogenous polynucleotide in the cell or progeny cell. 10 In an embodiment, the selected cell or progeny cell comprises: i) a ﬁrst exogenous polynucleotide encoding a WRIl and a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, ii) a first ous cleotide encoding a WRIl, a second ous polynucleotide encoding a DGAT, preferably a DGATl, and a third exogenous 15 polynucleotide encoding an oleosin, iii) a ﬁrst exogenous polynucleotide encoding a WRII, a second exogenous polynucleotide encoding a DGAT, preferably a DGATI, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous cleotide encoding an MGAT, preferably an MGATZ, 20 iv) a first exogenous polynucleotide encoding a WRIl, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide ng an oleosin, and a fourth exogenous polynucleotide encoding LEC2 or BBM, V) a ﬁrst exogenous polynucleotide encoding a WRII, a second exogenous 25 polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide ng an MGAT, preferably an MGATZ, and a ﬁfth exogenous polynucleotide ng LEC2 or BBM, vi) a ﬁrst exogenous polynucleotide encoding a WRIl, a second exogenous 30 polynucleotide encoding a DGAT, preferably a DGATI, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding an RNA molecule which ts sion of a gene encoding a lipase such as a CGi58 polypeptide, vii) a ﬁrst exogenous cleotide encoding a WRIl, a second exogenous 35 polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an 53 RNA molecule which inhibits expression of a gene encoding a lipase such as a CGiS 8 polypeptide, and a fifth exogenous cleotide encoding LEC2 or BBM, viii) a first exogenous polynucleotide encoding a WRIl, a second exogenous cleotide encoding a DGAT, ably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an RNA le which inhibits sion of a gene encoding a lipase such as a CGiS 8 polypeptide, and a ﬁfth exogenous polynucleotide encoding an MGAT, preferably an MGATZ, or ix) a ﬁrst exogenous cleotide encoding a WRIl, a second exogenous 10 polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, a ﬁfth exogenous polynucleotide encoding an MGAT, preferably an MGATZ, and a sixth exogenous polynucleotide encoding LEC2 or BBM. 15 In a further embodiment, the selected cell or progeny cell is a cell of a plant seed and comprises a ﬁrst exogenous polynucleotide encoding a WRIl, a second exogenous polynucleotide encoding a DGAT, preferably a DGATl, a third exogenous cleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding an MGAT, preferably an MGATZ. Preferably, the seed further comprises a fifth 20 ous polynucleotide encoding a GPAT.

In a preferred embodiment, the one or more exogenous cleotides are stably integrated into the genome of the cell or progeny cell.

In a preferred embodiment, the process further comprises a step of regenerating a transgenic plant from the cell or progeny cell comprising the one or more exogenous 25 polynucleotides. The step of regenerating a transgenic plant may be performed prior to the step of expressing the one or more exogenous polynucleotides in the cell or a progeny cell thereof, and/or prior to the step of analysing the lipid content of the cell or progeny cell, and/or prior to the step of selecting the cell or progeny cell having an increased level of one or more non-polar lipids. The process may further comprise a step of obtaining seed or a progeny plant from the transgenic plant, wherein the seed or progeny plant comprises the one or more exogenous polynucleotides.

The process of the eighth or ninth aspect may be used as a ing assay to determine r a ptide encoded by an exogenous polynucleotide has a desired function. The one or more exogenous polynucleotides in these aspects may 35 comprise a sequence as deﬁned above. Further, the one or more exogenous polynucleotides may not be known prior to the process to encode a WRIl transcription 54 factor and a DGAT, a WRll transcription factor and a MGAT, a WRIl transcription factor, a DGAT and a MGAT, a WRIl transcription factor, a DGAT, a MGAT and an Oleosin, a WRIl transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, a WRll transcription factor, a DGAT and an Oleosin, a DGAT and an Oleosin, or a MGAT and an Oleosin, but rather may be candidates therefor. The process therefore may be used as an assay to identify or select cleotides encoding a WRIl transcription factor and a DGAT, a WRIl ription factor and a MGAT, a WRIl transcription factor, a DGAT and a MGAT, a WRIl transcription factor, a DGAT, a MGAT and an Oleosin, a WRll transcription factor, a DGAT, a MGAT, an Oleosin 10 and a GPAT, a WRll transcription , a DGAT and an n, a DGAT and an Oleosin, or a MGAT and an Oleosin. The candidate polynucleotides are introduced into a cell and the products analysed to determine Whether the candidates have the desired function.

In an tenth aspect, the invention provides a enic cell or transgenic plant 15 obtained using a process of the invention, or a vegetative plant part or seed obtained therefrom which comprises the one or more exogenous polynucleotides.

In a eleventh , the invention provides a use of one or more polynucleotides encoding, or a genetic construct comprising polynucleotides encoding: i) a ed 1 (WRIl) ription factor and a DGAT, 20 ii) a WRll transcription factor and a DGAT and an n, iii) a WRll transcription factor, a DGAT, a MGAT and an Oleosin, iv) a monoacylglycerol acyltransferase (MGAT), V) a diacylglycerol acyltransferase 2 (DGATZ), vi) a MGAT and a glycerol—3—phosphate acyltransferase (GPAT), 25 Vii) a MGAT and a DGAT, viii) a MGAT, a GPAT and a DGAT, ix) a WRIl transcription factor and a MGAT, x) a WRIl transcription factor, a DGAT and a MGAT, xi) a WRll transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, 30 xii) a DGAT and an n, or xiii) a MGAT and an Oleosin, and xiv) ally, a silencing suppressor polypeptide, for producing a transgenic cell, a transgenic non-human organism or a part thereof or a transgenic seed having an enhanced ability to produce one or more non-polar lipids 35 relative to a corresponding cell, non-human organism or part thereof, or seed lacking the one or more polynucleotides, wherein each of the one or more polynucleotides is 55 exogenous to the cell, non-human organism or part thereof, or seed and is operably linked to a er which is capable of directing expression of the polynucleotide in a cell, a non-human organism or a part thereof or a seed, respectively.

In an embodiment, the invention provides a use of a ﬁrst cleotide encoding an RNA or transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a cell, a non-human organism or a part thereof, or a seed, together with a second polynucleotide that encodes an RNA or polypeptide involved in biosynthesis of one or more non-polar , for producing a transgenic cell, a transgenic non-human organism or part thereof, or a transgenic seed 10 having an enhanced ability to e one or more non-polar lipids relative to a corresponding cell, non-human organism or part thereof, or seed lacking the ﬁrst and second polynucleotides, n the ﬁrst and second polynucleotides are each exogenous to the cell, non-human organism or part thereof, or seed and are each operably linked to a promoter which is capable of directing expression of the 15 polynucleotide in the transgenic cell, transgenic non-human sm or part thereof, or transgenic seed, respectively.

In a further embodiment, the invention provides a use of one or more polynucleotides for producing a transgenic cell, a transgenic non-human organism or part thereof, or a transgenic seed having an ed ability to produce one or more 20 non-polar lipid(s) relative to a corresponding cell, non-htiman organism or part thereof, or seed lacking the one or more exogenous polynucleotides, wherein each of the one or more polynucleotides is exogenous to the cell, non—human organism or part thereof, or seed and is operably linked to a promoter which is e of directing expression of the cleotide in a cell, a non-human organism or a part thereof, or a seed, 25 respectively, and wherein the non—polar lipid(s) comprise a fatty acid which comprises a hydroxyl group, an epoxy group, a ropane group, a double carbon-carbon bond, a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a methylated or hydroxylated ed chain, or a combination of two or more f, or any of two, three, four, five or six of the aforementioned groups, bonds or branched 3O chains. Such uses also have utility as screening assays.

In a twelfth aspect, the invention provides the use of one or more polynucleotides encoding, or a genetic construct comprising cleotides encoding: i) a Wrinkled 1 (WRll) transcription factor and a DGAT, ii) a WRIl transcription factor and a DGAT and an Oleosin, 35 iii) a WRll ription factor, a DGAT, a MGAT and an Oleosin, iv) a MGAT and a glycerol—3—phosphate acyltransferase (GPAT), 56 v) a MGAT and a DGAT, vi) a MGAT, a GPAT and a DGAT, vii) a WRIl transcription factor and a MGAT, viii) a WRIl transcription factor, a DGAT and a MGAT, ix) a WRII transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, and X) optionally, a silencing suppressor polypeptide, for producing a recombinant plant cell having an enhanced ability to produce one or more lar lipids relative to a corresponding cell lacking the one or more 10 polynucleotides, wherein each of the one or more polynucleotides is exogenous to the cell and is operably linked to a promoter which is capable of directing expression of the polynucleotide in a cell, wherein the cell has a total non-polar lipid content of at least about 7% (w/w dry weight), and wherein, if the WRIl transcription factor polypeptide is present, expression of the ﬁrst fatty acid acyltransferase ptide 15 and the transcription factor ptide in the cell has an effect on non-polar lipid accumulation in the cell that is larger than an additive effect of the individual effects of each polynucleotide alone.

In a enth aspect, the invention provides the use of a ﬁrst polynucleotide encoding a fatty acid ansferase polypeptide, together with a further one or more 20 polynucleotides encoding one or more of the following: (i) an RNA molecule which inhibits expression of a gene encoding a polypeptide involved in the degradation of lipid and/or which reduces lipid content such as a , (ii) a transcription factor polypeptide that ses the expression of one or 25 more glycolytic or fatty acid biosynthetic genes in a plant or part thereof, or alga, (iii) a second fatty acid ansferase polypeptide, or (iv) an RNA molecule which inhibits expression of a gene encoding a polypeptide ed in starch biosynthesis such as a AGPase polypeptide, for producing a transgenic plant or part thereof, or alga, having an enhanced ability to 3O produce one or more non-polar lipids relative to a corresponding plant or part thereof, or alga, lacking the polynucleotide(s), wherein the polynucleotide(s) are exogenous to the plant or part thereof, or alga, and are each ly linked to a promoter which is capable of directing expression of the polynucleotide(s) in the transgenic plant or part thereof, or alga, wherein a vegetative part of the plant, has a total non-polar lipid 35 content of at least about 7% (w/w dry weight), and n, if the transcription 56A factor polypeptide of (ii) is present, the plant or part thereof, or alga, comprises the RNA molecule of (i).

In a fourteenth aspect, the invention provides a process for producing seed, the process comprising: i) g a plant, multiple plants, or non-human organism according to the invention, and ii) harvesting seed from the plant, plants, or non—human organism.

In a preferred embodiment, the process ses growing a population of at least about 1000 such plants in a ﬁeld, and harvesting seed from the population of plants. 10 The harvested seed may be placed in a container and transported away from the ﬁeld, for example ed out of the country, or stored prior to use.

In a ﬁfteenth aspect, the invention provides a fermentation process comprising the steps of: i) providing a vessel containing a liquid composition comprising a non-human 15 organism of the invention which is suitable for fermentation, and constituents required for fermentation and fatty acid biosynthesis, and ii) providing conditions conducive to the fermentation of the liquid composition contained in said vessel.

In a sixteenth aspect, the invention provides a recovered or extracted lipid 20 obtainable by a process of the invention, or obtainable from a vegetative plant part, man organism or part thereof, cell or progeny cell, enic plant, or seed of the invention. The recovered or extracted lipid, ably oil such as seedoil, may have an enhanced TAG content, DAG content, TAG and DAG content, MAG content, PUFA content, speciﬁc PUFA content, or a speciﬁc fatty acid content, and/or total 25 lar lipid t. In a preferred embodiment, the MAG is 2-MAG. The extent of the increased TAG content, DAG t, TAG and DAG content, MAG content, PUFA t, speciﬁc PUFA content, speciﬁc fatty acid content and/or total non- polar lipid content may be as deﬁned in Feature (i).

In a seventeenth aspect, the invention provides an industrial product produced 30 by a process of the invention, preferably which is a hydrocarbon product such as fatty acid , preferably fatty acid methyl esters and/or a fatty acid ethyl esters, an alkane such as methane, ethane or a longer-chain alkane, a mixture of longer chain alkanes, an , a biofuel, carbon monoxide and/or hydrogen gas, a ohol such as ethanol, propanol, or butanol, biochar, or a ation of carbon monoxide, 35 hydrogen and biochar. 57 In an eighteenth aspect, the invention provides a use of a plant, vegetative plant part, non-human organism or a part thereof, cell or progeny cell, transgenic plant produced by a s of the invention, or a seed or a recovered or extracted lipid of the invention for the manufacture of an industrial product. The industrial product may be as deﬁned above.

In a nineteenth aspect, the invention provides the use of the plant or part thereof, or alga of the invention for the manufacture of an industrial product, such as a hydrocarbon product such as fatty acid esters, fatty acid methyl esters and/or a fatty acid ethyl esters, an alkane such as e, ethane or a longer-chain alkane, a 10 e of longer chain alkanes, an alkene, a biofuel, or carbon monoxide and/or hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen and biochar.

In a twentieth , the invention provides a s for ing fuel, the process sing: 15 i) reacting a lipid of the invention with an alcohol, optionally in the ce of a catalyst, to produce alkyl esters, and ii) optionally, blending the alkyl esters with petroleum based fuel. The alkyl esters are preferably methyl esters. The fuel produced by the process may comprise a m level of the lipid of the invention or a hydrocarbon product produced 20 therefrom such as at least 10%, at least 20%, or at least 30% by volume.

In a twenty-ﬁrst , the invention provides a process for ing a synthetic diesel fuel, the process comprising: i) converting lipid in a vegetative plant, non—human sm or part thereof of the invention to a syngas by gasiﬁcation, and 25 ii) converting the syngas to a biofuel using a metal catalyst or a microbial catalyst.

In a -second aspect, the invention provides a process for producing a biofuel, the process comprising converting lipid in a vegetative plant part, non—human organism or part thereof of the invention to bio-oil by pyrolysis, a bioalcohol by 30 fermentation, or a biogas by gasification or anaerobic digestion.

In a further aspect, the invention provides a process for producing a feedstuff, the process sing admixing a plant, vegetative plant part thereof, non-human organism or part thereof, cell or progeny cell, transgenic plant produced by a process of the invention, seed, red or extracted lipid, or an extract or portion f, 35 with at least one other food ingredient.

In a further aspect, the invention provides feedstuffs, cosmetics or chemicals comprising a plant, vegetative part thereof, non-human organism or part thereof, cell or progeny cell, transgenic plant produced by a process of the invention, seed, or a recovered or ted lipid of the invention, or an extract or portion thereof. 57A Natutally, when vegetative al of a plant is to be harvested because of its oil t it is desirable to harvest the material when lipid levels are as high as possible. The t inventors have noted an association between the glossiness of the vegetative tissue of the plants of the invention and oil content, with high levels of 5 lipid being associated with high gloss. Thus, the glossiness of the vegetative material can be used as marker to assist in determining when to harvest the material.

In a further aspect, the invention provides a recombinant cell comprising one or more exogenous polynucleotide(s) and an increased level of one or more non-polar lipid(s) relative to a corresponding cell lacking the one or more exogenous 10 polynucleotide(s), wherein each of the one or more exogenous polynucleotides is operably linked to a promoter which is capable of ing expression of the polynucleotide in a cell, and wherein one or more or all ofthe following features apply: (a) the one or more ous polynucleotide(s) comprise a first exogenous 15 polynucleotide which encodes an RNA or transcription factor polypeptide that increases the expression ofone or more glycolytic or fatty acid biosynthetic genes in a non-human organism or a part thereof, and a second exogenous polynucleotide which encodes an RNA or polypeptide involved in biosynthesis of one or more non-polar lipids, 20 (b) if the cell is a cell of a vegetative part of a plant, the cell has a total nonpolar lipid content of at least about 3%, more preferably at least about 5%, preferably at least about 7%, more ably at least about 10%, more preferably at least about 11%, more preferably at least about 12%, more preferably at least about 13%, more preferably at least about 14%, or more preferably at least about 15% (w/w), 25 (c) the cell is an alga selected from the group consisting of diatoms (bacillariophytes ), green algae (chlorophytes), blue-green algae (cyanophytes), golden-brown algae (chrysophytes), haptophytes, brown algae and heterokont algae, (d) the one or more non-polar s) comprise a fatty acid which comprises a yl group, an epoxy group, a cyclopropane group, a double carbon-carbon bond, 30 a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a ated or hydroxylated ed chain, or a combination of two or more thereof, or any of two, three, four, five or six ofthe aforementioned groups, bonds or branched chains, (e) the total fatty acid content in the non-polar s) comprises at least 2% 35 more oleic acid and/or at least 2% less ic acid than the non-polar lipid(s) in the corresponding cell lacking the one or more exogenous polynucleotides, 578 (f) the lar lipid(s) comprise a modiﬁed level of total sterols, preferably free (non-esterified) sterols, steroyl esters, steroyl ides, relative to the non-polar lipid(s) in the corresponding cell lacking the one or more exogenous polynucleotides, (g) the non-polar lipid(s) comprise waxes and/or wax esters, and (h) the cell is one member of a population or tion of at least about 1000 such cells.

In an embodiment, the one or more exogenous polynucleotide(s) se the ﬁrst ous polynucleotide and the second exogenous polynucleotide, and wherein one or more or all of the features (b) to (h) apply. 10 In a further aspect, the ion provides a inant plant cell comprising two or more exogenous polynucleotide(s) and an increased level of one or more non- polar lipid(s) relative to a corresponding cell lacking the two or more exogenous polynucleotide(s), n the two or more exogenous polynucleotide(s) comprise a ﬁrst exogenous polynucleotide which encodes a ﬁrst fatty acid acyltransferase 15 polypeptide and one or more further ous polynucleotide(s) encoding one or more of the following: (i) an RNA molecule which inhibits expression of a gene encoding a polypeptide involved in the degradation of lipid and/or which reduces lipid content such as a lipase, 20 (ii) or transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a plant cell, (iii) a second fatty acid acyltransferase polypeptide, or (iv) an RNA molecule which inhibits expression of a gene encoding a polypeptide involved in starch biosynthesis such as a AGPase polypeptide, 25 wherein the cell has a total non—polar lipid content of at least 7% (w/w dry weight), and wherein, if the transcription factor polypeptide of (ii) is present, the plant or part thereof, or alga, comprises the RNA le of (i).

In a further aspect, the present invention provides a method of determining when to harvest a plant to optimize the amount of lipid in the vegetative tissue of the plant at 3O harvest, the method sing i) measuring the gloss of the vegetative tissue, ii) comparing the measurement with a pre-determined minimum glossiness level, and 35 57C iii) optionally harvesting the plant.

In another aspect, the present invention provides a method of predicting the quantity of lipid in vegetative tissue of a plant, the nmethod comprising measuring the gloss of the vegetative tissue.

In a preferred embodiment of the two above aspects the vegetative tissue is a leaf(leaves) or a n thereof.

In a further aspect, the present invention provides a method of trading a plant or a part thereof, comprising obtaining the plant or part comprising a cell of the ion, and trading the obtained plant or plant part for pecuniary gain. 10 In an ment, the method further comprises one or more or all of: i) cultivating the plant, ii) harvesting the plant part from the plant, iii) storing the plant or part thereof, or iv) transporting the plant or part f to a different location. 15 In a further aspect, the present invention provides a process for producing bins of plant parts comprising: a) harvesting plant parts comprising a cell of the ion by collecting the plant parts from the plants, or by separating the plant parts from other parts of the , 20 b) optionally, sifting and/or sorting the harvested plant parts, and c) loading the plant parts of a) or the sifted and/or sorted plant parts of b) into bins, thereby ing bins of the plant parts.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless speciﬁcally stated otherwise. 25 In a further aspect, the invention provides an industrial product, an extracted lipid, a fuel or a biofuel produced by the s of the ion.

In a further aspect, the invention provides a process for feeding an animal comprising providing an animal with the plant or part thereof, or alga of the invention, or the feedstuff as described herein. 3O The t invention is not to be limited in scope by the speciﬁc embodiments bed herein, which are intended for the purpose of exempliﬁcation only.

Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this cation, unless speciﬁcally stated otherwise or the 35 context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. 57D The invention is hereinafter described by way of the following non-limiting Examples and with nce to the accompanying ﬁgures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1. A representation of various lipid sis pathways, most of which converge at DAG, a central molecule in lipid synthesis. This model includes one le route to the formation of sn-2 MAG which could be used by a bi—functional MGAT/DGAT for DAG formation from g1ycerol—3-phosphate ). Abbreviations are as follows: 10 GP; glycerol-3—phosphate LysoPA; lysophosphatidic acid PA; phosphatidic acid MAG; monoacylglycerol DAG; diacylglycerol 15 TAG; triacylglycerol Acyl-CoA and FA~COA; acyl-coenzyme A and fatty acyl—coenzyme A PC; phosphatidylcholine GPAT; glycerol-B-phosphate acyltransferase; glycerol-S-phosphate 0- acyltransferase; acyl-CoA:sn-glycerol—3-phosphate 1-O-acyltransferase; EC 2.3.1.15 20 GPAT4; g1ycerolphosphate acyltransferase 4 GPAT6; glycerolphosphate acyltransferase 6 [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ‘58 LPAAT; 1-acyl-glycerol-3~phosphate. acyltransferase; 1-acylglyeerol-3— phosphate O-acyltransferase; oA:l—acyl-sn-g1ycerolphosphate 2 acyltransferase; EC 2.3.1.51 PAP; phosphatidic acid atase; phosphatidate phosphatase; phosphatic acid phosphohydrolase; phosphatidic acid phosphatase; EC 4 MGAT; an acyltransferase having monoacylglycerol acyltransferase (MGAT; 2-acylglycerol O-acyltransferase acyl-CoAr2-acylglycerol O-acyltransferase; EC 2.3.1 .22) activity ‘ ' M/DGAT; an acyltransferase having monoacylglycerol acyltransferase 10 (MGAT; 2-acylglycerol O-acyltransferase; acyl-CoA22-acylglycerol O-' acyltransferase; EC 22) and/or diacylglycerol acyltransferase (DGAT; glycerol O-acyltransferase; acyl-CoA:1,2-diacyl—sn-glycerol O-acyltransferase; EC 2.3.1.20) activity LPCAT; oA:lysophosphatidylcholine acyltransferase; 1- 15 acylglycerophosphocholine O-aeyltransferase; acyl-CoA: l-acyl-sn-glycero phosphocholine O-acyltransferase; EC 2.3.1.23 , PLD-Z; Phospholipase D zeta; choline phosphatase; lecithinase D; lipophosphodiesterase 11; EC 3.1.4.4 CPT; GDP-choline:diacylglycerol cholinephosphotransferase; l 20 aCetylglycerol T cholinephosphotransferase; alkylacylglycerol cholinephosphotransferase; cholinephosphotransferase; phosphorylcholine—glyceﬁde ' ’ ‘ ’ transferase; EC 2.7.8.2 PDCT; phosphatidylcholine:diacylglycerol cholinephosphotransferase PLC; phospholipase C; EC 3.1 ..43 25 PDAT; phospholipid:diacylglycerol acyltransferase; phospholipid: 1 ,2-diacyl- - sn—glycerol O—acyltransferase; EC 2.3.1.158 Pi, inorganic phosphate Figure 2. Relative DAG and TAG increases in ana berithamiana leaf tissue 30 transformed with constructs encoding p19 (negative control), Arabidopsis thaliana DGAT], Mus musculus MGATl and a combination of DGATl and MGATI, each expressed from the 358 promoter. The MGATI enzyme was far more active than the DGATl enzyme in promoting both DAG and TAG accumulation in leaf tissue.

Expression of the MGATl gene resulted in twice as much DAG and TAG 35 accumulationin leaftlssue compared to expression ofDGATl alone.

Figure 3. ve TAG increases in N. benthamiana leaf transformed with cts encoding p19 (negative control), A. thaliana DGATl, M. musculus _ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk .\59 - MGATZ and a combination ofMGATZ and DGATl. Error bars denote standard error ' of triplicate samples.

Figure 4. Radioactivity (DPM) in MAG, DAG and TAG fractions isolated from transiently-transformed N. benthamiana leaf lysates fed with snMAG[14C] and unlabelled oleic acid over a time-course. The constructs used were as for Figure 3.

Figure 5. As for Figure 4 but fed [14C]Go3-P and unlabelled oleic acid. 10 Figure 6. Autoradiogram of TLC plate showing TAG-formation by A. thaliana ' DGAT] and‘M. musculus MGATl but not M. musculus MGATZ in yeast assays. The assay is described in Example 5.

Figure 7. TAG levels in Arabidopsis na T2 and T3 seeds transformed with a 15 chimeric DNA expressing MGATZ relative to parental (untransformed) control. Seeds were harvested at maturity cated). SW: desiccated seed weight. TAG levels are ' given as ug TAG per lOOug seed .

Figure 8. Total fatty acid content in seed of transformed Arabidopsis na ‘ plants transformed with constructs encoding MGATl or MGATZ. 20 Figure 9. Relative TAG level in transiently-transformed N. bentliamiana leaf tissue ed to Arabidopsis thaliana DGAT] loverexpression.

Figure 10. ,TAG conversion from sn—l,2-DAG in DGAT assay from microsomes of 25 N. benthamiana leaf tissues expressing P19 control, opsis thaliana DGATl and Arabidopsis thaliana DGATZ Figure 11. Total FAME quantiﬁcation in A. na seeds transformed with ‘ pJP3382 and pJP3383. < 30' Figure 12. Maximum TAG levels obtained for different gene combinations transiently expressed in N. benthamiana leaves. The V2 negative control represents . » the average TAG level based on 15 ndent repeats. 35 Figure 13. Co-expression of the genes coding for the Arabidopsis na DGAT] acyltransferase and A. thaliana WRI] transcription factor resulted in a synergistic effect on TAG levels in ana benthamiana leaves. Data shown are averages and Ddard deviations of ﬁve independent inﬁltrations.

Substitute Sheet (Rule 26) RO/AU L [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 60 Figure 14 TAG levels in stably-transformed N. benthamiana aerial seedling tissue.

Total lipids were extracted from aerial tissues of N. benthamiana seedlings and analysed by TLC-'FID using an internal DAGE standard to allow accurate comparison . between samples.

. Figure 15. Total fatty acid levels of A. thaliana T2 seed populations transformed with control vector (pORE04), M. musculus MGATl GAT1) or M. musculus - MGAT2 (3SS:MGAT2). a , 10 Figure 16. Map of the insertion region between the left and right borders of pJP3502. TER Lectin denotes the e max lectin terminator; Arath- ’ WRIl, Arabidopsis thaliana WRIl. ription factor coding ; PRO Arath- Rubisco SSU, A. na rubisco small subunit er; Sesin-Oleosin, Sesame 15 indicum oleosin coding region; PRO CaMV3SS-Ex2, cauliﬂower mosaic virus 35$ promoter having a duplicated enhancer region; Arath-DGA’I‘I, A. thaliana DGATI acyltransferase coding region; TER Agrtu-NOS, Agrobacterium tumefaciens nopaline synthase terminator. 20 Figure 17. Schematic representation of the construct pJP3503 including the insertion region between the left and right borders of pJP3503. TER Agrtu-NOS denotes the Agrobacterium tumefaciens nopaline synthase terminator; Musmu— MGAT2, Mus Musculus MGAT2 acyltransferase; PRO CaMV24S-Ex2, cauliﬂoWer mosaic virus 35$ duplicated enhancer region; TER Glyma-Lectin, e max lectin 25 terminator; Arath-WRIl , Arabidopsis’thaliana WRIl transcription factor; PRO Arath- Rubisco SSU, A. thaliana rubisco small subunit promotor; Sesin-Oleosin, Sesame indicum oleosin;Arath-DGAT1, A. thaliana DGATl. acyltransferase Figure 18. TAG yields in different aged leaves of three wild type tobacco plants 30 ) and three pJP3503 primary tran'sformants (4, 29, 21). Leaf stages are indicated by ‘G’, green; ‘YG’, yellow—green; ‘Y’, . Plant stages during sampling were budding, wild type 1; ﬁrst ﬂowers appearing, wild type 2; ng, wild type 3; producing seed pods 503 transformants). 35 Figure 19A. DNA insert containing expression tes for the Umbelopsis ramanniana DGATZA expressed by the Glycine max alpha' subunit beta-conglycinin promoter, Arabidopsis thaliana WRIl expressed by the Glycine max kunitz trypsin Dbitor 3 promoter and the Mus musculus MGAT2 expressed by the Glycine max Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 61v ’alpha' subunit beta-conglycinin promoter. Gene coding regions and expression cassettes are excisable by restriction digestion.

Figure 198. DNA insert containing expression cassettes for the Arabidopsis thaliana LEC2 and WRII transcription factor genes expressed by inducible illus ach pr'omoters,the Arabidopsis thaliana DGATl expressed by the constitutive CaMV-‘ 35S er and the Aspergillus alcR gene expressed by the constitutive CsVMV promoter. Expressed of the LEC2 and WRIl transcription factors is induced by ethanol or an analagous compound. 10 Figure 20. 7 map Figure 21. pJP3569 map 15 KEY TO THE CE LISTING SEQ ID N0:l Mus musculus codon optimised MGATI SEQ ID N012 Mus musculus codon optimised MGAT2 SEQ ID N033 Ciona intestinalis codon optimised MGATI 20 SEQ ID N024 Tribolium castaneum codon optimised MGATI SEQ ID N0:5 Danio rerio codon sed MGATI SEQ ID NO:6 Danio rerio codon optimised MGAT2 SEQ ID N027 Homo sapiens MGATl polynucleotide (AF384163) SEQ ID N028 Mus us MGATl polynucleotide (AF384162) 25 SEQ ID N029 Pan troglodytes MGAT] polynucleotide transcript variant ' (XM_OOl 166055) SEQ. ID N0:10 Pan dytes MGATl polynucleotide transcript variant 2 (XM_0526044.2) , SEQ ID N021 l Canisfamiliaris_MGATl polynucleotide (XM_545667.2) 30 SEQ ID N0:12 Bos taurus MGATI polynucleotide (NM_001001153.2) SEQ ID NO:1 3 Rattus norvegicus MGATl polynucleotide 1108803.1)> SEQ ID N0:l4 Danio rerio MGAT] polynucleotide (NM_001122623.1)' SEQ ID N0:15 Caenorhabditis elegans MGATl cleotide (NM_073012.4) SEQ ID N0216 Caenorhabditis elegans MGATI polynucleotide (NM_182380.5) 35 SEQ ID N0:l7 Caenorhabditis elegans MGATl polynucleotide (NM_065258.3) SEQ ID NO:18 Caenorhabditis elegans MGATI polynucleotide (NM_075068.3) , SEQ ID N0219 Caenorhabditis s MGATI polynucleotide (NM_072248.3) mID N0:20 Kluyveromyces lactis MGATl polynucleotide (XM_455588.1) Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 62 SEQ ID NO:21 Ashbya gossypii MGATI polynucleotide (NM_208895.1) SEQ ID NO:22 Magnaporthe oryzae MGATl polynucleotide (XM;368741.1) SEQ ID NO:23 Ciona intestinalis MGATl cleotide (XM_002120843.1) SEQ ID NO:24 Homo sapiens MGAT2 polynucleotide (AY157608) SEQ ID NO:25 Mus musculus MGAT2 polynucleotide (AY157609) SEQ ID NO:26 Pan troglodytes MGAT2 polynucleotide(XM_522112.2) SEQ ID NO:27 Canisfamiliaris MGAT2 polynucleotide (XM_542304.1) SEQ ID NO:28 Bos taunts MGAT2 polynucleotide (NM_001099136.1) SEQ ID NO:29 Rattus norvegicus MGAT2 polynucleotide (NM_001109436.2) 10 SEQ ID NO:30 Gallus gallus MGAT2 polynucleotide (XM_424082.2) SEQ ID NO:31 Danio rerio MGAT2 polynucleotide (NM_001006083.1) SEQ ID NO:32 Drosophila melanogaster MGAT2 polynucleotide (NM_136474.2) SEQ ID NO:33 Drosophila melanogaster MGAT2 cleotide (NM_136473.2) SEQ ID NO:34 Drosophila melanogaster MGAT2 polynucleotide (NM_136475.2) 15 SEQ ID NO:35 les e MGAT2 polynucleotide (XM_001688709.1) . SEQ ID NO:36 Anopheles gambiae MGAT2 polynucleotide (XM_315985) SEQ ID NO:37 Trzbolzum castaneum MGAT2 polynucleotide 0053.1) SEQ ID NO:38 Homo sapiens MGAT3 polynucleotide (AY229854) SEQ ID NO:39 Pan troglodytes MGAT3 polynucleotide transcriptt variant 1 20 (XM_001154107.1) SEQ ID NO:40 Pan troglodytes MGAT3 cleotide transcript variant 2 (XM_001154171.1) V .

SEQ ID NO:41 Pan troglodytes MGAT3 polynucleotide transcript variant 3 (XM_5278422) 25 SEQ ID NO:42 Canisfamiliaris MGAT3 polynucleotide (XM_845212 1) SEQ ID NO.43 Bos taurus MGAT3 polynucleotide (XM_870406.4) SEQ ID N0:44 Danio rerio MGAT3 polynucleotide (XM_688413.4) SEQ ID N0:45 Homo sapiens MGATI polypeptide (AAK84178.1) SEQ ID N0:46 Mus musculus MGATI ptide 177.1) '30. SEQ ID N0:47 Pan troglodytes MGATl polypeptide isoform 1 (XP_001166055.1) SEQ ID N0:48 Pan troglodytes MGATlpolypeptide isoform 2 6044.2) SEQ ID N0:49 Canisfamiliaris MGATI polypeptide 5667.2) SEQ ID N0250 Bos taurus MGATI polypeptide (NP_001001153.1) SEQ ID NOISI Rattus norvegicus MGATl polypeptide (NP_001102273.1) I .35 SEQ ID N0352 Danio rerio MGATI polypeptide (NP_001116095.1) SEQ ID NO:53 Caenorhabditis elegans MGATI polypeptide (NP_505413.1) SEQ ID NO:54 Caenorhabditis elegans MGATI polypeptide (NP_872180.1) a) ID NO:55 Caenorhabditis elegans IMGATI polypeptide (NP_497659.1) Substitute Sheet Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 63 SEQ-ID NO:56 Caenorhabditis elegans MGATI polypeptide (NP_507469.1) SEQ ID NO:57 Caenorhabditis s MGATl polypeptide 4649.1) ' SEQ ID NOz58 Kluyveromyces lactis MGAT] polypeptide (XP4555881) SEQ ID NO:59 Ashbya gossypii MGATI polypeptide (NP_983542.1) 5 SEQ ID NO:60 Magnaporthe oryzae MGATI polypeptide (XP_368741 1) SEQ ID NO:61 Ciona intestinalis MGATl polypeptide (XP_002120879) SEQ ID NO:62 Homo sapiens MGAT2 polypeptide (AA023672.1) SEQ'ID NO:63 Mus musculus MGAT2 polypeptide (AA023673.1) SEQ ID NO:64 Pan dytes MGAT2 polypeptide (XP_5221 12.2) 10 SEQ ID NO:65 amiliaris MGAT2 polypeptide (XP_542304.1) SEQ ID NO:66 Bos taurus MGAT2 polypeptide (NP_001092606.1) SEQ ID NO:67 Rattus norvegicus MGAT2 polypeptide (NP0011029062) SEQID NO:68 Gallus gallus MGAT2 polypeptide (XP_424082.2) SEQ ID NO:69 Danio rerioMGAT2 polypeptide (NP0010060831) 15 SEQ ID NO:70 Drosophila melanogaster MGAT2 polypeptide (NP_610318.1) SEQ ID NO:71 Drosophila melanogaster MGAT2 polypeptide (NP_610317.1) SEQ ID NO:72 hila melanogaster MGAT2 polypeptide 0319.2) SEQ ID NO:73 Anopheles e MGAT2 ptide (XP_001688761) SEQ ID NO:74 Anopheles gambiae MGAT2 polypeptide (XP_315985.3) 20 \SEQ ID NO:75 1Tribolium castaneum MGAT2 polypeptide (XP_975146) SEQ ID NO:76 Homo sapiens MGAT3 polypeptide (AA063579.1) SEQ ID NO:77 Pan troglodytes MGAT3 polypeptide m 1 (XP_001154107.1) SEQ ID NO:78 Pan troglodytes MGAT3 polypeptide isofonn 2 (XP_001154171.1) ' SEQ ID NO:79 Pan troglodytes MGAT3. m 3 (XP_527842.2) 25 SEQ ID NO:80 Canisfamiliaris MGAT3 polypeptide 0305.1) SEQ ID NO:81 Bos ”taurus MGAT3 ptide (XP_875499.3) .

SEQ ID NO:82 Danio rerio MGAT3 polypeptide (XP_693505.1) SEQ ID NO:83 Arabidopsis thaliana DGATl polypeptide (CAB44774.1) SEQ ID NO:84 Arabidopsis thaliana GPAT4 polynucleotide (NM_100043.4) 30 SEQ ID NO:85 Arabidopsis thaliana GPAT6 polynucleotide 9367.3) SEQ ID NO:86 Arabidopsis thaliana GPAT polynucleotide (AF1951 15.1) SEQ ID NO:87 Arabidopsis thaliana GPAT polynucleotide (AY062466.1) SEQ ID NO:88 Oryza sativa GPAT polynucleotide (AC118133.4) , SEQ ID NO:89 Picea sitchensis GPAT polynucleotide (EF086095.1) 35 SEQ ID NO:90 Zea mays GPAT polynucleotide (BT067649.1) SEQ ID NO:91 Arabidopsis thaliana GPAT polynucleotide (AK228870.1) . SEQ ID NO:92 Oryza sativa GPAT polynucleotide (AK241033.1) D) ID NO:93 Oryza sativa GPAT polynucleotide (CM000127.1) Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 64 SEQ ID NO:94 Oryza sativa GPAT polynucleotide (CM000130.1) ' SEQ ID NO:95 Oryza sativa GPAT polynucleotide (CM000139.1) SEQ ID NO:96 Oryza sativa GPAT polynucleotide (CM000126.1) SEQ ID NO:97 Oryza sativa GPAT polynucleotide (CM000128.1) SEQ ID NO:98 Oryza sativa GPAT cleotide (CM000140.1) SEQ ID NO:99 Selaginella moellendorﬂii GPAT polynucleotide (GL377667.1) SEQ ID N0:100 Selaginella moellendorjﬁi GPAT polynucleotide (GL377667.1) SEQ ID NO:101 Selaginella moellendotﬂii GPAT polynucleotide (GL377648.1) SEQ ID NO: 102 Selaginella moellehdorﬂ’ii GPAT polynucleotide (GL377622.1) 10 SEQ ID NO: 103 nella moellendorﬂii GPAT polynucleotide (GL377590.1) SEQ ID NO: 104 Selaginella ndorﬂii GPAT polynucleotide (GL377576. 1) SEQ ID NO: 105 Selaginella moellendorﬂii GPAT polynucleotide 576.1) SEQ ID NO: 106 Oryza sativa GPAT polynucleotide (NM_00105 1 374.2) SEQ ID NO: 107 Oryza sativa GPAT polynucleotide (NM_001052203.1) 15 SEQ ID N0:108: Zea mays GPAT8 polynucleotide (NM_001153970.1) SEQ ID NO: 109: Zea mays GPAT polynucleotide 1155835.1) SEQ ID N0:1 10: Zea mays GPAT polynucleotide (NM_001174880.1) SEQ ID N02111 Brassica napus GPAT4 polynucleotide (JQ666202.1) SEQ ID N0:112 Arabidopsis-thaliana GPAT8 polynucleotide 6264.5) 20 SEQ ID N0:113 Physcomitrella patens GPAT polynucleotide (XM_001764949. 1) SEQ ID N02114 Physcomitrella patens GPAT polynucleotide (XM_001769619.1) SEQ ID N0:115 mitrella patens GPAT polynucleotide (XM_001769672.1) SEQ ID N0:116 mitrella patens GPAT cleotide (XM_001771 134.1) SEQ ID N0:1l7 Physcomitrella patens GPAT polynucleotide (XM_001780481.1) 25 SEQ ID N0:118 Vitis vinifera GPAT polynucleotide (XM_002268477.1) SEQ ID N0:119 Vitis vini'fera GPAT polynucleotide (XM_002275312.1) SEQ ID N0:120 Vitis vinifera GPAT polynucleotide'(XM_002275996.1) SEQ ID N0:121 Vitis viniﬁzra GPAT polynucleotide (XM_002279055J) SEQ ID N0:122 Populus trichocarpa GPAT cleotide (XM_002309088.1) 30 SEQ ID N0:123 Populus trichocarpa GPAT polynucleotide (XM_002309240.1) SEQ ID N0:124 Populus trichocarpa GPAT polynucleotide (XM_002322716. l) SEQ ID N0:125 Populus trich_ocarpa GPAT polynucleotide (XM_002323527.1) SEQ ID N0:126 Sorghum bicolor GPAT polynucleotide (XM_002439842.1) SEQ ID NO: 127 Sorghum bicolor GPAT polynucleotide 2458741. 1) 35 SEQ ID N0:128' Sorghum bicolor GPAT polynucleotide (XM_002463871 . l) SEQ ID N0:129 Sorghum bicolor GPAT polynucleotide (XM_002464585.1) SEQ ID N0:130 Ricinus communis GPAT polynucleotide (XM_00251 ) DQ ID N0:131 Ricinus communis GPAT cleotide (XM_002517392.1) Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 65 SEQ ID NO:132 Ricinus communis GPAT cleotide (XM_002520125.1) SEQ ID NO:133 Arabidopsis lyrata GPAT cleotide 2872909.1) SEQ ID NO:134 Arabidopsis lyrata GPAT6 polynucleotide (XM_002881518.1) SEQ ID NO 135 . Verniciafordii putative GPAT8 polynucleotide (FJ479753.1) SEQ ID NO 136 Oryza sativa GPAT polyn'ucleotide (NM_001057724.1) SEQ ID NO:137 Brassica napus GPAT4 polynucleotide (JQ666203.1)SEQ ID NO- 138 Populus trichocarpa GPAT polynucleotide (XM_002320102.1) ‘ SEQ ID NO:1'39 Sorghum bicolor GPAT polynucleotide 2451332.1) SEQ ID NO:140 Ricinus communis GPAT polynucleotide (XM_002531304.1) 10 SEQ ID NO:141 Arabidopsis Iyrata GPAT4 polynucleotide (XM_002889315.1) SEQ ID NO:142 Arabidopsis thaliana GPATl polynilcleotide (NM_100531.2) SEQ ID NO 143 Arabidopsis thaliana GPAT3 polynucleotide (NM_116426.2) SEQ ID NO:144 Arabidopsis thaliana GPAT4 polypeptide (NP_171667.1) SEQ ID NO:145 Arabidopsis thaliana GPAT6 ptide (NP_181346.1) 15 SEQ ID NO:146 Arabidopsis thaliana GPAT polypeptide 784.1) ' SEQ ID NO:147 .Arabidopsis thaliana GPAT polypeptide (AAL32544.1) ' SEQ ID NO:148 Oryza sativa GPAT polypeptide (AAP03413.1) SEQ ID NO:149 Picea sitchensis GPAT polypeptide (ABK25381.1) SEQ ID NO:150 Zea mays GPAT polypeptide (ACN34546.1) 20 SEQ NO ID: 1 51 Arabidopsis thaliana‘GPAT polypeptide 762.1) I SEQ ID NO:152 Oryza sativa GPAT polypeptide 933.1) SEQ ID NO:153 Oryza sativa GPAT polypeptide (EAY84189.1) SEQ ID NO:154 Otyza eativa GPAT polypeptide (EAY98245.1) SEQ ID NO:155 Oryza sativa GPAT polypeptide (EA221484.1) 25 SEQ ID NO:156 Oryza sativa GPAT polypeptide (EEC71826.1) SEQ ID N011 57 Oryza sativa GPAT polypeptide 137.1) SEQ ID NO:158 Oryza sativa GPAT polypeptide (EEE59882.1) p SEQ ID NO:159 Selaginella moellendorﬂii GPAT polypeptide (EFJ08963.1) SEQ ID NO:160 Selaginella moellendorﬂii GPAT polypeptide 964.1) 30 SEQ ID NO:161 Selaginella ndorﬂii GPAT polypeptide (EF111200.1) SEQ ID NO:162 Selaginella moellendarﬂii GPAT polypeptide (EF11566411) SEQ ID NO:163 Selaginella moellendorﬂii GPAT polypeptide (EFJZ4086.1) SEQ ID NO:164 Selaginella moellendorﬂii GPAT polypeptide 816.1) SEQ ID NO:165 Selaginella moellendorﬂ’ii GPAT polypeptide (EFJ29817.1) 35 SEQ ID NO:166 Oryza sativa GPAT polypeptide (NP_001044839:1) SEQ ID NO:167 Oryza sativa GPAT polypeptide (NP_001045668.1) . SEQ ID NO:168 Zea mays GPAT 8 polypeptide (NP_001147442.1) D) ID NO:169 Zea mays GPAT polypeptide (NP_001149307.1) Substitute Sheet .

(Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 66' SEQ ID NO: 170 Zea mays protein GPAT polypeptide (NP_001168351.1) SEQ ID NO:171 Brassica napus GPAT4 polypeptide (AFH02724.1) SEQ ID NO: 172 Arabidopsis thaliana GPAT8 polypeptide (NP_1 91950.2) SEQ ID NO: 173 Physcomz'trella patens GPATpolypeptide (XP_001765001.1) SEQ ID NO:174 Physcomitrella patens GPAT polypeptide 1769671.1) SEQ ID NO 175 Physeomitrella patens GPAT polypeptide (XP_001769724.1) SEQ ID NO:176 Physcomitrella pateris GPAT polypeptide (XP_001771186.1) SEQ ID NO:177 Physcomitrella patent GPAT polypeptide'(XP_OOl 780533.1) SEQ ID NO: 178 Vitis vinifera GPAT polypeptide (XP_002268513J) 10 SEQ ID NO:179 Vitis vinifera GPAT polypeptide (XP_002275348.1) SEQ ID NO:180 Vitis vinifera GPAT polypeptide (XP_002276032J) , SEQ ID N01‘18I Vitis ra GPAT polypeptide (XP_002279091.1) SEQ ID NO:182 Populus trichocarpa GPAT polypeptide (XP_002309124.1) SEQ ID NO:183 Populus trichocarpa GPAT polypeptide (XP_002309276.1) 15 SEQ ID NO:184 Populus trichocarpa GPAT polypeptide (XP_002322752._1) SEQ ID NO:185 Populus trichocarpa GPAT polypeptide (XP_002323563.1) ' SEQ ID NO: 186 Sorghum bicolor GPAT polypeptide (XP_002439887.1) SEQ ID NO: 187 Sorghum r GPAT polypeptide (XP_002458786.1) SEQ ID NO: 188 Sorghum bicolor GPAT polypeptide (XP_002463916.1) 20 SEQ ID NO:189 Sorghum bicolor GPAT polypeptide (XP_002464630.1) SEQ ID NO:190 Ricinus communis GPAT ptide (XP_002511873.1) SEQ ID NO:191 Ricinus communis GPAT polypeptide (XP_002517438.1) SEQ ID NO:192 s is GPAT polypeptide (XP_002520171.1) SEQ ID NO:193 Arabidopsis lyratd GPAT polypeptide (XP_002872955.1) 25 SEQ ID NO: 194 Arabidopsis lyrata GPAT6 polypeptide (XP_002881564.1) SEQ ID NO:195- Verniciafordii GPAT polypeptide (ACT32032.1) SEQ ID NO:196 Oryza sativa GPAT polypeptide (NP_001051189.1) SEQ ID NO:197 Brgssica napus GPAT4 polypeptide (AFH02725.1) SEQ ID NO:198 .Populus carpa GPAT polypeptide (XP_002320138.1) 30 SEQ ID NO: 199 Sorghum bicolor GPAT polypeptide 2451377.1) SEQ ID N02200 Ricinus communis GPAT polypeptide (XP_00253 1350.1) SEQ ID NO:201 Arabidopsis lyrata GPAT4 ptide 2889361.1) SEQ ID NO:202 Arabidopsis thaliana GPATl polypeptide-(NP_563768.1) SEQ ID N02203 Arabidopsis thaliana GPAT3 polypeptide (NP__192104.1) ' ‘35 SEQ ID NO:204 Arabidopsis thaliana DGATZ polynucleotide (NM_115011.3) SEQ ID N02205 Ricinus communis DGAT2 polynucleotide (AY916129.1) SEQ ID NO:206 Vernicia‘fordii DGATZ polynucleotide (DQ356682.1) a) ID NO:207 rella ramanniana DGAT2 polynucleotide (AF3910893) 5 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 67 SEQ ID NO:208 Homo sapierts DGAT2 polynucleotide (NM_032564.I) SEQ ID NO:209 Homo sapiéns DGAT2 polynucleotide (NM_001013579.2) SEQ ID NO:210 Bos taurus DGAT2 polynucleotide (NM_205793.2) SEQ ID NO:211 Mus musculus DGAT2 polynucleotide (AF384160.1) SEQ ID NO:212 Arabidopsis thaliana DGAT2 polypeptide (NP_566952.1) SEQ ID NO:213 Ricinus is DGAT2 polypeptide (AAY16324.1) SEQ ID NO:214 Verniciaﬁirdii DGAT2 polypeptide 474.l) SEQ ID NO:215 Mortierella ramanniana DGAT2 polypeptide (AAK84179.1) SEQ ID NO:216 Homo s DGAT2 polypeptide (Q96PD7.2) 10 SEQ ID NO:217 Homo sapiens DGAT2 polypeptide (Q58HT5.1) SEQ ID NO:218 Bos taurus DGAT2 polypeptide (Q70VZ8.1) SEQ ID NO:219 Mus musculus DGAT2 polypeptide (AAK84175.I) SEQ ID NO:220 YFP tripeptide — conserved DGAT2 and/or 2 ce motif 15 SEQ ID NO:221 HPHG tetrapeptide — conserved DGAT2 and/or MGATl/2 sequence motif .

SEQ ID NO:222 EPHS tetrapeptide — conserved plant DGAT2 sequence motif _ SEQ ID NO:223 RXGFX(K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q) — long 1 conserved sequence motif of DGAT2 which is part of the putative glycerol 20 phospholipid domain ‘ SEQ ID NO:224_ XN— conserved ce motif of mouse DGAT2 and Z which is a putative neutral lipid binding domain SEQ ID NO:225 plsC acyltransfetase domain (PF01553) ofGPAT _ SEQ ID NO:226 HAD-like hydrolase (PF12710) superfamily domain ofGPAT 25 SEQ ID NO:227 Phosphoserinc phosphatase domain (PF00702). GPAT4—8 contain a ' N-tenninal region homologous to this domain SEQ ID NO:228 Conserved GPAT amino acid sequence GDLVICPEGTTCREP SEQ ID NO:229 Conserved GPAT/phosphatase amino acid sequence (Motif I) SEQ ID NO:230 Conserved GPAT/phosphatase amino acid sequence (Motif III)‘ 30 SEQ ID NO:231 Arabidopsis thaliana WRII polynucleotide 2701.2) SEQ ID NO:232 Arabidopsis thaliana WRIl polynucleotide (NM_001035780.2) .

SEQ ID NO:233 Arabidopsis thaliana WRII polynucelotide (NM_115292.4) I SEQ ID NO:234 Arabidopsis lyrata subsp. lyrata polynucleotide (XM_002876205.1) SEQ ID NO:235 Brassica napus WRII polynucelotide (DQ370141.1) 35 SEQ ID NO:236 Brassica napus WRII polynucleotide-(HM370542.1) SEQ ID NO:237 Glycine max WRIlpolynucelotide (XM_003530322.1) SEQ ID NO:238 Jatropha curcas WRIl polynucleotide (JF703666J) a) ID NO:239 Ricinus communis WRIl cleotide (XM_002525259.1) Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 68 SEQ ID NO:240 Populus trichocarpa WRII polynuCleotide (XM_002316423.1) SEQ ID NO:241 Brachypodium distachyon WRIIpolynucleotide 3578949.1) SEQ ID NO:242 Hordeum vulgare subsp. vulgare WRII polynucleotide (AK355408.1) SEQ IDVN02243 Sorghum bicolor WRII polynucelotide (XM_002450149.1) SEQ ID NO:244 Zea mays .WRII polynucleotide (EU960249.1) ' I SEQ ID NO:245» Brachypodium distachyon WRII polynucelotide .

(XM_003561141.1) ' ' .

SEQ ID NO:246 Sorghum r WRII polynucleotide (XM_002437774.1) 10 SEQ ID NO:247 Sorghum bicolor WRII polynucleotide (XM_002441399.1) SEQ ID NO:248 Glycine max WRII polynucleotide (XM_00353063 8.1) SEQ ID NO:249 Glycine max WRII cleotide(XM_003553155.1) SEQ ID NO:250 'Populus trichocarpa WRII polynucIeotide 2315758.1) SEQ ID NO:251 Vitis ra WRII polynucleotide(XM_002270113.1) 15 SEQ ID NO:252 Glycine max WRII polynucleotide (XM_OO3533500.1) SEQ ID NO:253 Glycine max WRII polynuqleotide (XM_003551675.1) SEQ ID NO‘2254 Medicago truncatula WRII polynucleotide 3621069.1) SEQ ID NO:255 Populus trichocarpa WRII polynucleotide (XM_002323800. 1) SEQ ID NO:256 Ricinus communis WRII polynucleotide (XM_OOZS 17428.1) 20 SEQ ID NO:257 WRII . Brachypodium distachyon polynucleotide (XM_003572188.1) SEQ ID NO:258 m bicolor WRII polynucleotide (XM_0024443 84. l) - SEQ ID NO:259 Zea mays WRII polynucleotide (NM_001 176888 .1) SEQ ID NO:260 Arabidopsis lyrata subsp. lyrata WRIl polynucleotide 25 (XM_002889219.1) ‘ SEQ ID NO:261 opsis thaliana WRII polynucleotide (NM_106619.3) SEQ ID NO:262 Arabidopsis lyrata subsp. lyrata WRII polynucleotide ' (XM_002890099.1) ' ' ‘ SEQ ID NO:263 Ihellungiella halophila WRII polynucleotide (AK352786.1) 3O SEQ ID NO:264 Arabidopsis thaliana WRIl cleotide (NM_101474.2) SEQ ID NO:265 Glycine max WRII polynucleotide 3530302.1) SEQ ID NO:266 Brachypodium distachyon WRII polynucleotide ~ (XM_003578094.1) SEQ ID NO:267 Sorghum r WRII polynucleotide (XM_002460191.1) 35 SEQ ID NO:268 Zea mays WRIl' polynucleotide (NM_001152866.1) SEQ ID NO:269 Glycine max WRII polynucleotide (XM_003519119.1) SEQ ID NO:270 Glycine max WRIl polynucleotide (XM_003550628.1) D).ID NO:271 Medicago tr'uncatula WRII polynucleotide (XM_003610213. 1) Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 69 SEQ ID NO:272 Glycine max WRII polynucleotide.(XM_003523982.1) SEQ ID NO:273 Glycine max WRII polynucleotide.(XM_003525901.1) SEQ ID NO:274 Populus trichocarpa WRIl polynucleotide (XM_002325075.1) SEQ ID NO:275 Vitis vinifera WRII polynucleotide (XM_002273010.2) SEQ ID NO:276 Populus trichocarpa WRII polynucleotide ( XM__002303 830.1) SEQ ID NO:277 Lupim's angustifolius WRII polynucleotide, partial sequence (NA- 080818_Plate14f06.bl) ’ SEQ ID NO:278 Lupinis angustifolius WRII polynucleotide ‘SEQ ID NO:279 Arabidopsis thaliana WRII polypeptide (A8MSS7) 10 SEQ ID NO:28O A'rabidopsis thaliana WRIl polypeptide (Q6X5Y6) _ ' SEQ ID _ opsis lyrata subsp. lyrata WRII polypeptide (XP__002876251.1) SEQ ID NO:282 ca napus WRII polypepetide 282.1) SEQ ID NO:283 Brassica napus WRII polyppetide 346.1_) 15 SEQ ID NO:284 Glycine max WRII polypeptide (XP_003530370.1) SEQ ID NO:285 Jatropha curcas WRII polypeptide (AE02213 1 .1) ’ SEQ ID NO:286 Ricinus communis WRII polypeptide (XP_0025253\05.1) SEQ ID NO:287 Populus carpa WRII polypeptide (XP_002316459.1) SEQ ID NO:288 Vitis vinifera WRII polypeptide (CBIZ9147..3) 20 SEQ ID NO:289 Brachypodium hyon WRII polypeptide (XP_003578997.1) SEQ ID NO:290 m e subsp. ’vulgare WRII polypeptide (BAJ86627.1) SEQ ID NO:291 OIyza sativa WRII polypeptide (EAY79792.1) SEQ ID NO:292 Sorghum bicolor WRII polypeptide (XP_00245Q194.1) SEQ ID NO:293 Zea mays WRII polypeptide (ACG32367.I) 25 SEQ ID NO:294 Brachypodium distachyon WRIl polypeptide (XP_003561 189.1) SEQ ID NO:295 Brachypodium sylvaticum WRII polypeptide (ABL85061.1) SEQ ID NO:296 Oryza sativa WRII polypeptide (BAD68417.1) SEQ ID NO:297 Sorghumvbicolor WRIl polypeptide (XP__0024378_19.1) SEQ ID NO:298 Sorghum bicolor WRII polypeptide (XP_0024414_44.1) 30 SEQ ID NO:299 , Glycine max WRIl polypeptide 3530686.1) SEQ ID NO:300 Glycine max WRII polypeptide (XP_003553203.1) SEQ ID NO:301 Populus trichocarpa WRII ptide (XP_002315794.1) SEQ ID N02302 Vitis vinifera WRIl polypeptide (XP_002270149.1) SEQ ID N01303 Glycine max WRII ptide (XP_003533548.1) 35 SEQ‘ID N01304 Glycine max WRII polypeptide (XP_0035517.23.1) SEQ ID N02305 Medicago truncatula WRII polypeptide (XP_003621117.1) SEQ ID N02306 Populus trichocarpa WRII polypeptide (XP_002323836.1) D) ID NO:307 Ricinus communis WRII polypeptide (X_P_002517474.1) Subeﬁmte Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 70 SEQ ID NO:308 Vitis vinz‘fera WRII polypeptide (CAN79925.1) SEQ ID NO:309 Brach/ypodium distachyon WRII polypeptide 3572236.1) SEQ ID NO:310 Oryza sativa WRII polypeptide (BAD10030.1) SEQ ID NO:311 Sorghum bicolor WRII polypeptide (XP_002444429.1) ' SEQ ID NO:312 Zea mays WRII polypeptide (NP_001170359.1) SEQ ID NO:313 Arabidopsis lyrata subsp. lyrata WRII polypeptide (XP_002889265.1) ' i _ SEQ ID NO:314 Arabidopsis thaliana WRII polypeptide (AAF68121.1) SEQ ID NO:315 ArabidOpsis thaliana WRII polypeptide (NP_178088.2) IO SEQ ID NO:316 Arabidopsis lyrata subsp. lyrata WRII ptide ' (XP_0028_90145.1) SEQ ID NO:317 ungiella halophila WRII polypeptide (BAJ33872.1) SEQ ID NO:3,18 Arabidopsis thaliana WRII polypeptide (NP_563990.I) SEQ ID NO:319 Glycine max WRII polypeptide (XP_003530350.1) 15. SEQ ID NO:320 Brachypodium distqchyon WRII polypeptide (XP_003578142.1) i SEQ ID NO:321 Otjyza saliva WRII polypeptide (EAZO9147.1) SEQ ID NO:322 Sorghum bicolor WRII polypeptide (XP_002460236.1) SEQ ID NO:323 Zea mays WRII polypeptide (NP_001146338.1) SEQ ID NO:324 Glycine max WRII polypeptide (XP_003519167.1) 20 SEQ ID NO:325 Glycine max WRII polypeptide (XP_003550676.1) SEQ ID NO:326 Medicago truncatula WRII polypeptide (XP_003610261.1) SEQ ID NO:327 Glycine max WRII ptide (XP_003524030. l) SEQ ID NO:328 e max WRII polypeptide 3525949.1) SEQ ID NO:329 Populus carpa WRII polypeptide (XP_002325111.1) '25 SEQ ID NO:330 Vitis vinifera WRII ptide (CBI36586.3) SEQ ID NO:331 Vitis vinifera WRII polypeptide (XP_002273046.2) SEQ ID NO:332 Populus trichocarpa WRII polypeptide (XP_002303866.1) SEQ ID NO:333 Vitis vinifera WRII ptide (CBIZSZéI .3) SEQ ID NO:334 Sorbi-WRLI 30 SEQ ID NO: 335 Lupan-WRLI SEQ ID NO:336 Ricco-WRLI ' SEQ ID NO:337 Lupin angustifolius WRIl polypeptide SEQ ID NO:338 Aspergillusfumigatus DGAT polynucleotide (XM_750079.1) SEQ ID NO:339 Ricinus communis DGAT polynucleotide 496.1) 35 SEQ ID NO:340 Verniciafordii DGATI polynucleotide (DQ356680.1) SEQ ID NO:341 Vernonia galamensis DGAT] polynucleotide (EF6S3276.1) SEQ ID NO:342 Vernonia galamensis DGATI polynucleotide (EF653277.1) ‘ D) ID NO:343 us alatus DGAT] polynucelotide (AY751297. 1) Substitute Sheet ' (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 71 SEQ ID NO:344 Caenorhabditis s DGATI polynueelotide (AF221132.1) SEQ ID NO:345 Rattus norvegicus DGATI polynucelotide (NM_053437.1) SEQ ID NO:346 VHomo‘ sapiens DGATlpolynucleotide (NM_012079.4) SEQ ID NO:347 Aspergillusﬁtmigdtus DGATI polypeptide 5172.1) 5 SEQ ID NO:348 Ricinus communis DGATl polypeptide (AAR11479.1) SEQ ID NO:349 Verniciafordii DGATI polypeptide (ABC94472.1) SEQ ID NO:350 Vernom'a galamensisDGATl polypeptide (ABV21945.1) SEQ ’ID NO:351 Vernonia galamensis DGATI polypeptide (ABV21946.1) SEQ ID-N02352 us alatus DGATI polypeptide (AAV31083.1) 10 SEQ ID NO:353 Cdenorhabditis elegans DGATI polypeptide (AAF82410.1) SEQ ID NO:354 RattusTnorvegicus DGATI polypeptide 5889.1) i SEQ ID NO:355 Homo sapiens DGATl polypeptide (NP_036211.2) SEQ ID NO:356 WRIl inotif(R G V T/S R H R W T G R) SEQ ID NO:357 WRll motif(F/Y E A H L W D K) 15' SEQ ID NO:358 WRIl motif(D L A A L K Y W G) SEQ ID NO:359 WRIl motif(S X G F S/A R G X) SEQ ID NO:360 WRIl motif(H H H/Q N G R/K W E A R I G R/K V) SEQ ID NO:361 WRIl motif(Q E E A A A X Y D) SEQ ID NO:362 Brassica napus oleosin polypeptide (CAA57545. 1) '20 SEQ 363 Brassica napus oleosin'SI-l ptide (ACG69504.1) SEQ ID NO:364 Brassica napus oleosin 82-1 polypeptide (ACG69503.1) SEQ ID NO:365 Brassica napus oleosin S3-l polypeptide (ACGG9513.1) SEQ ID NO:366 Brassica napus oleosin S4-l polypeptide 507.1) SEQ ID NO:367 ica napus oleosin S5-1 polypeptide (ACG69511L1) 25 SEQ ID NO:368 Arachis hypogaea oleosin 1 ptide (AAZ20276.1) SEQ ID NO:369 Arachis hypogaea oleosin 2 polypeptide (AAU21 500.1)- SEQ ID NO:370 Arachis hypogaea n 3 polypeptide (AAU21501;1) SEQ ID NO:37‘1 Arachis hypogaea oleosin 5 polypeptide (ABC96763.1) SEQ ID NO:372 Ricinus communis oleosin 1 polypeptide (EEF40948.1) 30 SEQ ID NO:373 Ricinus cammuhis oleosin 2 polypeptide (EEFS 1616.1) SEQ ID NO:374 Glycine max oleosin isoform a polypeptide (P295302) SEQ ID NO:375 Glycine max oleosin isoform b polypeptide (P2953 1 .1) SEQ ID NO:376 Linum iusitatissimum oleo'siri low molecular weight isoform polypeptide (ABB01622.1) , 35 SEQ ID NO:377 amino'acid ce ofLinum usitatissimum oleosin high molecular weight isoform ptide (ABB01624.1) SEQ ID NO:378 Helianthus annuus oleosin polypeptide (CAA44224.1) D) ID NO:379 Zea mays n polypeptide (NP__001105338.1) Substitute Sheet (Rule'26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 72 SEQ ID NO:380 Brassica napus steroleosin polypeptide (ABM30178LI) SEQ ID NO:381 ca napus steroleosin SLOl-l- polypeptide (ACG69522.1) SEQ ID NO:382 Brassica napus steroleosin SL02-1 polypeptide (ACG69525.1) SEQ ID NO:383 Sesamum indicum steroleosin polypeptide (AAL13315.1) SEQ ID NO:384 Zea mays steroleosin polypeptide (NP_001 152614.1) SEQ ID NO:385 ica napus caleosin CLO-1 polypeptide ‘(ACG69529.1) SEQ ID NO:386 Brassica napus caleosin CLO—3 polypeptide (ACG69527.1) SEQ ID NO:387 Sesamum indicum caleosin polypeptide (AAF 1) SEQ ID NO:388 Zea mays caleosin polypeptide (NP_001 151906.1) 10 SEQ ID NO:389 Brassica napus oleosin polynucleotide (X82020.1) SEQ ID NO:390 Brassica napus oleosin S 1-1 cleotide (EU678256.1) SEQ ID NO:391 Brassica napus oleosin SZ-l polynucleotide (EU678255.1) SEQ ID NO:392 ca hapus oleosin 83-1 polynucleotide 265.1) SEQ ID N02393: Brassica napus oleosin S4-1 polynucleotide (EU678259.1) , 15 SEQ ID NO:394‘ Brassica napus oleosin SS-l polynucleotide (EU678263.1) SEQ ID NO:395 Arachis hypogaea oleosin l polynucleotide 716.1) SEQ ID NO:396 Arachis hypogaea oleosin 2 polynucleotide (AY722695.1) SEQ ID NO:397 Arachis hypogaea oleosin 3 polynucleotide (AY722696.1) SEQ ID NO:398 Arachis hypogaea oleosin 5 polynucleotide (DQ368496.1) 20 SEQ ID NO:399 Helianthus annnus oleosin polynucleotide (X62352.1) SEQ ID NO:400 Zea mays oleosin polynucleotide (NM_0011 1) SEQ ID NO:401 Brassica napus' steroleosinpolynucleotide (EF143915.1) SEQ ID NO:402 Brassica napus steroleosin SLOl-l polynucleotide (EU678274. 1) ' SEQ ID NO:403 ca napus steroleosin SL02-l polynucleotide (EU678277.1) 25 SEQ ID NO:404 Zea mays steroleosin polynucleotide (NM_001159142.1) SEQ ID NO:405 Brassica napus caleosin CLO-1 polynucleotide (EU678281.1) SEQ ID NO:406 Brassica napus in CLO-3 polynucleotide (EU678279.1) SEQ ID NO:407 Sésamum m in polynucleotide 921.1) SEQ ID NO:408 Zea mays caleosin polynucleotide (NM~_001158434.1) 30 SEQ ID NO:409 pJP3502 entire vector sequence (three—gene) SEQ ID NO:410 pJP3503 entire vector sequence (four-gene) SEQ ID NO:411 pJP3502 TDNA (inserted into genome) sequence sEQ ID NO:412 pJP3503 TDNA (inserted into genome) sequence SEQ ID NO:413 pJP3507 vector sequence 35 SEQ ID NO:414 Linker sequence SEQ ID NO:415 Soybean Synergy ’ SEQ ID NO:416 12ABFJYC_pJP3569_‘insert D.

Substitute Sheet- (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 73 SEQ ID NO:417 Partial N. benthamiana CGI-58 sequence selected for hpRNAi silencing (pTV46) SEQ ID NO:418 Partial N. tabacum AGPase sequence selected for hpRNAi silencing (pTV35) ' , I SEQ ID NO:419 GXSXG lipase motif SEQ ID NO:420 HX(4)D acyltransferase motif SEQ ID NO:421 GF probable lipid binding motif SEQ ID NO:422 Arabidopsis thaliana CGi58 polynucleotide (NM_118548.1) SEQ ID NO:423: Brachypodium distachyon CGi58 polynucleotide ' 10 (XM_003578402.1) ' , SEQ ID NO:424 Glycine max CGi58 polynucleotide (XM_003523590.1) SEQ ID NO:425 Zea mays CGi58 polynucleotide (NM_001155541.1) ' SEQ ID NO:426 Sorghum bicolor CGi58 polynucleotide (XM_002460493.1) SEQ ID NO:427 Ricinus coinmunis CGi58 cleotide (XM_002510439.1) 15 SEQ ID'NO:428 Medicago truncatula CGi58 cleotide (XM_003603685.1) SEQ ID NO:429 opsis na CGi58 polypeptide (NP_194147.2) SEQ ID NO:430 Brachypodium disiachyon CGi58 polypeptide (XP_003578450.1) SEQ ID NO:431‘ Glycine Max CGi58 polypeptide (XP_003523638J).

SEQ ID NO:432‘ Zea Mays CGi58 polypeptide (NP_001149013.1) 20 SEQ ID NO:433 Sorghum bicolor CGi58 polypeptide (XP_002460538.1) SEQ ID NO:434 Ricinus communis CGi58 polypeptide (XP_002510485.1) SEQ ID NO:435 Medicago truncatula CGi58 polypeptide (XP_003603733.1) SEQ ID NO:436 Oryza sativa CGi58 polypeptide (EAZO9782.1) SEQ ID NO:437 Arabidopsis thaliana LEC2 polynucleotide (NMé102595.2) 25 SEQ ID NO:438 Medicago truncatula LEC2 polynucelotide 7.l) SEQ ID NO:439 Brassica napus LEC2 celotide(HM370539.1) SEQ ID NO:440 Arabidopsis na BBM polynucleotide (NM_121749.2) i' SEQ ID NO:441 Medicago truncatula BBM polynucleotide (AY899909.1) SEQ ID NO:442 Arabidopsis thaliana LEC2 polypeptide 4304.1) 30 SEQ ID NO:443 Medicago truncatula LEC2 ptide (CAA42938.1) SEQ ID NO:444 Brassica napus LEC2 ptide (AD016343.1) SEQ ID NO:445 Arabidopsis thaliana BBM polypeptide (NP_197245.2) SEQ ID NO:446 Medicago truncatula BBM polypeptide (AAW82334.1) 1 SEQ ID NO:447 Inducible Aspergilus niger ach promoter 35 SEQ ID NO:448 AlcR inducer that activates thevAch promoter in the presence of ethanol D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 74 DETAILED DESCRIPTION OF THE INVENTION General Technigues and Deﬁnitions Unless speciﬁcally deﬁned otherwise, all technical and scientiﬁc terms used ‘ herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, lipid and fatty acid try, biofeul V production, and biochemistry). ' Unless otherwise indicated, the recombinant protein, cell culture, and logical techniques ed in the present invention are standard proCedures, 10 well known to those skilled in the art. Such techniques are described and explained throughout the ture in sources such as, J. Perbal, A cal Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL 15 Press (1991), D.M. Glover and RD. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), RM. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, ing all updates until present), Ed Harlow and David Lane (editors) dies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), 20 and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

Selected ions The term "transgenic non-human organism" refers to, for example, a whole 25 plant, alga, non-human animal, or an organism suitable for fermentation such as a yeast or fungus, comprising an ous polynucleotide (transgene) or an exogenous polypeptide. In an embodiment, the transgenic non-human organism is not an animal or part thereof. In one embodiment, the transgenic non-human organism is a phototrophic organism (for e, a plant or alga) capable of obtaining energy from ‘ .30 sunlight to synthesize organic compounds for nutrition. In r embodiment, the enic man organism is a yntheic bacterium.

The term "exogenous" in the context of a polynucleotide or polypeptide refers to the polynucleotide or ptide when present in a cell which does not naturally comprise the cleotide or polypeptide) Such a cell is referred to herein as a 35 “recombinant cell” or a “transgenic cell”. In an embodiment, the exogenous polynucleotide or ptide is from a different genus to the cell comprising the exogenous polynucleotide or polypeptide. In another embodiment, the exogenous Dynucleotide or polypeptide is from a different species. In one embodiment the Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 75 exogenous polynucleotide or polypeptide is_expressed in a host plant or plant cell and the exogenous cleotide or polypeptide is from a different species or genus. The ~ exogenous polynucleotide or ptide may be non-naturally occurring, such as for example, a synthetic DNA molecule which has been produced by recombinant DNA methods. The DNA molecule may, often preferably, include a protein coding region which has been codon—optimised for expression in the cell, thereby producing a polypeptide which has the same amino acid sequence as a naturally ing polypeptide, even though the nucleotide sequence of the protein coding region is urally occurring. The exogenous polynucleotide may encode, or the exogenous 10 polypeptide maybe: a diacylglycerol ansferase ) such as a DGATI or a DGATZ, a glycerol-B-phosphate acyltransferase (GPAT) such as a GPAT which is capable of synthesising MAG, a Wrinkled l (WRIl) transcription factor, an Oleosin, or a silencing suppressor polypeptide. In one embodiment, the exogenous polypeptide ‘ is an exogenous MGAT such as an MGATl or an MGAT2. 15 As used herein, the term "extracted lipid" refers to a composition extracted from a transgenic organism or part thereof which comprises at least 60% (w/w) lipid.

As used herein, the term "non~polar lipid" refers to fatty acids and derivatives thereof which are e in c solvents but insoluble in water. The fatty acids may be ‘free fatty acids and/or in an esteriﬁed form. Examples of esteriﬁed forms 20 include, but are not limited to, triacylglycerol (TAG), diacylyglycerol (DAG), monoacylglycerol (MAG). Non-polar lipids also include sterols, sterol esters and wax esters. Non-polar lipids‘ are also known as "neutral lipids". Non-polar lipid is typically a liquid at room temperature. Preferably, the non-polar lipid predominantly (>50%) comprises fatty acids that are at least 16 carbons in length. More preferably, 25 at least 50% of the total fatty acids in the non-polar lipid are'Cl8 fatty acids for example, oleic acid. In an embodiment, at least 50%, more preferably at least 70%, more preferably at least 80%, 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 30 preferably at least 97%, more preferably at least 98%, more ably at least 99% of the fatty acids in nonepolar lipid" of the ion can be found as TAG. The non- polar lipid may be r puriﬁed or treated, for example by hydrolysis with a strong base to release the free fatty acid, or by fractionation, distillation, or the like. Non— polar lipid may be present in or obtained from plant parts such as seed, leaves or fruit, 35 from recombinant cells or from non—human organisms such as yeast. Non-polar lipid of the ion may form part of "seedoil" if it is obtained from seed. The free and esteriﬁed sterol (for e, sitosterol, campesterol, stigmasterol, brassicasterol, A5- Dnasterol, sitostanol, campestanol, and cholesterol) concentrations in the extracted Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 76 'lipid may be as described in Phillips et a1., 2002. Sterols in plant oilsare present as free alcohols, esters with fatty acids (esteriﬁed sterols), glycosides and acylated glycosides of sterols. Sterol concentrations in naturally occurring vegetable oils ils) ranges up to a maximum of about 1100mg/100g. HydrOgenated palm oil has one of the lowest concentrations of naturally occurring vegetable oils at about 60mg/100g. The recovered or extracted seedoils of the invention preferably have between about 100 and about 1000mg total sterol/100g of oil. For use as food or feed, it is preferred that sterols are t primarily as free or esteriﬁed forms rather than glycosylated forms. In the seedoils of the present invention, preferably at least 50% 10 of the s in the oils are present as esteriﬁed s, except for soybean seedoil which has about 25% of the sterols esteriﬁed. The canola seedoil and rapeseed oil of the invention preferably have between about 500 and about 800 mg total /100g, with sitosterol the main sterol and campesterol the next most nt. The corn seedoil of the invention preferably has between about 600 and about 800 mg total 15 / 100g, with sitosterol the main sterol. The soybean seedoil of the invention preferably has between about 150 and about 350 mg total sterol/100g, with sitosterol the main sterol and stigrnasterol the next most abundant, and with more free sterol than esteriﬁed sterol. The cottonseed oil of the invention preferably has between about 200 and about 350 mg total sterol/100g, with sitosterol the main sterol. The '20 coconut oil and palm oil of the invention ably have n about 50 and about 100mg total sterol/ 100g, with sitosterol the main sterol. .The safﬂower seedoil of the invention preferably has between about 150 and about 250mg total sterol/100g, with sitosterol the main sterol. The peanut l of the invention preferably has between about 100 and about 200mg total sterol/100g, with sitosterol the main sterol. The ' 25 sesame seedoil of the invention preferably has between about 400 and about 600mg total sterol/100g, with sitosterol the main sterol. The sunﬂower seedoil of the invention preferably has between about 200 and 400mg total sterol/100g, with sitosterol the main sterol. Oils ed from vegetative plant parts according to the - invention ably have less than 200mg total sterol/100g, more preferably less than 30 100mg total sterol/100 g, and most preferably less than 50mg total sterols/100g, with the majority of the sterols being free sterols.

As used herein, the term "seedoil" refers to a composition obtained from the rain of a plant which comprises at least 60% (w/w) lipid, or obtainable from the seed/grain if the seedoil is still present in the seed/grain. That is, seedoil of the 35 invention includes seedoil which is present in the seed/grain or portion thereof, as well as seedoil which has been ted from the seed/grain. The seedoil is ably extracted seedoil. Seedoil is typically a liquid at room temperature. ly, the total fatty acid (TFA) content in the seedoil predominantly (>50%) ‘ Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 77 comprises fatty acids that are at least 16 s in length. More preferably, at least 50% of the total fatty acids in the seedoil are C18 fatty acids for example, oleic acid.

The fatty acids are typically in an ﬁed form such as for example, TAG, DAG, acyl-CoA or phospholipid. The fatty acids maybe free fatty acids and/or in an ﬁed form. In an ment, at least 50%, more preferably at least 70%, more preferably at least 80%, 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 ably at least 96%, more preferably at least 97%, more preferably at least 98%,‘more preferably at least 99% of the fatty 10 acids in seedoil of the invention can be found as TAG. In an embodiment, seedoil of the invention is "substantially puriﬁed" or ed" oil'that has been separated from one .or more other lipids, nucleic acids, polypeptides, or other contaminating molecules with which it is associated in the seed or in a crude extract. It is preferred that the substantially puriﬁed seedoil is at least 60% free, more preferably at least 15 75% free, and more preferably, at least 90% free from other components with which it is associated in the seed or extract. Seedoil of the invention may further compriSe tty acid molecules such as, but not limited to, sterols; In an embodiment, the l is canola oil (Brassica sp. such as Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus) mustard oil (Brassica juncea), other Brassica 20 oil (e.g.‘, Brassica napobrassica,IBrassica camelina), sunﬂower oil (Helianthus Sp. such z'anthus annuals), linseed oil (Linum usitatissimum), soybean oil (Glycine max), safﬂower ‘oil (Carthamus tinctorius), corn oil (Zea mays), tobacco ' oil (Nicotiana sp. such as Nicotiana tabacum or Nicotiana benthamiana), peanut oil (Arachis hypogaea), palm oil (Elaeis guineensis), cottonseed oil (Gossypium 25 hirsutum), coconut oil (Cocos nucifera), avocado oil (Persea americana), olive oil (Olea europaea), cashew oil (Anacardium occidentale), macadamia oil (Macadamia intergrifolia), almond oil (Prunus amygdalus), oat seed oil (Avena sativa), rice oil (Oryza sp. such as» Oryza sativa and Oryza glaberrima), opsis seed oil (Arabidopsis thaliana), or oil from the seed of Acrocomia aculeata (macauba palm), 30 Aracinis Vhypogaea (peanut), Astrocaryum mummuru (mummuru), Astrocaryum vulgare (tucuma), Attalea geraensis (Indaia-rateiro), A'ttalea humilis (American oil palm), Attalea oleifera (andaia), a ata (uricuri), Attalea speciosa (babassu), Beta vulgaris' (sugar beet), Camelina sativa (false ﬂax), Caryocar brasiliense (pequi), Crambe abyssinica (Abyssinian kale), Cucumis melo ), 35 Hordeum vulgare (barley), Jatropha curcas (physic nut), Joannesia princeps (arara nut-tree), Licania rigida (oiticica), 'Lupinus angustifolius (lupin), Mauritia ﬂexuosa (buriti palm), Maximiliana maripa (inaja palm), thus sp. such as Miscanthus x Danteus and Miscanthus is, Oenocarpus bacaba (bacaba-do-azeite), Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 78 rpus bataua (pataua), Oenocarpus distichus (bacaba—de-leque), Panicurn virgatum. (switchgrass), Paraqueiba paraensis (mari), Persea amencana (avocado), Pongamia pinnata (Indian beech), Populus trichocarpa, Ricinus is (castor), Saccharum sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum sp. such as Sorghum bicolor, Sorghum vulgare, Theobroma ﬁJrum (cupuassu), Triﬁylium sp., Trithrinax brasiliensis (Brazilian needle palm) and Triticum sp. (wheat) such as Triticum aestivum. Seedoil may be ted from seed/grain by any method known in the art. This typically involves extraction with nonpolar solvents such as diethyl ether, petroleum ether, chloroform/methanol or butanol' 10 mixtures, generally associated with ﬁrst crushing of the seeds. Lipids associated with the starch in the grain may be extracted with water-saturated butanol. The seedoil may be "de-gummed" by methods known in the art to remove polysaccharides or d in other ways to remove contaminants or improve purity, stability, or .

The TAGS and other esters in the seedoil may be hydrolysed to release free fatty 15 acids, or the seedoil hydrogenated, treated chemically, or enzymatically as known in ' the art. _ As used herein, the term "fatty acid" refers to a carboxylic acid with a long aliphatic tail of at least 8 carbon atoms in , either saturated or unsaturated.

Typically, fatty acids have a carbon-carbon bonded chain of at least 12 carbons in 20 . Most naturally occurring fatty acids have an even number of carbon atoms because their thesis es acetate which has two carbon atoms. The fatty acids may be in a free state (non-esteriﬁed) or in an esterified form such as part of a TAG, DAG, MAG, acyl-CoA (thio-ester)_bound, or other covalently bound form.

When covalently bound in an ﬁed form, the fatty acid is referred to herein as an 25 "acy " group. The fatty acid may be esteriﬁed as a phospholipid such as a phosphatidylcholine (PC), phosphatidylethanolamine, .phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or diphosphatidylglycerol. Saturated fatty acids do not n any double. bonds or other functional groups along the chain.

The term "saturated" refers to hydrogen, in that all carbons (apart from the carboxylic 30 acid [-COOH] group) contain as many hydrogens as possible. In other words, the omega (to) end contains 3 hydrogens (CH3-) and each carbon within the chain contains 2 hydrogens ). Unsaturated fatty acids are of similar form to saturated fatty acids, except that one or more alkene functional groups exist along the chain, with each alkene substituting a -bonded "-CH2—CH2—" part of the chain with a 35’ doubly-bonded "-CH=CH—" portion (that is, a carbon double bonded to another carbon). The two next carbon atoms in the chain that are bound to either side of the double bond can occur in a cis or trans conﬁguration.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 79 ' As used herein, the terms "polyunsaturated fatty acid" or "PUFA" refer to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and at least two alkene groups (carbon-carbon double bonds). The PUFA content of the vegetative plant part, or the non-human organism or part thereof of the ion may be increased or sed depending on the combination of exogenous pOlynucleotides expressed in the vegetative plant part, or non~human organism or part thereof, or seed of the invention. For example, when an MGAT is expressed the PUFA level typically increases, whereas when DGATl is expressed alone for in combination with WRIl, the PUFA level is typically decreased due to an increase in 10 the level of oleic acid. Furthermore, ifA12 rase activity is d, for example by silencing an endogenous A12 desaturase, PUFAI content is ly 'to increase in the absence Of an exogenous polynucleotide encoding a different A12 desaturase. . cylglyceride" or "MAG" is glyceride in which the glycerol is esteriﬁed with one fatty acid. As used herein, MAG comprises a hydroxyl group at an sn-l/3 15 (also ed to herein as sn-l MAG or l—MAG or l/3-MAG) or sn-2 position (also referred to herein as 2-MAG), and therefore MAG does not include phosphorylated molecules such as PA or PC. MAG is thus a component of neutral lipids in a cell.

"Diacylglyceride" or "DAG" is glyceride in which the glycerol is esteriﬁed . with two fatty acids which may be the same or, preferably, different. As used , 20 DAG comprises a hydroxyl group at a sn-l,3 or sn-2 on, and therefore DAG does not include phosphorylated molecules such as PA or PC. DAG is thus a component of neutral lipids in a cell. In the Kennedy pathway of DAG synthesis e l), the precursor sn-glycerolphosphate (GP) is esteriﬁed to two acyl groups, each coming from a fatty acid coenzyme A ester, in a ﬁrst reaction catalysed 25 by a glycerol-S-phosphate acyltransferase (GPAT) at position sn-l to. form LysoPA, followed by a second acylation at position sn-2 catalysed by a lysophosphatidic acid acyltransferase (LPAAT) to form phosphatidic acid (PA). This intermediate is then de—phosphorylated to form DAG. In an alternative ic pathway (Figure 1), DAG may be formed by the acylation of either sn-l MAG or preferably sn-2 MAG, 30 catalysed by MGAT. DAG may also be formed from TAG by removal of an acyl group by a , or from PC essentially by removal of a choline headgroup by any of the enzymes CPT, PDCT or PLC (Figure l).

"Triacylglyceride" or "TAG" is glyceride in which the glycerol is esteriﬁed - 'with three fatty acids which may be the same (e. g. as in tri-olein) or, more commonly, 35 different. In the Kennedy pathway of TAG synthesis, DAG is formed as described above, and then a third acyl group is esteriﬁed to the glycerol backbone by the activity of DGAT. Alternative pathways for formation of TAG include one sed by the Dyme PDAT and the MGAT pathway bed herein.

- Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 80 As used herein, the term "acyltransferase" refers to a protein which is capable of transferring an acyl group from acyl-CoA onto a substrate and includes MGATs, , ' GPATs and DGATs. _ As used herein, the term "Wrinkled 1" or "WRII" or "WRLI" refers to a transcription factor of the AP2/ERWEBP class which regulates the expression of several enzymes involved in glycolysis and de novo fatty acid biosynthesis. WRll has two plant-speciﬁc (AP2/BREE) DNA-binding domains. WRII in at least Arabidopsis also, regulates the breakdown of sucrose via glycolysis thereby regulating _ the supply of precursors for fatty acid biosynthesis. In other words, it controls the 10 carbon ﬂow from the ynthate to storage lipids. wriI mutants have wrinkled seed phenotype, due to a defeCt in the incorporation of sucrose and glucose into , ' TAGS.

. ' Examples of genes which are trancribed by WRIl include, but are not d to, one or more, preferably all, of te kinase 2920, At3 g22960), pyruvate 15 dehydrogenase (PDH) Elalpha subunit (Atlg01090), acetyl-CoA carboxylase (ACCase), BCCP2 subunit (At5g15530), enoyl—ACP ase (At2g05990; EAR), phosphoglycerate mutase (Atlg22170), cytosolic fructokinase, and cytosolic _ oglycerate mutase, sucrose synthase (SuSy) (see, for example, Liu et al., 2010b; Baud et al., 2007; Ruuska et al., 2002). 20 WRLl contains the conserved domain AP2 (cd00018). AP2 is a DNA-binding domain found in transcription regulators in plants such as AZ and EREBP (ethylene responsive element binding protein). In EREBPs the domain speciﬁcally binds to the llbp GCC box of the ne response element (ERE), a promotor element essential for ethylene responsiveness. EREBPs and the C-repeat binding .25 factor CBFl, which is involved in stress response, contain a single copy of the AP2 domain. APETALAZ-like proteins, which play a role in plant development n ‘ two .

. Other sequence motifs in WRIl and its onal ho‘mologs include: R G V T/S R H R W T G R (SEQ ID NO:356). 30 !”7‘ F/Y E A H L w D K (SEQ ID NO:357).

D L A A L K Y W G (SEQ ID NO:358).

S X G F S/A R G X (SEQ ID ).

GR/KWEARIGR/KV(SEQIDNO:360).

Q EEAAAXYD (SEQ ID NO:361). 35 As used herein, the term "Wrinkled' 1" or "WRII" also includes "Wrinkled 1- like" or "WRIl-like" proteins. Examples of WRIl proteins include Accession Nos: Q6X5Y6, (Arabidopsis thaliana; SEQ ID N03280), XP_002876251.1 (Arabidopsis Data subsp. Lyrata; SEQ ID N02281), ABDl6282.l (Brassica napus; SEQ ID Substitute Sheet RO/AU . (Rule 26) [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 81 NO:282), ADOI6346.1 (Brassica napus; 'SEQ ID NO:283), 530370.1 ‘ (Glycine max; SEQ ID NO:284), AE022131.1 (Jatropha curcas; SEQ ID ), XP__002525305.1 (Ricinus 'communie; SEQ ID NO:286), 316459.1 (Populus carpa; SEQ ID NO:287),' CB129147.3 (Vitis vinifera; SEQ ID NO:288), XP__003578997.I ypodium distachyon; SEQ ID NO:289), BA186627.1 um vulgare subsp. vulgare; SEQ ID NO:290), EAY79792.1 (Oryza sativa; SEQ ID NO:291), XP_002450194.1 (Sorghum bicolor; SEQ ID NO:292), 67.1 (Zea mays; SEQ ID NO:293), XP_003561189.1 (Brachypodium hyon; SEQ ID NOE294), ABL85061.1 (Brachypodium sylvaticum; SEQ ID 10 NO:295), BAD68417I1 (Oryza sativa; SEQ ID NO:296), XP_0024378I9.1 (Sorghum bicolor; SEQ ID NO:297), XP_002441444.1 (Sorghum bicolor; SEQ ID NO:298), XP_003530686.1 (Glycine max; SEQ ID NO:299), XP_003553203.1' (Glycine max; SEQ ID NO:300), XP_002315794.1 (Populus trichocarpa; SEQ ID NO:301), XP_002270149.1 (Vitis vinifera; SEQ ID NO:302), XP_003533’548.1 (Glycine max; '15 SEQ ID NO:303), XP_003551723.1 (Glycine max; SEQ ID NO:304), XP_003621117.] (Medicago truncatula; SEQ ID ), XP_002323836.1 (Populus trichocarpa; SEQ ID NO:306), XP_002517474.1 (Ricinus communis; SEQ ID NO:307), CAN79925.1 (Vitis vinifera; SEQ ID NO:308), XP_003572236.1 ypodium distachyon; SEQ ID NO:309), BADIOO30.1 (Otyza sativa; SEQ ID 20 NO:310), XP_002444429.1 (Sorghum bicolor; SEQ ID NO:3II), VNP_001170359.1 (Zea mays; SEQ ID NO:312), XP4002889265.1 (Arabidopsis lyrata subsp. lyrata; SEQ ID ), AAF68121.1 (Arabidopsis thaliana; SEQ ID NO:314), NP_178088.2 dopsis thaliana; ‘ SEQ ID NO:315), 890145.1 (Arabidopsis lyrata subsp.‘ lyrata; SEQ ID NO:316), BAJ33872.1 (Thellungiella 25 halophila; SEQ ID NO:317), NP_563990.1 (Arabidopsis thaliana; SEQ ID NO:318), XP_003530350.1 (Glycine max; SEQ ID NO:319), XP__003578142.1 (Brachypodium distachyon; SEQ ID NO:320), EAZO9147.1 (Oryza sativa; SEQ ID NO:321), XP_002460236.1 (Sorghum bicolor; SEQ ID ), NPé001146338.1 (Zea mays; ‘ 'SEQ ID NO:323), XP_003519167.71 (Glycine max; SEQ ID NO:324); 30 VXP_003550676.I (Glycine max; SEQ ID NO:325), 610261.1 (Medicago truncatula; SEQ ID NO:326), XI’__003524030.I (Glycine max; SEQ ID NO:327), XP_003525949.1. ne max; SEQ ID NO:328), XP__002325111.1 (Populus trichocarpa; SEQ ID NO:329), CBI36586.3 '(Vitis vinifera; SEQ ID NO:330), XP_002273046.2 (Vitis viniﬁzra; SEQ ID ), XP_002303866.1 (Populus' '35 trichocarpa; SEQ ID NO:332), and CBI25261.3 (Vitis vim'ﬂzra; SEQ ID NO:333).

Further examples include Sorbi-WRL] (SEQ ID NO:334), WRLI (SEQ ID NO:335), Ricco-WRLI (SEQ ID NO:336), and Lupin angustifolz'us WRII (SEQ ID D337). , Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 82 As used herein, the term "monoacylglycerol acyltransferase" or "MGAT" refers to a protein which transfers a fatty acyl group from acyl—CoA to a. MAG substrate to e DAG. Thus, the term "monoacylglycerol acyltransferase activity" at least refers to the transfer of an acyl group from acyl-CoA to MAG to produce DAG. MGAT is best known for its role in fat absorption in the intestine of mammals, where the fatty acids and sn-2 MAG generated from the digestion of dietary fatare resynthesized into TAG in enterocytes for chylomicron synthesis and V ion. MGAT catalyzes the ﬁrst step ofthis process, in which the acyl group from fatty acyl—CoA, formed from fatty acids and CoA, and sn‘-2 MAG are covalently 10 joined. The term "MGAT" as used herein includes enzymes that act on sn-l/3 MAG and/or sn-Z MAG substrates to form sn-1,3 DAG‘ and/or sn-1,2/2,3-DAG, respectively. In a preferred embodiment, the MGAT has a preference for sn-2 MAG substrate ve to sn-l MAG, or substantially uses only sn-2 MAG as substrate (examples include MGATs described in,Cao et a1., 2003 (speciﬁcity of mouse 15 MGATl for sn2-l8zl—MAG > snl/3MAG (Figure 5)); Yen and Farese, 2003 (general activities of mouse MGAT] and human MGAT2 are higher on 2-MAG than on l-MAG acyl-acceptor substrates (Figure 5); and Cheng et al., 2003 (activity of human MGAT3 on Z—MAGs is‘ much higher than on 1/3-MAG substrates (Figure 2D)). .20 As used herein, MGAT does not include enzymes which transfer an acyl group preferentially to LysoPA relative to, MAG, such s are known as LPAATs.

That is, a MGAT preferentially uses osphorylated monoacyl substrates, even though they may have low catalytic activity on . A preferred MGAT does not have detectable activity in acylating LysoPA. As Shown herein, a MGAT (i.e.,'M. 25 musculus MGAT2) may also have DGAT function but predominantly ﬁmctions as a MGAT, i.e., it has greater catalytic activity as a MGAT than as a DGAT when the enZyme ty is expreSsed in units ofnmoles product/min/mg protein (also see Yen ' ‘ et al.,2002).

There are three known classes ofMGAT, referred to as, MGATl, MGAT2 and . 30 MGAT3, respectively. gs of the human MGAT] gene (AF384163; SEQ ID NO:7) are present (i.e. sequences are known) at least in chimpanzee, dog, cow, mouse, rat, zebraﬁsh, Caenbrhabditis elegans, Schizosaécharomyces pombe, Saccharomyces‘ cerevisiae, Kluyveromyces lactis, Eremothecium gossypii, orthe grisea, and Neurospora crassa. Homologs of the human MGAT2 gene 35 (AY157608) are present at least in chimpanzee, dog, cow, rat, chicken, zebraﬁsh, fruit ﬂy, and mosquito. Homologs of the human MGAT3 gene (AY229854) are present at least in chimpanzee, dog, cow, and zebraﬁsh. However, D . 1 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 83 homologs from other organisms can be readily identiﬁed by methods known in the art for fying homologous sequences. , _ Examples ofMGATI polypeptides include proteins encoded by MGAT] genes from Homo sapiens (AF384163; SEQ ID NO:7), ‘Mus' musculus (AF384162; SEQ ID N028), Pan troglodytes II66055 and XM_0526044.2; SEQ ID N09 and SEQ ID NO:10, respectively), Canisfamiliaris 5667.2; SEQ ID, N01] 1), Bos taurus 1001153.2; SEQ ID NO:12), Rattus norvegicus (NM_001108803.1; SEQ ID NO:13), Danio rerio MGAT] (NM_001122623.1; SEQ ID NO:14), Caenorhabditis elegans (NM__0730 1 2.4, NM~182380.5, NM_065258.3 , 1'0 . NM_075068.3, and NM_072248.3; SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID‘ NO:18, and SEQ ID NO:19,. respectively), Kluyveromyces lactis (XM_455588.1; SEQ ID NO:20), Ashbya' gossypii (NM_208895.1; SEQ ID NO:21), Magnaporthe oryzae (XM_368741.1; SEQ ID NO:22), Ciona intestinalis ted (XM_002120843.1 SEQ ID NO:23). Examples of MGAT2 polypeptides include 15 proteins encoded by MGAT2 genes from Homo sapiens (AY157608‘; SEQ ID NO:24), Mus musculus (AY157609; SEQ ID NO:25), Pan troglodytes (XM_522112.2; SEQ ID NO:26), Cam's familiaris (XM_542304.1; SEQ ID NO:27), Bos taurus (NM_OOIO99I36.1; SEQ ID NO:28), Rattus norvegicus (NM_00110943§.2; SEQ ID NO:29), Gallus gallus (XM_424082.2; SEQ ID NO:30), 20 Danio rerio 1006083.1 SEQ ID NO:31), Drosophila melanogaster 6474.2, NM_136473.2, and NM_I36475.2; SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, ctively), Anopheles gambiae 1688709.1 and XM_315985; SEQ ID N035 and SEQ ID NO:36, respectively), Tribolium castaneum _ (XM_970053.1; SEQ ID NO:37). Examples of MGAT3 ptides include 25 proteins encoded by MGAT3 genes from Homo sapiens (AY229854; SEQ ID , NO:38), Pan troglodytes 1154107J, AXM_001154171.1, and XM_527842.2; SEQ ID NO:39, SEQ ID NO:40, and 'SEQ' ID NO:41), Cam's familiaris 5212.1; SEQ ID , Bos taurus (XM_870406.4; SEQ ID NO:43), Danio rerio (XM_688413.4; SEQ ID NO:44). '30 As used herein "MGAT pathway" refers to an anabolic pathway, different to the Kennedy pathway for the formation of TAG, in which DAG is formed by the acylation of either sn-l MAG or preferably sn-2 MAG, catalysed by MGAT; The DAG may subsequently be used to form TAG or Other lipids. The MGAT pathway is exempliﬁed in Figure 1. 35 As used , the term "diacylglycerol acyltransferase" (DGAT) refers to a protein which transfers a. fatty acyl group from acyl-CoA to a DAG substrate to produce TAG. Thus, the term "diacylglycerol acyltransferase activity" refers to the Disfer of an acyl grOup from oA to DAG to produce. TAG. A DGAT may Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 84 also have MGAT on but predominantly ons as a DGAT, i.e., it has greater tic activity as a DGAT than as a MGAT when the enzyme activity is expressed in units of nmoles product/min/mg protein (see for example, Yen et al., 2005).

There are three known types of DGAT, referred to as DGATl, DGAT2 and DGAT3, respectively.‘ DGATl polypeptides typically have 10 transmembrane domains, DGAT2 polypeptides typically have 2 transmembrane domains, whilst DGAT3 polypeptides lly have none and are thought to be soluble in the cytoplasm, not integrated into nes. Examples of DGATl polypeptides include ns encoded by DGAT] genes from Aspergillus fumigatus 10 5172.1; SEQ ID ), Arabidopsis thaliana (CAB44774.1; SEQ ID NO:83), Ricinus communis (AAR11479.1; SEQ ID N03348), Vernicia fordii (ABC94472.1; SEQ ID NO:349),. Vernonia galamensz‘s (ABV21945.1 and ABV21946.1; SEQ ID NOZ350 and SEQ ID NO:351, respectively), Euonymus alatus ' (AAV31083.1; SEQ ID N02352), Caenorhabditis elegans (AAF82410.1; SEQ ID .15 NO:353), Rattus norvegicus (NP_445889.1; SEQ ID NOZ354), Homo sapiens (NP_036211.2; SEQ ID N02355), as well as variants and/or mutants thereof.

Examples of DGAT2 polypeptides include proteins encoded by DGAT2 genes from Arabidopsis thaliana (NP_566952.1; SEQ ’ID NO:212), Ricinus communis (AAY16324.1; SEQ ID NO:213), Vernicia fordii (ABC94474.1; SEQ ID'NO:214), 20 Mortierella ramarmiana (AAK84179.1; SEQ ID NO:215), Homo sapiens (Q96PD7.2; SEQ. ID ) (Q58HT5.1; SEQ ID NO:217), Bos taurus (Q7OVZ8.1; SEQ ID NO:218), Mus musculus (AAK84175.1; SEQ. ID NO:219), as well as variants and/or mutants thereof. _ Examples of DGAT3 polypeptides include proteins encoded by DGAT3 genes 25 from peanut (Arachis hypogaea, Saba, et al., 2006), as well as variants and/or mutants thereof. A DGAT has little or no detectable MGAT activity, for example, less than 300 pmol/min/mg protein, preferably less than 200 pmol/min/mg protein, more preferably less than 100 pmol/min/mg protein.

DGAT2 but not DGATl shares high sequence homology with the MGAT 30- s, suggesting that DGAT2 and MGAT genes likely share a common genetic . Although multiple rns are involved in catalysing the same step in TAG - synthesis, they may play distinct functional roles, as suggested by differential tissue distribution and lular localization of the DGAT/MGAT family of enzymes. In ‘ - s, MGAT] is mainly expressed in stomach, kidney, adipOSe tissue, whilst 35 MGATZ and MGAT3 show highest sion in the small intestine. In mammals, DGATl is ubiquitously expressed in many tissues, with highest expression in small intestine, whilst DGAT2 is most abundant in liver. MGAT3 only exists in higher D'nmals and humans, but not in s from bioinformatic analysis. MGAT3 SubStitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 85 shares higher sequence homology to DGAT2 than MGAT] and MGAT3. MGAT3 exhibits signiﬁcantly higher DGAT activity than MGATI and MGATZ enzymes (MGAT3 > MGATl > MGAT2) when either MAGS or DAGs were used as I substrates, suggesting MGAT3 functions as a putative TAG synthase.

Both MGATl and MGATZ belong to the same class of ansferases as DGAT2. Some of the motifsthat have been shown to be important for DGAT2 - tic activity in some DGATZS are also conserved in MGAT acyltransferases. Of particular interest is a ve l binding domain with the concensus a sequence FLXLXXXN (SEQ ID NO:224) where each X is independently any amino 10 acid other than proline, and N is any nonpolar amino acid, located within the N- terminal transmembrane region followed by a putative glycerol/phospholipid acyltransferase domain. The FLXLXXXN motif (SEQ ID NO:224) is found in the mouse DGAT2 (amino acids 81—88) and MGATl/Z but not in yeast or plant DGATZS.

It is important for activity of the mouse DGAT2. Other DGAT2 and/or MGAT1/2 '15 sequence motifs include: I. A highly conserved YFP tripeptide (SEQ ID NO:220) in most DGAT2 polypeptides and also in MGAT 1' and MGAT2, for example, present as amino acids 139-141 in mouse DGAT2. Mutating this motif within the yeast DGAT2 with non- i conservative substitutions rendered the enzyme non-functional. 20 2. HPHG tetrapeptide (SEQ ID NO:221), highly conserved in MGATs as well as in DGAT2 sequences from animals and fungi, for example, present as amino acids 161-164 in mouse DGAT2, and important for catalytic activity at least in yeast and mouse DGAT2. Plant DGAT2 acyltransferases have a EPHS (SEQ ID NO:222) conserved ce instead, so conservative changes to the ﬁrst and fourth amino 25 acids can be tolerated. 3. A longer conserved motif which is part of the putative glycerol phospholipid domain. .An example , of this motif is K/R)XAXXXGXXX(L/V)VPXXXFG(E/Q) (SEQ ID NO:223), which is t as amino acids 304-327 in mouse DGAT2. This motif is less conserved in 30 amino acid sequence than the others, as would be expected from its length, but homologs can be recognised by motif ing. The g may vary between the more conserved amino acids, i.e., there may be additional X amino acids within the ' i motif, or less X amino acids compared to the sequence above.

As used herein, the term rolphosphate acyltransferase" or "GPAT" 35 refers to a protein which acylates ol—3-phosphate (GP) to form LysoPA and/or MAG, the latter product forming if the GPAT also has atase activity on LysoPA. The acyl group that is transferred is typically from acyl-CoA. Thus, the term Dicerol-3—phosphate acyltransferase activity" refers to the acylation of GP to Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 86 form LysoPA and/or MAG. The term "GPAT" encompasses enzymes that e G- 3-P to -l LPA and/or sn-2 LPA, preferably sn-2 LPA. In a preferred embodiment, the GPAT has phosphatase activity. In a most preferred embodiment, the GPAT is a sn-2 GPAT having phosphatase activity which produces sn-Z MAG.

As used herein, the term "sn-l glycerol—3-phosphate acyltransferase" (sn-l GPAT) refers to a n which acylates sn-glycerolphosphate (GP)‘ to preferentially form l-acyl-sn~glycerolphosphate (sn-l LPA).- Thus, the term "sh-1 glycerolphosphate acyltransferase activity" refers to the acylation of sn-glycerol phosphate to form 1-acyl-sn-glycerolphosphate (sn-l LPA). 10 As used herein, the term "sn-2 glycerolphosphate acyltransferase" (sn—2 GPAT) refers to a protein which acylates cerolphosphate (Ge3-P) to preferentially form 2-acyl-sn-glycerolphosphate (sn-2 LPA). Thus, the term "sh-2 glycerolphosphate acyltransferase activity" refers to the acylation of sn-glycerol—3- phosphate to. form 2-acyl-sn-glycerolphosphate (sn-2 LPA). 15 The GPAT family is large and all known members contain two ved domains, a plsC acyltransferase domain (PF01553; SEQ ID N02225) and a HAD-like hydrolase (PF12710; SEQ ID NO:226) superfamily domain. In addition to this, in Arabidopsis thaliana, GPAT4-8 all contain a N-terminal region homologous to a phosphoserine phosphatase domain (PF00702; SEQ ID N02227). GPAT4 and 20. GPAT6 both contain conserved residues that are known" to be critical to "phosphatase ty, speciﬁcally conserved amino acids (shown in bold) in Motif I TN][L/V]; SEQ ID NO:229) and Motif 111 (K-[G/S][D/S]XXX[D/N]; SEQ I ID NO:330) located at the therrninus (Yang et al., 2010). Preferably, the GPAT has sn-Z preference and phosphatase activity to produce sn-2 MAG (also referred to 25 herein as "2-MAG") from olphosphate (GP) (Figure 1), for example,- GPAT4 (NP__171667.I; SEQ ID NO:144) and GPAT6 1346.1; SEQ ID NO:145) from Arabidopsis. More preferably, the GPAT uses acyl-CoA as a fatty acid V substrate. ‘ Homologues of GPAT4' (NP_171667.1; SEQ ID NO:144) and GPAT6 ‘30 (NP_l8’1346.1; SEQ ID NO:145) include AAF02784.1 (Arabidopsis thaliana; SEQ ID NO:146), 44.I dopsis thaliana; SEQ ID NO:147), AAP03413.1 (Oryza sativa; SEQ ID NO:148), ABK25381.l (Picea sitchensis; SEQ ID NO:149), ACN34546.l (Zea Mays; SEQ ID NO:150), BAF00762.1 (Arabidopsisthalianq; SEQ ID NO:151), BAH00933.1 (Oryza sativa; SEQ ID NO:152), EAY84189.l (Oryia .35 sativa; SEQ ID NO:153), EAY98245.1 (Oryza sativa; SEQ ID NO:154), EAZZI484.1 (0ryza sativa; SEQ ID NO:155), EEC71826.I (Oryza ; SEQ ID NO:156), EEC76137.1 (Oryza sativa; SEQ ID NO:157), EEE59882.1 (Oryza sativa; SEQ ID D158), 63.1 inellq moellendorﬂii; SEQ ID NO:159), EFJ08964.1 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 87 (Selaginella moellendorﬂii; SEQ ID NO:160), EFJ11200.1 (Selaginella 'moellendorfﬁi; SEQ ID NO:161), EFJ15664.1 inella moellendorﬂii; SEQID NO:162), EFJZ4086.) (Selaginella moellendorfﬁi; SEQ ID NO:163), EFJ29816.1 (Selagt'nella moellendorﬂii; SEQ ID NO:164), 17.1 (Selaginella moellendotﬂii; SEQ ID NO:165), NP_001044839.1 (Oryza‘sativa; SEQ ID NO:166), NP_001045668.1 (Oryza sativa; SEQ ID NO:167), NP_001147442.1 (Zea mays; SEQ ID NO:168), NP_001149307.1 (Zea mays; SEQ ID NO:169), NP_001168351.1 (Zea lmays; SEQ ID NO:170), AFH0272421 ica napus; SEQ ID NO:171)‘ NP_191950.2 (Arabidopsis thali'ana; SEQ ‘_ID NO:172), XP_00176_5001.1 10 omitrella patens; SEQ ID NO:1’73), 1769671.1 (Physcomitrella patens; , SEQ ID NO:174), XI’4001769724J (Physcomitrella patensg'SEQ ID NO:175), XP_001771186.1 omitrella patens; SEQ ID NO:176), XP_001780533.1 (Physcomitrella patens; SEQ ID NO:177), XP_002268513.1 (Vitis vim'fera; SEQ ID NO:178), XP_002275348.1 (Vitis vinifera; SEQ ID NO:179), XP_002276032.1 (Vz'tis . 15 vinifera; SEQ ID. NO:180), XP__002279091.1 (Vitis vinifera; SEQ ID NO:181), ' XP_002309124.1 (Populus trichocarpo; SEQ ID NO:182), XP_002309276.1 (Populus trichocarpa; SEQ ID NO:183), XP_002322752.1 (Populus carpa; SEQ ID NO:184), XP_002323563.1 (Populus trichocarpa; SEQ ID NO:185), 439887.1 (Sorghum bicolor; SEQ ID NO:186), XP_002458786.1 (Sorghum 20 bicolor; SEQ ID NO:187), XP_002463916.1 (Sorghum bicolor; SEQ I DNO:188), . XP_002464630.1 um bicolor; SEQ I DNO:189), XP_002511873.1 (Ricinus communis; SEQ ID' NO:190), XP_002517438.1 (Ricinus communis; SEQ ID NO:191), XP*002520171.1 (Ricinus_ communis; SEQ ID NO:192), XP_0I)2872955.1 (Arabidopsis lyrata; SEQ ID NO:193), XP_002881564.1 (Arabidopsis lyrata; SEQ 25 ID NO:194), ACT32032.1 (Vernicia fordii; SEQ. ID NO:195), 051‘189J (Oryza sativa', SEQ ID ), AFH02725.1 (Brassica napus; SEQ ID NO:197), XP_002320138.1 (Populus trichocarpa; SEQ ID ), XP_002451377.1 (Sorghum bicolor; SEQ ID NO:199), XP_002531350.1 (RiCinus communis; SEQ ID ), and XP_002889361.1 (Arobidopsis lyrata; SEQ ID NO:201). 30 ved motifs and/or residues can be used as a sequence-based diagnostic for the ﬁcation of bifunctional GPAT/phosphatase enzymes. Alternatively, a more ent function-based assay could be utilised. Such an assay involves, for example, feeding labelled glycerolphosphate to cells or microsomes and quantifying the levels of labelled produCts by thin-layer chromatography or a similar 35 que. GPAT activity results in the production of labelled LPA whilst GPAT/phosphatase activity results in the production of labelled MAG. ' ‘ As used herein, the term "Oleosin" refers to an amphipathic protein present in Dmembrane of oil bodies in the storage tissues of seeds (see, for e, Huang, ‘ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 88 1996; Lin et al., 2005; Capuano et al., 2007; Lui et al., 2009; a and Hara- Nishimura, 2010). ' Plant seeds accumulate TAGvin subcellular structures called oil bodies. These organelles consist of a TAG core surround by a phospholipid monolayer ning several embedded proteins including oleosins (Jolivet et al., 2004). Oleosins - represent the most abundant protein in the membrane of oil bodies.

Oleosins are of low Mr ,000).I Within each seed species, there are usually two or more oleosins of different Mr. Each oleosin molecule contains a vely hydrophilic N-terminal domain (for example, about 48 amino acid ’10 residues), a central totally hydrophobic domain (for example, of. about 70-80 amino acid residues) which is particularly rich in aliphatic amino acids such as alanine, glycine, leucine, isoleucine and valine, and an amphipathic a—helical domain (for' example, about of about 33 amino acid residues) at or near the C-terminus. Generally, the central stretch of hydrophobic residues is ed into the lipid core and the 15 Iamphiphatic N—terminal and/or haticC-tenninal are located at the surface of the oil“ bodies, with vely charged residues embedded in a phospholipid monolayer and the vely d ones exposed to the exterior. , As used herein, the term "Oleosin'l encompasses eosins which have multiple oleosin polypeptides fused together as a single ptide, for example 2x, 20 4x or 6x oleosin peptides, and caleosins which bind calcium sard et al., 2009), and steroleosins which bind sterols. However, generally a large proportion of the oleosins of oil bodies will not be caleosins and/or steroleosins.

A substantial number of oleosin protein sequences, and nucleotide sequences encoding or, are known from a large number of different plant Species. 25 Examples include, but are not limited to, oleosins from Arabidposis, canola, com, rice, peanut, castor, soybean, ﬂax, grape, cabbage, cotton, er, sorghum and barley. Examples of oleosins (with their Accession Nos) include Brassica napus oleosin (CAA57545.1; SEQ ID NO:362), Brassica napus oleosin S] -1 (ACG69504.1; SEQ ID NO:363), Brassica napus n 82-1 (ACG69503.1;’SEQ ID NO:364), 30 ca napus oleosin S3-1 (ACG69513.1; SEQ ID NO:365), Brassica napus oleosin S4-l (ACG69507.1; SEQ ID NO:366), Brassica napus oleosin SS—l (ACG6951 1.1; SEQ ID NO:367), Arachis hypogaea oleosin 1 (AAZ20276.1; SEQ ID NO:368), Arachis hypogaea oleosin 2 (AAU21500.1;‘SEQ ID NO:369), Arachis hypogaea oleosin 3 (AAU21501.1; SEQ ID NO:370), Arachis hypogaea oleosin 5 35_ (ABC96763.1; SEQ ID NO:371), Ricinus communis oleosin 1 (EEF40948.1; SEQ ID NO:372), Ricinus communis oleosin 2 (EEF51616.1; SEQ ID NO:373), Glycine max oleosin isoform a (P295302; SEQ ID NO:374), Glycine max oleosin isoform b D9531J; SEQ ID NO:375), Linum issimum n low molecular weight Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 89 isoform (AB301622.1; SEQ ID NO:376), Linum usitatissinium oleosin high molecular weight isoform (ABBOl624.l; SEQ ID NO:377), Helianthus annuus oleosin (CAA44224.1; SEQ ID NO:378), Zea mays oleosin (NP_001105338.1; SEQ ID NO:379), Brassica napus steroleosin (ABM30178.1; SEQ ID NO:380), Brassica napus steroleosin SLOl-l (ACG69522.1; SEQ ID NO:381), Brassica napus steroleosin SL02-1 (ACG69525.1; SEQ ID NO:382), Sesamum indicum steroleosin (AAL13315.1; SEQ ID NO:383), Zea mays steroleosin (NP_001152614.1;I SEQ ID NO:384), Brassica napus caleosin CLO-1 (ACG69529.1; SEQ ID NO:385), Brassica napus caleosin‘CLO-3 (ACGG9527.1; SEQ ID NO:386), Sesamum m in 10 (AAF13743.1; SEQ ID NO:387), Zea mays caleosin (NP_001151906.1; SEQ ID ‘ NO:388).

. As used herein, the term a "polypeptide involved in starch biosynthesis" refers to any polypeptide, the downregulation of which in a cell below normal (wild-type) I levels s in a reduction in the level of starch synthesis and an se in the 15 levels of starch. An example of such a polypeptide is AGPase. i As used herein, the term "ADP-glucose phosphorylase" or e" refers to . an enzyme which regulates starch thesis, catalysing conversion of glucose-1— phosphate and ATP to ADP-glucose which serves as the building block for starch polymers. The active form of the AGPase enzyme consists of 2 large and 2 small i 20 subunits. _ The ADPase enzyme in plants exists primarily as a tetramer which consists of 2 large and _2 small subunits. Although these ts differ in their catalytic and regulatory roles depending on the species (Kuhn et al., 2009), in plants the small subunit generally ys catalytic activity. The molecular weight of the small 25 subunit is approximately 50-55 kDa. The molecular weight of the large large subunit is approximately 55-60 kDa. The plant enzyme .is ly activated by 3- phosphoglycerate (PGA), a product of carbon dioxide ﬁxation; in the e of PGA, the enzyme exhibits only about 3% of its activity. Plant AGPase is also strongly inhibited by inorganic phosphate (Pi). In contrast, bacterial and algal 30 AGPase exist as homotetramers of SOkDa. The algal enzyme, like its plant rpart, is activated by PGA and inhibited by Pi, whereas the ial enzyme is - activated by se-1, 6-bisphosphate (FBP) and inhibited by AMP and Pi.

As. used herein, the term "polypeptide involved in the degradation of lipid ' and/or which reduces lipid conten " refers to any polypeptide, the downregulation of 35 which in a cell below normal (wild-type) levels results an increase in the level of oil, such as fatty acids and/or TAGS, in the cell, preferably a cell of vegetative tissue of a plant. Examples of such polypeptides include, but are not limited, lipases, or a lipase D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 90 such as CGi58 polypeptide, DEPENDENTI triacylglycerol lipase (see, for e, Kelly et al., 2012) or a lipase deceribed in WO 2009/027335.

H As used herein, the term “lipase” refers to a protein which hydrolyzes fats into glycerol and fatty acids: Thus, the term “lipase activity” refers to the hydrolysis of fats into glycerol and fatty acids.

As used herein, the term “CGi58” refers to a soluble acyl-CoA-dependent ‘ lysophosphatidic acid acyltransferase also known in the art art as "At4g24160" (in plants) and “Ictlp” (in yeast). The plant gene such as that from Arabidopsis gene locus, At4g24160, is expressed ative transcripts: full-length ' as two a longer 10 isoform (At4g24l60.l) and a smaller isoform (At4g24160.2) missing a portion of the 3' end (see James et al., 2010; Ghosh et al., 2009; US 201000221400). Both mRNAs code for a protein that is homologous to the human CGI-58 protein and other orthologous members of this (x/B ase family (ABHD). In an embodiment, the C6158 (At4g24160) protein contains three motifs that are conserved'across plant , 15 species: a GXSXG lipase motif (SEQ ID NO:419), a HX(4)D acyltransferase motif (SEQ ID NO:420), and VX(3)HGF, a probable lipid binding motif (SEQ ID NO:42l).

' The human CGI—58 protein has lysophosphatidic acid acyltransferase (LPAAT) activity but not lipase activity. In contrast, the plant and yeast proteins possess a canonical lipase sequence motif GXSXG (SEQ ID NO:419), that is absent from 20 vertebrate s, mice, and zebraﬁsh) proteins. Although the plant and yeast CG158 proteins appear to possess detectable amounts 'of TAG lipase and phospholipase A activities in on to LPAAT activity, the human protein does not.

Disruption of the homologous CGI-58 gene in Arabidopsis thaliana s in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of 25 isolated lipid droplets ﬁom cgi-58 loss—of—function mutants showed they contain triacylglycerols with common leaf-speciﬁc fatty acids. Leaves of mature cgi-58 plants exhibit a marked increase in absolute tn'acylglycerol levels, more than 10—fold higher than in wild-type plants. Lipid levels in the oil~storing seeds of cgi-58 loss-of- on plants were unchanged, and unlike mutations in B-oxidation, the cgi—_58 seeds 30 germinated and grew normally, requiring no rescue with sucrose (James et al., 2010).

Examples of CGi58 polypeptides include proteinsfrom Arabidopsis thaliana 4147.2; SEQ ID NO:429), Brachypodium hyon (XP_003578450.1; SEQ ID ), Glycine max 35236318.1; SEQ ID NO:431), Zea mays (NP_001149013.1; SEQ ID NO:432), Sorghum bicolor (XP_002460538.1; SEQ ID 35 ), Ricinus communis 2510485.I; SEQ ID NO:434), Medicago truncatula (XP_003603733.1; SEQ ID NO:435), and Oryza sativa (EAZO9782.1; , ' SEQ ID NO:436). ' ' D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 91 As used herein, the term “Leafy Cotyledon 2” or “LEC2” refers to a B3 domain transcription factor which participates in zygotic and in somatic embryogenesis. Its ectopic expression facilitates the embryogenesis from vegetative plant tissues (Alemanno et al., 2008). LEC2 also comprises a DNA binding region found thus far only in plant proteins. Examples of LECZ polypeptides include ns from Arabidopsis thaliana (NP_564304.1) (SEQ ID N0:442), Medicago truncatula (CAA42938.I) (SEQ ID N0:443) and Brassica napus (AD016343.l) (SEQ ID N0:444). ‘ As used herein, the term “BABY BOOM” or “BBM” refers an AP2/ERF '10 transcription factor that induces" regeneration under culture conditions that normally do not support ration in wild-type plants; Ectopic expression of Brassica napus ' BBM (BnBBM) genes in B. napus and Arabidopsz's induces spontaneous somatic genesis and genesis from seedlings grown on hormone-free basal medium lier et al., 2002). In tobacco, ectopic BBM expression is sufﬁcient to ‘15 induce adventitious shoot and root regeneration on basal medium, but exogenous cytokinin is required for somatic embryo (SE) formation (Srinivasan et al., 2007).

Examples of BBM polypeptides include proteins from Arabidopsis thaliana (NP_197245.2) (SEQ ID N0:445) and Medicago truncatula (AAW82334.1) (SEQ ID N0:446).

I 20 As used herein, the term "FADZ" refers to a membrane bound delta-12 fatty acid ase that desaturates oleic acid 08:1”) to produce ic acid (C1832A9'12).

As used herein, the term 'fepoxygenase" or "fatty acid epoxygenase" refers to an enzyme that introduces an epoxy group into a fatty acid resulting in the production of an epoxy fatty acid. In preferred embodiment, the epoxy group is uced at the 25 12th carbon on a fatty acid chain, in which case the epoxygenase is a A12- epoxygenase, especially of a C16 or C18 fatty acid chain. The epoxygenase may be a A9-epoxygenase, a A15 epoxygenase, or act at a different position in the acyl' chain as known in the art. The epoxygenase may be of the P450 class. red epoxygenases are ,of the mono-oxygenase class as described in WO98/46762. Numerous . 30 epoxygenases or presumed enases have been cloned and are known in the art.

'Further examples of expoxygenases include proteins comprising an amino acid in SEQ ID N022] of WC 2009/129582, polypeptides d by . sequence provided genes from Crepis paleastina (CAA76156, Lee et al., 1998), Stokesia laevis (AAR23815, Hatanaka et al., 2004) (monooxygenase type), Euphorbia lagascae 35 (AAL62063) (P450 type), human CYPZJ2 (arachidonic acid epoxygenase, U37143); human CYP1A1 (arachidonic acid epoxygenase, K0319l), as well as variants and/or mutants thereof. ‘ D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 92 As used herein, the term, "hydroxylase" or "fatty acid ylase" refers to an enzyme that uces a hydroxyl group into a fatty acid resulting in the production of a ylated fatty acid. In a preferred embodiment, the hydroxyl group is introduced at the 2nd, 12th and/or 17th carbon on a C18 fatty acid chain. Preferably, the hydroxyl group is introduced at the 12th carbon, in which case the hydroxylase is a A12-hydroxylase. In another preferred embodiment, the hydroxyl group is introduced at the 15th carbon on a C16 fatty acid chain. Hydroxylases may also have enzyme activity as a fatty acid desaturase. Examples of genes encoding A12-hydroxylases include those from Ricinus cbmmunis (AAC9010, van de L00 1995); Physaria 10 lindheimeri, (ABQ01458, Dauk et al., 2007); rella fendleri, (AAC32755, ’Broun et al., 1998); Daucus carota, (AAK30206); fatty acid hydroxylases which ‘ hydroxylate the termimis of fatty acids, for example: A. na CYP86A1 (P48422, fatty acid w—hydroxylase); Vicia sativa 1 (P98188, fatty acid co-hydroxylase); mouse CYP2E1 (X62595, lauric acid w-l hydroxylase); rat CYP4A1 (M57718, fatty _ ‘ 15 acid w7hydroxylase), as well as as variants and/or mutants thereof.

As used herein, the term "conjugase" or "fatty acid conjugase" refers to. an enzyme e of forming a conjugated bond in the acyl chain of a fatty acid.

Examples of conjugases e those encoded by genes from ula oﬁ‘icinalis (AF343064, Qiu et al., 2001); Vernicia fordii (AAN87574, Dyer et al., 2002); Punica 20 granatum (AY178446, Iwabuchi et al., 2003) and Trichosanthes kiriloivii (AY178444, Iwabuchi et al., 2003); as. well as as variants and/or‘mutants thereof.

As used herein, the term "acetylenase" or "fatty acid acetylenase" refers to an enzyme that introduces a triple bond into a fatty acid resulting in the production of an acetylenic fatty acid. In a preferred embodiment, the triple bond is introduced at the _25 2nd, 6th, 12th and/or 17th carbon on a C18 fatty acid chain. Examples acetylenases include those from Helianthus annuus (AAO3 8032, ABC59684), as well as as variants and/or mutants thereof.

Examples of such fatty} acid modifying genes include proteins according to the ing Accession Numbers which. are grouped by putative on, and 30 homologues from other species: A12 acetylenases ABC00769, CAA76158, AAO38036, AAO38032; A12 conjugases AAG42259, AAG42260, AAN87574; A12 desaturases P46313, 16, AASS7577, AAL61825, 93, 94; A12 epoxygenases XP_001840127, CAA76156, 15; A12 hydroxylases ‘ 'ACF37070, AAC32755, ABQ01458, AAC49010; and A12 P450 enzymes such as 35 AF406732.

As used herein, the term "vegetative tissue" or "vegetative plant part" is any plant tissue, organ or part other than organs for sexual reproduction of plants, Dciﬁcally seed bearing organs, ﬂowers, pollen, fruits and seeds. Vegetative tissues Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 93 and parts include at least plant leaves, stems (including bolts and tillers but excluding the heads), tubers and roots, but excludes ﬂowers, pollen, seed including the seed coat, embryo and endosperm, fruit including mesocarp tissue, seed-bearing pods and seed-bearing heads. In one embodiment, the vegetative part of the plant is an aerial plant part. In another or further embodiment, the vegetative plant part is a green part . such as a leaf orstem.

As used herein, the term "wild-type" or variations thereof refers to a cell, or non-human organism or part f that has not been genetically modiﬁed.

The term "corresponding" refers to a vegetative plant part, a cell, or non- 10 human organism or part thereof, or seed that has the same or similar genetic background as a vegetative plant part, a cell, or non-human organism or part f, or seed of the invention but that has not been d as described herein (for e, a vegetative plant part, a cell, or man organism or part thereof, or seed lacks an exogenous polynucleotide encoding a MGAT or an exogenous MGAT). 15 In a preferred embodiment, a vegetative plant part, a cell, or non-human organism or part thereof, or seed is at the same developmental stage as a vegetative plant part, a‘ cell, or non-human organism or part thereof, or seed of the invention. For example, if the non-human organism is a ﬂowering plant, then preferably the corresponding plant is also ﬂowering. A ponding a vegetative plant part, a cell, or non-human 20 organism or part thereof, or seed can be used as a control to e levels of nucleic acid or protein expression, or the extent and nature of trait modiﬁcation, for example . non-polar lipid production and/or content, with a vegetative plant part, a cell, or non- human sm or part thereof, or seed modiﬁed as described herein. A person skilled in the art is readily able to determine an appropriate "corresponding" cell, 25 tissue, organ or sm for such a comparison. _ As used herein "compared with" refers to comparing levels of a non-polar lipid or total non-polar lipid t of the enic non-human sm or part thereof expressing the one or more exogenous polynucleotides or exogenous polypeptides with a transgenic non-human organism or part thereof lacking the one or more ‘ 30 exogenous polynucelotides or polypeptides.

As used herein, "enhanced ability to prodube non-polar lipid" is a relative term which refers to the total‘ amount of lar lipid being produced by a cell, or non- human organism or part thereof of the invention being sed relative to a corresponding cell, or man organism or part thereof. In one embodiment, the 35 TAG and/or polyunsaturated fatty acid content of the non-polar lipid is increased.

As used herein, i'germinate at a rate ntially the same as for a corresponding wild-type plant" refers to seed of a plant of the invention being Dtively fertile when compared to seed of a wild type plant lacking the deﬁned Substitute Sheet.

(Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 94 exogenous polynucleotide(s). In one embodiment, the number of seeds which germinate, for instance when grown under optimal greenhouse conditions for the plant species, is at least 75%, more ably at least 90%, of that when compared to corresponding wild-type seed. In another embodiment, the seeds which germinate, for ce when grown under optimal greenhouse conditions for the plant species, grow at a rate which, on average, is at least 75%, more preferably at least 90%, of that ' when compared to corresponding wild-type plants.

As used herein, the term "an iSolated or recombinant polynucleotide which down regulates the production and/or ty of an nous enzyme" or variations 10‘ thereof, refers to a cleotide that encodes an RNA molecule that down regulates ’ the production and/or activity (for example, encoding an siRNA, hpRNAi), or itself down regulates the production and/or ty (for example, is an siRNA which can be delivered directly to, for example, a cell) of an endogenous enzyme for example, DGAT, sn-l' glycerolphosphate ansferase (GPAT), , l-acyl-glycerol 15 phosphate acyltransferase (LPAAT), acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), phosphatidic acid phosphatase (PAP), AGPase, or delta-12 fatty acid desturase (FADZ), or a combination of two or more thereof.

As used herein, the term "on a weight basis" refers to the weight of a substance (for example, TAG, DAG, fatty acid) as a percentage of the weight of the composition 20 comprising the substance (for example, seed, leaf). For example, if a transgenic seed has 25. pg total fatty acid per 120 ug seed weight; the tage of total fatty aCid on a weight basis is 20.8%.

As used , the term "on a ve basis" refers to the amount of a substance in a composition comprising the substance in comparison with a 25 corresponding composition, as a percentage.

As used herein, the term "the relative non-lipid content" refers to the expression of the non-polar lipid content of a cell, organism or part thereof, or extracted lipid therefrom, in comparison with a corresponding cell, organism or part thereof, or the lipid extracted from the corresponding cell, organism or part thereof, as 30 a percentage, For e, if a transgenic seed has 25 pg total fatty acid, whilst the corresponding seed had 20 pg total fatty acid; the increase in non-polar lipid content on a relative basis equals 25%.

As used herein, the term "biofuel" refers to any type of fuel, typically as used to power machinery such as automobiles, trucks or petroleum powered motors, whose 35 energy is derived from biological carbon n. ls include fuels derived-from biomass conversion, as well as solid biomass, liquid fuels and biogases. Examples of biofuels include bioalcohols, biodiesel, synthetic diesel, vegetable Oil, bioethers, Dgas, syngas, solid biofuels, algae-derived fuel, biohydrogen, biomethanol, 2,5- Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 95 Dimethylfuran (DMF), ethyl ether (bioDME), Fischer-Tropsch, diesel, biohydrogen diesel, mixed alcohols and wood diesel.

As used herein, the term ‘fbioalcohol" refers to biologically produced alcohols, for e, l, propanol and butanol. Bioalcohols are produced by the action of microorganisms and/or enzymes through the fermentation of sugars, hemicellulose or cellulose.

As used , the term "biodiesel" refers to a composition comprising fatty ' acid methyl- or ethyl,- esters derived from non-polar lipids by transesteriﬁcation.

As used herein, the term "synthetic diesel" refers to a form of diesel fuel-which 10 is derived ﬁ'om renewable feedstock rather than the fossil feedstock used in most diesel fuels. _ As used herein, the term "vegetable oil" includes a pure plant oil (or straight vegetable oil) or a waste vegetable oil (by product of other industries).

As used herein, the term ‘bioethers" refers to nds that act as octane I 1'5 rating enhancers.

As used herein, the term sl' refers to methane or a ﬂammable e of methane and other gases produced by anaerobic digestion of c material by ‘ anaerobes. .

As used herein, the term "syngas" refers to a gas mixture that contains varying 20 amounts of carbon monoxide and hydrogen and possibly other hydrocarbons, produced by partial combustion of biomass.

As used herein, the term “solid biofuels" includes wood, t, grass trimmining, and non-food energy crops.

As used herein, the term "cellulosic ethanol" refers to ethanol ed from I 25 cellulose or hemicellulose.

As used herein, the term "algae fue " refers to a biofuel made from algae and includes algal biodiesel, biobutanol, biogasoline, methane, l, and the equivalent of vegetable oil made from algae.

As used herein, the term "biohydrogen" .' refers to hydrogen produced i 30 biologically by, for example, algae. _ , As used herein, the term "biomethanol' refers to methanol produced biologically. Biomethanol may be produced by gasification of organic materials to ed by conventional methanol synthesis. . syngas As used herein, the term "2,5-Dimethylﬁ1ran" or "DMF" refers to a 35 heterocyclic compound with the formula C4H20. DMF is a derivative of furan ’ that is derivable from cellulose.

, As used herein, the term "biodimethyl ether" or "bioDME", also known as Dioxymethane, refers to am organic compound with the formula CH30CH3.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 96 Syngas may be converted into methanol in the presence of catalyst (usually copper- based), with subsequent methanol dehydration in the presence of a different catalyst (for e, -alumina) resulting in the production of DME. _ As used , the term er—Tropsch" refers to a set of chemical reactions that convert a mixture of carbon monoxide and hydrogen into liquid hydrocarbons. The syngas can ﬁrst be conditioned using for example, a water gas shift to achieve the required Hz/CO ratio. The conversion takes place in the presence of a catalyst, usually iron or cobalt. The temperature, re and catalyst determine r a light or heavy syncrude is produced. For example at 330°C mostly gasoline _1o and oleﬁns, are produced whereas at 180° to 250°C mostly diesel and waxes are produCed. The liquids ed ﬁom the . , which se various hydrocarbon fractions, are very clean ur free) straight-chain hydrocarbons.

Fischer-Tropsch diesel can be produced directly, but a higher yield is achieved if ﬁrst ' Fischer-Tropsch wax is produced, followed by hydrocracking. ' 15 As used herein, the term "biochar" refers to charcoal made from biomass, for example, by pyrolysis of the biomass. _ As used herein, the term "feedstock" refers to a material, for example, biomass or a. conversion product thereof (for example, syngas) when used to produce a t, for example, a biofuel such as biodiesel, or a synthetic diesel. 20 As used , the term "industrial product" refers to carbon product > which is predominantly made of carbon and en such as fatty acid methyl- and/or ethyl-esters or alkanes ‘such as methane, mixtures of longer chain alkanes 'which are typically liquids at ambient temperatures, a l, carbon monoxide and/or hydrogen, or a bioalcohol such as ethanol, propanol, or butanol, or biochar. 25 The term trial product" is intended to include intermediary products that can be converted to other industrial products, for example, syngas is itself considered to be an industrial product which can be used to synthesize a hydrocarbon product which is} also considered. to be an industrial product. The term industrial product as used herein includes both pure forms of the above compounds, or more commonly a mixture of 30 various nds and components, for example the hydrocarbon product may‘ ‘ contain a range ofcarbon chain lengths, as well understood in the art. \ As used herein, "gloss" refers to an optical phenomenon caused when evaluating the appearance of a surface. The evaluation of gloss describes the capacity of a surface to reﬂect directed light. 35 Throughout this speciﬁcation the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion any other element, integer or step, or group of elements, integers or steps. L I Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 97 The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings ' or for either meaning.

‘ As used herein, the term about, unless stated to the contrary, refers to +/- 10%, more ably +/- 5%, more preferably +/- 2%, more preferably +/- 1%, even more preferably +/— 0.5%, of the designated value.

, Production of Diacylgylerols and Triacylglycerols In one ment, the vegetative plant part, transgenic non-human organism 10 or part thereof of the invention es higher levels of non-polar lipids such as DAG or TAG, preferably both, than a corresponding vegetative plant part, non-human sm or part f. In one example, enic plants of the invention produce seeds, leaves, leaf portions of at least lcm2 in surface area, stems and/or tubers having an increased non-polar lipid content such as DAG or TAG, preferably both, when 15 compared to corresponding seeds, leaves, leaf portions of at least 1cm2 in surface area, stems or tubers. The non-polar lipid content of the vegetative plant part, non- human organism or part thereof is at 0.5% greater on a weight basis when compared to a corresponding non-human organism or part thereof, or as further deﬁned in Feature (i). 20 'In another embodiment, the tive plant part, transgenic non-human sm or part thereof, preferably a plant or seed, produce DAGs and/or TAGs that are enriched for one or more particular fatty acids, A wide spectrum of fatty acids can be incorporated into DAGs and/or TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids. Some non-limiting es of fatty 25 acids that can be incorporated into DAGs and/or TAGS and which may be increased in level include: capric (10:0), lauric (12:0), myristic , palmitic (16:0), palmitoleic (16:1), stearic , oleic (18:1), vaccenic (18:1), ic (18:2), eleostearic (18:3), lenic “(1823), a~linolenic 03), stearidonic (18:4m3), arachidic (20:0), eicosadienoic (20:2), dihomo-y-linoleic (20:3), eicosatrienoic (20:3), 3O arachidonic (20:4), eicosatetraenoic (20:4), eicosapentaenoic (20:5(03), behenic ‘ (22:0), docosapentaenoic (22:50)), docosahexaenoic (22:603), lignoceric (24:0), nervonic (24:1), cerotic (26:0), and montanic (28:0) fatty acids. In one embodiment of the present invention, the vegetative plant part, transgenic-organism or parts thereof is enriched for DAGs and/or TAGS comprising oleic acid, or polyunsaturated fatty '35 acids. _ In one embodiment of the invention, the vegetative plant part, transgenic non- human organism or part thereof, ably a plant or seed, is transformed with a Dienc DNA which encodes an MGAT which may or may not have DGAT activity.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 9.8 Expression of the MGAT preferably results in higher levels of non-polar lipidssuch as DAG or TAG and/or increased non-polar lipidlyield in said vegetative plant part, transgenic man organism or part thereof. In a preferred embodiment, the I transgenic non-human organism is a plant.

In a further embodiment, the vegetative plant part, transgenic non-human organism or part thereof is transformed with a chimeric DNA which encodes a GPAT or a DGAT. Preferably, the vegetative plant part or transgenic non-human organism is transformed with both chimeric DNAs, which are preferably ntly linked on one DNA molecule such as,‘for example, a single T-DNA molecule. 10 Yang et al. (2010) describe two glycerolphosphate acyltransferases (GPAT4 and GPAT6) from Arabidopsis with sn-2 preference and phosphataseactivity that are able to e sn-2 MAG from glycerolphosphate (GP) (Figure 1). These I s are proposed to be part of the cutin sis y. Arabidopsis GPAT4 I and GPATG have been shown to use acyl-CoA as a fatty acid substrate (Zheng et al., ' 15 2003) .

, Combining a tional GPAT/phosphatase with a MGAT yields a novel ‘ DAG synthesis pathway using G-3—P as one substrate and two acyl groups d from acyl-CoA as the other substrates. Similarly, combining such a bifunctional GPAT/phosphatase with a MGAT which has DGAT ty yields a novel TAG 20 synthesis pathway using glycerolphosphate as one substrate and three acyl groups . derived from acyl-CoA as other substrates.

. Accordingly, in one embodiment of the ion, the vegetative plant part, . transgenic non-human organism or part thereof is co-transformed with a bifunctional GPAT/phosphatase and with a MGAT which does not have DGAT activity. This 25 would result in the production ofMAG by the bifunctional GPAT/phosphatase which [would then be converted to DAG by the MGAT and then TAG by a native DGAT or other activity. Novel DAG production could be Conﬁrmed and selected for by, for e, performing such a co-transformation in a yeast strain ning lethal SLCI + SLC4 knockouts such as that described by Benghezal et al. (2007; Figure 2). Figure 30‘ 2 of Benghezal et al. (2007) shows that knocking out the two yeast LPATS (SLCl & SLC4) is lethal. The S'LCI + SLC4 double yeast mutant can only be maintained because of a complementing plasmid which provides one of the slc genes (SLCl in their case) in trans. Negative selection by adding FOA to the medium results in the loss of this complementing plasmid (counterselecticn of the Ura selection ) and I renders the cells non viable.

In another embodiment of the invention, the vegetative plant part, transgenic non-human organism or part thereof, preferably a plant or seed, is co-transformed ' D1 chimeric DNAs encoding a bifunctional GPAT/phosphatase, and a MGAT which Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 99. has DGAT activity. This would result in the production of MAG by the bifunctional GPAT/phosphatase which would then be converted to DAG and then TAG by the MGAT. ' ’ ' In a further embodiment, one or more endogenous GPATs with no detectable phosphatase activity are silenced, for example one or more genes encoding GPATs that acylate glycerol-S-phosphate to form LPA in the Kennedy Pathway, (for example, ArabidOpsis GPATl) is silenced.

In another embodiment, the tive plant part, transgenic non-human organism or part thereof, preferably a plant or seed, is transformed with a ic 10 DNAs encoding a DGAT], a DGAT2, a Wrinkled 1 (WRIl) transcription‘factor, an Oleosin, or a silencing suppressor polypeptide. The chimeric DNAs are preferably covalently linked on one DNA le such as, for example, a single T-DNA molecule, and the , vegetative plant or _ part, transgenic non-human organism part thereof is preferably homozygous for the one DNA le inserted into its genome. 15 Substrate preferences could be engineered into the novel DAG and TAG synthesis pathways by, for example, supplying transgenic H1246 yeast strains expressing MGAT variants with a concentration of a particular free fatty acid (for e, DHA) that prevents complementation by the wildtype MGAT gene. Only the variants able to use the supplied free fatty acid would grow. Several cycles of 20 MGAT engineering would result in the production of a MGAT with increased . preference for particular fatty acids.

‘ The various Kennedy Pathway. complementations and _ supplementations described above could be performed in any cell type due to the ubiquitous nature of the initial substrate ‘glycerol—3-phosphate. In one embodiment, the use of transgenes 25 results in increased oil yields.

Polynucleotides ' The terms "'polynucleotide", and "nucleic acid"‘are used interchangeably.

They. refer to. a ric form of nucleotides of any length, either 30 ibonucleotides or ribonucleotides, or analogs thereof. A polynucleotide of the invention may be of c, cDNA, nthetic, or synthetic origin, double- stranded or single-stranded and by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, ‘ 35 or (3) does not occur in nature.‘ The following are. miting examples of polynucleotides: coding or non-coding s of a gene or gene fragment, loci (locus) deﬁned from linkage analysis, exons, introns, messenger RNA (mRNA), Dsfer RNA. (tRNA), ribosomal RNA , ribozymes, cDNA, recombinant Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 100 polynucleotides, ed polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNA of any ce, nucleic acid probes, and primers. A polynucleotide may comprise modiﬁed nucleotides such as methylated tides and nucleotide analogs. If present, modiﬁcations to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modiﬁed after polymerization such as by conjugation with a labeling ent.

By ted polynucleotide" it is meant a polynucleotide which has generally 10 been separated from the polynucleotide Sequences with which it is associated or linked1n its native state. Preferably, the isolated polynucleotideis at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from the * polynucleotide sequences with. which it is naturally associated or linked.

As used herein, the term "gene" is to be taken in its broadest context and 15 includes the deoxyribonucleotide sequences comprising the transcribed region and, if translated, the protein coding region, of a structural gene and including sequences located adjacent to the coding region on both the S' and 3' ends for a distance of at least about 2 kb on either end and which are involved in expression of the genes In this regard, the gene includes control signals such as promoters, enhancers, 20 termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control Signals, in which case, the gene is referred to as a "chimeric gene". The ces which are located 5' of the protein coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are d 3' or ream of the protein coding region and which 25 are present on the mRNA are referredto as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A c form or ' clone of a gene contains the coding region which may be interrupted with non-coding sequences termed "introns", "intervening regions", or "intervening sequences." .

Introns are segments of a gene which are transcribed into nuclear RNA . 30 Introns may contain regulatory elements such as enhancers. Introns are d or ed out" from the nuclear or primary transcript; introns therefore are absent in the mRNA transcript. The mRNA ﬁmctions during translation to specify the sequence or order of amino acids in a nascent ptide. The term "gene" includes a synthetic or fusion le encoding all or part of the proteins of the invention described '35! herein and a complementary nucleOtide sequence to any one of the above.

As used herein, "chimeric DNA" refers to any DNA le that is not naturally found1n nature; also referred to herein as a "DNA construct" Typically, Dneric DNA comprises regulatory and ribed or protein coding sequences that Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 101,' are not naturally found together in nature. Accordingly, chimeric DNA may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences d from the same source, but arranged in a manner different than that found in nature. The open g frame may or may not be linked to its natural upstream and downstream regulatory elements.

The open reading frame may be incorporated into, for example, the plant genome, in a non-natural location, or in a replicon or vector where it is not naturally found such as a ial plasmid or a viral vector. The term "chimeric DNA" is not limited to DNA molecules which are replicable in a host, but includes DNA capable of being ligated 1.0 . into a replicon by, for example, speciﬁc adaptor sequences.

A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The term includes a gene in a progeny cell, plant, seed, non-human organism or part thereof which was introducing into the genome of a itor cell thereof. Such progeny cells etc may be at least a 3rd or 4th generation 15 progeny from the progenitor cell which was the primary transformed cell. Progeny may be produced by sexual reproduction or vegetatively' such as, for example, from tubers in potatoes or ratoons in ane. The term ically modiﬁed", and " variations f, is a broader term that includes introducing a gene into a cell by transformation or transduction, mutating a gene in a cell and genetically altering or 20 modulating the regulation of a gene in a cell, or the progeny of any cell modiﬁed as bed aboVe.

I A "genomic region" as used herein refers to a position within the genome where a transgene, or group of enes (also referred to herein as a'cluster), have been ed into a cell, or predecessor thereof. Such regions only comprise 25 nucleotides that have been incorporated by the ention of man such as by i I ‘ methods described herein.

A "recombinant polynucleotide" of the invention refers to a nucleic acid - molecule which has been constructed or d by artiﬁcial recombinant methods.

The inant polynucleotide may be present in a cell in an altered amount or 3O expressed at an altered rate (e.g., in the case of mRNA) ed to its native state.

. In one embodiment, the cleotide is introduced into a cell that does not naturally comprise the polynucleotide. Typically an exogenous DNA is used as a template for ~ transcription of mRNA which is then translated into a continuous sequence of amino acid es coding fora polypeptide of the invention within the transformed cell. In ,35 another ment, the polynucleotide is endogenousto the cell and its expression is altered by recombinant means, for example, an exOgenous control sequence is introduced upstream ofan endogenous gene of interest to enable the transformed cell Express the polypeptide encoded by the gene.

Substitute Sheet (Rule ’26) RO/AU .

[Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 102 A recombinant polynucleotide of the invention includes polynucleotides which have not been separated from other components of the cell-based or cell-free expression system, in which it is present, and polynucleotides produced in said cell- based or cell-free s which are subsequently puriﬁed away from at least some other components. The polynucleotide can be a uous stretch of nucleotides existing in , or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single cleotide. Typically, such chimeric polynucleotides comprise at least an open g frame encoding a polypeptide of the invention operably linked to a promoter 10 suitable of driving transcription of the open reading frame in a cell of st.

With regard to the deﬁned polynucleotides, it will be appreciated that % ~identity ﬁgures higher than those provided above will, encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity ﬁgures, it is preferred that the polynucleotide comprises a polynucleotide ce which is at 15 least 60%, more preferably at least 65%, more ably at least 70%, more- preferably at least 75%, more preferably at least 80%, more preferably at least 85%, 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 ably at least 95%, more ably at least 96%, more preferably at least 97%, more 20 preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. 25 A polynucleotide of, or useful for, the t invention/may selectively hybridise, under stringent conditions, to a polynucleotide deﬁned herein. As used herein, stringent conditions are those that: (I) employ during isation a denaturing agent such as formamide, for example, 50% (v/v) forrnamide with 0.1% (w/v) bovine serum albumin, 0.1% Ficoll, 0.1% nylpyrrolidone, 50 mM sodium 30 phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (2) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt‘s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS, and/or (3) employ low ionic strength and high 35 temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% ' SDS at 50°C.

Polynucleotides of the invention may possess, when compared to naturally During molecules, one or more mutations which are deletions, insertions, or ' Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 103 substitutions ofnucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either lly occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shufﬂing on the nucleic acid as bed above).

Polmucleotide for Reducing Expression Levels of Endogenous Proteins I RNA Interference. .

. RNA interference (RNAi) is particularly useful for speciﬁcally ting the '10 production .of a ular n. Although not wishing to be limited by , Waterhouse et a1. (1998) have provided a model for the mechanism by which dsRNA (duplex RNA) can be used to reduce protein production. This technology relies on the presence of dsRNA molecules that contain a ce that is essentially identical to the mRNA of the gene of interest or part thereof. Conveniently; the dsRNA can be 15 produced from a single promoter in a recombinant vector or host( cell, where the sense and anti-sense sequences are ﬂanked by an ted sequencelwhich enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules is well within'the capacity of a person skilled in the art, particularly 20 considering Waterhouse et al. , Smith et a1. (2000), W0 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.

' In one example, a DNA is introduced that directs the synthesis of an at least partly double stranded RNA product(s) with homology to the target gene to be inactivated. The DNA therefore ses both sense and antisense sequences that, 25 when transcribed into RNA, can hybridize to form the double stranded RNA region.

In one embodiment of the invention, the sense and antisense sequences are separated by a spacer region that comprises an intron which, when transcribed into RNA, is d out. This arrangement has been shown to result in a higher efﬁciency of gene ing. The double stranded region may comprise one or two RNA molecules, 30 transcribed from either one DNA region or two. The presence of the double stranded molecule is thought to trigger a response from an endogenous system that destroys both the double stranded RNA and also the homologous RNA transcript from the . target gene, efﬁciently reducing or eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridize should each be '35 at least l9pcontiguous tides.L The full-length sequence corresponding to the entire gene transcript may be used. The degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, at least 90%, or at least D100%. ‘The RNA molecule may of course comprise unrelated sequences which ‘ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 104 may function to stabilize the molecule. The RNA le may be expressed under the control of a RNA polymerase II or RNA polymerase III promoter. Examples of the latter e tRNA or snRNA promoters.

. ' Preferred small interfering RNA ("siRNA") molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA.

Preferably, the siRNA sequence ces with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more ably about 4S%-55%), and does not have a high percentage identity to any tide ce other than the target in the genome of the sm in which it is 10 to be introduced, for example, as determined by standard BLAST search. microRNA p MicroRNAs (abbreviated miRNAs) are generally 19—25 nucleotides (commonly about 20-24 nucleotides in plants) ding RNA molecules that are 15 derived from larger precursors that form imperfect stem—loop structures. miRNAs bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually ing in translational repression or target degradation and gene silencing. \ .

In plant cells, miRNA precursor molecules are believed to be largely processed 20 in the nucleus. The pri—miRNA (containing one or more local -stranded or "hairpin" regions as well as the usual 5' "cap" and polyadenylated tail of an mRNA) is ' processed to a shorter miRNA precursor molecule that also includes a stem-loop or fold-back structure and is termed the iRNA". In plants, the pre-miRNAs are cleaved by distinct DICER-like (DCL) enzymes, yielding miRNA:miRNA* duplexes. 25 Prior to transport out of the nucleus, these duplexes are methylated.

, In the cytoplasm, the miRNA strand from the miRNA duplex is selectively orated into an active RNA-induced silencing complex (RISC) for target recognition.The RISC— complexes contain a particular subset Of Argonaute (see, for example, Millar and . proteins that exert sequence-speciﬁc gene repression 30 Waterhouse, 2005; Pasquinelli et al., 2005; Almeida and re, 2005).

Cosuppression .

Genes can suppress the expression of related endogenous genes and/or transgenes already present in the genome, a enon termed homology-dependent 35 gene silencing. Most of the instances of homologydependent gene silencing fall into two classes - those that function at the level of transcription of the transgene, and those that operate post-transcriptionally.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 105 Post-transcriptional homology—dependent gene silencing (i.e.,‘ cosuppression) describes the loss of expression of a transgene and related endogenous or viral genes in transgenic plants. ression often, but not always, occurs when ene transcripts are abundant, and it is generally thought to be triggered at the level of rnRNA processing, localization, and/or degradation. Several models exist to explain how cosuppression works (see in Taylor, 1997). ' One model, the "quantitative" or "RNA threshold" model, proposes that cells can cope with the accumulation of large amounts of ene ripts, but only up to a point. Once that al threshold has been crossed, the sequence-dependent 10 degradation of both transgene and related endogenous gene transcripts is initiated. It has been proposed that this mode of cosuppression may be triggered following the synthesis of copy RNA (cRNA) molecules by reverse transcription of the excess transgene mRNA, presumably by nous RNA-dependent RNA polymerases.

These cRNAs may hybridize with transgene and endogenous mRNAs, the unusual 15 hybrids targeting gous ripts for ation. However, this model does not account for reports suggesting that cosuppression can apparently occur in the . absence of transgene transcription and/or t the detectable accumulation of transgene transcripts. ' To account for these data, a second model, the "qualitative" or "aberrant RNA" . 20 model, proposes that interactions between transgene RNA and DNA and/or between endogenous and introduced DNAs lead to the methylation of transcribed s of the genes. The methylated genes are proposed to produce RNAs that are in some way aberrant, their anomalous features triggering the speciﬁc degradation of all related transcripts. Such aberrant RNAs may be ed by complex transgene loci, 25 particularly those that contain inverted repeats. _ A third model proposes that olecular base. pairing between transcripts, rather than RNA hybrids generated through the“ action of an RNA-dependent RNA rase, may trigger cosuppression. Such base pairing maybecome more common as transcript levels rise, the putative double-stranded regions triggering the 3O targeted degradation of homologous ripts. A similar model proposes olecular base pairing instead of intermolecular base g between transcripts- Cosuppression involves introducing an extra copy of a gene or a fragment thereof into a plant in the sense orientation with respect to a promoter for its expression. A skilled person would appreciate that the size of the sense fragment, its 35 correspondence to target gene regions, and its degree of sequence identity to the target ' gene can vary. In some instances, the additional copy of the gene sequence interferes with the expression of the target plant gene. Reference is made to WO 97/20936 and.

D0465572 for methods of implementing co-suppression approaches.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 106 Antisense Polynucleotides The term "antisense polynucletoide" shall be taken to mean a DNA or RNA, or combination thereof, molecule that is complementary to at least a portion of a specific ‘ mRNA molecule encoding an endogenous polypeptide and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is well known in the art (see for e, G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)). The use of antisense techniques in plants has been ed by Bourque (1995) and Senior (1998). Bourque (1995) lists a large , 10 number of examples of how nse sequences have been utilized in plant systems as a method of gene inactivation. Bourque also states that attaining 100% inhibition ofany enzyme activity may not be necessary as partial tion will more than likely I result in measurable change in the system. Senior (1998) states that antisense methods are now a very well established technique for manipulating gene expression.

I I 15 " In one embodiment, the antisense polynucleotide hybridises under logical conditions, that is, the nse polynucleotide (which is fully or partially single stranded) is at least capable of forming a double stranded polynucleotide with mRNA encoding a protein such as an endogenous enzyme, for e, DGAT, GPAT, LPAA, LPCAT, PAP, AGPase, under normal conditions in V 20 acell; - , Antisense les may e sequences that correspond to the structural genes or for sequences that effect control over the gene expression or splicing event.

For example, the antisense sequence may correspond to the targeted coding region of endogenous gene, or the 5'-untranslated region (UTR) or the 3'-UTR or combination 25' of these. It may be complementary in part to intron sequences, which may be spliced out during or aﬁer transcription, preferably only to exon sequences of the target gene.

In view of the generally greater divergence of the UTRs, targeting these regions ‘ provides greater speciﬁcity of gene inhibition.

The length of the antisense sequence should be at least 19 uous 30 nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides. The full-length sequence complementary to the entire gene transcript may be used. The length is most preferably 00 nucleotides. The degree of ty of the antisense sequence to the targeted transcript should be at least , 90% and more preferably %.‘ The antisense RNA molecule may of course 35' comprise unrelated sequences which may function to ize the molecule.

,D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 107 Catalytic Polynucleotides The term "catalytic polynucleotide" refers to a, DNA molecule or DNA— containing molecule (also‘k'nown in the art as a. yribozyme") or an RNA or RNA-containing molecule (also knbwn as a "ribozyme") which speciﬁcally _ recognizes a distinct substrate and catalyses the chemical modiﬁcation of this substrate. The nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T ' (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence for speciﬁc recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity 10 (also referred to herein as the "catalytic domain"). The types of ribozymes that are particularly useful in this invention are hammerhead mes (Haseloff and I Gerlach, 1988; Perriman et al., .1992) and hairpin ribozymes ukhin et al., 1996; Klein eti'al., 1998; Shippy et al., 1999).

Ribozymes useful in the invention and DNA encoding the ribozymes can be '15 chemically synthesized using methods well known'in the art. The ribozymes can also be prepared from a DNA molecule (that upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, for example, the promoter for T7 RNA polymerase or SP6 RNA polymerase. In a te embodiment, the DNA can be ed into an sion cassette or transcription cassette. Aﬂer synthesis, the 20' RNA molecule can be modiﬁed by ligation to a DNA molecule having the ability to ize the me and make it resistant to RNase.

As with nse oligonucleotides, small interfering RNA and microRNA described herein, catalytic polynucleotides. useful in the ion should be capable of "hybridizing" the target c acid molecule under "physiological conditions", ‘25 namely those conditions within a plant, algal or fungal cell. ‘ Recombinant Vectors .

One embodiment of the present invention includes a inant vector, which comprises at least one cleotide deﬁned herein and is capable of 30 delivering the polynucleotide into a host cell. Recombinant yectors include expression vectors. Recombinant vectors contain heterologous polynucleotide sequences, that is, polynucleotide sequences that are not naturally found adjacent to a polynucleotide deﬁned herein, that ably, are derived from a ent species.

The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically 35 is a viral vector, derived from a virus, or a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, ampliﬁcation, and transformation of the expression cassette in prokaryotic cells, e.g., pUC-derived Dtors, pSK-derived vectors, pGEM-derived vectors, rived s, pBS- Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 108 derived vectors, or binary vectors containing one or more T-DNA regions. Additional c acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the c acid construct, and sequences that enhance transformation of yotic and eukaryotic (especially plant) cells.

"Operably linked" as used herein, refers to a functional relationship between . two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the ﬁmctional 10 relationship of transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence of a . polynucleotide deﬁned herein, ifit stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory ts that are operably linked to a ribed sequence are physically contiguous 1'5 to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter may independently be the same or different. ’ 20 Recombinant vectors may also contain: (a) one or more secretory signals which encode signal peptide sequences, to enable an sed polypeptide deﬁned herein to be ed ﬁ'om the cell that produces the polypeptide, or which e for localisation of the expressed polypeptide, for example, for retention of the polypeptide ' in the asmic reticulum (ER) in the cell, or transfer into a d, and/or (b) 25 contain fusion sequences which lead to the expressibn of c acid molecules as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion or localisation of a polypeptide deﬁned .

Preferred signal segments include, but are not d to, Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, or the soy n oil body binding 30 protein signal. Recombinant vectors may also include intervening and/or untranslated sequences nding and/or Within the nucleic acid sequence of a polynucleotide deﬁned .

To facilitate identiﬁcation of transformants, the recombinant vector desirably comprises a selectable or screenable marker gene as, or in addition to, the nucleic acid 35 sequence of a polynucleotide deﬁned herein. By r gene" is meant a gene that imparts'a distinct phenotype to cells expressing the marker gene and thus, allows such transformed cells to be distinguished from cells that do not have the marker. A notable marker gene confers a trait for which one can "select“ based on resistance Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 109 to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, that is, by "screening" (e.g., ﬁ-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells). The markergene and the nucleotide sequence of interest do not have to be linked, since nsformation of unlinked genes as for e, described in US 4,399,216, is also an efﬁcient s in for example, plant transformation. The actual choice of a marker is not crucial as long as it is functional (i.e., selective) in combination with the cells of choice such as a plant cell. 10 Examples of bacterial able markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol, or ycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selection of plant transformants include, but arevnot limited to, a 'hyg gene which encodes hygromycin B resistance; a neomycin otransferase ) gene 15 ring resistance to kanamycin, paromomycin, G418; a glutathione-S-transferase gene from rat liver ring resistance to hione derived herbicides as for example, described in EP ; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothn'cin _ as for example, described in W0 , 87/05327; an acetyltransferase gene from 20 Streptomyces viridochromogenes conferring resistance .to the selective agent phosphinothricin as for example, described in EP 275957; a gene encoding a 5- enolshikimatephosphate synthase ) conferring tolerance to N- phosphonomethylglycine as for e, described by Hinchee et a1. (1988); a bar- gene conferring resistance against bialaphos as for example, described in 25‘ WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers ‘ resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea, or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that .30 confers resistance to 5-methyl phan; or a dalapOn dehalogenase gene that I confers resistance to the herbicide. ' .

Preferred screenable markers include, but are not limited to, a uidA gene encoding a B—glucuronidase (GUS) enzyme for which s chromOgenic substrates are known; a B-galactosidase gene encoding an enzyme for which chromogenic 35 substrates are known; an aequorin gene (Prasher et al., 1985) which may be employed in calcium-sensitive bioluminescence detection; a green ﬂuorescent protein gene (Nin et al., 1995) or derivatives thereof; or a rase (luc) gene (Ow et al., 1986) Dch allows for bioluminescence detection. By "reporter molecule" it is meant a Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 110 molecule that, by its chemical nature, provides. an analytically identiﬁable signal that facilitates determination ofpromoter activity by reference to protein product.

Preferably, the recombinant vector is stably incorporated into the genome of the cell such as the plant cell. Accordingly, the recombinant vector may comprise riate elements which allow the vector to be incorporated into the genome, or into a chromosome of the cell.

Expression Vector As used herein, an "expression vector" is a DNA or RNA vector that is capable 10 of transforming a host cell and of effecting expression of one or more ed polynucleotides. Preferably, the sion vector is also capable of replicating within the host cell. Expression s can beeither prokaryotic or eukaryotic, and are typically, viruses or plasmids. Expression vectors of the present invention include any vectors that on (i.e., direct gene expression) in host cells of the present 15 invention, including in bacterial, fungal, endoparasite, arthropod, , algal, and plant cells. Particularly preferred expression vectors of the present invention can direct gene expression in yeast, algae and/or plant cells.

" Expression s of the present invention contain tory sequences such ‘ as transcription control sequences, translation l sequences, s of 20 repliCation, and other regulatory sequences that are compatible with the host cell and that l the expression of cleotides of the present invention. In particular, expression vectors of the present invention include transcription control sequences.

Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control 25 ces are those which control transcription tion such as promoter, enhancer, operator and repressor sequences. Suitable transcription l sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. The choice of the regulatory sequences used-depends on the target organism such as a plant and/or target organ or tissue of interest. Such 30 regulatory sequences may be obtained from any otic organism such as plants or plant viruses, or may be chemically synthesized. A variety of such transcription control sequences are known to those skilled in the art. Particularly preferred transcription control sequences are promoters active in directing transcription in plants, either constitutively or stage and/or tissue speciﬁc, ing on the use of the 35 plant or part(s) thereof.

A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in for example, Pouwels et al., Dning Vectors: A Laboratory Manual, 1985, supp. 1987, Weissbach and Weissbach, Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 111 Methods for Plant lar Biology, Academic Press, 1989, and _Gelvin et al., Plant Molecular y Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.'g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-speciﬁc expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a enylation signal. 10 A number of constitutive promoters that are active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, the cauliﬂower mosaic virus (CaMV) 35$ promoter, the Figwort mosaic virus (FMV) 358, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of 15 the se-l,5-bis-phosphate carboxylase, the rice cytosolic Vtriosephosphate isomerase er, the adenine phosphoribosyltransferase promoter of Arabidapsis, the rice actin 1 gene promoter, the mannopine synthase and ne se promoters, the Adh promoter, the sucrose se promoter, the R gene complex er, and the phyll or/B binding protein gene promoter. These promoters '20 have been used to create DNA vectors that have been expressed in plants, see for example, WO 84/02913. All of these promoters have been used to create various. types of plant-expressible recombinant DNA vectors.

For the purpose ofexpression in source tissues of the plant such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention 25 have relatively high expression in these c tissues. For this e, one may choose from a number of promoters for genes with tissue- or cell-speciﬁc, or - enhanced expression. Examples of such promoters reported in the literature include, ‘ I the chloroplast glutamine synthetase GSZ promoter from pea, the chloroplast fructose- 1,6-biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS] er 30 from potato, the serine/threonine kinase promoter and the glucoamylase (CHS) er from Arabidopsis thaliana. Also reported to be active in photosynthetically active s are the ribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larix laricina), the promoter for the Cab gene, Cab6, from pine, the promoter for the Cab-l gene from wheat, the promoter for the Cab—1 gene from spinach, the 35 promoter for the Cab 1R gene from rice, the te, orthophosphate se (PPDK) promoter from Zea mays, the promoter for the tobacco Lhcbl*2 gene, the Arabidopsis thaliana Suc2 sucrose—H30 symporter promoter, and the er for the Dakoid membrane protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, Substitute Sheet ,‘ ‘ (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 112 AtpD, Cab, Rch). Other ers for the phyll cup—binding proteins may also be utilized in the present invention such as the promoters for Lth gene and PsbP gene from white mustard (Sinapis alba).

A variety of plant gene promoters that are regulated in response to environmental, hormonal, al, and/or developmental signals, also can be used for expression cf RNA-binding protein genes in plant cells, including promoters ted by (1) heat, (2) light (e. g., pea Rch-3A promoter, maize Rch promoter), (3) es such as abscisic acid, (4) ng (e.g., WunI), or (5) chemicals such as methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO 10 97/06269), or it may also'be advantageous to employ (6) organ-speciﬁc promoters.

As used herein, the term “plant e organ speciﬁc promoter" refers to a promoter that preferentially, when compared to other plant tissues, directs gene transcription in a storage organ of a plant. Preferably, the promoter only directs expression of a gene of interest in the storage organ, and/or expression of the gene of '15 interest in other parts of the plant such as leaves is not detectable by Northern blot analysis and/or RT-PCR. TypiCally, the promoter drives expression of genes during growth and development of the storage organ, in ular during the phase of . synthesis and accumulation of storage compounds in the e organ. Such promoters may drive gene sion in the entire plant storage organ or only part 20 thereof such as the seedcoat, embryo or cotyledon(s) in seeds of ledonous plants or the endosperm or aleurone layer of seeds ofmonocotyledonous .

For the purpose of sion in sink tissues of the plant such as the tuber of the potato plant, the fruit of tomato, or the seed of soybean, canola, cotton, Zea mays, wheat, rice, and barley, it is preferred that the'promoters utilized in the present 25 invention have relatively high expression in these speciﬁc tissues.‘ A number of promoters for genes with tuber-speciﬁc or -enhanced expression are known, including the class I patatin promoter, the promoter for the potato tuber ADPGPP genes, both the large and small subunits, the sucrose synthase promoter, the promoter for the major tuber proteins, including the 22 kD protein complexesand proteinase inhibitors, 30 the promoter for the granule bound starch synthase gene (GBSS), and other class I and 1 II patatins promoters. Other promoters can also be used to express a protein in speciﬁc tissues such asseeds or fruits. The promoterfor B—conglycinin or other seed- speciﬁc promoters such as the napin, zein, linin and phaseolin promoters, can be used.

Root speciﬁc promOters may also be used. An example of such a promoter is the 35 promoter for the acid ase gene. Expression in root tissue could also be accomplished by utilizing the root speciﬁc subdomains of the CaMV 358 promoter that have been identiﬁed.

D Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 113 In a particularly preferred embodiment, the promoter directs expression in tissues and organs in which lipid biosynthesis take place. Such promoters may act in » seed development at a suitable time for modifying lipid composition in seeds.

In an embodiment, the promoter is a plant storage organ c promoter. In one embodiment, the plant storage organ speciﬁc promoter is a seed speciﬁc promoter. In a more preferred embodiment, the promoter entially s expression in the cotyledons of a ledonous plant or in the endosperm of a monocotyledonous plant, relative to expression in the embryo of the seed or relative to other organs in the plant such as leaves. Preferred promoters for seed-speciﬁc 10 expression include: I) promoters from genes ng enzymes involved in lipid ‘ biosynthesis and accumulation in seeds such as desaturases and elongases,- 2) promoters from genes encoding seed storage proteins, and 3) promoters from genes encoding enzymes involved in carbohydrate biosynthesis and accumulation in seeds.

Seed speciﬁc promoters which are suitable are, the d rape napin gene er 15 » (US 5,608,152), the Viciafaba USP promoter (Baumlein et al., 1991), the Arabidopsis n promoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980), or the legurhin B4 promoter ein et al., 1992), and promoters which lead to the seed-speciﬁc expression in ts such as maize, barley, wheat, rye, rice and the like. Notable promoters '20 which are suitable are the barley 1pt2 or lptl gene promoter (W0 95/15389 and WO 95/23230), or the promoters described in W0 99/16890 (promoters from the barley ‘ hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene). Other promoters include those 25 described by Broun et a1. (1998), a et al. (2004), US 20070192902 and US 20030159173. In an is ment, the seed _ speciﬁc promoter preferentially expressed in deﬁned parts of the seed such as the cotyledon(s) or the endosperm”.

Examples of cotyledon speciﬁc promoters e,,but are not limited to, the FPl promoter (Ellerstrom et al., 1996), the pea Iegumin promoter (Perrin et al., 2000), and 30 the bean phytohemagglutnin er (Perrin et al., 2000). Examples of erm speciﬁc promoters include, but are not limited to, the maize zein—l er (Chikwamba et al., [2003), the rice glutelin-1 promoter (Yang et al., 2003), the barley D-hordein promoter (Horvath et al., 2000) and wheat HMW glutenin promoters (Alvarez et al., 2000). In a further embodiment, the seed speciﬁc promoter is not 35 expressed, or is only expressed at a low level, in the embryo and/or after the seed germinates. p p In r embodiment, the plant storage organ speciﬁc promoter is a tuber Diﬁc promoter. Examples include, but are not limited to, the potato patatin B33, Substitute Sheet- (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 114 PATZl and GBSS promoters, as well as the sweet potato sporamin promoter (for review, see Potenza et al., 2004). In a preferred embodiment, the promoter directs expression preferentially in the pith of the tuber, relative to the outer layers (skin, bark) or the embryo of the tuber.

, In r embodiment, the plant storage organ speciﬁc promoter is a hit . c er. Examples include, but are not limited to, the tomato polygalacturonase,lE8 and Pds promoters, as well as the apple ACC oxidase promoter (for review, see Potenza et al., 2004). In a preferred embodiment, the promoter preferentially directs expression in the edible parts of the ﬁ‘uit, for example the pith of 10 the fruit, relative to the skin of the fruit or the seeds within the mm.

In an embodiment, the ble promoter is the Aspergillus nidulans alc system. Examples of inducible expression systems which can be used instead of the Aspergillus nidulans alc system are described in a review by Padidam (2003) and Corrado and Karali (2009). These include tetracycline repressor (TetR)-based and tetracycline I 15 inducible systems (Gatz, 1997), tetracycline repressor-based and ycline-inactivatable systems (Weinmann et al., 1994), glucocorticoid receptor- based (Picard, 19,94), estrogen receptor-based and other steroid-inducible systems s (Bruce et al., 2000), glucocorticoid receptor-, tetracycline repressor-based dual control systems (Bohner et al., 1999), ecdysone receptor-based, insecticide- 20 inducible systems (Martinez et al., 1999, Padidam et al., 2003, Unger et a1, 2002, ord ct al., 2000, Dhadialla et al., 1998, ez and , 1999), AlcR— based, ethanol-inducible systems bok, 1991) and ACEl-based, copper-inducible i A systems (Mett et al., 1993).

In another embodiment, the inducible promoter is a safener inducible promoter 25 such as, for example, the maize Vln2-I or ln2-2 er (Hershey and Stoner, 1991), the safener inducible promoter is the maize GST—27 promoter (Jepson et al., 1994), or the soybean GH2/4 promoter (Ulmasov et al., 1995).

‘ Safeners are a group of structurally diverse chemicals used to increase the plant’s tolerance to the toxic effects of an herbicidal compound. Examples of 30 these compounds include naphthalic anhydride and N,N—diallyl-2,2-dichloroacetamide (DDCA), which protect maize and sorghum t thiocarbamate herbicides; cyometn'nil, which protects sorghum against metochlor; triapenthenol, which protects ns against metribuzin; and substituted benzenesulfonamides, which improve the tolerance of several cereal crop species to sulfonylurea herbicides. 35 In r embodiment, the inducible er is a seneScence inducible promoter such as, for example, senescence-inducible promoter SAG (senescence associated gene) 12"and SAG 13 from opsis (Gan, 1995; Gan and Amasino, D5) and LSC54 from Brassica napus (Buchanan—Wollaston, 1994).

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 115 For sion in vegetative tissue leaf-speciﬁc promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters, can be used. For example, the tomato RBCSl, RBCSZ and RBCS3A genes are expressed in leaves and light grown seedlings (Meier et al., 1997). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka et a1. (1994), can be used. Another leaf-speciﬁc promoter is the light ting chlorophyll a/b binding protein gene promoter (see, Shiina et al., 1997). The Arabidopsis thaliana myb-related gene promoter (Atmbe) described by Li et a1. (1996), is leaf-speciﬁc. The Atmbe promoter is expressed in~ 10 developing leaf trichomes,‘ stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. A leafpromoter identiﬁed in maize by Busk et al. (1997), can also be used.

In some instances, for example when LEC2 or BBM is recombinantly expressed, it may be desirable that the.transgene is not-expressed at high levels. An 15 example of a promoter which can be used in such circumstances is a truncated napin A er which retains the seed-speciﬁc expression pattern but with a reduced expression level (Tan et al., 2011).

The 5' non-translated leader sequence can ,be derived from the promoter selected to express the heterologous gene sequenceof the polynucleotide of the 20 present invention, or may be heterologous with respect to the coding region of the enzyme to be ed, and can be speciﬁcally modiﬁed if desired so as to increase translation of mRNA. For a review of zing expression of transgenes, see Koziel et a1. (1996). The 5’ non-translated regions so be obtained from plant viral RNAs co mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, 25 Alfalfa mosaic virus, among ) from suitable eukaryotic genes, plant genes (wheat and maiZe chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence. The present ion is not limited to constructs n the non~ translated region is derived from the 5' non-translated sequence that accompanies the. er sequence. The leader sequence could also be derived from an unrelated 30 promoter or coding sequence. Leader ces useful in context of the present invention comprise the maize Hsp70 leader (US 5,362,865 and US 5,859,347), and the TMV omega t.

‘ The termination of transcription is accomplished by a 3' non-translated DNA sequence operably linked in the expression vector to the polynucleotide of st. 35 The 3' non-translated region of a recombinant DNA molecule ns a polyadenylation signal that functions in plants to cause the on of. adenylate nucleotides to the 3' end of the RNA. The 3' non-translated region can be obtained U1 various genes that are expressed in plant cells. The nopaline synthase 3', Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 116 untranslated region, the ‘3' untranslated region from pea small subunit ‘Rubisco. gene, the 3' slated region from soybean 7S seed storage protein gene are commonly used in this capacity. The 3' transcribed, non-translated regions containing the enylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also I suitable. i Recombinant DNA technologies can be used‘to improve expression of a transformed polynucleotide by manipulating for example, the number of copies of the polynucleotide within a host cell, the ncy with which those polynucleotide are transcribed, the efﬁciency with which the resultant transcripts are translated, and the 10 efﬁciency of post-translational modiﬁcations. Recombinant techniques useful for increasing the expression of polynucleotides deﬁned herein include, but are not limited to, operatively linking the polynucleotide to a high—copy number plasmid, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to the d, substitutions or modiﬁcations of 1,5 transcription control signals (e.g., ers, operators, enhancers), substitutions or modiﬁcations of ational control s (e.g., me binding sites, Shine- Dalgamo sequences), ation of the polynucleotide to correspond to the codon ' usage of the host cell, and the deletion of sequences that destabilize transcripts. 20 Transfer Nucleic Acids _ Transfer nucleic acids can be used to deliver an exogenous cleotide to a cell and comprise one, preferably two, border setwences and a polynucleotide of interest. The transfer c acid may or may not encode a selectable marker.

Preferably, the transfer nucleic acid forms part of a binary Vector in a bacterium, ’ 25 where the binary vector further comprises elements which allow replication of the vector in the bacterium, selection, or nance of bacterial cells containing the binary vector. Upon transfer to a eukaryotic cell, the transfer nucleic acid component of the binary vector is capable of integration into the genome ofthe eukaryotic cell.

As used herein, the term "extrachromosomal transfer nucleic acid“ refers to a 30 nucleic acid molecule that is capable of being transferred from a bacterium such as Agrobacteriam sp., to a eukaryotic cell such as a plant leaf cell. An extrachromosomal transfer nucleic acid is a c element that is nown as an element capable of being transferred, with the subsequent integration of a nucleotide sequence contained within its borders into the genome of the recipient cell. In this 35 respect, a‘ transfer nucleic acid is ﬂanked, typically, by two "border" sequences, although in some instances a single border at one end can be used and the second end of the erred nucleic acid is generated randomly in the transfer process. A’ ”nucleotide of st is typically positioned between the left border-like sequence.

’ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 117 and the right border-like sequence of a transfer nucleic acid. The polynucleotide contained within the transfer nucleic acid may be operably linked to a variety of different promoter and terminator regulatory elements that facilitate its expression, that is, transcription and/or translation of the polynucleotide. Transfer DNAs (T- DNAs) from cterium sp. 'such as Agrobacterium tumefaciens or 'Agrobacterium rhizogenes, and man made ts/mutants thereof are probably the best characterized examples of transfer nucleic acids. Another example is P-DNA I ("plant-DNA") which comprises T-DNA border-like sequences from plants.

As used , ”T-DNA" refers to for example, T—leA of an Agrobacterium 10, tumefaciens Ti plasmid or from an Agrobacterium rhizbgenes Ri plasmid, or man made variants thereof which function as T-DNA. The T-DNA may comprise an entire T-DNA including both right and left border sequences, but need only comprise the minimal sequenCes required in cis for transfer, that is, the right and T-DNA border sequence. The T-DNAs of the invention have inserted into them, anywhere between 15 the right and left border sequences (if t), the polynucleotide of interest ﬂanked by target sites for a site-speciﬁc recombinase. The sequences encoding factors required in trans for transfer of the T—DNA into a plant cell such as vir genes, may be ed into the T—DNA, or may be present on the same on as the T-DNA, or preferably are in trans on a compatible replicon in the cterium host. Such . 20 y vector systems" are well known in the art.

'- As used herein, "P—DNA" refers to a transfer nucleic acid isolated from a plant genome, or m'anmade variants/mutants thereof, and comprises at each end, or at only one end, a T-DNA border—like sequence. The border-like sequence preferably shares at least 50%, at least 60%,4at least 70%, at least 75%, at least 80%, at' least 90% or at 25 least 95%, but less than 100% sequence identity, with a T-DNA border sequence from an Agrobacterium sp. such‘ as Agrobacterium tumefaciens or Agrobacterium rhizogenes. Thus, P-DNAs‘can be used instead of T-DNAs to transfer a nucleotide sequence contained within the P-DNA from, for example Agrobacterium, to another cell. The P-DNA, before insertion of the exogenous cleotide which is to be '30 transferred, may be modiﬁed to tate cloning and should preferably not encode any proteins. The P-DNA is characterized in that it contains, at least a right border sequence and preferably also a left border sequence.) As used , a "border" ce of a transfer nucleic acid can be isolated ' from- a selected organism such as a plant or bacterium, or be a man made 35 variant/mutant thereof. The border ce promotes and facilitates the transfer of the polynucleotide to which it is linked and may tate its integration/in the recipient cell genome. In an embodiment, a border-sequence is between'S-IOO, base _ Dr's (bp) in length, 10-80 bp in length, 15—75 bp in length, 15-60 bp in length, 15-50 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 118 bp in length, 15-40 bp in length, 15-30 bp in length, 16-30 bp in length, 20—30 bp in , length, 21-30 bp in length, 22-30 bp in length, 23—30 bp in length, 24-30 bp in length, . 25-30 bp in length, or 26-30 bp in length. Border sequences from T-DNA from Agrobacterium sp. are well known in the art and include those described in Lacroix et a]. (2008), Tzﬁra and Citovsky (2006) and Glevin (2003).

Whilst traditionally only cterium sp. have been used to transfer genes to plants cells, there are now a large number of systems which have been. identiﬁed/developed which act in a similar manner to Agrobacterium sp. Several non- Agrobacterium species have recently been genetically modiﬁed to be' competent for 10 gene transfer (Chung et al., 2006; Broothaerts et al., 2005). These e Rhizobium sp. NGR234, izobium meliloti and Mezorhizobium loti. The bacteria are made competent for gene transfer by providing the bacteria with the machinery needed for the transformation process, that is, a set of virulence genes encoded by an Agrobacterium Ti-plasmid and the T-DNA segment residing on a separate, small 15 binary plasmid. Bacteria engineered in this way are e of transforming different plant tissues (leaf disks, calli and oval tissue), monocots or dicots, and various different plant species (e.g., tobacco, rice).

Direct er of eukaryotic expression plasmids from bacteria to eukaryotic hosts was ﬁrst achieved several decades ago by the fusion of mammalian cells and 20 protoplasts of plasmid-carrying Escherichia coli (Schaffner, 1980). Since then, the number of ia capable of delivering genes into mammalian cells has steadily increased (weiss, 2003), being discovered by four groups ndently (Sizemore et a1. 1995; Courvalin et al., 1995, Powell ct al., 1996; Darji et al., 1997).

Attenuated Shigella ﬂexneri, Salmonella typhimurium or E. coli that had been 25 rendered invasive by the virulence plasmid (pWRl 00) of S. i have been shown to be able to transfer sion ds after invasion‘of host cells and intracellular death due to metabolic attenuation. Mucosal application, either nasally or orally, of such inant Shigella or Salmonella d immune ses against the ‘ antigen that was encoded by the expression plasmids. In the me, the list of 30 bacteria that Was shown to be able to transfer expression plasmids to‘mammalian host cells in vitro and in vivo has been more then doubled and has been documented for S. typhi, S. choleraesuis, Listeria monocytogenes, Yersinia tuberculosis, and Y. enterocolitica (Fennelly et al., 1999; Shiau et al., 2001; Dietrich et al., 1998; Hense et al., 2001; Al-Mariri et al.,2002). 35 In general, it could be assumed that all bacteria that are able to enter the cytosol of the host cell (like S. ﬂexneri or L. monacytogenes) and lyse within this » ar compartment, should be able to transfer DNA. This is known as 'abortive' or Dcidal‘ invasion as the bacteria have to lyse for the DNA er to occur (Grillot- Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 119.

Courvalin et al., . In addition, even many of the bacteria that remain in the phagocytic vacuole (like S. typhimurium) may also be able to do _so. Thus, , recombinantlaboratory strains of E. coli that have been engineered to be ve but are unable of phagosomal eScape, could deliver their plasmid load to the nucleus of the ed mammalian cell nevertheless (Grillot-Courvalin et al., 1998).

Furthermore, Agrobacterium tumefaciens has recently also been shown to uce enes into mammalian cells (Kunik et al., 2001).

As _used , the terms "transfection", "transformation" and variations f are generally used interchangeably. "Transfected" or "transformed" cells may 10 have been manipulated to introduce the polynucleotide(s) of interest, or may be ‘ progeny cells derived thereﬁom.

V Recombinant Cells The invention also provides a recombinant cell, for example, a recombinant 15 plant cell, which is a host cell transformed with one'or more polynucleotides or vectors deﬁned herein, or ation thereof. The term "recombinant cell" is used interchangeably with the term "transgenic cell " herein. Suitable cells of the invention include any cell that can be ormed with a polynucleotide or recombinant vector of the invention, encoding for e, a polypeptide or enzyme bed herein. _20' The cell is preferably a cell which is thereby capable of being used for producing lipid. The recombinant cell may be a cell in culture, a cell in vitro, or in an organism such as for example, a plant, or in an organ such as, for example, a seed or a leaf.

Preferably, the cell is in a plant, more preferably in the seed of a plant. In one embodiment, the recombinant cell is a non-human cell. 25 Host cells into which the polynucleotide(s) are introduced can be either untransformed cells or cells that are already ormed with at least one c acid. Such nucleic acids may be related to lipid synthesis, or unrelated. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptide(s) deﬁned herein, in which case the recombinant cell derived therefrom 30 has an enhanced capability of producing the polypeptide(s), or can be capable of producing said polypeptide(s) only aﬁer being transformed with at least one polynucleotide of the invention. In an embodiment, a recombinant cell of the ' invention has an enhanced capacity to produce non-polar lipid.

‘ Host cells ofthe present invention can be any cell capable of producing at least 35 one protein described herein, and include bacterial, fungal (including yeast), parasite, arthropod, animal, algal, and plant cells. The cells may be prokaryotic or eukaryotic.

Preferred host cells are yeast, .algal and plant cells. In a preferred embodiment, the nit cell is a seed cell, in particular, a cell in a cotyledon or endosperm ofa seed. In Substitute Sheet (Rulé >26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 120 one embodiment, the cell is an animal cell. The animal cell may be of any type of animal such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or cells of aquatic s such as ﬁsh or crustacea, "invertebrates, insects, etc. Non ng examples of arthropod cells include insect cells such as Spodoptera frugiperda (Sf) cells, for example, Sf9, Sf21, Trichoplusia ni cells, and Drosophila S2 cells. An e of a bacterial cell useful as a host cell of the present invention is Synechococcus spp. (also known as Synechocystis spp.), for example Synechococcus elongatus. Examples of algal cells useful as host cells of the present invention e, for example, . Chlamydomonar sp. (for example, 10 Chlamydomonas reinhardtii), Dunaliella sp., Haematococcus sp., Chlorella sp., Thraustochytrium sp., Schizochytrium sp., and Volvox sp.

‘ Host cells for expression of the instant nucleic acids may include microbial hosts that grow on a variety of feedstocks, including simple or complex ydrates, organic acids and alcohols and/or hydrocarbons over a wide range of 15 temperature and pH values. Preferred microbial hosts are oleaginous organisms that are naturally capable of non-polar lipid sis.

The host cells may be of an organism suitable for a fermentation process, such as, for example, Yarrowia lipolytica or other yeasts. 20 Transgenic Plants ‘ The invention also es a plant comprising an exogenous polynucleotide or polypeptide of the invention, a cell of the invention, a vector of the invention, or a combination thereof. The term "plant" refers to whole plants, whilst the term "part thereof" refers to plant organs (e.g., leaves, stems, roots, ﬂowers, , single cells 25 (e.g., pollen), seed, seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue such asvascular tissue, plant cells and progeny of the same. As used . herein,‘plant parts comprise plant cells. . n As used herein, the term "plan " is used in it broadest sense. It es, but is not limited to, any species of grass, ornamental or decorative plant, crop or cereal 30 (e.g., oilseed, maize, soybean), fodder or forage, fruit or vegetable plant, herb plant, woody plant, ﬂower plant, or tree. It is not meant to limit a plant to any particular ure. It also refers to a unicellular plant (e.g., microalga). The term "part thereof' in reference to a plant refers to a plant cell and progeny of same, a plurality ofplant cells that are largely differentiated into a colony (e.g., volvox), a structure that 35 is present at any stage of a plant's development, or a plant . Such ures include, but are not limited to, leaves, stems, ﬂowers, fruits, nuts, roots, seed, seed .‘coat, embryos. The term "plant tissue" includes differentiated and erentiated D185 of plants including those present in leaves, stems, ﬂowers, fruits, nuts, roots, Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 121 seed, for example, embryonic , endosperrn, dermal tissue (e.g., epidermis, periderm), vascular tissue (e.g., xylem, phloem), or ground tissue ising- parenchyma, chyma, and/or sclerenchyma cells), as well as cells in culture (e.g, single cells, protoplasts, callus, embryos, etc.). Plant tissue may be in planta, in organ culture, tissue e, or cell Culture.

A "transgenic plant", "genetically modiﬁed plant" or variations thereof refers to a plant that contains a transgene not found in a wild-type plant of the same Species, variety or cultivar. Transgenic plants as deﬁned in the context of the present invention include plants and their progeny which have been genetically modiﬁed 10 using recombinant techniques to cause production of at least one polypeptide deﬁned herein, in the desired plant or part thereof. Transgenic plant parts has a corresponding meaning, I ‘ The terms "seed" and 1"grain" are used interchangeably herein. "Grain" refers to ' mature grain such as harvested grain or grain which is still on a plant but ready for 15 harvesting, but can also refer to grain after imbibition or ation, according to the context. Mature grain ly has a moisture content of less than about 18—20%.

In a preferrd embodiment, the moisture t of the grain is at a level which is generally regarded as safe for storage‘,preferab1y between 5% and 15%, n 6% and 8%, nt8% and 10%, or between 12% and 15%. "Developing seed" as used 20 herein refers to a seed prior to maturity, typically found in the reproductive structures of the plant after fertilisation or anthesis, but can also refer to such seeds prior to maturity which are isolated from a plant. Mature seed commonly has a re content of less than about 18-20%. In a preferrd embodiment, the moisture content of the seed is at a level which is generally regarded as safe for storage, preferably 25 between 5% and 15%, between 6% and 8%, between 8% and 10%, or between 12% ' ' and 15%.

As used herein, the term "plant storage organ" refers to a part of a plant specialized to store energy in the form of for example, proteins, carbohydrates, lipid.

Examples of plant storage organs are seed, fruit, tuberous roots, and tubers. A 30 preferred plant storage organ of the invention is seed.

As used herein, the term "phenotypically normal" refers to a cally- modiﬁed plant or part thereof, particularly a e organ such as a seed, tuber or - fruit of the invention not having a signiﬁcantly reduced ability to grow and reproduce when compared to an ﬁed plant or plant thereof. In an embodiment, the 35 genetically modiﬁed plant or part thereof which is phenotypically normal comprises a inant polynucleotide encoding a silencing suppressor operably linked to a plant storage organ speciﬁc promoter and has an ability to grow or reproduce which is ly the same as a ponding plant or part thereof not comprising said Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 122 polynucleotide. Preferably, the biomass, growth rate, germination rate, storage organ size, seed size and/or the number of viable seeds produced is not less than 90% of that of a plant lacking said recombinant polynucleotide when grown under identical conditions. This term does not encompass features of the plant which may be different to the wild-type plant but which do not effect the ness of the plant for commercial purposes such as, for example, a ballerina phenotype of seedling leaves.

Plants provided by or contemplated for Use in the practice of the present invention include both monocotyledons and ledons. In red embodiments; the plants of the present invention are crop plants (for example, cereals and pulses, 10 maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or pea), or other legumes. The plants may be grown for production of edible roots, , leaves, stems, ﬂowers or fruit. The plants may be ble or ornamental plants.

The plants of the invention may be: mia aculeata (macauba palm), Arabidopsis thaliana, Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru), 15 Astrocaryum vulgare (tucuma), Attalea ggeraensis (Indaia-rateiro), Attalea humilis (American oil- palm), Attalea oleifera (andaié), Attalea phalerata (uricuri), Attalea speciosa (babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica sp. such as Brassicav carinata, Brassica' juncea, ca napobrassica, Brassica napus (canola), Camelina sativa (false ﬂax), is sativa (hemp), Carthamus tinctorius 20 (safﬂower), Caryocar brasiliense ), Cocos nucifera (Coconut), Crambe abyssim‘ca (Abyssinian kale), Cucumis melo ), Elaeis guineensis an palm), Glycine max (soybean), Gassypium hirsutum (cotton), Helianthus sp. such as Helianthus annuus wer), Hordeum vulgare (barley), Jatropha curcas c nut), Joannesia princeps (arara nut-tree), Lemna ' Sp. (duckweed) such as Lemna 25 aequinoctialis, Lemna ma, Lemna ecuadorierzsis, Lemna gibba (swollen duckweed), Lemna japom'ca, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna , Lemna trisulca, Lemna turionifera, Lemna valdiviana, Lemna yungénsis, Licania rigida (oiticica), Linum usitatissimum (ﬂax), Lupinus angustifolius (lupin), Mauritia ﬂexuosa (buriti palm), Maximiliana 30 maripa (inaja palm), Miscanthus sp. such as Miscanthus x giganteus and Miscanthus sinensz‘s, an‘a sp. (tabacco) such as Nicotiana tabacum or Nicotiana benthamiarza, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua (pataua), Oenocarpus distichus (bacaba-dc-leque), Oryza sp. (rice) such as Oryza sativa and Oryza glaberrima, Panicum virgatum (switchgrass), Paraqueiba sis , 35 Persea amencana (avocado), Pongamia pinnata n beech), Populus trichocarpa, Ricinus communis (castor), Saccharum sp. (sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum sp. such as Sorghum bicolor, Sorghum agare, Theobroma grandiforum (cupuassu), Trifolium sp., Trithrinax' brasz'liensis Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 123 ' (Brazilian needle palm), Triticum sp. (wheat) such as um aestivum, Zea mays (com), alfalfa (Medicago sativa), rye e cerale), sweet potato (Lopmoea s), cassava (Manihot esculenta), coffee (Cofea spp.), pineapple (Anana comosua), citris tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado (Persea americana), ﬁg (Ficus basica), guava um guajava), mango (Mangifer indica), olive (Olea’ europaea), papaya a papaya), cashew (Anacardium occidentale), macadamia (Macadamia rifolia) and almond (Prunus amygdalus). plants include C4 i Other preferred grasses such as, in addition to those 10 mentioned above, Andropogon 'i, Bouteloua curtipendula, B. gracilis, Buchloe dactyloides, chyrium scoparium, Sorghastrum nutans, Sporobolus cryptandrus; C3 grasses such as Elymus. canadensis, the legumes Lespedeza capitata and stemum villosum, the forb Aster azureus; and woody plants such as Quercus ellipsoidalis and Q. macrocarpa. Other preferred plants include C3 grasses. 15 In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an "oilseed plant" is a plant species used for the commercial tion of lipid from the seeds of the plant. The oilseed plant may be, for example, oil-seed rape (such as canola), maize, sunﬂower, saﬂlower, soybean, 20 sorghum, ﬂax (linseed) or sugar beet. rmore, the oilseed plant may be other Brassicas, cotton, peanut, poppy, rutabaga, mustard, castor bean, sesame, safﬂower, or nut producing plants. The plant may produce high levels of lipid in. its fruit such as olive, oil palm or coconut. Horticultural plants to which the present invention may be applied are lettuce, endive, or vegetable Brassicas including cabbage, broccoli, or 25 cauliﬂower. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper.

In a preferred embodiment, the transgenic plant is homozygous for each and every gene that has been uced (transgene) so that its progeny do not segregate ' for the d phenotype. The transgenic plant may also be heterozygous for the ‘30 introduced transgene(s), preferably uniformly heterozygous for the transgene such as for example, in F1 progeny which have been. grown from hybrid seed. Such plants 7 may provide advantages such as hybrid vigour, well known in the art.

Where relevant, the enic plants may also comprise additional transgenes encoding enzymes involved in the tion of lar lipid such as, but not 35 limited to LPAAT, LPCAT, PAP, or a phospholipid:diacylglycerol ansferase (PDATl, PDAT2 or PDAT3; see for example, Ghosal et al., 2007) , orta combination of two or more thereof. The transgenic plants of the invention may also express ‘- ' ‘ Disin ﬁ’om an exogenous polynucleotide.

Substitute Sheet .

(Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 124 Transformation ofplants Transgenic plants can be produced using techniques known in the art, such as those generally described in Slater et al., Plant Biotechnology - The Genetic, Manipulation of Plants, Oxford University Press (2003), and Christou and. Klee, I i 5 Handbook of Plant Biotechnology, John Wiley and Sons (2004).

. As used herein,’ the terms "stably orming", "stably. transformed" and variatiOns thereof refer to the ation of the polynucleotide into the genome of the cell such that they are transferred to y cells during cell division without the need for positively selecting for their presence. Stable transformants, or progeny 10 thereof, can be selected by any means known in the art such as Southern blots on chromosomal DNA, or in situ hybridization of genomic DNA.

Agrobacterium-rnediated transfer is a widely applicable system for introducing genes into plant cellsbecause DNA can be introduced into cells in whole plant tissues, plant organs, or explants in tissue culture, for either transient expression, or for stable 15 integration of the DNA in the plant cell genome. The use ofAgrobacterium-mediated plant integrating'vectors to introduce DNA into plant cells is well known in the art (see for e, US 5177010, US 5104310, US 5004863, or US 5159135). The region of DNA to be transferred is defined by the border sequences, and the intervening DNA (T-DNA) is usually inserted into the plant genome. Further, the , 20 integration of the T-DNA - is a relatively precise process resulting in few , rearrangements. In those plant varieties where Agrobacterium-mediated transformation is nt, it is the method of choice because of the facile and deﬁned nature of the gene transfer. Preferred Agrobacterium transformation s are capable of replication in E. coli as well as Agrobacterium, allowing for convenient 25 manipulations as described (Klee et al., In: Plant DNA Infectious Agents, Hahn and Schell, eds., Springer-Verlag, New York, pp. 3 ). ’ Acceleration methods that may be used e for e, microprojectile bombardment and the like. One e of a method for delivering transforming _ nuc1eic acid molecules to plant cells is microprojectile bombardment. This method 30 has been reviewed by Yang et al., Particle Bombardment logy for'Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in 35 addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. An rative embodiment of a method for ring DNA into Zea Dys cells by acceleration is a biolistics et-particle delivery system, that can be used Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 125 to propel particles coated with DNA through a screen such as a stainless steel or Nytex screen, onto a ﬁlter surface covered with corn cells cultured in suspension. A particle delivery system suitable for use with the present invention is the helium acceleration PDS-l GOO/He gun available from d Laboratories.

For the dment, cells in suspension may be concentrated on ﬁlters.

Filters containing the cells to be bombarded are positioned at an riate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded.

Alternatively, immature s or other target cells may be arranged on solid 10 culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the ration device and the cells to be bombarded. Through the use of techniques set forth herein, one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus that express the 15 gene product 48 hours post-bombardment often range from one to ten and e one to three.

. In bombardment transformation, one may optimize the pre—bombardment ing conditions and the bombardment parameters to yield the maximum numbers of stable transfonnants. Both the physical and biological parameters for 20 bombardment are ant in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the ﬂight and ty of either the macro— or microprojectiles.’ Biological factors include all steps involved in manipulation of cells before and immediately aﬁer‘ bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with 25 bombardment, and also the nature of the transforming DNA such as linearized DNA or intact oiled plasmids. It is believed that pre~bombardment manipulations are especially important fOr sful transformation of re embryos.

In another alternative embodiment, plastids can be stably ormed.

Methods disclosed for plastid transformation in higher plants e particle gun 30 delivery of DNA containing a selectable marker and ing of the DNA to the , plastid genome through homologous recombination (US 5,451,513, US 5,545,818, US 5,877,402, US 5,932479, and WO 99/05265). _ I Accordingly, it is contemplated that one may wish to adjust various aspects of - the bombardment parameters in small scale studies to fully optimize the conditions. 35 One may particularly wish to adjust physical parameters such as gap distance, ﬂight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors by modifying conditions that inﬂuence the physiological state of the D'pient cells and that may therefore inﬂuence transformation and integration Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 126 efﬁciencies. For example, the osmotic state, tissue ion and the subculture stage, or cell cycle of the recipient cells, may be adjusted for m transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

I Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. ation of these systems to different plant ies, depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative s for the regeneration of cereals from protoplasts are 10 described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah etal., 1986).

Other methods of cell transformation can also be used and include but are not limited tothe introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos. ' 15 ' .

The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular y, Academic Press, San Diego, Calif, (1988)). This regeneration and growth process typically includes the 20 steps of selection of transformed cells, culturing those dualized, cells through the usual stages of embryonic “development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are fter planted in an appropriate plant growth medium such as soil.

‘ The pment or regeneration of plants ning the foreign, exogenous 25 gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-groWn plants of agronomically important lines.

Conversely, pollen from plants of these important lines is used .to pollinate regenerated plants. A transgenic plant of the present invention containing a desired 30 polynucleotide is cultivated using methods well known to one d in the art.

Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants have been published for cotton (US 5,004,863, US 5,159,135, US 5,518,908), soybean (US 5,569,834, US 5,416,011), Brassica (US 5,463,174), peanut (Cheng et al., 1996), and pea (Grant et al., 1995). 35 MethOds for transformation of cereal plants such as wheat and barley for ucing genetic variation into the plant by introduction of an exogenous nucleic ' acid and for regeneration of plants from protoplasts or immature plant embryos are D1 known in the art, see for example, CA 588, AU 61781/94, AU 667939, US ' Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 127. 6,100,447, WO 97/048814, US 5,589,617, US' 6,541,257, and other methods are set out in W0 99/14314. Preferably, transgenic wheat or barley plants are produced by Agrobacterium tumefaciens mediated transformation procedures. Vectors carrying the desired polynucleotide may be introduced into regenerable wheat cells of tissue ' cultured plants or explants, or suitable plant systems such as protoplasts.

The rable wheat cells are preferably from the scutellum of re . embryos, mature embryos, callus derived from these, or the meristematic tissue.

To conﬁrm the presence of the transgenes in transgenic cells and plants, a , polymerase chain reaction (PCR) ampliﬁcation or Southern blot analysis can be 10 performed using methods known to those skilled in the art. sion products of the enes can be detected in any of a y of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene such as GUS. Once transgenic plants have been obtained, 15 they may be grown to produce plant tissues or parts having the desired phenotype The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics. Preferably, the vegetative plant are harvested at a time When the yield of non—polar lipids are at their highest. In one ment, the 20 vegetative plant-parts are harvested, about at the time of ﬂowering.

A transgenic plant formed using 'Agrobacterium or other transformation methods typically contains single locus ' _a c on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene(s). More preferred is a enic plant that is homozygous for the added ), that is, a 25 transgenic plant that contains’two added genes, one gene at the same locus on each chromosome of a chromosome pair. .A homozygous transgenic plant can be obtained by self-fertilising a gous transgenic plant, germinating some of the seed I produced and analyzing the resulting plants for the gene of interest.

It is also to be understood that two different enic plants that contain two 30 independently segregating exogenous genes or loci can also be crossed (mated) to ‘ produce offspring that contain both sets of genes or loci. Selﬁng of appropriate F 1 y can produce plants that are homozygous for both exogenous genes or loci.

Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. ptions of other breeding methods 35 that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., an Society of Agronomy, Madison Wis. (1987).

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 128 TILLING In one embodiment, G (Targeting Induced Local Lesions IN Genomes) ' can be used to produce plants. in which nous genes are knocked out, for example genes encoding a DGAT, sn-l g1ycerolphosphate acyltransferase (GPAT), -glycerolphosphate acyltransferase - (LPAAT), acyl- CoA:lysophosphatidylcholine acyltransferase _(LPCAT), atidic acid I phosphatase (PAP), or a combination of two or more thereof.

In a ﬁrst step, introduced mutations such as novel single base pair changes are ' induced in a tion of plants by treating seeds (or pollen) "with a chemical 10 mutagen, and then advancing plants to a generation where ons will be stably inherited. DNA is extracted, and seeds are stored from all members of the population to create a ce that can be accesSed repeatedly over time; ' For a TILLING assay, PCR primers are designed to speciﬁcally amplify a single gene target of interest. Speciﬁcity is especially important if a target is a 15 member of a gene family or part of a polyploid genome. Next, dye-labeled primers can be used to amplify PCR products from pooled DNA of multiple individuals.

These PCR products are denatured and reannealed to allow the formation of . mismatched base pairs." Mismatches, or heteroduplexes, represent both naturally occurring single nucleotide polymorphisms (SNPs) (i.e., several plants ﬁ'om the ‘ 20 population arelikely to carry the same polymorphism) and induced SNPs (i.e., only _ rare individual plants are likely to display the mutation). Aﬁer heteroduplex ion, the use of an endonuclease, such as Cell, that izes and cleaves mismatched DNA is the key to discovering novel SNPs within a TILLING i I population. . , ~ ' .25 Using this approach, many thousands of plants can be screened to identify any individual with a single base change as well as small ions or deletions (1-30 bp) in any gene or speciﬁc region of the genome. Genomic fragments being assayed can range in size anywhere from 0.3 to 1.6 kb. At 8¥fold pooling, 1.4 kb fragments (discounting the ends of fragments where SNP detection is problematic due to noise) { 3o and 96 lanes per assay, this combination allows up to a million base pairs of genomic DNA to be screened per single assay, making TILLING a high-throughput technique.

TILLING is further described in Slade and Knauf (2005), and Henikoff et a]. (2004).

In addition to allowing efﬁcient detection of mutations, high-throughput TILLING technology is ideal for the detection of natural polymorphisms. ore, 35. interrogating an unknown homologous DNA by heteroduplexing to a known sequence reveals the number and position of polymorphic sites. Both nucleotide s and small insertions and ons are identiﬁed, including at least some repeat number hisms. This has been called ling (Comai et a1., 2004).

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 129 Each SNP is recorded by its approximate position within a few, nucleotides.

Thus, each haplotype can be archived based on its ty. sequence data can be obtained with a relatively small incremental effort using aliquots of the same ampliﬁed DNA that is used for the mismatch—cleavage assay. The leﬁ or right sequencing primer for a single reaction is chosen by its proximity to the polymorphism. Sequencher software performs a multiple ent and discovers the base change, which in each caSe conﬁrmed the gelvband. g Ecotilling'can be performed more cheaply than full sequencing, the method currently used for most SNP discovery. Plates containing arrayed ecotypic DNA can '10 be screened rather than pools of DNA from mutagenized plants. Because detection is on gels with nearly base pair resolution and background patterns are uniform across lanes, bands that are of identical size can be matched, thus discovering and genotyping SNPs in a single step. In this way, ultimate sequencing of the SNP is simple and efﬁcient, made more so by the fact that thegaliquots of the same PCR ' 15 products used for screening can be subjected to DNA sequencing.

Enhancing Exogenous RNA Levels and Stabilized Expression Post-transcriptional gene silencing (PTGS) is a tide ce-speciﬁc defense mechanism that can target both cellular and viral mRNAs for degradation. ’20 PTGS occurs in plants or fungi stably or transiently transformed with a recombinant polynucleotide(s) and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced polynucleotide. transcriptional" refers to a mechanism ing at least partly, but not necessarily exclusively, after production of an initial RNA transcript, for example during processing of the initial RNA 25 transcript, or concomitant with splicing or export of the RNA to the cytoplasm, or within the cytoplasm by xes associated with ute proteins.

RNA molecule levels can be increased, "and/or RNA molecule levels stabilized over numerous generations or under different environmental conditions, by limiting the expression of a silencing suppressor in a storage organ of a plant or part thereof. 30 As used , a cing suppressor" is any polynucleotide or polypeptide that can be expressed in a plant cell that enhances the level of expression product from a different transgene in the plant cell, particularly, over repeated generations from the initially transformed plant; In an embodiment, the silencing suppressor is a viral silencing suppressor or mutant thereof. A large number of viral ing suppressors 35 are known in-the art and include, but are not limited to P19, V2, P38, Pe-Po and RPV- PO. Exarhples of suitable viral silencing suppressors e those described in W0 , 2010/057246. A silencing ssor may be stably expressed in a plant or part 2 Deofof the present invention. ' Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 130 As used herein, the term “stably expressed" or variations thereof refers to the level of the RNA molecule being essentially the same or higher in progeny plants over ' repeated generations, for e, at least three, at least ﬁve, or at least ten generations, when compared to corresponding plants lacking the exogenous polynucleotide encoding the silencing suppressor. However, this term(s) does not exclude the possibility that over repeated tions there is some loss of levels of the RNA molecule when ed to a previous generation, for example, not less than a 10% loss per generation.

. The suppressor can be selected from any source e.g. plant, viral, mammal, etc. 10 The suppressor may be, for example, ﬂock house virus BZ, pothos latent virus P14, pothos latent virus AC2, African a mosaic virus AC4, bhendi yellow vein mosaic disease C2, bhendi yellow vein mosaic disease C4, bhendi yellow vein mosaic disease 3C1, tomato sis virus p22, tomato chlorosis virus CP, tomato chlorosis virus CPm, tomato golden mosaic virus ALZ, tomato leaf curl Java virus BCI, tomato 15 yellow leaf curl virus V2, tomato yellow leaf curl virus-China C2, tomato yellow leaf curl China virus Y10 isolate BCl, tomato yellow leaf curl Israeli isolate V2, ‘mungbean yellow mosaic virus-Vigna AC2, hibiscus chlorotic ringspot virus CP, turnip crinkle virus P38, turnip crinkle virus CP, cauliﬂower mosaic virus P6, beet yellows virus p21, citrus tristeza virus p20, ,citrus tristeza virus p23, citrus tristeza 20 virus CP, cowpea mosaic virus SCP, sweet potato chlorotic stunt virus p22, cucumber ‘ mosaic virus 2b,tomato aspenny virus HC-Pro, beet curly top virus L2, soil borne wheat mosaic virus 19K, barley stripe mosaic virus Gammab, poa semilatent virus Gammab, peanut clump irus P15, rice dwarf virus Pnle, curubit aphid home yellows virus PO, beet western yellows virus P0, potato virus X P25, er vein 25 ing virus Plb, plum pox virus HC-Pro, sugarcane mosaic virus , potato virus Y strain HC-Pro, tobacco etch virus Pl/HC—Pro, turnip mosaic virus Pl/HC— ‘ Pro, cocksfoot mottle virus P1, cocksfoot mottle virus-Norwegian isolate Pl, rice yellow mottle virus Pl, rice yellow mottle Nigerian isolate P1, rice hoja blanca‘ virus N83, rice stripe virus N83, crucifer infecting tobacco mosaic virus 126K, '30 crucifer infecting tobacco mosaic virus p122, tobacco mosaic virus p122, tobacco mosaic virus 126, tobacco mosaic virus 130K, tobacco rattle virus 16K, tomato bushy stunt virus P19, tomato spotted wilt virus NSs, apple chlorotic leaf spot virus P50, grapevine virus A p10, grapevine leafroll associated virus-2 g of BYV p21, as ' well as variants/mutants thereof. The list above provides the virus from which the '35 suppressor can be obtained and the protein'(e.g;, 82, P14, etc.), or coding region ation for the suppressor ﬁom each particular virus. Other candidate silencing suppressors may be obtained by examining viral genome sequences for polypeptides Doded at the same position within the viral genome, relative to the structure of a Substitute Sheet (Rule 26) RO/AU .

[Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk .131 related viral genome comprising a known silencing ssor, as is appreciated by a ' V person of skill in the art.

Silencing suppressors can be categorized based on their mode of action. suppressors such as V2 which preferentially bind to a double-stranded RNA le which has overhanging 5 ends relative ' to a corresponding double-stranded RNA I molecule having blunt ends are particularly useful for enhancing transgene expression when used in combination with gene silencing (exogenous polynucleotide encoding a dsRNA). Other suppressors such as p19 which preferentially bind a dsRNA molecule which is 2] base pairs in length relative to a dsRNA molecule of a different length can i 10 also allow transgene expression in the presence of an exogenous polynucleotide encoding a dsRNA, but generally to a lesser degree than, for e, V2. This allows the selection of an optimal combination of dsRNA, silencing suppressor and over-expressed transgene for a particular e. Such optimal combinations can be - ﬁed using a method of the invention. 15 In an embodiment, the silencing suppressor preferentially binds to a double- ed RNA molecule which has overhanging 5'1 ends relative to a corresponding -stranded RNA molecule having blunt ends. In this context, the corresponding double-stranded RNA molecule preferably has the same nucleotide sequence as the molecule with the 5' overhanging ends, but without the nging 5' ends. Binding .20 assays are routinely performed, for example in in vitro assays, by any method as.

I I I known to a person of skill in the art.

Multiple copies of a suppressor may be'used. Different suppressors may be used er (6. g., in tandem).

Essentially any RNA le which is desirable to be expressed in a plant 25 \Storage organ can be co-expressed with the silencing suppressor. The RNA molecule may inﬂuence an agronomic trait, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and the like. The encoded polypeptides may be involved in metabolism of lipid, starch, carbohydrates, nutrients, et‘c., or may be responsible for the synthesis of proteins, peptides, lipids, waxes, es, sugars, 30 carbohydrates, ﬂavors, odors, toxins, noids. hormones, polymers, ﬂavonoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, oses, glycoproteins, ' ' glycolipids, etc.

In a particular example, the plants produced increased levels of enzymes for lipid production in plants such as Brassicas, for example d rape or sunﬂower, '35 safﬂower, ﬂax, cotton, soya bean or maize.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 132 Plant Biomass An se in the total lipid content of plant biomass equates to greater energy content, making its use in the tion of l more economical.

Plant biomass is the c als produced by plants, such as leaves, roots, seeds, and stalks. Plant biomass is a complex mixture of organic materials, such as carbohydrates, fats, and ns, along with small amounts of minerals, such as sodium, orus, calcium, and iron. The main components ofplant biomass are carbohydrates (approximately 75%, dry weight) and lignin (approximately 25%), which can vary with plant type. The carbohydrates are mainly cellulose or 10 llulose ﬁbers, which impart strength to the plant structure, and lignin, which holds the fibers together. . Some plants also store starch (another carbohydrate polymer) and fats as sources of energy, mainly in seeds and roots (such as corn,. soybeans, and potatoes).

Plant biomass typically has a low energy density as a result of both its physical 15 form and moisture content. This makes it inconvenient and inefﬁcient for storage and transport, and also usually unsuitable for use without some kind of pre-processing.

There are a range of processes available to convert it into a 'more convenient form including: 1) physical pre—processing (for example, grinding). or 2) conversion by thermal (for example, combustion, gasiﬁcation, sis) or chemical (for 20 example, - anaerobic digestion, fermentation, composting, transesteriﬁcation) _ ses. In this way, the biomass is converted into what can be described as a ‘ I biomass fuel. stion 25 Combustion is the process by which flammable als are allowed to burn in the presence of air or oxygen with the release of heat. The basic process is oxidation. Combustion is the simplest method by which s can be used for energy, and has been used to provide heat. This heat can itselfbe used in a number of ‘ ways: 1) spaceheating, 2) water (or other ﬂuid) heating for central or district heating 30 or process heat, 3) steam raising for electricity tion or motive force. When the ﬂammable fuel material is a form of biomass the oxidation is of predominantly the carbon (C) and hydrogen (H) in the cellulose, hemicellulose, lignin, and other molecules t to form carbon dioxide (C02) and water (H20). 35 Gasiﬁéation Gasiﬁcation is a'partial oxidation process whereby a carbon source such as plant biomass, is broken down into carbon monoxide (CO) and hydrogen (H2), plus Dbon dioxide (C02) and possibly hydrocarbon molecules such as methane (CH4). If / Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 133 the gasiﬁcation takes place at a relatively low temperature, such as 700°C to 1000°C, the t gas will have a relatively high level of hydrocarbons compared to high temperature gasiﬁcation. As a result it may be used directly, to be burned for heat or electricity generation 'via a steam turbine or, with suitable gas clean up, to run an 5 internal combustion engine for electricity generation. The combustion chamber for a simple boiler may be close coupled with the gasiﬁer, or the producer gas may be cleaned of longer chain hydrocarbons (tars), transported, stored and burned remotely.

A gasiﬁcation system may beclosely integrated with a combined cycle gas turbine for electricity generation (IGCC - integrated gasiﬁcation combined cycle). Higher 10 temperature gasiﬁcation (1200°C to 1600°C) leads to few hydrocarbons in the product gas, and a higher proportion of CO and H2. This is known as synthesis gas s or biosyngas) as it can be used to synthesize longer chain hydrocarbons using techniques such as Fischer-Tropsch (FT) synthesis. If. the-ratio of H2 to CO is correct (2:1) FT synthesis can be used to convert syngas into high quality Synthetic diesel biofuel 15 which is compatible with tional fossil diesel and diesel engines.

Pyrolysis As used , the term ysis" means a process that uses slow heating in the absence of oxygen to produce gaseous, oil and char products from biomass. 20 sis is a thermal or thermo-chemical conversion ~of lipid-based, particularly triglyceride-based, materials. The products of pyrolysis include gas, liquid and a sold char, with the proportions of each depending upon the parameters of theprocess.

Lower atures. (around 400°C) tend to produce more solid char (slow pyrolysis), whereas somewhat higher atures (around 500°C) produce a much higher 25 proportion of liquid (bio-oil), provided the vapour residence time is kept down to around ls or less. After this, secondary ons take place and increase the gas yield. The bio-oil produced by fast (higher temperature) pyrolysis- is a dark brown, mobile liquid with a heating. value about half that of conventional fuel oil. It can be burned directly, co-ﬁred, upgraded to other fuels or gasiﬁed. 30 Pyrolysis involves direct thermal cracking of the lipids or a combination of w thermal and catalytic cracking. At temperatures of about 0°C, cracking , producing short chain arbons "such as alkanes, alkenes, alkadienes, aromatics, oleﬁns and carboxylicacid, as well as carbon monoxide and carbon dioxide.

Four main catalyst types can be used including transition metal sts, 35 molecular sieve type catalysts, activated alumina and sodium carbonate (Maher et al., 2007). Examples are given in US 4102938. Alumina (A1203) activated by acid is an eﬁ'ective st (US 5233109). Molecular sieve catalysts are porous, highly Distalline structures that exhibit size selectivity, so that molecules of only certain Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 134 '\ sizes can pass through. These include zeolite catalysts such as ZSM—S or HZSM-S . which. are crystalline materials comprising A104 and SiO4 and. other silica-alumina catalysts. The ty and selectivity of these sts depends on the acidity, pore size and pore shape, and typically operate at 300-500°C. Transition metal catalysts are described for examplein US 4992605. Sodium carbonate catalyst has been usedin the pyrolysis of oils (Dandik and Aksoy, 1998).

Transesteriﬁcation p "Transesteriﬁcation" as used herein is the conversion of lipids, principally 10 - triacylglycerols, into fatty acid methyl esters or ethyl esters using short chain alcohols such as methanol or ethanol, in the presence of- a st such as alkali or acid.

MethanolIS used more commonly due to low cost and availability. The catalysts may be homogeneous catalysts, heterOgeneous catalysts or enzymatic catalysts; Homogeneous catalysts e ferric sulphate folloWed by KOH. Heterogeneous 15 catalysts include CaO, K3PO4, and 02. Enzymatic, catalysts include Novozyme 435 produced from Candida antarctica. ‘ Anaerobic Digestion Anaerobic digestion is the s whereby bacteria breakdown organic 20 material in the absence of air, yielding a biogas containing methane. The products of this process are biogas ipally methane (CH4) and carbon dioxide ), a solid ', residue (ﬁbre or digestate) that is similar, but not identical, to compost and a liquid liquor that can be used as a fertilizer. The methane can be burned for heat or electricity generation. The solid residue of the anaerobic digestion process can be 25 used as a soil conditioner or alternatively can be burned as a fuel, or d.

Anaerobic digestion is typically performed on biological material in an aqueous slurry. However there are an increasing number of dry digesters. Mesophilic digestion takes place n 20°C and 40°C and can take a month or two to complete. Thermophilic digestion takes place ﬁom 50-65°C and is , but the 30 bacteria are more sensitive.

Fermentation tional fermentation processes for the production of bioalcohol make use of the starch and sugar components of plant crops. Second generation bioalcohol 35 precedes this with acid and/or enzymatic hydrolysis of hemicellulose and Cellulose - into table 'saccharide's to make use of a much larger proportion of available biomass. More detail is ed below under the g "Fermentation processes . Dlzpidproduction”.

Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 135 Composting Composting is the aerobic decomposition of organic matter by rganisms. It is typically performed on relatively dry material rather than a slurry. Instead of, or in on to, collecting the ﬂaminable biogas emitted, the exothermic nature of the composting process can be exploited and the heat produced used, usually using a heat pump. tion ofNon-Polar Lipids 10 Techniques that are ely practiced in the art can be used to extract, process, purify and analyze the non-polar lipids produced by cells, organisms or parts thereof of the instant invention. Such techniques are described and explained throughout the literature in sources such as, Fereidoon Shahidi, Current Protocols in Food Analytical Chemistry, John Wiley &. Sons, Inc. (2001) Dl.l.l-D1.l.ll, and 15 Vich et a]. (1998). ‘ tion ofseedoil Typically, plant seeds are , pressed, and/or extracted to e crude seedoil, which is then degummed, refined, bleached, and deodorized. Generally, techniques for crushing seed are known'in the art. For example, oilseeds can be 20 tempered by spraying them with water to raise the moisture content to, for example, 8.5%, and ﬂaked using a smooth roller with a gap setting of 0.23 to 0.27 mm.

Depending on the type of seed, water may not be added prior to crushing. Application of heat deactivates enzymes, facilitates further: cell rupturing, coalesces'the lipid droplets, and agglomerates protein particles, all of which facilitate the extraction 25 process.

In an embodiment, the majority of the seedoil is released by passage through a screw press. Cakes expelled from the screw press are then solvent extracted for example, With hexane, using a heat traced column. Alternatively, crude seedoil ed by the pressing operation can be passed through a settling tank with a 30 slotted wire drainage top to remove the solids that are expressed with the seedoil during the pressing operation. The clariﬁed seedoil can be passed through a plate and frame ﬁlter to remove any ing ﬁne solid les. If desired, the seedoil recoVered from the extraction process can be combined With the clariﬁed seedoil to \ e a blended crude seedoil. ‘ 3,5 Oncethe solvent is stripped from the crude seedoil, the pressed and extracted portions are combined and subjected to normal lipid sing procedures (i.e., degumming, caustic ng, bleaching, and deodorization).

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 136 In an embodiment, the oil and/or protein content of the seed is analysed by I near-inﬁ'ared ance spectroscopy as described in Horn et al. (2007).

Degumming Degumming is an early step in the reﬁning of oils and its primary purpose is the removal of most of the phospholipids from the oil, which may be present as approximately 1-2% of the total extracted lipid. Addition. of ~2% of water, typically containing phosphoric acid, at 70—80°C to the crude oil results in the separation of most of the olipids accompanied by trace metals and pigments. The insoluble 10 material that is removed is mainly amixture of phospholipids and triacylglycerols and is also known as lecithin. Degumming can be performed by addition of concentrated phosphoric acid to the crude seedoil to t non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present. Gum is ted from the seedoil by centrifugation. The l can be reﬁned by addition of a sufﬁcient 1-5 amount of a sodium hydroxide on to titrate all of the fatty acids and ng I the soaps thus formed.

Alkali reﬁning , \ Alkali reﬁning is one of the g processes for treating crude oil, 20 sometimes also referred to as neutralization. It usually 'follows degumming and precedes bleaching. Following degumming, the seedoil can treated by the addition of a sufﬁcient amount of an alkali solution to, titrate all of the fatty acids and phosphoric acids, and removing the soaps thus formed. Suitable alkaline materials include sodium hydroxide, ium hydroxide; sodium carbonate, m ide, 25 calcium hydroxide, calcium ate and ammonium hydroxide. This s is typically carried out at room temperature and removes the free fatty acid fraction.

Soap is removed by centrifugation or by extraction into a solvent for the soap, and the neutralised oil is washed with water. If required, any excess alkali in the oil may be neutralized with a suitable acid such as hydrochloric acid or sulphuric acid. 3O Bleaching Bleaching is a g process in which oils are heated at 90—120°C for 10-30 minutes in the presence of a bleaching earth (02—20%) and in the absence of oxygen by operating with nitrogen or steam or in a vacuum. This step in oil processing is 35 designed to remove unwanted pigments (carotenoids, chlorophyll, gossypol etc), and the process also removes oxidation products, trace metals, sulphur compounds and traces of soap.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 137 Deodorization Deodorization is'a treatment of oils and fats at a high temperature (200— 260°C) and low pressure (0.1—1 mm Hg). This is typically achieved by introducing steam into the seedoil at a rate of about 0.1 ml/minute/100 ml of seedoil.

Deodorization can be performed by g the seedoil to 260°C under vacuum, and slowly introducing steam into the seedoil at a rate of about 0.1 ml/minute/100 ml of seedoil. After about 30 minutes of sparging, the seedoil is allowed to 0001 under vacuum. The seedoil is typically transferred to a glass container and ﬂushed with argon before being stored under refrigeration. If the amount of seedoil is limited, the 10 seedoil can be placed under vacuum for example, in a Parr reactor and heated to 260°C for the same length of time that .it would have been deodorized. This treatment improves the colour of the seedoil and removes a majority of the volatile substances or s compounds including any ing free fatty acids, monoacylglycerols and oxidation products. 15 Winterisatiori Winterization is a process sometimes used in commercial production of oils for . the separation of oils and fats into solid (stearin) and liquid (olein) fractions by crystallization at sub-ambient temperatures. It was applied originally to cottonseed oil 20 to produce a free t. It is typically used .to decrease the saturated fatty acid content of oils. t.

Plant biomassfor the production oflipids Parts of plants involved in ynthesis (e.g., and stems and leaves of higher 25 plants and aquatic plants such as algae) can also be used to e lipid.

Independent of the type of plant, there are several methods for extracting lipids from green biomass. One way is physical extraction, which often does not use solvent extraction. It is a "traditional" way using several different types of mechanical extraction. Expeller pressed extraction is a common type, as are the screw press and 30 ram press extraction methOds.. The amount of lipid extracted using these methods varies widely, depending upon the plant al and the mechanical process employed. Mechanical extraction is lly less efﬁcient than solvent extraction ‘ described below.

Insolvent extraction, an organic solvent (e.g., hexane) ' is mixed with at least 35 the genetically modiﬁed plant green biomass, preferably aﬁer the green biomass is dried and ground. Of course, other parts of the plant besides the green biomass (e.g., containing seeds) can be ground and mixed in as well. The t dissolves the Di in the s and the like, which solution is then separated from the s by Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk ionNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 138 mechanical action (e.g., with the pressing processes above). This separation step can also be performed by.\ ﬁltration (e.g., with a ﬁlter press or r device) or I centrifugation etc. The organic solvent can then be separated from the lar lipid (e.g., by distillation). This second separation step yields non—polar lipid from the plant and can yield a ble solvent ifone employs conventional vapor recovery.

If, for instance, vegetative tissue as described herein, is not to be used immediately to extract, and/0r process, the lipid it is preferably handled post-harvest to ensure the lipid content does not decrease, or such that any decrease in lipid content is minimized as much as possible (see, for example, Christie, 1993). In one 10 embodiment, the vegetative tissue is frozen as soon as possible after harvesting using, for example, dry ice or liquid nitrogen. In another embodiment, the tive tissue is stored at a cold temperature, for example -20°C or -60°C in'an atmosphere of nitrogen. .15 Algaefor the production oflipids ’ Algae can produce 10 to 100 times as much mass as terrestrial plants in a year.

In addition to being a proliﬁc organism, algae are also capable of producing oils and starches that can be converted into biofuels.

The speciﬁc algae most useful for biofuel production are known as microalgae, 20 Consisting of small, oﬁen unicellular, types. These algae can grow almost re.

With more than 100,000 known species of diatoms (a type of alga), 40,000 known species ofgreen plant-like algae, and smaller numbers of other algae species, algae will grow rapidly in nearly any. environment, with almost . any kind of water.

Speciﬁcally, useful algae can be grown in marginal areas with limited or poor quality 25 water, such as in the arid and mostly empty regions of the American SouthWest.

These areas also have abundant sunshine for photosynthesis. In short, algae can be an ideal organism for production of biofuels - efﬁcient growth, needing no premium land or water, not competing with food crops, needing much smaller amounts of land than food crops, and storing energy in a desirable form. V. 30 Algae can store energy in its cell structure in the form of either oil or starch.

Stored oil can be as much as 60% of the weight of the algae. n species which are highly proliﬁc in oil or starch tion have been identiﬁed, and growing conditions have been tested. ses for extracting and converting these materials to fuels have also been deVeloped. 35 The most common oil-producing algae can generally include, or consist. essentially of, the diatoms (bacillariophytes), green algae (chlorophytes), blue-green algae (cyanophytes), and -brown algae (chrysophytes). - In addition a ﬁfth Dip known as haptophytes may be used. Groups include brown algae and Substitute Sheet (Rule 26) RO/AU ' [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 139 heterokonts. c non-limiting es algae include the Classes: phyceae, Eustigmatophyceae, Prymnesiophyceae, Bacillariophyceae.

Bacillariophytes capable of oil tion e the genera Amphipleura, Amphora, Chaetoceros, Cyclotella, Cymbella, Fragilaria, chia, Navicula, Nitzschia, 5 Phaeodactylum, and Thalassiosira. Speciﬁc miting examples of chlorophytes "capable of oil production include Ankistrodesmus, Botryococcus, Chlorella, Chlorococcum, Dunaliella, Monographidium, Oocystis, Scenedesmus, and Tetraselmis.

In one , the chlorophytes can be Chlorella or Dunaliella. Speciﬁc non-limiting ‘ examples of cyanophytes capable of oil production include Oscillatoria and 10 Synechococcus. A c example of chrysophytes capable of oil production includes Boekelovia. Speciﬁc non-limiting examples of haptophytes include ' Isochysis and Pleurochysis.

Speciﬁc algae useful in the present invention include, for e, Chlamydomonas sp. 'such as Chlamydomonas reinhardtii, Dunaliella sp. such as 15 Dunaliella salina, Dunaliella tertiOlecta, D: acidophila, D. bardawil, D. ,bioculata, D. 'lateralis, D. maritima, D. minuta, D. parva, D. peircei, D. polymorpha, D. primo-lecta. D. pseudosalina, D. quartolecta. D. viridis, Haematococcus sp., Chlorella sp. such as Chlorella vulgaris, ChIOrella sorokz'niana or Chlorella protothecoides, Thraustochytrium sp., Schizochytrium sp., Volvox Sp, Nannochloropsis sp., 20 Botryococcus braunii which can contain over 60wt°/o lipid,- Phaeodactylum tricomunim, Thalassiosira nana, Isochrysz‘s sp., a sp., coccum sp, Ellipsoidion sp., Neochloris sp., Scenedesmus sp.

Further, the oil-producing algae of the present invention can include a combination of an effective amount of two or more strains in order to maximize 25 benefits from each strain. As a practical matter, it can be difﬁcult to achieve 100% purity of a single strain of. algae or a combination of desired algae s. However, when discussed herein, the oil-producing algae is intended to cover intentionally introduced s of algae, while foreign strains are preferably minimized and kept below an amount which would entally affect yields of desired oil-producing 30 algae and algal oil. Undesirable algae strains can be controlled and/or eliminated using any number of techniques. For example, careful l of the growth environment can reduce introduction of foreign strains. Alternatively, or in addition to other techniques, a virus selectively chosen to speciﬁcally target only the n strains can be introduced into the growth reservoirs in an amount which is ive to 35 reduce and/or eliminate the foreign strain. An appropriate virus can be readily identiﬁed using conventional techniques. For example, a sample of the foreign algae will most often include small amounts of a virus' which targets the foreign algae. This DIS can be isolated and grown in order to produce amounts which'would effectively Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 140 control or eliminate the foreign algae population among the more desirable oil- ' producing algae. , lture is a form of aquaculture involving the farming of species of algae (including microalgae, also referred to as phytoplankton, microphytes, or planktonic algae, and lgae, commonly known as seaweed); Commercial and rial algae cultivation has numerous uses, including production of food ingredients, food, and algal fuel.

' Mono or mixed algal cultures can be cultured in open-ponds (such as raceway- ' ‘ type ponds and lakes) or photobioreactors. 10 Algae can be harvested using microscreens, by centrifugation, by ﬂocculation (using for e, chitosan, alum and ferric chloride) and by froth ﬂotation.

Interrupting the carbon dioxide supply can cause algae to ﬂocculate on its own, which is called occulation". In froth on, the cultivator aerates the water into a froth, and then skims the algae from the top. Ultrasound and other harvesting 15 methods are currently under development.

Lipid may be separated from the algae by ical crushing. When algae is . dried it retains its lipid content, which can then be "presSed" out with an oil press.

Since different s of algae vary widely in their physical attributes, various press conﬁgurations (screw, expeller, piston, etc.) work better for speciﬁc algae types. 20 Osmotic shock is sometimes used to release cellular components such as lipid from algae. Osmoticshock is a sudden reduction in osmotic pressure and can cause cells in a solution to rupture.

, ' Ultrasonic extraction can accelerate extraction prbcesses, ’ in particular enzymatic extraction processes employed to extract lipid from algae; Ultrasonic 25 waves are used to create cavitation bubbles in a t material. When these bubbles * collapse near the cell walls, the resulting shock waves and liquid jets cause those cells walls to break and release their contents into a solvent.

Chemical solvents (for e,.hexane, benzene, petroleum ether) are oﬁen used in the extraction of lipids from algae. Soxhlet extraction can be use to extract 30' lipids from algae through repeated washing, or percolation, with an organic solvent under reﬂux in a special glassware.

' Enzymatic extraction may be used to extract lipids from algae. Ezymatic ' extraction uses enzymes to degrade the cell walls with water acting as the solvent.

I The enzymatic extraction can be "supported byultrasonication. 35 Supercritical C02 can also be used as a solvent. In this method, C02 is liqueﬁed under pressure and heated to the point that it becomes .supercn'tical (having properties ofboth a liquid and a gas), allowing it to act as a solvent.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk .141 Fermentation processesfor lipidproduction As used herein, the term the "fermentation process" refers to any fermentation process or any process comprising a fermentation step. A fermentation process es, without limitation, fermentation processes used to produce alcohols (e.g., ethanol, methanol, butanol), c acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid), s (e.g., acetone), amino acids (e.g., glutamic acid), gases (e.g., H2 and C02), antibiotics (e.g., penicillin and tetracycline), enzymes, vitamins (e.g., riboﬂavin, beta-carotene), and hormones. Fermentation processes also include fermentation processes used in the consumablealcohol industry (e.g., beer 10 and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred fermentation processes include alcohol. fermentation processes, as are well known in the art. Preferred fermentation processes are anaerobic tation processes, as are well known in the art. ‘Suitable fermenting cells, typically microorganisms that are able to ferment, that is,convert, sugars such 15 as glucose or maltose, ly or indirectly into the desired fermentation product.

Examples of fermenting microorganisms include fungal organisms such as yeast, ably an oleaginous organism. As used herein, an "oleaginous organism" is one which accumulates at least 25% of its dry weight as triglycerides. As used herein, "yeast" includes Saccharomyces ' spp., Saccharomyces 'cerevisiae, 20 Saccha'romyces carlbergensis, Candida spp., omyces spp., Pichia spp., Hansenula spp., derma spp., Lipomyces Starkey, and Yarrowia ytica.

Preferred yeast include Yarrowia lipolytica or other nous yeasts and strains of the Saccharomyces spp., and in particular, Saccharomyces cerevisiae.

In one embodiment, the fermenting rganism is a transgenic sm 25 that comprises one or more exogenous polynucleotides, wherein the transgenic organism has an increased level of One or more non-polar lipids when compared to a corresponding sm lacking the one or more exogenous polynucleotides. The transgenic microorganism is preferably grown under conditions that optimize activity of fatty acid biosynthetic genes and fatty ‘acid acyltransferase genes. This leads to 3O production of the greatest and the most economical yield of lipid. In general, media ions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the lipid accumulation phase and the‘time of cell harvest. 35 Fermentation media must contain a suitable carbon source. Suitable carbon sources may include, but are not limited to: monosaccharides (eg.,- glucose, fructose), harides (e.g.., lactose, e), oligosaccharides, polysaccharides (e.g., starch, Dulose or es thereof), sugar alcohols (e.g., glycerol) or mixtures from Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 142 renewable feedstocks _(e.g.,' cheese whey permeate, comsteep liquor, sugar beet molasses, barley malt). Additionally, carbon sources may include alkanes, fatty acids, esters of fatty acids, monoglycen'des, diglycerides, triglycerides, phospholipids and various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) and animal fats. Additionally, the carbon substrate may include one-carbon substrates (e.g., carbon dioxide, methanol, formaldehyde, e, —containing amines) for which lic conversion into key mical intermediates has been demonstrated. Hence it is plated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing substrates and 10 will only be limited by the choice of the host microorganism. gh all of the above mentioned carbon substrates and mixtures thereof are expected to be suitable in . the present invention, preferred carbon substrates are sugars and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NI-102804) or organic source (e.g., 15 urea, glutamate). In on to appropriate carbon and nitrogen sources, the fermentation media may also contain suitable minerals, salts, ors, buffers, vitamins and other components known to those d in the art suitable for the growth of the microorganism and ion of the enzymatic pathways necessary for lipid production. . 20 A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5‘ to pH 7.0 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of lipid in the cells of oleaginous 25 microorganisms requires a age process, since the metabolic state must be "balanced" between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of lipids in microorganisms. In this approach, the ﬁrst stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth 3.0 and cell division. In the second stage of the fermentation, it is preferable to establish, conditions of nitrogen ation in the culture to promote high levels of lipid accumulation. The effect of this nitrogen deprivatiOn is to reduce the effective concentration of AMP in the cells, thereby reducing the activity of the NAD- dependent isocitrate ogenase of mitochondria. When this , citric acid 35 will accumulate, thus forming abundant pools of acetyl-CoA in the cytoplasm and g fatty‘acid synthesis. Thus, this phase is characterized by the Cessation of cell division followed by the synthesis of fatty acids and accumulation ofTAGS.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 143 Although cells are typically grown at about 30°C, some studies have shown increased synthesis of unsaturated fatty acids at lower temperatures. Based on process economics, this temperature shift should likely occur after the ﬁrst phase of the two- stage tation,when the bulk of the microorganism's growth has occurred.

It is contemplated that a variety of fermentation process s may be applied, where commercial production of lipids using the instant nucleic acids is desired. For example, commercial tion of lipid from a recombinant ial host may be produced by a batch, tch or continuous fermentation process.

A batch fermentation process is a closed system wherein the media 10 composition is set at the beginning of the process and not subject to further additions beyond those required for maintenance of pH and oxygen level during the process. i Thus, at the beginning of the culturing process the media is inoculated with the desired organism and growth or metabolic activity is permitted to occur without adding additional ates (i.e., carbon and nitrogen sources) to the medium. In 15 batch processes the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. In a typical batch process, cells moderate through a static lag phase to a high-growth log phase and ﬁnally to a stationary phase, wherein the growth rate is diminished or halted. Left untreated, cells in the stationary phase will eventually die. A variation of the standard batch process 20 “is the fed-batch process, n the substrate is continually added to the fermentor .\ over the course of the fermentation process. A fed-batch process is also suitable in the t ion. Fed-batch processes are useful when catabolite repression is _ apt to inhibit the metabolism of the cells or where it is desirable to have d amounts of substrate in the media at any one time. ement of the substrate 25 concentration in fed-batch systems is difﬁcult and therefore may be estimated on the basis of the changes of measurable factors such as pH,'dissolved oxygen and the partial pressure of waste gases (e.g., C02). Batch and fed-batch culturing s are common and well known in the art and examples may be found in Brock, In Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd ed., Sinauer 30 Associates, Sunderland, Mass., (1989); or Deshpande.(l992).

Commercial tion of lipid using the instant cells may also be accomplished by a continuous fermentation precess, wherein a deﬁned media is continuously added to a bioreactor while an equal amount of culture volume is removed simultaneously for product recovery. Continuous cultures generally 35 maintain the cells in the log phase of growth at a constant cell density. Continuous or semi-continuous culture methods permit the modulation of one factor or any number ‘ . of factors that affect cell growth or end product concentration. For example, one ' . nroach may limit the carbon source and allow all other parameters to moderate Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 144 metabolism. In other systems, a number of factors ing growth may be altered continuously while the cell. concentration, measured by media turbidity, is kept constant. Cdntinuous s strive to maintain steady state growth and thus the cell growth rate must be balanced against cell loss due to media being drawn off the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of t formation, are well known in the art of industrial iology and a variety of methods are detailed by Brook, supra.

Fatty acids, including PUFAs, may be found in the host microorganism as free 10 fatty acids or in esteriﬁed forms such as acylglycerols, phospholipids, sulfolipids or glyColipids, and may be ted from the host cell through a variety of means well- known in the art.

In general, means for the puriﬁcation of fatty acids, including PUFAs, may include extraction with organic solvents, sonication, supercritical ﬂuid tion 15 (e.g., using carbon dioxide), ﬁcation and physical means such as presses, or combinations thereof. Of particular st is tion with methanol and chloroform in the presence of water (Bligh and Dyer, 1959).. Where desirable, the s layer can be acidiﬁed to protonate negatively-charged moieties and thereby increase partitioning of desired products into the organic layer. After extraction, the 20 organic solvents can be removed by evaporation under a stream of nitrogen. When isolated'in conjugated forms, the products may be enzyrnatically or chemically cleaved to release the Rec fatty acid or a less complex conjugate of interest, and can then be t to further manipulations to produce a desired end product. Desirably, conjugated forms of fatty acids are cleaved with potassium‘hydroxide.’ 25 If r puriﬁcation is necessary, standard methods can be employed. Such s may include extraction, treatment with urea, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high-speed 'centrifugation or distillation, or combinations of these ques. Protection of reactive groups such . as the acid or alkenyl groups, may be done at any step through ‘known techniques 30 (e.g., alkylation, iodination). s used include methylation of the fatty acids to produce methyl esters. Similarly, protecting groups may be removed at any step.

Desirably, puriﬁcation of fractions containing GLA, STA, ARA, DHA and EPA may be accomplished by treatment with urea and/or fractional distillation.

An example of the use of plant biomass for the production of a biomass slurry 35 using yeast is described in W0 201 1/ 100272.

Uses of Li ids D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 145 The lipids produced by the methods described have a variety of uses. In some embodiments, the lipids are used as food oils. In other embodiments, the lipids are reﬁned and used as lubricants or for other industrial uses such as the synthesis of cs. In some preferred embodiments, the lipids are reﬁned to produce biodiesel.

Biofuel As used herein the term "biofuel" es biodiesel and bioaleohol. Biodiesel can be made from oils derived from plants, algae and fungi. Bioalcohol is produced from the fermentation of sugar. This sugar can be extracted directly from plants (e.g., 10' sugarcane), derived from plant starch (e.g., maize or wheat) or made ﬁom cellulose (e.g., wood, leaves or stems).

Biofuels currently cost more to 'produce than petroleum fuels. In addition to processing costs, biofuel crops require planting, ising, pesticide and herbicide applications, harvesting and ortation. Plants, algae and fungi of the present ,15 invention may reduce production costs ofbiofuel.

. General methods for the production of bioﬁJel can be found in, for example, Maher and Bressler, 2007; Greenwell et al., 2010; Karmakar et al., 2010; Alonso et al., 2010; Lee and d, 2010; Liu et al., 2010a; Gong and Jiang, 2011; Endalew ' et al., 2011; Semwal et al., 2011. 20 Bioalcohol The production of biologically ed alcohols, for example, ethanol, propanol and butanol is well known. Ethanol is the most common bioalcohol.

The basic steps for large scale production of ethanol are: 1) microbial (for example, yeast) fennentation of sugars, 2) lation, 3) dehydration, and Optionally 25‘ 4)’ denaturing. Prior to fermentation, some crops require sacchariﬁcation or hydrolysis of carbohydrates such as cellulose and starch into sugars. Sacchariﬁcation of cellulose is called cellulolysis. s can be used to convert starch into sugar. 3O Fermentation Bioalcohol is produced by microbial fermentation of the sugar. Microbial fermentation will currently only work directly with sugars. Two major ents of plants, starch and cellulose, are both made up of , «and can in principle be I converted to sugars for fermentation. 35 lation D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 146 For the ethanol to be usable as a fuel, the ty of the water must be removed. Most of the water is d by distillation, but the purity is limited to 95- 96% due to the formation of a low-boiling ethanol azeotrope with maximum (95.6% m/m (96.5% v/v) ethanol and 4.4% m/m (3.5% v/v) water). This mixture is called hydrous ethanol and can be used as a fuel alone, but unlike anhydrous ethanol, _ hydrous ethanol is not miscible in all ratios with gasoline,‘so the water fraction is typically removed in further treatment in order to burn in combination with gasoline ‘ ’ in gasoline engines. 10 Dehydration , ' . Water can be removed from from an azeotropic ethanol/water mixture by dehydration. Azeotropic distillation, used in many early fuel ethanol plants, consists - of adding benzene or cyclohexane to the mixture. When these components are added to the mixture, it forms a heterogeneous azeotropic mixture in vapor-liquid-liquid 15 equilibrium, which when distilled produces anhydrous l in the column bottom, and a vapor mixture of Water and cyclohexane/benzene. When sed, this becomes a two-phase liquid mixture. r early method, called extractive distillation, consists of adding a y component which will increase ethanol's relative lity. When the ternary mixture is distilled, ”it will produce anhydrous 20 ethanol on the top stream of the column. s A third method has emerged and has been adopted by the majority of modern ethanol plants. This new process uses” molecular sieves to remove water from fuel ethanol. In this process, l vapor under pressure passes through a bed of molecular sieve beads. The bead‘s pores are sized to allow absorption of Water while 25 excluding ethanol. After a period of time, the bed is regenerated under vacuum or in the ﬂow of inert atmosphere (e.g. N2) to removethe absorbed water. Two beds are often used so that one is available. to absorb water while the other is being regenerated. 30 Biodiesel .

The production of biodiesel, or alkyl , is well known. There are three . basic routes to ester tion from lipids: 1) Base catalysed transesteriﬁcation of the lipid with alcohol; 2) 'Direct acid catalysed esteriﬁcation of the lipid with methanol; and 3) Conversion of the lipid to fatty acids, and then to alkyl esters with 35 acid catalysis.

I Any method for preparing fatty acid alkyl esters and glyceryl ethers (in which one, two or three of the hydroxy groups on glycerol are etheriﬁed) can be used. For ‘ Dmple, fatty acids can be prepared, for example, by hydrolyzing' or saponifying Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 147 triglycerides with acid or base catalysts, respectively, or using an enzyme such as a lipase or an esterase. Fatty acid alkyl esters can be prepared by reacting a fatty acid with an alcohol in the presence of an acid catalyst. Fatty acid alkyl esters can also be prepared by reacting a triglyceride with an alcohol in the presence of an acid or base catalyst. Glycerol ethers can be prepared, for example, by reacting glycerol with an alkyl halide in the presence of base, or with an olefin or alcohol in the presence of an I I acid catalyst.

In some preferred embodiments, the lipids are transesteriﬁed to produce methyl esters and glycerol. In some preferred embodiments, the lipids are reacted 10 with an alcohol (such as methanol or ethanol) in the presence of a catalyst (for e, ium or sodium hydroxide),to produce alkyl . The alkyl esters canlbe'used for biodiesel or blended with petroleum based fuels.

V The alkyl esters can be ly blended with diesel fuel, or washed with water or other aqueous solutions to remove various impurities, including the catalysts, 15 before blending; It is possible to neutralize acid catalysts with base. However, this process produces salt. To avoid engine corrosion, it is preferable to minimize the salt ' concentration in the fuel additive ition. Salts can be ntially removed from the Composition, for example, by washing the composition with water.

' In another embodiment, the ition is dried after it is , for 20 example, by passing the composition through a drying agent such as calcium sulfate.

‘ In yet another embodiment, a neutral ﬁre] additive is obtained without producing salts or using a washing step, by using a ric acid, such as Dowex 50"", which is a resin that contains sulfonic acid groups. The catalyst is easily removed by ion aﬁer the esteriﬁcation and etheriﬁcation reactions are complete. 25 Plant triacylglycerols as a biofuel source Use of plant triacylglycerols for the production of biofuel is reviewed in t et al. oils are primarily of various , (2008). Brieﬂy, plant compdsed _ triacylglycerols (TAGS), les that consist of three fatty acid chains (usually 18 30 or 16 ‘carbons long) esteriﬁed to glycerol. The fatty acyl chains are chemically similar to the aliphatic hydrocarbons that make up the bulk of the molecules found in petrol and diesel. The arbons in petrol contain between 5 and 12 carbOn atoms per molecule, and this volatile fuel, is mixed with air and ignited with a spark in a conventional engine. In contrast, diesel fuel components typically have 10—1 5 carbon 35 atoms per molecule and are ignited by the very high compression obtained in a diesel engine. However, most plant TAGs‘ have a viscosity range that is much higher than that of conventional diesel: 2.9 mmzs'l ed to 1.9—4.1, mmzs‘l, Dectively (ASTM D975; Knothe and Steidley, 2005). This higher'viscosity results Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 148 in poor fuel atomization in modern diesel engines, leading to problems derived from - incomplete combustion such as carbon deposition and coking (Ryan et a1., 1984). To ' me this problem, TAGS are converted to less viscous fatty acid esters by ﬁcation with a primary alcohol, most ly methanol. The resulting fuel is commonly referred to as biodiesel and has a dynamic viscosity range from 1.9 to 6.0 mmzs'l (ASTM D6751). The fatty acid methyl esters (FAMEs) found in sel, have a high energy y as ed by their high heat of combustion, which is similar, if not greater, than that of conventional diesel e, 2005). Similarly, the cetane number (a measure of diesel ignition quality) of the FAMEs found in biodiesel 10 exceeds that of conventional diesel (Knothe, 2005).

Plant oils are mostly composed of ﬁve common fatty acids, namely palmitate (16:0), stearate (18:0), oleate (18:1), ate (18:2) and linolenate (1823), although, depending on the particular species, longer or shorter fatty acids may also be major constituents. These fatty acids differ from each other in terms of acyl chain length 15 and number of double bonds, leading to different physical properties. Consequently, the fuel properties of biodiesel derived from a mixture of fatty acids are dependent on that ition. Altering the fatty aCid proﬁle can therefore improve fuel properties of biodiesel such as cold-temperature ﬂow characteristics, oxidative stability and NOx emissions. Altering the fatty acid composition of TAGS may reduce the viscosity of 20 the plant oils, eliminating the need for chemical modiﬁcation, thus ing the ' ' ffectiveness of biofuels. ' Most plant oils are derived from triacylglycerols stored in seeds. However, the present invention provides methods for also increasing oil. content in vegetative tissues. The plant tissues of the present invention have an increased total lipid yield. 25 Furthermore, the level of oleic acid is increased signiﬁcantly while the saturated fatty acid alpha linolenic acid was reduced. .

Once a leaf is developed, it undergoes a developmental change from sink (absorbing nutrients) to source (providing sugars). In food crops, most sugars are translocated out of source leaves to support growth of new leaves, roots and fruits. '30 e translocation of carbohydrate is an active process, there is a loss of carbon and energy during translocation. Furthermore, aﬁer the developing Seed takes up carbon from the plant, there are additional carbon and energy losses associated with the conversion of carbohydrate into the oil, protein or other major components of the seed (Goffman etal., 2005). Plants of the. present invention increase the energy . 35 content of leaves and/or stems, such that the whole above-ground plant may be . harvested and used to produce biofuel.

D 1 Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 149 Algae as a l source _ Algae store oil inside the cell body, sometimes but not always in vesicles. This oil can be recovered in several relatively simpleways, including solvents, heat, and/or pressure. However, these methods typically recover only about 80% to 90% of the stored oil. Processes which offer more effective oil extraction methods which can recover close to 100% of the stored oil at low cost as known in the art. These processes include or consist of depolymerizing, such as biologically breaking the walls of the algal cell and/or on vesicles, if present, to release the oil from the oil- producing algae. 10 In addition, a large number of viruses exist whiCh invade and rupture algae cells, and can thereby release the contents of the cell in particular stored oil or starch.

Such viruses are an integral part of the algal ecosystem, and many of the s are speciﬁc to a single type of algae. Speciﬁc es of. such viruses include the Chlorella virus PBCV—l ecium Bursaria Chlorella Virus) which is speciﬁc to 15 certain Chlorella algae, and hages such as SM—l, P-60, and AS-l c to the blue-green algae Synechococcus. The particular virus ed will depend on the particular species of algae to be used in the growth process. One aspect of the present invention is the use of such a virus to rupture the algae so that oil contained inside the algae cell wall can be recovered. In another detailed aspect of the present invention, a 20 mixture of biological agents can be used to e the algal cell wall and/or oil vesicles.

I Mechanical crushing, for example, an expeller or press, a hexane or butane selvent recovery step, supercritical ﬂuid extraction, or the like can also be useful in extracting the oil from 'oil es of the oil-producing algae. Alternatively, 25 mechanical approaches can be used in' combination with biological agents in order to improve reaction rates and/or separation of materials. Regardless of the particular biological agent or agents chosen such can be uced in amounts which are sufﬁcient to serve as the primary mechanism by which algal oil is released from oil es in the oil-producing algae, i.e. not a merely incidental ce of any of ' 30 these.

Once the oil has been released from the algae itcan be recovered or separated 16 from a slurry of algae debris material, for example, cellular residue, oil, enzyrhe, by—products, etc. This can be done by sedimentation or centrifugation, with centrifugaticn generally being faster. Starch production can follow similar separation 35. processes.

An algal feed can be formed from a biomass feed source as well as an algal feed source. Biomass from either algal or tenestrial sources can be merized in Danety of ways such as, but not limited to sacchariﬁcation, hydrolysis or the like.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk I .150 The source material can be almost any sufﬁciently voluminous cellulose, lignocellulose, polysaccharide or carbohydrate, glycoprotein, or other material making up the cell wall of the source material.

The tation stage can be conventional in its use of yeast to ferment sugar to alcohol. The fermentation process produces carbon dioxide, l, and algal husks. All of these products can be used elsewhere in the process and systems of the present invention, with substantially no unused al or wasted heat. Alternatively, . if ethanol is so produced, it can be sold as a product or used to produce ethyl aCetate for the transesteriﬁcation process. r considerations would apply to alcohols , 10- other than ethanol. - .

I Algal oil can be ted to sel through a process of direct hydrogenation or transesteriﬁcation of the algal oil. Algal oil is in a similar form as most vegetable oils, which are in the form of triglycerides. A triglyceride consists of three fatty acid chains, one attached to each of the three carbon atoms in a glycerol ‘15 backbone. This form of oil can be burned directly. However, the properties of the oil in this form are not ideal for use in a diesel , and without modiﬁcation, the engine will soon run poorly or fail. In accordance with the present invention, the ceride is converted into biodiesel, which is similar to but superior to petroleum diesel fuel in many respects. 20 One process for converting the triglyceride to biodiesel is transesteriﬁcation, and es reacting the ceride with alcohol or other acyl acceptor to produce free fatty acid esters and glycerol. The free fatty acids are in the form of fatty acid ' alkyl esters (FAAE).

With the chemical s, additional steps are needed to separate the catalyst 25 and clean the fatty acids. In addition, if ethanol is used as the acyl acceptor, it must be essentially dry to prevent production of soap via saponiﬁcation in the process, and the glycerol must be d. The biological process, by comparison, can accept ethanol in a’less dry state and the cleaning and puriﬁcation of the biodiesel and glycerol are I much easier. ' 30 .Transesteriﬁcation often uses a simple alcohol, typically methanol derived from petroleum. When methanol is used the ant biodiesel is called fatty acid methyl ester (FAME) and most biodiesel sold today, especially in Europe, is FAME.

However, ethanol can also be used as the alcohol in transesteriﬁcation, in which case the biodiesel is fatty acid ethyl ester (FAEE). In the US, the two types are usually not 35 guished, and are collectively known as fatty acid alkyl esters (FAAE), which as ' a generic term can apply regardless of the acyl acceptor used. Direct hydrogenation can also be utilized to convert at least a'portion of the algal 'oil to a biodiesel. As an, in one aspect, the biodiesel product can include an alkane.

' Substitute Sheet (Rule 26) RO/AU ' [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 151 The algal triglyceride can also be converted -to sel by direct hydrogenation. In this process, the ts are alkane chains, propane, and water.

The glycerol backbone 7is hydrogenated to propane, so there is substantially no glycerol produced as a byproduct. Furthermore, no alcohol or transesteriﬁcation catalysts are . All of the biomass can be used as feed for the oil-producing algae with none needed for fermentation to produce alcohol for transesteriﬁcation.

The ing alkanes are pure hydrocarbons, with no oxygen, so the biodiesel produced in this way has a slightly higher energy content than the alkyl esters, degrades more slowly, does not attract water, and has other desirable chemical 10 properties. ' Feedstuﬁfs The, present invention includes compositions which can be used as feedstuffs.

For purposes of the present invention, "feedstuﬂ‘s" include any food or preparation for 15 human or animal consumption (including for enteral and/or parenteral consumption) which when taken into the body: (I) serve to h or,buildiup tissues or supply energy, and/or (2) maintain, e or support adequate nutritional status or metabolic function. Feedstuffs of the invention include nutritional compositions for babies and/or young children. 20 Feedstuffs of the invention comprise for example, a cell of the ion, a plant of the invention, the plant part of the invention, the seed of the invention, an extract of the invention, the product of a method of the invention, the product of a . fermentation process of the invention, or a composition along with a suitable carrier(s). The term “carrier" is used in its broadest sense to ass any 25 component which may or may not have nutritional value. As the person d in the art will appreciate, the carrier must be suitable for use (or used in a sufﬁciently low concentration) in a feedstuff, such that it does not have deleterious effect on an i organism which consumes the uff. , g V The feedstuff of the present invention comprises a lipid produced directly or 30 indirectly by use of the methods, cells or organisms disclosed herein. The composition may either be in a solid or liquid form. Additionally, the composition may e edible macronutrients, vitamins, and/or minerals in amounts desired for a particular use. The amounts of these ingredients will vary depending on whether the composition nded for use with normal individuals or for use with individuals 35 having specialized needs such as individuals ing from metabolic ers and the like.

Examples of suitable caniers with nutritional value include, but are not limited Dnacrbnutrients such as edible fats, carbohydrates and proteins. Examples of such 1” Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 152 edible fats include, but are not limited to, coconut oil, borage oil, fungal oil, black current oil, soy oil, and mono- and di-glycerides. Examples of such carbohydrates include, but are not limited to, glucose, edible lactose, and hydrolyzed starch.

Additionally, examples of proteins which may be utilized in the nutritional composition of the invention include, but are not limited to, soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the ing may be added to the feedstuff compositions of the present invention, calcium, phosphorus, potassium, 10 sodium, chloride, magnesium, manganese, iron, copper, zinc, um, iodine, and 'vitamins A, E, D, C, and the B complex. Other such vitamins and ls may also be added. ' _ The components utilized in the feedstuff compositions of the present invention can be of semi-puriﬁed or d origin. By semi-puriﬁed or puriﬁed is meant a 15 al which has eparedby puriﬁcation of a natural material or by de novo . synthesis.

A feedstuff composition of the present invention may also be added to food even when supplementation of the diet is not required. For e, the composition may be added to food of any type, including, but not limited to, margarine, modiﬁed 20 butter, cheeses, milk, yogurt, chocolate, candy, snacks, salad oil’s, cooking oils, cooking fats, meats, ﬁsh and ges.

The genus Saccharomyces spp is used in both brewing of beer and wine ‘ making and also as an agent in baking, particularly bread. Yeast is a major of vegetable extracts. . constituent Yeast is also used as an additive in animal feed. It '25 will be apparent that genetically modiﬁed yeast strains can be provided which are adapted to synthesize lipid as described herein. These yeast strains can then be used in food stuffs and in wine and beer making to‘provide products which have enhanced lipid content.

. Additionally, lipid produced in accordance with the present invention or host 3O cells transformed to n andexpress the subject genes may also be used as animal food supplements to alter an animal‘s tissue or milk fatty acid composition to‘one more desirable for human or animal consumption. Examples of such animals include I sheep, cattle, horses and the like.

. Furthermore, feedstuffs of the invention can be used in aquaculture to increase . 35 the levels of fatty acids in ﬁsh for human or animal ption.

V Preferred feedstuffs of the invention are the , seed and other plant parts such as , fruits and stems which may be used directly as food or feed for Dians or other animals. For example, animals may graze directly on such plants Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 153 grown in the field, or be fed more measured amounts in lled feeding. The invention includes the use of such plants and plant parts as feed for increasing the polyunsaturated fatty acid’levels in humans and other animals.

Compositions ‘ ' The present invention also encompasses itions, particularly pharmaceutical compositions, comprising one Or more lipids produced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the lipids, in combination with a rd, well-known, non-toxic pharmaceutically-acceptable 10 carrier, adjuvant or vehicle such as phosphate—buffered saline, water, ethanol, polyols, vegetable oils, a wetting agent, or an on such as a water/oil emulsion. The composition may be in either a liquid or solid form. For example, the composition may be in the form of a tablet, capsule, ingestible liquid, powder,topical ointment or cream. Proper ﬂuidity can be maintained for e, by the maintenance of the 15 required particle size in the case of dispersions and by the use of surfactants. It may also be ble to include isotonic agents for example, sugars, sodium chloride, and . the like. s such inert ts, the composition can also include adjuvants such ' as wetting , emulsifying and suspending agents, sweetening agents, ﬂavoring agents and perfuming agents. 20 Suspensions, in addition to the active compounds, may comprise suspending I agents such as lated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of‘ these substances.

Solid dosage forms such as tablets and capsules can be prepared using 25 techniques well known in the art. For example, lipid produced in accordance with the present invention can be tableted with conventional tablet bases such, as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate. Capsules can be prepared by incorporating 30 these excipients“ into a n capsule along with idants and the relevant 1ipid(‘s).

For intravenous administration, the lipids produced in accordance with the present ion or derivatives thereof may be incorporated into commercial formulations. ' , 35 A typical dosage of a particular fatty acid is from 0.1 mg to 20 g, taken from one to ﬁve times per day (up to 100 g daily) and is preferably in the range of from about lO’mg to about 1, 2, 5, or 10 g daily (taken in one or multiple doses). As known Dhe art, a minimum of about 300 mg/day of fatty acid, especially polyunsaturated Substitute Sheet .

(Rule 26) RO/AU .- [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 154 fatty acid, is desirable. However, it will be iated that any amount of fatty acid. will be beneﬁcial to the subject.

Possible routes of administration of the pharmaceutical compositions of the present invention e for example, enteral and parenteral. For example, a liquid preparation may be administered orally. onally, a homogenous mixture can be completely dispersed in water, admixed under e conditions with physiologically acceptable diluents, preservatives, buffers or propellants'to form a spray or inhalant.

The dosage of the composition to be administered to the subject may be determined by'one of ordinary skill in the art and depends upon various factors such 10 as weight, age, overall health, past history, immune status, etc., of the subject.

Additionally, the compositions of the t invention may be utilized for cosmetic purposes. The compositions may be added to pro-existing cosmetic itions, such that a mixture is formed, or a fatty acid ed according to the ' invention may be used as the sole "active" ingredient in a cosmetic composition. 15 LOW , The terms "polypeptide" and "protein" are generally used interchangeably.

A polypeptide or class of polypeptides may be deﬁned by the extent ofidentity of its amino acid sequence to a nce amino acid sequence, or by' _ (% identity) 20 having a greater. % identity to one reference amino acid sequence than to another.

The ,% ty of a polypeptide to a nce amino acid sequence is typically determined by GAP analysis (Needleman and WunsCh, 1970; GCG program) with parameters of a gap creation penalty = 5, and a gap extension penalty = 0.3. The _ query sequence is at least 100 amino acids in length and the GAP analysis aligns the .25 two sequences over a region of at least 100 amino acids.- Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even, more preferably, the GAP analysis aligns two sequences over their entire length. The polypeptide or class of polypeptides may have the same enzymatic activity as, or a different activity than, 30 or lack the ty of, the reference polypeptide. Preferably, the polypeptide has an enzymatic activity of at least 10% of the ty of the reference polypeptide.

. As used herein a "biologically active fragment" is a portion of a ptide of the ion which maintains a deﬁned activity of a full-length reference polypeptide for example, MGAT activity. Biologically active fragments as used herein exclude 35' the full-length polypeptide. ically active fragments can be any size portion as long as, they maintain the deﬁned activity. Preferably, the biologically active fragment maintains at least 10% of the activity of the full length ptide.

D Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 155 With regard to a deﬁned polypeptide or enzyme, it will be appreciated that % identity ﬁgures higher than these provided ' herein will encompass preferred embodiments. Thus, where applicable, in light ofthe minimum % identity ﬁgures, it is preferred that the polypeptide/enzyme comprises an amino acid sequence which is at least 60%, more preferably at least, 65%,. more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more ably at least 94%, more preferably at least 95%, more ably at least 96%, more ably at least 97%, more 10 preferably at least 98%, more preferably at least 99%, more ably.at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. 15 Amino acid sequence mutants of the polypeptides deﬁned herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid deﬁned herein, 'or by_ in Vitro synthesis of the desired polypeptide. Such mutants include for example, ons, insertions, or substitutions of es within the amino acid ce. A combination of deletions, insertions and substitutions can be made to 20 arrive at the ﬁnal construct, ed that the ﬁnal polypeptide product possesses the desired characteristics.

Mutant (altered) ptides can be prepared using any technique knoWn in the art, for example,using directed evolution or nal design strategies (see below). Products derived from mutated/altered DNA can readily be screened using 25 techniques described herein to determine if they possess fatty acid acyltransferase activity, for example, MGAT, DGAT, o'r GPAT/phosphatase activity.

In designing amino acid sequence mutants, the location of the'mutation site and the nature of the mutation will depend on characteristic(s) to be modiﬁed. The sites for mutation can be modiﬁed individually or in series for example, by (1) 30 substituting ﬁrst with conservative amino acid s and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) ' inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 es and lly about 1 to 5 contiguous residues.- 35 Substitution mutants have at least one amino acid residue in the polypeptide removed and a different residue‘inserted in its place. The sites of greatest interest for substitutional nesis include sites identiﬁed as the active site(s). Other sites of "Best are those in which particular residues obtained from various strains or species Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 156 are identical, These positions may be important for biological activity. These sites, especiallythose falling within a sequence of at least three other identically conserved sites, are preferably. substituted in a relatively conservative manner. Such conservative substitutions are shown in Table I under the heading of "exemplary substitutions".

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four Conservative amino acid changes when ed to a naturally occurring polypeptide. s of conservative amino acid changes are provided in Table 1. As the skilled person would be aware, such minor changes can 10 reasonably be predicted nOt to alter the activity of the polypeptide when expressed in a inant cell.

‘ Table 1; Exemplary tutions, Original Exemplary Residue Substitutions Ala A) val § leu 116 l __ __ ”——ser __asI J M—Ieu; val ala Leu L ile, val; met;'ala; he __ l—_ __ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 157 Directed Evolution ’ In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used to pick out variants that have the d qualities, for example, increased fatty acid acyltransferase activity. Further rounds of mutation and selection are then applied. A l ed evolution gy involves three steps: 1) ifiCation: The geneencoding the protein of interest is mutated and/or recombined at random to create a large y of gene variants. Variant gene libraries can be constructed through error prone PCR (see, for example, Leung, 1989.; Cadwell and Joyce, 1992), from pools of DNaseI ed fragments prepared from 1‘0 parental templates (Stemmer, 1994a; Stemmer, 1994b; Crameri et al., 1998; ‘Coco et al., 2001) from rate oligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures of both, [or even from undigested parental templates (Zhao et al., 1998; Eggert et al., 2005; Je’zéquek et al., 2008) and are usually assembled through PCR. ies can also be made from parental ces recombined in vivo or in vitro by 15 either homologous or non-homologous recombination meier et al., 1999; Volkov et al., 1999; Sieber et al., 2001). Variant gene libraries can also be constructed by sub-cloning a gene of interest into a suitable vector, transforming the vector into a "mutator" strain suCh as the E. coli XL—l red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. Variant 20 gene libraries can also be constructed by subjecting the gene of interest to DNA shufﬂing (i.e., in vitro homologous recombination'of pools of selected mutant genes ' by random fragmentation and reassembly) as broadly described by Harayama (1998). 2) Selection: The library is tested for the presence of mutants (variants) possessing the desired property using a screen or selection. ’ Screens enable the '25 identiﬁcation and isolation of high-performing mutants by hand, while selections autOmatically eliminate all ctional mutants. A screen may e screening forthe presence ofknown conserved amino acid motifs. Alternatively, or in on, a screen may involve expressing the mutated polynucleotide in a host, organsim or part thereof and assaying the level of fatty acid acyltransferase activity by, for example, 30 quantifying the level of resultant product in lipid extracted from the organism or part thereof, and determining the level of product in the extracted lipid from the organsim or part thereof relative to a corresponding organism or part thereof lacking the mutated polynucleotide and optionally, expressing the parent (upmutated) polynucleotide. Alternatively, the screen may involve feeding the organism or part I 35 f labelled substrate and determining the level of substrate or product in the organsim or part thereof relative to a corresponding organism or part thereof lacking the mutated polynucleotide and optionally, expressing the parent (unmutated) Dmucleotide. , 'Substitute Sheet ' (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 158 3) Ampliﬁcation: The variants identiﬁed in the selection or screen are replicated many fold, enabling researchers to ce their DNA in order to understand what ons have occurred.

Together, these three steps are termed a "round" of directed evolution. Most experiments will entail more than one round. In these experiments, the "winners" of the previous round are diversiﬁed in the next round to create a new library. At the, end of the experiment, all evolved protein or polynucleotide mutants are characterized ' using biochemical methods. . -10 Rational Design ‘ = A protein can be ed ally, on the basis of known information about protein structure and folding. This can be lished by design from scratch (de novo design) or by redesign based on native scaffolds (see, for example, Hellinga, 19.97; and Lu and Berry, Protein Structure Design and Engineering, Handbook of ‘ 15 ns 2, 1153-1157 (2007)). Protein design lly involves identifying ' sequences that fold into a given or target structure and can be accomplished using computer models. Computational protein design algorithms search the sequence— conformation for sequences that are ‘low in energy when folded to thetarget strueture. ational protein design algorithms use models of protein energetics 20' to evaluate how mutations womd affect a protein's structure and function. These energy functions typically include a combination of molecular mechanics, statistical (i.e. knowledge-based), and other empirical terms. Suitable available software ' includes IPRO (Interative Protein Redesign and Optimization), EGAD (A Genetic Algorithm for Protein Design), Rosetta , Sharpen, and Abalone. 25 Also included within the scope ofthe invention are polypeptides deﬁned herein which are differentially modiﬁed during or after synthesis for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatizationiby known protecting/blocking groups, proteolytic cleavage, linkage to. 30 an antibody molecule or other cellular ligand, etc. These ations may serve to increase the stability and/or bioactivity of the polypeptide of the invention.

\ Identiﬁcation-ofFatt Acid Ac ltransferases in one aspect, the invention provides a method for identifying a nucleic acid 35, molecule encoding a fatty acid acyltransferase having an increased ability to produce ' MAG, DAG and/or TAG in a cell.

The method comprises obtaining a cell sing a nucleic acid le Doding a fatty acid acyltransferase operably linked to a promoter which is active in Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 159 the cell. The nucleic acid molecule may encode a naturally occurring fatty acid acyltransferase such as MGAT, GPAT and/or DGAT, or a mutant(s) thereof. Mutants may be engineered using standard procedures in them (see above) such as by random mutagenesis, targeted mutagenesis, or saturation mutagenesis on . ming known genes of interest, or by subjecting ent genes to DNA shufﬂing. For example, a polynucleotide sing a sequence selected from any one of SEQ ID ‘ - NOs:l to 44 which encodes a MGAT may be mutated and/or recombined at random to create a large y of gene variants ts) using for example, error-prone PCR and/or DNA shufﬂing. Mutants may be selected for further investigation on the. 10 basis that they comprise a conserved amino acid motif. For example, in the caseof a candidate nucleic acid encoding 'a MGAT, a skilled person may determine whetherit comprises a sequence as ed in SEQ ID NOs1220, 221', 222, 223, and/or 224 before testing r the nucleic acid encodes a onal MGAT mutant (by for example, transfection into a host cell, such as a plant cell and assaying for fatty acid .15 acyltransferase (i.e., MGAT) activity as described herein).

Direct PCR sequencing of the nucleic acid or a fragment thereof may be used to determine the exact nucleotide sequence and deduce the corresponding arninoacid sequence and thereby identify conserved amino acid sequences. Degenerate primers based on conserved amino acid sequences may be used to direct PCR ampliﬁcation. 20 Degenerate primers can also be used as probes in DNA hybridization assays.

Alternatively, the conserved amino acid sequence(s) may be. ed in protein hybridization assays that utilize for example, an antibody that specifically binds to the conserved amino acid sequences(s), or a substrate that speciﬁcally binds to‘ the conserved amino acid such for a that binds. ‘ sequences(s) as, example, lipid 25 FLXLXXXN (a-putative neutral lipid binding domain; SEQ ID N01224).

In one embodiment, the nucleic acid molecule comprises a sequence of tides encoding a MGAT. The sequence of nucleotides may i) comprise a sequence selected from any one of SEQ ID NOs:1 to 44', ii) encode a polypeptide comprising amino acids having'a sequence as ed in any one of SEQ ID NOsz45 .30 to 82, or a biologically active fragment thereof, iii) be at least 50% identical to i) or ii), or iv) hybridize to any one of i) to iii) under stringent conditions. In another or additional embodiment, the nucleic acid molecule comprises a sequence of nucleotides encoding one or more ved DGAT2 and/or MGAT1/2 amino acid sequences as ed in SEQ ID 'NOsz220, 221, 222, 223, and 224. In a preferred 35 ment, the nucleic acid molecule comprises a sequence of tides encoding the conserved amino acid sequences provided in SEQ ID N02220 and/or SEQ ID N02224.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk .160 In another embodiment, the nucleic acid le comprises a sequence of nucleotides encoding a GPAT, preferably a GPAT which has phosphatase activity.

The sequence of nucleotides may i) comprise a sequence selected from any one of SEQ ID INOs284 to 141, ii) encode a polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOszl44 to 201, or a biologically active ' fragment f, iii) be at least 50% cal to i) or ii), or iv) hybridize to any one of i) to iii) under stringent conditions. In another or additional embodiment, the nucleic acid molecule comprises a sequence of nucleotides encoding one or more conserved GPAT amino acid sequences as provided in SEQ ID NOs:225, 226, and IO 227, or a sequence of amino acids which is at least 50%, preferably at least 60%, more preferably at least 65% identical thereto.

In another embodiment, the c acid le comprises a sequence of nucleotides encoding a DGAT2. The sequence of nucleotides may se i) a sequence of nucleotides selected from any one of SEQ ID NO:204 to 211, ii) encode a 15 ptide comprising amino acids having a sequence as provided in any one of SEQ ID NO:212 to 219, or a biologically active fragment thereof, iii) be at least 50% identical to i) or ii), or iv) hybridize to any one of i) to” iii) under stringent conditions.

In a preferred embodiment, the DGAT2 comprises. a sequence of nucleotides of SEQ ID NO:204 and/or a Sequence of nucleotides encoding a polypeptide comprising 20 amino acids having a sequence as provided in SEQ ID NO:212.

A cell comprising a nucleic acid molecule encoding a fatty acid acyltransferase operably linked to a promoter which is active in the cell may be]. obtained using standard procedures in the art such as by introducing the nucleic acid molecule into a ' cell by, for example, calcium phosphate precipitation, polyethylene glycol treatment, 25 electroporation, and combinations of these treatments. Other methods of cell transformation can also be used and include, but are not d to, the introduction of ’ DNA into plants by direct DNA transfer or injection. Transformed plant cells may also be obtained using Agrobacterium-mediated er and acceleration methods as bed herein. ' 30 The method further comprises determining if the level of MAG, DAG and/or TAG ed in the cell is increased when compared to a corresponding cell lacking the nucleic acid using known techniques in the art such as those exempliﬁed in e 1. For instance, lipids can be extracted in a chloroform/methanol solution, dried and separated by thin layer chromatography (TLC). ties of TAG, DAG, 35 MAG, free fatty acid, and other lipids can be veriﬁed with internal lipid standards after staining with iodine vapor, The resultant chromatograms can ed using a Phosphorlmager and the amount of MAG, DAG and TAG quantiﬁed on the basis of - Dknown amount of internal standards, or atively, the cells may be fed sn-2 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 161 monooleoyl'glycerol[”C] or [”C]glycerolphosphate and associated ctivity quantitated by liquid scintillation counting (i.e., the amount of ed MAG, DAG and TAG is ﬁed). ' _ The method further ses identifying a nucleic acid le encoding a fatty acid acyltransferase having an increased ability to produce MAG, DAG and/or TAG in a cell. In a preferred embodiment, the fatty acid acyltransferase catalyzes an enzyme reaction in the MGAT pathway. In a further preferred embodiment, DAG is increased via the MGAT pathway (i.e., acylation of MAG with fatty acyl-CoA is - catalysed by a MGAT to form DAG). In another or additional embodiment, the 10 substrate MAG is produced by a GPAT which also has phosphatase activity and/or DAG is acylated with fatty acyl-CoA by a DGAT and/or a MGAT having DGAT activity to form TAG.

Gloss 15 Certain aspects of the invention relate to measuring the glossiness of vegetative material as a marker for the level of lipid in the material, with higher glossiness levels" being associated with higher lipid levels.

' The gloss of the vegetative material can be determined using known procedures. Glossmeters (reﬂectometers) provide a quantiﬁable way of measuring 20 gloss intensity ng consistency of ement by deﬁning the precise illumination and viewing conditions. The conﬁguration of both nation source and observation reception angles allows measurement over a small range of the overall reﬂection angle. The measurement results of a eter are related to the amount of reﬂected light from a black glass stande with a‘deﬁned refractive index. 25' The ratio of reﬂected to incident light for the-specimen, compared to the ratio for the gloss standard, is recorded as gloss units.

The measurement scale, Gloss Units (GU), of a glossmeter is a scaling based on a highly polished reference black glass rd with a deﬁned refractive index having a specular reﬂectance of lOOGU at the speciﬁed angle. This standard is used 30‘ to establish an upper point calibration of 100 with the lower end point ished at 0 , on a perfectly matt surface. This scaling is suitable for most non—metallic materials.

The optimal or ed level of glossiness of vegetative material is likely to- vary between plant species. The skilled person can readily e the lipid content of , vegetative material of ent plants of the invention and identify a suitable pre- 35 determined level of glossiness that can be used as a standard in the ﬁeld for assessing the best time to havest a vegetative material from a particular plant species.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 162' EXAMPLES Example 1. General materials and methods Expression of genes in plant cells-in a transient expression system Genes were expressed in plant cells using a transient expression system essentially as described by Voinnet et a1. (2003) and Wood et al. (2009). Binary vectors containing the coding region to be expressed by a strong tutive e358 promoter containing a duplicated enhancer region were introduced into Agrobacterium ,tumefaciens strain AGLl. A chimeric binary vector, 3SSzpl9, for expression of the p19 viral silencing suppressor waspseparately introduced into AGLl , 1‘0" as bed in W02010/057246. A chimeric binary , 3SS:V2, for expression of the V2 viral silencing suppressor was separately introduced into AGLl. The recombinant cells were grown to stationary phase at 28°C in LB broth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin. The bacteria were then pelleted by centrifugation at 5000 g for 5 min at room temperature before being resuspended to 15. OD600 = 1.0 in an ation buffer containing 10 mM MES pH 5.7, 10 mM MgCl2 and 100 uM acetosyringone. The cells were then ted at 28°C with g for 3 hours after which the OD600 was measured and a volume of each culture, including the viral suppressor construct 9 or 3SS:V2, required to reach a ﬁnal concentration of OD600 = 0.125 added to a fresh tube. The ﬁnal volume Was made 20 up with the above buffer; Leaves were then ated with the culture mixture and the plants were typically grown for a further three to ﬁve days after ation before leaf discs were recovered for either puriﬁed cell lysate preparation or total lipid isolation. 25' Puriﬁed leaf lysate assay .

. Nicotiana benthamiana leaf s previously inﬁltrated as described above were ground in a solution containing 0.1 M potassium phosphate buffer (pH 7.2) and . 0.33 M sucrose using a glass homogenizer. Leaf homogenate was centrifuged at 20,000 g for 45 minutes at 4°C after which each supernatant was collected. Protein 30 content in each supernatant was measured according to Bradford (1976) using a Wallacl420 multi-label counter and a Bio—Rad Protein Assay dye reagent (Bio-Rad , Laboratories, Hercules, CA USA). Acyltransferase assays used 100 ug n according to Cao et a1. (2007) with somevmodiﬁcations. The on medium contained 100 mM Tris-HCl (pH 7.0), 5 mM Mng, 1 mg/mL BSA (fatty ree), . 35 200 mM sucrose, 40 mM cold oleoyl-CoA, 16.4 uM sn-2 monooleoylglycerol[”C] (55mCi/mmol, American Radiochemicals,‘ Saint Louis, MO USA) or 6.0 uM [I4C]glycerolphosphate (G—3-P) disodium salt (150 mCi/mmol, American micals). The assays were carried out for 7.5, 15, or 30 minutes.I Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 163 Lipid analysis \ y ‘ In summary, the methods used for analysing lipids in seeds or vegetative tissues were as follows: ogsis seed and any other r sized seed: -(i) Fatty acid composition -direct methylation of fatty acids in seeds, without crushing of seeds. _ (ii) Total‘fatty acid. or TAG quantitation - direct methylation of fatty acids in seeds, without crushing of seeds, with use of a 17:0 TAG standard. 10 Canola seed, Camelina seed, and any other larger sized seeds; (i) Single seed fatty acid composition - direct methylation of fatty acids in seed after breaking seed coat. (ii) Pooled seed—fatty acid composition of total extracted lipid — crushing seeds 15 in CHCl3/ MeOH and methylation ofaliquots of the extracted lipid. (iii) Pooled seed-total lipid content (seed oil content) - two times lipid extraction for complete recovery of seed lipids aﬁer crushing seeds from known amount of dessicated seeds, with ation of lipids from known amount of seeds together with 17:0 fatty acids as internal standard, 20 (iv) Pooled seed-puriﬁed TAG tation - tWo times lipid extraction for complete recovery ‘of seed lipids after crushing seeds, from known amount of dessicated seeds, TAG fractionation from the lipid using TLC, and direct methylation ofTAG in silica using 17:0 TAG as internal standard.

I 25 Leafsamples: (i) Fatty acid composition of total lipid - direct methylation of fatty acids in freeze—dried s. (ii) Total lipid quantitation - direct methylation of fatty acids in known weight offreeze-dried samples, with 17:0 FFA. ' p 30 (iii) TAG quantitation - because of the presence of substantial amounts of polar lipids in leaves, TAG was fractionated by TLC from extracted total lipids, and methylated in the presence of 17:0 TAG internal standard. Steps: Freeze dry samples, weighing, lipid extraction, fractionation of TAG from known amount of total lipids, direct ation ofTAG in silica together with 17:0 TAG as internal standard. 35 The methods are detailed as follows: D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 164 Analysis ofoil content in Arabidposis seeds Where seed oil t was to be determined in small seeds such as Arahidopsis' seeds, seeds were dried in a desiccator for 24 hours and approximately 4 mg of seed was transferred to a 2 ml glass vial ning lined screw cap. 0.05 mg triheptadecanoin dissolved in 0.1 ml toluene was added to the vial as internal standard. Seed FAME were ed by adding 0.7 ml of 1N methanolic HCl (Supeleo) to the vial containing seed material. Crushing of the seeds was not necessary with small seeds Arabidopsis seeds. The mixture was ed brieﬂy and incubated at 80°C for 2 hours. Aﬁer cooling to room temperature, 0.3 ml 10 of 0.9% NaCl (w/v) and 0.1 ml hexane was added to the vial and mixed well for 10 minutes in a.Heidolph Vibramax 110. The FAME was collected into a 0.3 ml glass insert and analysed by GC with a ﬂame ionization detector (FID) as mentioned I earlier. .

. The peak area of individual FAME Were ﬁrst corrected on the basis of the peak .15 area} responses of a known amount of the same FAMEs present in a commercial rd GLC-4ll (NU-CHEK PREP, INC, USA). GLC-4ll contains equal amounts of 31 fatty acids (% by weight), ranging from C820 to C22:6. In case of fatty . acids which Were not present in the standard, the peak area responses of the most similar FAME was taken. For example, the peak area response of FAMEs of 16:1d9 20 was used for 16:1d7 and thelFAME-response of C22:6 was used for C22:S. The corrected areas were used to calculate the mass of each FAME in the sample by comparison to the internal standard mass. Oil is stored mainly in the form of TAG and its weight was calculated based on FAME weight. Total moles of glycerol was determined by calculating moles of each FAME and dividing total moles of FAMEs 25 by three. TAG was calculated as the sum of glycerol and fatty acyl moieties using a relation: % oil by weight = 100x ((411: total mol FAME/3)+(total g FAME~ (15x total mol FAME)))Ig seed, where 41 and 15 are molecular weights of glycerol moiety and methyl group, tively. 30 Analysis ofoil content-in Camelina seedsand canola seeds by extraction After harvest at plant maturity, Camelina or canola seeds were dessicated by storing the seeds for 24 hours at room temperature in a ator containing silica gel as dessicant. re content of the seeds is typically 6-8%. Total lipids were extracted from known weights of the dessicated seeds by crushing the seeds using a _35 mixture of chloroform and methanol (2/1 v/v) in an orf tube using a Reicht tissue lyser (22 frequency/seconds for 3 minutes) and a metal ball. One volume of 0.1M KCl was added and the mixture shaken for 10 minutes. The lower non-polar De Was collected after centrifuging the e for 5 minutes at 3000 rpm. The ‘ Substitute Sheet , (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 165 remaining upper (aqueous) phase was washed with 2 volumes of chloroform by mixing for 10 minutes. The second non-polar phase was also collected and pooled with the ﬁrst. The solvent was ated from the lipids in the extract under nitrogen ﬂow and the total dried lipid was dissolved in a known volume of chloroform. ' . _ , To measure the amount of lipid in the extracted material, a known amount’of V 17:0—TAG was added as al standard and the lipids from the known amount of seeds incubated in l N methanolic—HCl (Silpelco) for 2 hours at 80°C. FAME thus made were extracted in hexane and analysed by GC. Individual FAMEs were 10 quantiﬁed on the basis of the amount of 17:0 ME. Individual FAMEs weights, aﬁer subtraction of weights of the esteriﬁed methyl groups from FAME, were Converted into moles by dividing by molecular s of individual FAMEs.

I Total moles of all FAMEs were divided by three to calculate moles of TAG and therefore glycerol. Then, moles of TAG were converted in to weight of TAG. '15 Finally, the percentage oil content on a seed'weight basis was calculated using seed weights, ng that all of the extracted lipid is TAG or equivalent to TAG for the purpose of calculating oil content. This method Was based on Li et al., (2006). Seeds other than Camelina or canola seeds that are of a similar size can also be analysed by ' this method. 20 - Canola and other seed oil content can also be measured by nuclear magnetic ‘ resonance techniques (Rossell and Pritchard,‘ 1991), for e, by a pulsed wave NMS 100 Minispec (Bruker Pty Ltd Scientiﬁc Instruments, Germany), or by near infrared" reﬂectance spectroscopy such as using a NIRSystems Model 5000 monochromator. T he NMR method can simultaneously measure moisture content. 25 Moisture content can ‘also be ed on a sample from a batch of seeds by drying the seeds in the sample for 18 hours at about 100°C, according to Li et al., (2006).

Where, fatty acid composition is to be determined for the oil in canola seed, the direct methylation method used for Arabidopsis seed (above) can be modiﬁed with the addition of cracking of the canola at. This method extracts suﬁicient 30 oil from the seed to allow fatty acid composition analysis. ' Analysis oflipidsfrom leaflysate assays Lipids from the lysate assays were extracted using chloroform:methanol:0.l M KC1 (2:1 :1) and recovered. The ent lipid Classes in the samples were separated 35 on Silica gel 60 thin layer tography (TLC) plates (MERCK, Dermstadt, Germany) impregnated with 10% boric acid. The solvent system used to fractionate TAG from the lipid extract consisted of chloroform/acetone (90/10 v/v). Individual a} s were visualized by exposing the plates to iodine vapour and identiﬁed by \ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 166 running parallel tic standards on the same TLC plate. The plates were exposed to phosphor imaging screens overnight and analysed by a Fujiﬁlm 00 phosphorimager before liquid scintillation counting for DPM quantiﬁcation.

Total lipid isolation ctionation Tissues including leaf samples were freeze-dried, weighed (dry weight) and total lipids extracted as described by Bligh and Dyer (1959) or by using chloroforrnzmethan0120.1 M KCl (CMK; 2:1:1) as a solvent. Total lipids were extracted from N. benthamiana leaf samples, aﬁer ﬁ'eeze dying, by adding 900 pL of 10 a chloroform/methanol (2/1 v/v) mixture per 1 cm diameter leaf sample. 0.8 pg DAGE was added per 0.5 mg dry leaf: weight as internal standard when ID is was to be performed. Samples were homogenized using an IKA ultra-turrax tissue lyser after which 500 pL 0.1 M KCl was added. Samples were vortexed, centrifuged for 5 min and the lower phase was collected. The remaining upper phase 15 was extracted a second time by adding 600 uL chloroform, vortexing and centrifuging for 5 min. The lower phase was recovered and pooled into the previous collection.

Lipids were dried under a nitrogen ﬂow and ended in 2 “L chloroform per mg leaf dry weight. Total lipids of N. tabacum leaves or leaf samples were extracted as above with some modiﬁcations. If 4 or 6 leaf discs (each approx 1 cm2 surface area) 20 were combined, 1.6 ml ofCMK solvent was used, whereas if 3 or less leaf discs were ’ combined, 1.2 m1 CMK was used. Freeze dried leaf tissues were homogenized in an eppendorf tube containing a metallic ball using a Reicht tissue lySer (Qiagen) for 3 ‘ minutes at 20 frequency/sec. ' 25 Separation ofneutral lipids via TLC and transrriethylation Known volumes Of total leaf extracts such as, for example, 30 pL, were loaded on a TLC silica gel 60 plate (1x20 cm) (Merck 'KGaA, Germany). The neutral lipids were separated via TLC in an equilibrated development tank containing a hexane/DEE/acetic acid /1 v/v/v/) solvent system. The TAG bands were so visualised by iodine vapour, scraped from the TLC plate, transferred to 2 mL GC vials and dried with N2. 750 uL of 1N methanolic-HCl (Supelco analytical, USA) was added to each vial er with a known amount of C1720 TAG, such as, for example, 30 pg, as al standard for quantiﬁcation.

When analysing the effect on oleic acid levels of speciﬁc gene combinations, 35 TAG and polar lipids bands were collected from the TLC plates. Next, 15 pg of Cl7:0' internal standard wasadded to samples such as TAG samples, polar lipid samples and 20 uL of the total lipid extracts. ing drying under N2, 70 uL Dene and 700 uL methanolic HCl were added.

Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 167 ' Lipid samples for fatty acid composition analysis by GC were transmethylated by incubating the mixtures at 80°C for 2 hours in the presence of the methanolic4HC1.

After g samples to room temperature, the reaction was stopped by adding 350 pl H20. Fatty acyl methyl esters (FAME) were extracted from the mixture by adding ‘350 pl hexane, vortexing and centrifugation at 1700 rpm for 5 min. The upper hexane phase was collected and transferred into GC vials with 300 pl conical inserts. Aﬂer evaporation, the samples were resuspended in 30 pl . One pl was injected into ' ' mch.

The amount of individual and total fatty acids (TFA) present in the lipid 10 fractions was quantiﬁed by GC by determining the area under each peak and calculated by comparison with the peak area for the known amount of internal standard. TAG content in leaf was calculated as the sum of glycerol and fatty acyl moieties in the TAG fraction using a relation: % TAG by weigh = 100x ((4lx total mol FAME/3)+(total g FAME- (15x total mol FAME)))/g leaf dry weight, where 41 15 and 15 are molecular weights of ol moiety and methyl group, respectively.

Capillary gas-liquid tography (GC) , FAME were ed by GC using an Agilent Technologies 7890A GC (Palo Alto, California, USA) equipped with an SGE BPX70 (70% cyanopropyl 20 polysilphenylene—siloxane) column (30m x 0.25 mm i.d., 0.25 pm ﬁlm thickness), an FID, a split/splitless injector and an Agilent Technologies 7693 Series auto sampler and injector. Helium was used as the carrier gas. Samples were injected in split mode (50:1 ratio) at an oven temperature of 150°C. Aﬁer injection, the oven temperature was held at 150°C for 1 min, then raised to 210°C at 3°C.min'l and ﬁnally to 240°C '25 at 50°C.min". Peaks were quantiﬁed with t Technologies ChemStation re (Rev B.04.03 (1.6), Palo Alto, California, USA) based on the response of the known amount of the external standard GLC-4ll (Nucheck) and Me al standard. 30 ﬁcation ofTAG via Iatroscan , ' One uL of lipid extract was loaded on one Chromarod-SII for TLC—FID IatroscanTMtMitsubishi Chemical MedienceCorporation — Japan). The Chromarod rack was then transferred into an equilibrated developing tank ning 70 mL of a hexane/CHC13/2-propanol/formic acid (85/10.716/0.567/0.0567 v/v/v/v) t 35' system. After 30 min ofincubation, the Chromarod rack was dried for 3 min at 100°C and immediately scanned on an Iatroscan MK-6s TLC-FID analyser (Mitsubishi Chemical Medience Corporation — Japan). Peak areas ofDAGE internal standard and 9 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 168 TAG were integrated using SIC-48011 integration soﬁware (Version:7.0-E SIC l I System instruments Co., LTD —- Japan).

TAG quantiﬁcation was carried out in two steps. First, DAGE was scanned in all samples to correct the extraction yields after which concentrated TAG samples were selected and diluted. Next, TAG was quantiﬁed in d s with a second scan ing to the external calibration using glyceryl trilinoleate as external standard (Sigma-Aldrich).

Quantiﬁcation ofTAG in leafsamples by GC , 10 The peak area of individual FAME were ﬁrst corrected on the basis of the peak area responses of known amounts of the same FAMEs present in a commercial, standard GLC-411 (NU-CHEK PREP, Inc., USA). The ted areas were used to calculate the mass of each FAME in the sample by, comparison to the internal rd. Since oil is stored primarily in the fenn of TAG, the amount of oil was 15 calculated based on the amount of' FAME in each sample. Total moles of glycerol were determined by calculating the number of moles of FAMEs and dividing total moles ofFAMEs by three. The amount ofTAG was calculated as the sum of glycerol. and fatty acyl moieties using the formula: % oil by weight = 100x ((41x total mol , FAME/3)+(total g FAME-(15x total mol FAME)))/g leafdry weight, where 41 and 15 20 were the molecular weights of glycerol moiety and methyl group, respectively.

DGAT assay in Saccharomxces cerevisiae H1246 Saccharomyces cerevisiae strain H1246 is completely devoid of DGAT activity and lacks TAG and sterol esters as a result of knockout mutations in four 25 genes (DGAl, LROl, ARE], ARE2). The addition of free fatty acid (e.g. 1 mM 18:1”) to H1246 growth media is toxic in the absence of DGAT activity. Growth on such media can therefore be .used as an indicator or selection for the ce of DGAT activity in this yeast strain.

S cerevisiae H1246 was transformed with the pYESZ construct (negative 30 control), a construct ng Arabidopsis thaliana DGATl in pYESZ, or a construct ng Mus 'rnusculus MGAT2 in pYESZ. Transformants were fed [14C]18:1A9 ﬁee 1 I fatty acids.

In a separate experiment, S cerevisiae H1246 was transformed with the pYESZ construct (negative control), a construct encoding Bernadia pulchella DGAT] in 35 pYESZ, or a construct encoding M. musculus MGATl in pYESZ and fed 18:1A9 free fatty acids. S. cerevisiae $2880 wild type strain transformed with pYESZ served as a positive control.

D Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 169 Yeast transformants were ended in sterile mQ water and diluted to i OD600=1. Samples were further diluted in four consecutive ons, each at 1/1 0. 2111 of each dilution was spotted on each of the plates (YNBD, YNBG, YNBG+FA) together with 2 pL mQ water and 2 pL of an untransformed H1246 cell suspension (OD600=1). Plates were incubated for 6 days at 30°C before scoring growth.

Plate medium, 40 mL media perplate - YNBD: minimal dropout medium g uracil and containing 2% e, 0.01% NP40 and 100 11L ethanol 10 0 YNBG: minimal dropout medium lacking uracil and containing 2% galactose, 0 1% rafﬁnose, 0.01% NP40 and 100 pL ethanol.

- YNBG+FA: minimal dropout medium lacking uracil and containing 2% galactose, 1% rafﬁnose, 0.01% NP40 and lmM C1821Ag dissolved in 100 111 ethanol. 15‘ Example 2. Constitutive expression of a monoacylglycerol acyltransferase in plant cells ‘ MGAT] The enzyme activity of the monoacylglycerol acyltransferase 1 (MGATl) d by the gene from M. musculus (Yen et al., 2002) and A. thaliana 20 diacylglycerol acyltransferase (DGATl) (Bouvier—Nave et a1.H2000)"used here as a comparison with MGATl, were demonstrated1n N. benthamiana leaf tissue using a transient expression system as describedin Example 1. _ A vector designated RE04 was made by ing a PstI fragment containing a 358 promoter into the SfoI site of vector pORE04 aﬁer T4 DNA 25 polymerase ent to blunt the ends (Coutu et al., 2007). A chimeric DNA encoding the M. musculus MGATl, codon-optimised for Brassica napus, was synthesized by Geneart and designated 0954364_MGATJMA. A chimeric DNA designated BSSzMGATl and encoding the M. musculus MGATl nk Accession No. ) for expression in' plant cells was made by inserting the entire coding 30 region of 4_MGAT_pMA, contained within an EcoRI fragment, into 358- pORE04 at the EcoRI site: The vector containing the 3SS:MGAT1 construct was designated as pJP3184. Similarly, a chimeric DNA 3SS:DGAT1 encoding the A. thaliana DGATI (Genbank Accession No. AAF19262) for expression in plant cells _ was made by ing the entire coding region of pXZP513E, contained within a 35 BamHI-EcoRV fragment, into 358-pORE04 at the BamHI-EcoRV site. The vector containing the 3SSzDGATl construct was designated pJP2078.

The chimeric vectors were introduced into A. tumefaciens strain AGLl and as from cultures of these inﬁltrated into leaf tissue of N. benthamiana plants in a Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 170 24°C growth room. In order to allow direct comparisons between samples and to reduce inter-leaf variatiOn, s being compared were inﬁltrated on either side of the same leaf‘ Experiments were performed in triplicate. ing inﬁltration, the plants were grown for a further three days before leaf discs were taken, freeze-dried, and lipids extracted from the samples were onated and ﬁed as described in Example 1. This analysis revealed that the MGATl and DGATl genes were functioning to increase leaf oil levels in N. benthamiana as follows.

' Leaf tissue transformed with the 3>SSzpl9i uct only (negative control) contained an average of 4 pg free fatty acid (FFA) derived from DAG/mg dry leaf 10 weight and 5 pg FFA derived from TAG/mg dry leaf . Leaf tissue transformed a with the 3SSzpl9 and 35S:DGAT1 constructs (control for expression of DGATl) contained an average of 4 pg FFA» derived from DAG/mg dry leaf Weight and 22 pg FFA derived from TAG/mg dry leaf weight. Leaf tissue transformed with the 3SSzp19 and 3SS:MGAT1 constructs contained an average of 8 pg FFA derived from 15 DAG/mg dry leaf weight and 44 pg FFA derived from TAG/mg dry leaf weight._ Leaf tissue transformed with thel3SSzp19,‘ 35$:DGAT1 and 3SSzMGAT1 constructs did not contain DAG or TAG levels higher than those observed in the-35821319 and 3SS:MGAT1 inﬁltration (Figure 2). Also, a decrease in the level cf saturates in seeds was noted after MGAT expression when compared with either the p19 control or 20 DGAT] s (Table 2). r , .

The data bed above demonstrated that the MGAT]. enzyme was far more active than the DGATl enzyme in promoting both DAG and TAG accumulation in leaf tissue. Expression of the MGAT] gene resulted in twice as much TAG and DAG accumulation in leaf tissue compared to when the DGATl was expressed. This result 25_ was highly surprising and unexpected, considering that the MGAT is an enzyme. expressed. in mouse intestine, a vastly different biological system than plant leaves.

This study was'the ﬁrst demonstration of ectopic MGAT expression in a plant cell.

*Leaf s inﬁltrated with M. musculus MGAT] accumulated double the DAG and TAG relative to leaf tissue inﬁltrated with A. thaliana DGATl alone. The 30 ency of the production ofTAG was also surprising and unexpected given that the mouse MGAT has only very low activity as a DGAT. ‘Leaf tissue inﬁltrated with genes. encoding both MGATl and DGATl did not accumulate signiﬁcantly more ‘TAG than the MGATl—only leaf sample. Figure 1 is a representation of s TAG lation pathways, most of which converge at DAG, a central molecule in lipid 35 synthesis. For instance, MAG, DAG and TAG can be inter-converted via various enzyme activities including transacylation, lipase, MGAT; DGAT and PDAT. A decrease in the level of saturates was also noted after MGAT expression.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 171 MGAT2 » A chimeric DNA designated AT2 and encoding the M. us MGAT2 for sion in plant cells was made by inserting the entire MGAT2 coding region, contained within an EcoRI fragment, into 358-pORE04 at the EcoRI site._ The enzyme activity of the monoacylglycerol acyltransferase 2 ) encoded by the gene from M. musculus (Yen, 2003) (Genbank Accession No. Q80W94) and A. thaliana DGATl (Bouvier-Nave et al., 2000), used here as a comparison with MGAT2, was also trated in N.' benthamiana leaf tissue using a transient expression system as described in Example 1. 10 Compared with controls, DGATl expression increased leaf TAG 5.9—fold, MGAT2 by 73-fold and the combination of MGAT2+DGAT1 by ld (Figure 3).

The ability of MGAT2 alone to yield such signiﬁcant increases in TAG was unexpected for a number of reasons. Firstly, the amount of substrate MAG present in leaf tissue is known to be low and large increases in TAG accumulation from this 15 substrate would not'be expected. Secondly, the addition of MGAT activity alone (i.e., addition of MGAT2 which does not have DGAT activity) would be expected to yield DAG, not TAG, especially in a leaf environment where little native-DGAT activity is ' usually present. 20 Discussion The present inventors have singly demonstrated that the transgenic expression of a MGAT gene results in signiﬁcant increases in lipid yield in plant cells.

The present inventors understand that Tumaney et al. had isolated a DGAT with some MGAT ty and that they were not successful in attempts to clone a gene encoding 25 a MGAT as deﬁned herein. Tumaney et al. (2001) reported MGAT activity in peanut and isolated an enzyme responsible for this activity. However, Tumaney et a1. did not publish results of tests for DGAT activity. and it therefore seems that the enzyme reported was a DGAT with some MGAT activity. , previous, work had failed to identify any MGAT activity in other species (Stobart et al., 1997). rmore, it 30 was surprising that the enzyme isolated by Tumaney era]. was a soluble, lic, ‘ enzyme rather than a membrane-bound enzyme. > Recently, :researchers have identiﬁed a microsomal membrane—bound monoacylglycerol acyltransferase (MGAT) from immature peanut (Arachis ea) seeds. The MGAT could be solubilized from microsomal membranes 35 using a combination of a chaotropic agent and a zwitterionic detergent, and a functionally active l4S multiprotein complex was isolated and characterized.

Oleosin3 (OLE3) was identiﬁed as part of the multiprotein complex, which is capable D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 172 of performing bifunctional activities such as acylating monoacylglycerol (MAG) to diacylglycerol (DAG) and phospholipase A2 (PLA2; Parthibane et al., 2012).

Example 3. Biochemical demonstration of transgenic MGAT activig in leaf extracts Cell lysates were made from N. benthamiana leaf tissue that had been inﬁltrated with AT1, 3SS:MGAT2 and BSSzDGATl, as described in e 1. Separate leaf inﬁltrations were med, each in triplicate, for strains containing ‘ the 3SSzp19 construct only (negative control), the 3SSzMGAT2 strain together with 10 the 3SS:p19 strain, and a mixture of the 3SS:MGAT2 and BSSzDGATl cterium strains with the 35$:pl9 strain. The triplicate samples were harvested after three days and a puriﬁed cell lysate prepared by mechanical tissue lysis and fugation. The MGAT activities of the puriﬁed cell lysates were compared by feeding [”C]MAG to the lysates as described in Example 1. The data are shown' in I ' 15 Figure 4. .

. Little MGAT ty was observed in' the 35$:p19 control , since most I i of the radioactivity remained in MAG throughout the assay. In contrast, the ty of the labelled MAG in the BSSIMGATZ sample was y converted to DAG (Figure 4, central panel), ting strong. MGAT activity expressed. from the 20 3SSzMGAT2 construct. Furthermore, a signiﬁcant amOunt of TAG was also ed. The TAG production observed in the 358tMGAT2 sample was likely due to native N. benthamiana DGAT activity, or produced by another TAG synthesis route. The amount of TAG production was greatly increased by the further addition of 3SS:DGAT1 (Figure 4, right hand panel), indicating that the MGAT2 enzyme 25 produced DAG which was accessible for conversion to TAG by DGAT] in plant vegetative tissues.

Example '4. Biochemical demonstration of the production of MGAT-accessible MAG in leaf ts _ 30 In the in vitro assays described in Example 3 using leaf s, the ates (sn-Z MAG and oleoyl—CoA) were exogenously supplied, whereas in vivo MGAT activity in intact plant tissues would require the native'presence of these substrates.

The presence of low levels of MAG is various plant tissues has been reported ' previously (Hirayama and Hujii, 1965; Panekina et al., 1978; Lakshminarayana et al., 35 1984; Perry & Harwood,‘ 1993). To test whether the MGAT2 could access MAG , produced by native plant pathways, the above experiment was repeated but this time feeding\[”C]G—3-P to the lysates. The resultant data are shown schematically in Gum 5.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 173 The production of labelled MAG was observed in all s, indicating the de 'novo production of MAG from the GP in plant leaf lysates. Labelled DAG and TAG products were also observed in all samples although these were relatively low in the 3SSzpl9 control sample, indicating that the production of these l lipids by the endogenous Kennedy pathway was relatively low in this sample. In contrast, the majority of the label in the MGAT2 and MGAT2 + DGATl samples appeared in the DAG and TAG pools, indicating that the exogenously added MGAT catalysed conversion of the MAG that had been ed from the ed GP by a native plant pathway. 6 10 Examples 2 to 4‘ demonstrate several key : 1) Leaf tissue can synthesise MAG from GP such that the MAG is accessible to an exogenous MGAT expressed in the leaf tissue; 2) Even an MGAT which is derived from mammalian intestine can function in plant tissues, not known to possess an endogenous MGAT, ing a successful interaction with other plant factors involved in lipid synthesis; 3) DAG 15 produced by the exogenous MGAT activity is accessible to a plant DGAT, or an exogenous DGAT, to produce TAG; and 4) the expression of an exogenous MGAT can yield ”greatly increased TAG levels in plant tissues, levels which are at least as A great as that yielded by exogenous A. thaliana DGATl expression. '20 Example 5. Expression of DGAT], MGAT] and MGAT2 in yeast Chimeric yeast expression vectors were ucted by inserting genes encoding the A. thaliana DGAT], M. musculus MGATl and M. musculus MGAT2 into the pYESZ vector to yield pYESZ:DGATl, pYESZ:MGATl and pYESZ:MGAT2. These constructs were ormed in Saccharomyces cer‘evisiae 25. strain H1246 which is completely devoid of DGAT activity and lacks TAG and sterol esters as a result of knockout mutations in four genes (DGAI, LROI, ARE], AREZ).

Yeast strain H1246 is capable of synthesizing DAG from exogenously added fatty acids, but is unable to convert'the DAG to TAG because of the knockout mutations.

The ormed yeast cultures were fed [MC]18:IA9 before total lipids were extracted 30 and fractionated by TLC as described in Example 1. An autoradiogram of a representative TLC plate is shown in Figure 6.

TAG ion, indicating the presence of DGAT activity, was observed for the yeast cells containing either DGATl (positive l) and the mammalian 'MGATl but not in cells containing the MGAT2 d by the native M. musculus , 35 coding region. It was concluded that MGAT] from mouse also had DGAT activity in yeast cells, and therefore functioned as a dual function MGAT/DGAT enzyme, whereas MGAT2 did not have detectable DGAT activity and was therefore solely an ‘ , EAT. A construct which included an MGAT2 coding region which was codon Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk ed set by jyk 174 optimization for expression in yeast ted MGAT activity (production ofDAG)- when tested in vitro using yeast microsomes and ed MAG'substrate, whereas a r construct which was eodon-optimised for expression in B. napu‘s did not show DAG production in the yeast microsomes. This experiment showed the beneﬁt of codon—optimisation for the organism in which heterologous. coding regions were to be ' expressed.

Example 6. Expression of a monoacylglycerol acyltransferase in plants, seeds 10 and fungi Expression ofMGAT] in Arabidogsis thaliana seeds A gene encoding M. inusculus MGATI and under the control of a seed-speciﬁc - promoter (FPl, a truncated Brassica napus napin promoter) was used to generate stably transformed A. thaliana plants and progeny seeds. The. vector designated 15 pJP3l74 was made by inserting a Sall fragment containing an EcoRI site ﬂanked by the PP] promoter and Glycine max lectin polyadenylation signal into the SalI-Xhol site of vector pCWl41. The pCWl4l vector also contained an FPl-driven, intron- interrupted, seed-secreted GFP as a' able marker gene. The chimeric gene designated FPl :MGATl-GFP was made by inserting the entire coding region of the 20 construct 0954364_MGAT_pMA, ned within an EcoRI fragment, into pJP3174 at the EcoRI site, generating pJP3179. This ic vector was introduced into A. 'tumefaciens strain AGLl and cells from culture of the ormed Agrobacterium used to treat A. thaliana (ecotype Columbia) plants using the ﬂoral dip method for . transformation (Clough and Bent, 1998). Aﬁer maturation, the seeds from the treated 25 plants were viewed under a Leica MZFLIII dissection microscope and ebq 100' mercury lamp. Fiﬁeen transgenic seeds (strongly GFP positive) and ﬁfteen non- transgenic (GFP negative) seeds were isolated and each set pooled. The GFP positive and GFP negative pools were analysed for total fatty acid content as described in Example 1. This analysis provided the average fatty acid content and composition for 30 seeds transformed with the MGAT construct, but in a population which may have contained both hemizygous and homozygous transformed seeds.

This is revealed; that the MGATI gene was functioning to increase seed oil levels in A. thaliana seed with the ﬁﬁeen non-transgenic seeds (control, the same as twild-type) ning an average cf 69.4 rig total fatty acids while the ﬁﬁeen' 35 transgenic seeds transformed with the GFP gene, and ore likely to contain the FPleGATI genetic construct, contained an average of 71.9 ug total fatty acids.

This was an increase of 3.5% in the oil content relative to the control (wild-type). The Dysis also revealed that the MGAT gene was functioning to enrich polyunsaturated Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 175 fatty acids in the seed, as seen from the fatty acid ition of the total extracted lipid obtained from the seeds. In particular, the amount of ALA present as a percentage of the total fatty acid extracted from the seeds increasing from 16.0 to 19.6%. Similarly, the percentage of the fatty acid 20:2n6 increased from 1.25% to 1.90% and the fatty acid 20:3n3 increased from 0.26% to 0.51% (Table 2).

Table 2. Effect ofMGAT expression on seed fatty acid composition. 10 FA’profile (% ongA) ' 9‘- ‘ S 3 ° % 3 ” 0 as as q; 3’:- 2-. :3 $2 23 ‘- " :2 0 a 0 0 co Sample U . . _ 5 I Control 7.41 0.36 0.12 3.00 15.26 1.98 30.93 15.98 MGAT1 7.11 0.32 0.11 2.95 13.86 1.51 28.87 19.59 O 1- 0 IO M O 1- O In a E 2 5 a a a a E 0 U 8' a a a U U , > a Sample Total Control 1.86 17.95 1.74 1.25. 0.26 0.57 0.98 0.20 0.17 100.00 MGAT1 1.90 17.22 1.71 1.90 0.51 0.57 1.52 0.19 0.17 100.00 A further experiment was performed where the FPleGATl-GFP chimeric DNA was modiﬁed to remove the GFP gene. This c construct, designated FPleGATl, was transformed into an A. thaliana line which was mutant for FAD2.

The total fatty acid t of the T2 seed from antibiotic resistant T. plants, as well as parental lines grown alongside these plants, was determined according to e 1.

The data is shown in Table 3. The average total fatty acids of the seed from the control lines was 347.9 pg/100 seeds whereas the average of the transgenic seeds was 381.0 ug/100 seeds. When the data for the control line C6 was excluded for determining the average, the average for'the controls was 370 pg/100 seeds. The oil content in the transgenic seeds represented an increase of about 3% in relative terms compared to the oil content in the sformed seeds.

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Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 177 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 178 The coding region ofthe mouse MGAT2 gene, codon-optimised for expression in plant cells, was tuted for the MGATl coding region in the constructs mentioned above, and introduced into Arabidopsis. Thirty plants of each transgenic line (T1 and T2 , giving rise to T2- and T3-generation seeds) were grown in the 5 ouse in a randomly arranged distribution and compared to control plants.

Seeds from the transgenic plants were increased in their oil content relative to the control seeds (Figure 7). The average TAG percentage of the T3 transgenic seeds represented a relative increase of about 8% compared to the TAG percentage in the untransformed seeds (Table 4). A signiﬁcant increase was observed in the level of 10 polyunsaturated fatty acids in the TAG of the transgenic seeds, in particular of ALA, and a decrease in saturated fatty acid levels such as palmitic and stearic acids.

Moreover, the increased TAG levels'and altered fatty acid composition was more pronounced in the T3 generation than in the T2 seeds, presumably due to the gous state ofthe transgene in the T3 seeds. 15 Table 4. TAG levels and fatty acid composition in TAG extracted from Arabidopsis thaliana T2 and T3 seeds expressing MGAT2 ed to untransformed control seed. --mm -m—m m -_284E_-_-_ ----25 .m— --- ---_ $55 55!% TAG by Seed ‘ --1.8 __M_ -:: __M_-m--_ 20 ' Ex ression ofMGATl in Brassica na us seeds The vector FPl:MGATl used for the expression of M. musculus MGATI in Arabidopsis thaliaria 'seeds was used to generate transformed B. napus . The, vector was uced into A. tumefaciens strain AGLl via. stande electroporation 25 procedures. Cultures were grown overnight at 28°C in LB medium with agitation at 150 rpm. The bacterial cells were collected by centrifugation at 4000 rpm for 5 ' minutes, washed with Winans' AB (Winans, 1988) and re-suspended in 10 mL of ﬁnans‘ AB medium (pH 5.2) and grown with kanamycin (50 mg/L) and rifampicin \ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 179 (25 mg/L) overnight with the addition of 100 uM acetosyringone. Two hours before infection of the Brassz'ca cells, spermidine (120 mg/L) was added e final density of the bacteria adjusted to an OD 600nm of 03,04 with fresh AB media. Freshly isolated cotyledonary petioles from 8-day old B. napus seedlings grown on 1/2 MS (Murashige—Skoog, 1962) or hypocotyl segments ditioned by 3-4 days on MS . media with 1 mg/L thidiazuron (TDZ) + 0.1 rug/L alpha-naphthaleneacetic acid (NAA) were infected with 10 mL Agrobacterium cultures for 5 minutes. Explants (cotyledonary petiole and hypocotyl) infected with Agrobacterium were then blotted on sterile ﬁlter paper to remove the excess Agrobacterium and transferred to co- 10 cultivation media (MS media with 1 mg/L TDZ +. 0.1 mg/L NAA + 100 uM acetosyringone) supplemented with or without different antioxidants (L—cysteine 50 mg/L and ascorbic 15 mg/L). All the plates were sealed with paraﬁlm and incubated in the dark at 23-24°C for 48 hours.

The co-cultivated explants (cotyledonary petiole and hypocotyl) were then V 15 washed with sterile distilled water + 500 mg/L cefotaxime + 50 mg/L timentin for 10 minutes, rinsed in sterile distilled water for 10 minutes, blotted dry on sterile ﬁlter paper,‘ transferred to pre-selection media (MS + 1 mg/L TDZ + 0.1 mg/L NAA + 20 - mg/L adenine sulphate (ADS) + 1.5 mg/L AgN03 + 250 mg/L cefotaxime and 50 mg/L timentin) and ed for ﬁve days at 24°C with a l6hour/8hour photoperiod. '20 They were then transferred to selection media (MS + 1 mg/L TDZ + 0.1 mg/L NAA + 20 mg/L ADS + 1.5 mg/L AgNO; + 250 mg/L cefotaxime and 50 mg/L timentin) with 1.5 mg/L glufosinate ammonium and ed for 4 weeks at 24°C with. /8hour , photoperiod With a ly subculture onto the same media. Explants with green callus were transferred to shoot initiation media (MS + 1 mg/L kinetin + 20 mg/L 25 ADS + 1.5 mg/L AgN03 + 250 mg/L cefotaxime + 50 mg/L timentin + 1.5 mg/L glufosinate ammonium) and cultured for another 2-3 weeks. Shoots emerging form the ant explants were transferred to shoot elongation media (MS media with 0.1 mg/L gibberelic acid + 20 mg/L ADS + 1.5 mg/L AgNO; + 250 mg/L ceftoxime + 1.5 mg/L glufosinate ammonium and cultured for another two weeks. Healthy shoots 2-3 30 cm long were selected and transferred to rooting media (1/2 MS with 1 mg/L NAA + ' 20 mg/L ADS + 1.5 mg/L AgNO; + 250 mg/L cefotaxime) and Cultured for 2-3 weeks. Well established shoots with roots were erred to pots (seedling g mix) and grown in a growth cabinet for two weeks and subsequently transferred to glasshouse. Sixteen individual transformants in the cultivar Oscar were conﬁrmedto 35 be transgenic for the FPleGATl construct and grew normally under ouse conditions. Plant growth ed normal and the plants were fertile, ﬂowering and a setting seed normally. The plants were grown to maturity and seeds obtained from transformed plants were ted. Seeds from some of the transformed plants were D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 180‘ sed for seed oil content and fatty acid composition. Data from these preliminary analyses showed variability in the oil content and fatty acid composition, probably ‘ due to the plants being grown at different times and under different environmental conditions. To reduce variability, Tl plants which express MGATl are ed and grown under the same conditions as control ‘(wild-type, cultivar Qscar) plants of the _ same genotype, and the oil content compared.

Expression ofMGATI in Gosszgium hirsutum seeds The same seed-speciﬁc chimeric gene used for the expression of M. musculus 10' MGATl in Arabidopsis ina seeds was used to generate transformed Gossypium hirsutum plants. The vector designated FPl :MGATI was uced into A. tumefaciens strain AGLI via standard oporationprocedures and cells from the Agrobacteriuni culture used to introduce the chimeric DNAs into cells of Gossypium hirsuium, variety Coker315. Cotyledons excised from 10-day old cotton seedlings 15' were used as explants and infected and co-Cultivated with A. tumefaciens for a period of two days. ‘This was followed by a six-week ion'on MS medium (Murashige and Skoog, 1962) containing 0.1 mg/L 2,4-D, 0.1 mg/L kinetin, 50 mg/L kanamycin sulphate, and 25 mg/L cefotaxime. Healthy calli derived from the cotyledon explants were transferred to MS medium containing 5 mg/L -dimethylallylamino)—purine 20 (Zip), 0.1 mg/L naphthalene acetic acid (NAA), 25 mg/L kanamycin, and 250 mg/L cefotaxime for a second period of six weeks at 28°C. Somatic embryos that formed after about six to ten weeks of incubation, were germinated and maintained on the same medium, but without added phytohorrnone or antibiotics. _ ets developed from the somatic s were transferred to soil and maintained in a glasshouse 25 once leaves and roots were developed, with 28°C/20°C ight) growth temperature. Ten independent primary transgenic plants‘(T0) containing the FPl— V MGATl construct were grown in the glasshouse, ﬂowered and produced bolls containing seeds. The seeds were harvested on maturity. To enhance the reliability of the oil content analysis, 5 plants were ished from each of the 10 primary 30 enic plants and the mature T2 seeds are ted to the analysis of oil content.

. The) seed-speciﬁc expression of MGATl increases oil content and ses the . percentage of polyunsaturated fatty acids in the cotton l.

Ex ression of a MGATl and MGATZ cues in N. benthamiana lants aﬁer stable 35 W N. benthamiana was stably transformed with the 3SSzMGATl construct described in Example 2. 35S:MGAT] was introduced into A. 'tumefaciens strain AGLl via standard electroporation procedure. The transformed cells were grown on D Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 18] solid LB media supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L) and ted at 28°C for two days. A single colony was used to initiate fresh 'culture. Following 48 hours vigorous culture, the cells "were collected by centrifugation at 2,000x g and the supernatant was removed. The cells were resuspended in fresh solution containing 50% LB and 50% MS medium at the y of ODsoo =05.

Leaf samples of N. benthamiana grown in, vitro were excised and cut into square sections around 0.5-1 cm2 in size with a sharp scalpel while immersed in the A. ciens solution. ‘The wounded N. benthamiana leaf pieces submerged in A. 10 tumefaciens were allowed to stand at room temperature for 10 minutes prior to being blotted dry on a sterile ﬁlter paper and transferred 'onto MS plates without supplement. Following a co-cultivation period of two days at 24°C, the explants were washed three times with sterile, liquid MS medium, then blotted dry with sterile ﬁlter paper and placed on the selective MS agar supplemented With 1.0 mg/L I 15 benzylaminopurine (BAP), 0:25 mg/L indoleacetic acid (1AA), 50 mg/L kanarnycin and 250 mg/L cefotaxime. The plates were incubated at 24°C for two weeks to allow for shoot development from the transformed N. benthamiana leaf .

' To establish rooted transgenic plants in vitro, healthy green shoots were cut off and transferred into 200 mL tissue e pots containing MS 'agar medium 20 mented with 25 ug/L 1AA, 50 mg/L kanamycin and 250 mg/L Cefotaxime.

Sufﬁciently large leaf discs were taken from transgenic shoots and freeze-dried for TAG fractionation and quantiﬁcation analysis as described in Example 1 (Table 5).

The best 3SS:MGAT1 N. benthamiana plant had a TAG content of 204.85 ug/100 mg dry weight leaf tissue ed v'vith an average TAG content of 85.02 ug/100 mg 25 ,dry weight leaf tissue in the control lines, enting an se in TAG content of ' ' 24196. ' p p N., benthamiana was also stably transformed with the 35S:MGAT2 construct described in Example 2 and a control binary veCtor pORE4 (Table 6). The best 35S:MGAT2 N. benthamiana plant had a TAG content of 79.0 pig/100 mg dry weight 30 leaf tissue compared with a TAG content of 9.5 ug/100 mg dry weight leaf tissue in the control line at the same pmental stage, representing an increase in TAG content of 731%. The fatty acid proﬁle of the TAG fractions was also altered with ‘ signiﬁcantly reduced levels of the saturated fatty acids 16:0 and 18:0, and increased levels of the polyunsaturated fatty acids, particularly 18:3033 (ALA) (Table 6). The 35 fatty acid proﬁle of the polar lipids from the same leaf samples were not signiﬁcantly affected, indicating that the changes in the fatty acid composition of the non-polar lipids was real. The control plants in this experiment were smaller and ent ‘ physiologically than in the previous experiment with the 3SS:MGAT1 construct, and D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 182 this may have explained the different oil contents of the l plants from one experiment to the other. ' ments to directly compare the 35$:MGATl and 35:MGA.T2 constructs with control plants are performed using plants of the same size and physiology.

A new set of constitutive binary sion vectors was made using a' 358 promoter with duplicated enhancer region (e358). 35S:MGAT1#2 (pJP3346),- 3SSzMGAT2#2 (pJP3347) and 3SS:DGAT1#2 (pJP3352) were made by ﬁrst cloning the e358 promoter, contained within a BamHI-EcoRI fragment, into pOREO4 at the BamHI-EcoRI sites to yield 3. pJP3346 and pJP3347 were then ed by 10 cloning the MGAT_1 and MGAT2 genes, respectively, into the EcoRI site of pJP3343. pJP3352 was ed by cloning the A. thaliana DGATl, contained within a Xhol— ' ' AsiSI site, into the XhoI—AsiSI sites oprP3343. 6, pJP3347 and pJP3352 in Agrobacterium strain AGLl were used to transform N. benthamiana as described above. Fourteen conﬁrmed transgenic plants 15- were recovered for p]P3346 and 22 for pJP3347. A number of kanamycin resistant, transformed shoots have been generated with pJP3352. Expression analysis of the enes was performed on the plants ormed with MGATl orMGATZ. Plants with high levels of expression were selected. Expression analysis on plants transformed [With the A. thaliana DGATl is performed. The plants grow normally 20 and are grown to maturity. Seed is harvested when mature; Seed ﬁ'om high- expressing progeny are sown directly onto- soil for lipid analysis of the T2 segregating population, which includes both homozygous and heterozygous plants. Oil content of leaves of plants expressing-high levels of either MGATl or MGAT2 is signiﬁcantly increased compared to plants transformed with A. thaliana DGATl or control plants. 25 MGAT2 transgenic plants showed a signiﬁcant se in the unsaturated fatty acid 18:1 and l '% relative increase in total fatty acid content compared to the null events ' (Table 7). ' . . p 6, pJP3347 and a control vector in AGLl were also used to transform _ A. thaliana as described above. Twenty-ﬁve conﬁrmed transgenic T2 plants 30 comprising the T-DNA from pJP3346 and 43 enic plants for pJP3347 were identiﬁed. Expression analysis was performed on the transgenic plants. Seeds from high-expressing y were harvested and sown directly onto soil. Lipid analysis including oil content of the leaves from T2 and T3 progeny was med, ing from segregants lacking the transgenes. The highest levels of TAG were obtained in 35 plants that are homozygous for the MGAT transgenes.

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Expression ofMGATl in stably transformed Trilblium regens plants A chimeric gene encoding M. musculus MGATl was used to transform Trifolium repens, another dicotyledonous plant. Vectors containing the chimeric genes AT1 and 35S:DGAT1 were introduced into A. tumefaciens via a 10 standard electroporation procedure. Both vectors also n a 35S:BAR selectable marker gene. The transfonned Agrobacterium cells were grown on solid LB media supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L) and ted at 28°C for two days. .A single colony was used to initiate a fresh culture for each construct. Follnwing 48 hours vigorous culture, the Agrobacterium cultures were 15 used to treat T. repens (cv. Haifa) cotyledons that had been dissected from imbibed seed as described by Larkin et a1. (1996). Following co-cultivation for three days the- explants were exposed to 5 mg/L PPT to select transformed shoots and then transferred to rooting medium to' form roots, before transfer to soil. A transformed plant containing MGATl was obtained. The 35S promoter is expressed constitutively 20 incells of the transformed plants. The oil content is increased in at least the vegetative tissues such as leaves.

Expression ofMGAT in stably transformed Hordeum vulgare I A chimeric vector including M. musculus MGATI was used to produce stably 25 transformed m vulgare, a monocotyledonous plant. Vectors containing the chimeric genes UbizMGATl and UbizDGATl were constructed by cloning the entire M. musculus MGATl and A. thaliana DGAT] coding regions separately into pWVECS-Ubi. Vectors containing the chimeric genes Ubi:MGAT1 and UbizDGATl were uced into A. tumefaciens strain AGLl via a rd electroporation 30 procedure. Transformed Agrobacterium cells were grown on solid LB media supplemented with cin (50 mg/L) and rifampicin (25 mg/L) and the plates incubated at 28°C for two days. A single colony of each was used to initiate fresh cultures. ing 48 hours us e, the cterium cultures were used to 35 transform cells in immature embryos of barley (cv. Golden Promise) according to - published methods (Tingay et al., 1997; Bartlett et al., 2008) with some modiﬁcations.

Brieﬂy, embryos between 1.5 and 2.5 mm in length were isolated from immature Eopses [land the embryonic axes removed. The resulting explants were co- Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 187 ated for 2-3 days with the transgenic Agrobacterium and then cultured in the dark for 4-6 weeks on media containing timentin and hygromycin to generate embryogenic callus before being moved to tion media in low light conditions for two weeks. Callus was then transferred to regeneration media to allow for the regeneration of shoots and roots before transfer to soil. Transformed plants were obtained and transferred to the greenhouse. The MGATl coding region was expressed constitutively under the control of the Ubi promoter in cells. of the transformed plants. Transgenic plants were generated and their tissues analysed for oil content. Due to “the low number of transgenic events obtained in a ﬁrst 10 transformation, no tically signiﬁcant conclusion could be drawn from the data.

The coding region of the mouse MGAT2 gene, codon optimised for expression in plant cells, is substituted for the MGATl in the constructs mentioned above, and introduced into Hordeum as described above. Vegetative s from the resultant ‘ transgenic plants are increased for oil t. 15 Expression ofMGAT in yeast cells A chimeric vector including M. musculus MGATl was used to transform ' yeast, in this example Saccharomyces cerevisiae, a fungal microbe suitable for production of oil by fermentation. A genetic construct ATl was made by ' 20 inserting the entire Coding region of a construct designated 0954364_MGAT_pMA, ’ contained within an EcoRI fragment, into pYESZ at the EcoRI site, generating pJP3301. Similarly, a genetic construct Gall :DGATl, used here as a comparison and separately encoding the enzyme A. thaliana DGATl was made by ing the entire A. ana DGATl coding region into pYESZ. These chimeric vectors were 25 introduced into S. cerevisiae strain S288C by heat shock and transformants were selected on yeast minimal medium (YMM) plates containing 2% rafﬁnose as the sole carbon source. Clonal inoculum es were established in liquid YMM with 2% rafﬁnose as the sole carbon source. mental cultures were inoculated from these I in YMM medium containing 1% NP-40, to an initial OD600 of about 0.3. Cultures 30 were grown at 28°C with shaking (about 100 rpm) until OD600 was approximately 1.0. At this point, galactose was added to a ﬁnal tration of2% (w/v). Cultures were incubated at 25°C With shaking for a further 48 hours prior to harvesting by centrifugation. Cell pellets were washed with water before being freeze-dried for lipid class fractionation and ﬁcation analysis as described in Example 1. The 35 Gal promoter is expressed inducibly in the transformed yeast cells, increasing the oil ‘ content in the cells.- The coding region‘of the mouse MGAT2 gene, codon optimised for expression in yeast cells, is substituted for the MGAT] in the constructs mentioned above, and D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 188 introduced into yeast. The resultant transgenic cells are increased for oil content. The genes are also introduced into the oleaginous yeast, Yarrowia lipolytica, to increase oil content. sion ofMGAT in algal cells domonas rdtii' ‘ ' A chimeric vector including M. musculus MGAT] is used to stably transform algal cells. The genetic constructs designated 3SSzMGAT1 is made by cloning the MGAT] coding region into a cloning vector containing a Cauliﬂower mosaic virus 3SS promoter cassette and a paramomycin-resistance gene (aminoglycoside—O- ’ by 10 phosphotransferase VIII) expressed a C. reinhardtii RBCSZ promoter. 3SS:MGAT1 is introduced separately into a logarithmic culture of 5x107 cc503, a cell-wall-deﬁcient strain of Chlamydomonas rdtii by a modiﬁed glass bead method (Kindle, 1990). Both vectors also contain the BLE resistance gene as a selectable marker gene. Brieﬂy, a colony of ansformed cells on a TAP agar 15' plate kept at about 24°C is grown to about 5x106 cells/mL Over four days, the resultant cells are pelleted at 3000 g for ‘ 3 minutes at room temperature and resuspended to produce 5x107 cells in 300 uL of TAP media. 300 uL of 0.6mm ‘ diameter glass beads, 0.6 pg plasmid in 5 pL and 100 ”L of 20% PEG MW8000 are added and the mix is vortexed at maximum speed for 30 seconds, then erred to 20 10 mL of TAP and incubated for 16 hours with shaking in the dark. The cells are pelleted, resuspended in 200 pL of TAP then plated on TAP plates containing 5mg/L zeocin and ted in the dark for 3 weeks. Transformed coloniesare subcultured to a fresh TAP + zeocin 5 mg/L plate after which they are grown up under standard media conditions with zeocin ion. Aﬁer harvesting by centrifugation, the cell 25 pellets are washed with water before being freeze-dried for lipid class fractionation and quantification analysis as described in Example 1. The SSSIMGATl promoter is expressed constitutively in the transformed algal cells. The oil t of the cells is signiﬁcantly increased. _ The coding region of the mouse MGATZ gene, codon sed for expression ‘ 30 in‘plant cells, is substituted for the MGATl in the construct ned above, and introduced into Chlamydomonas. Oil content in the resultant transgenic cells is signiﬁcantly increased. sion ofMGAT in stably transformed Luginus ang_ustitolius 35 A chimeric vector including M. musculus MGAT] is used to transform Lupinus angustifolius, a leguminous plant. Chimeric vectors 3SS:MGATl and SSS: DGATl in Agrobacterium are used to transform L. ifolius as described by Pigeaire et al. (1997). Brieﬂy, shoot apex- explants are co-cultivated with transgenic D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 189 Agrobacterium before being thoroughly wetted with PPT solution (2 mg/ml) and transferred onto a PPT-free regeneration medium. The multiple axillary shoots developing from the shoot apices are'excised onto a medium containing 20 mg/L PPT and the surviving shoots erred onto fresh medium ning 20 mg/L PPT. y shoots are then transferred to soil. The 358 promoter is expressed constitutively in cells of the transformed plants, sing the oil" content in the . vegetative tissues and the seeds. A seed speciﬁc promoter is used to further increase the oil content in transgenic Lupinus seeds.

The coding region of the mouse MGAT2 gene, codon optimised for expression 10 in plant cells, is substituted for the MGAT] in the constructs mentioned above, and introduced into Lupinus. Seeds and vegetative tissues from the resultant enic plants are increased for oil content.

Expression ofMGAT in stably transformed cells hum bicolor 15 A chimeric vector including M. musculus MGAT‘I is used to stably transform Sorghum r. UbitMGATl and Ubi:DGATl in A. tumefaciens strain AGLl are used to transform Sorghum bicoloi‘ as described by Gurel et a1. . The Agrobacterium is ﬁrst centrifuged at 5,000 rpm at 4°C for 5 minutes and diluted to QD550 = 0.4 with liquid co-culture medium. Previously isolated immature embryos ,- I 20 are then d completely with the Agrobacterium suspension for 15 minutes and then cultured, scutellum side up, on co-cultivation medium in the dark for 2 days at 24°C. The immature embryos are then transferred to callus-induction medium (CIM) with 100 mg/L carbenicillin to t the growth of the Agrobacterium and leﬁ for 4 weeks. Tissues are then transferred to regeneration medium to shoot and root. The 25 Ubi er is expressed tutively1n cells of the transformed plants, increasing the oil content in at least the vegetative tissues The coding region of the mouse MGAT2 gene, codon optimised for expreSsion in plant cells, is substituted for the MGATl in the constructs mentioned above, and introduced into Sorghum. Vegetative s from the resultant transgenic plants are 30 increased for oil content.

Ex ression 1n stabl transformed lants of G1 cine max A chimeric gene encoding M. musculus MGATl is used to stably transform Glycine max, another legume which may be used for oil production. 3SS:MGAT1 in 35 Agrobacterium is used to transform G. max as described by Zhang et al. (1999). The .

Agrobacterium is co-cultivated for three days with cotyledonary explants derived from ﬁve day old seedlings. Explants are then cultured on Gamborg's B5 medium supplemented with 1.67 mg/L BAP and 5.0 mg/L glufosinate for four weeks aﬁer D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 190 which explants are subcultured to medium Containing MS major and minor salts and \‘ BS vitamins (MS/B5) supplemented with 1.0 mg/L zeatin—riboside, 0.5 mg/L GA3 and 0.1 mg/L 1AA amended with 1.7 mg/L or 2.0 mg/L glufosinate. Elongated shoots are rooted on a MS/BS rooting medium supplemented with 0.5 mg/L NAA without further inate selection. The 358 promoter is expressed constitutively in cells of the transformed plants, increasing the oil content in the vegetative tissues and the I seeds. i.

The coding region of the mouse MGAT2 gene, codon optimised for expression in plant cells, is substituted for the MGATl in the constructs mentioned above,‘ and 10 introduced into Glycine. Vegetative tissues and seeds from the resultant transgenic plants are sed for oil content.

Expression ofMGAT in stably transformed Zea mays A ic gene encoding M. musculus MGATl is used to stably transform 15 Zea mays. The vectors comprising 3SSV:MGAT1 and 35S2DGAT1 are used to transform Zea mays as bed by Gould et a1. (1991). , shoot apex explants are (to-cultivated with transgenic Agrobacterium for two days before being transferred onto a MS salt media containing kanamycin and carbenicillin. After several rounds of lture, transformed shoots and roots spontaneously form and are transplanted to 20 soil. The 353 promoter is expressed in cells of the transformed plants, increasing the 'oil content in the vegetative s and the seeds.

The coding region of the mouse MGAT2 gene, codon optimised for expression in plant cells, is substituted for the MGAT] in the constructs mentioned above, and introduced into Zea mays. Vegetative tissues and seeds from the resultant transgenic 25 plants are increased for oil Content. Alternatively, the MGAT coding regions are‘ expressed'under the control of an endosperm speciﬁc promoter such asthe zein er, or an embryo speciﬁc promoter obtained from a monocotyledonous plant, for increased expression. and increased oil content in the seeds. A further chimeric gene encoding a GPAT with phosphatase activity, such as A. na GPAT4 or 30 GPAT6 is introduced into Zea mays in combination with the MGAT, airmen increasing the oil content in corn seeds.

Expression ofMGAT in stably transformed Elaeis nsis (palm oil) A chimeric geneencoding M. musculus MGATl is used to stably orm _ 35' Elaeis guineensis. Chimeric s ated Ubi:MGAT1 and UbizDGATl in Agrobacterium are used. Following 48 hours vigorous culture, the cells are used to transform Elaeis guineensis as described by Izawati et a1. (2009). The Ubi promoter .D- Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 191 is expressed constitutively in cells of the transformed plants, increasing the oil content V in at least the fruits and seeds, and may be used to obtain oil. ' The coding region of the mouse MGATZ gene, codon optimised for expression in plant cells, is tuted for the MGAT] in the constructs mentioned above, and introduced into Elaeis. Seeds from the resultant transgenic plants are increased for oil content.

Expression ofMGAT in stably transformed Avena sativa {cats} A 'chimeric gene encoding M. musculus MGAT] is used to stably transform 10 Avéna sativa, another tyledonous plant. Chimeric vectors designated Ubi: MGATl and Ubi: DGAT], as described above and both containing a R selectable. marker, are used to transform Avena sativa as described by Zhang et a1.

. The coding region of the mouse MGATZ gene, codon optimised for expression 15 in plant cells, is substituted for the MGATl in the constructs'mentioned above, and introduced into Avena. Seeds from the resultant transgenic plants are increased for oil content.

Example 7. Engineering a MGAT with DGAT activig ' 20 An MGAT with altered DGAT ty, ally increased DGAT activity and potentially increased MGAT activity may be engineered by performing random mutagenesis, targeted mutag'enesis,,or saturation mutagenesis on MGAT gene(s) of interest or by subjecting different MGAT and/or DGAT genes to DNA shufﬂing.

DGAT function can be positively screened for by using, for example, a yeast strain 25 that has an absolute requirement for TAG-synthesis complementation when fed free fatty acids, such as strain H1246 which contains mutations in. four genes (DGAI, _ LROI, ARE], AREZ). Transforming theMGAT variants in such a strain and then supplying the transformed yeast with a concentration of free fatty acids that prevents mentation by the wildtype MGAT gene will only allow the growth of variants 30' with increased TAG-synthesis capability due to improved DGAT activity. The MGAT activity of these mutated genes can be determined by feeding labelled sn-l or sn-2 MAG and quantifying the production of labelled DAG. Several rounds of directed evolution in combination with al protein design would result in the production of a novel MGAT gene with r MGAT and DGAT activities. 35 The gene coding for the M. musculus MGATl acyltransferase was subjected to error prone PCR using Taq DNA polymerase in the presence of 0.15 mM MnClz to introduce random mutations. The randomized coding regions were then used as imers to amplify the entire yeast expression vector using high ﬁdelity PC D ' ‘ I Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 192 reaction conditions. Sequencing of 9099 bp of recovered, mutagenised DNA revealed a mutational ncy of about 0.8 %, corresponding to 8 mutations per gene or, on . average, 5.3 amino acid substitutions per polypeptide. The entire mutagenised library was ormed into E. coli DHSo. for e at -80 °C and plasmid preparation.

The size of the MGATl library was estimated at 3.8356E6 clones. A copy of the MGATI library was transformed into the yeast strain H1246, resulting in a library size of 3E6 clones. The MGAT] library as well as a pYESZ negative control, transformed into S. siae H1246, were subjected to 8 selection rounds, each consisting of (re)diluting cultures in minimal induction medium (1 % rafﬁnose + 2 % 10 galactose; diluted to OD600 = 0.35—0.7) in the presence of C18:1 free fatty acid at a 1 mM ﬁnal concentration. Negative controls consisted of identical es grown simultaneously in minimal medium containing glucose (2%) and in theabsence of C18:1 free fatty acid. After 8 selection rounds, an t of the selected MGATl library was plated on minimal medium containing glucose (2%). A total 'of 120 15 colonies were groWn in 240 pl minimal ion medium in 96 microtiter plates and d for neutral lipid yield using a Nile Red ﬂuorescence assay as described by Siloto et a1. (2009). Plasmid minipreps were prepared from 113 clones (=top 6 %) that displayed the highest TAG .

The entire MGAT] coding region of the selected clones is sequenced to. 20 ' identify the number of unique mutants and to identify the nature of the selected ons. Unique MGATl mutants are retransformed into S. cerevisiae H1246 for in ' vitro MGAT and DGAT assays using labelled MAG and C1821 substrates respectively (see Example 5). Selected MGATl ts are found to exhibit increased DGAT activity compared to the wild type acyltransferase, whilst MGAT 25 activity is possibly increased as well. .

‘ MGATl variants displaying increased MGAT and/or DGAT activities are used as parents in a DNA shufﬂing reaction. The resulting library is subjected to a' similar selection system as described above ing in further improvement of general acyltransferase activity. In addition, free fatty acids other than C1821 are added to the 30 growth medium _to select for MGAT] variants displaying altered acyl-donor speciﬁcities.

" Exam 1e 8. Constitutive ' ex n of the A. thaliana diac l l cerol ac ltransferasez in lants . 35 Expression of the A. thaliana DGAT2 in yeast (Weselake et al., 2009) and insect cells (Lardizabal et al., 2001) did not demonstrate DGAT activity. Similarly, the DGAT2 was not able to complement an A. thaliana DGATl knockout (Wesclake et al., 2009). The enzyme activity of the A. thaliana DGAT2 in leaf tissue was D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 193 determined using a N. benthamiana transient sion system as described in Example 1. The A. thaliana DGAT2 (accession Q9ASU1) was obtainedvby genomic PCR and cloned into a binary sion vector under the control of the 358 er to generate 358:DGAT2. This chimeric vector was introduced into A. tumefaciens strain AGLl and cells from es of these ated into leaf tissue of N. benthamiana plants in a 24°C growth room using 35$:DGAT1 as a' control. Several direct isons were inﬁltrated with the samples being'compared located on either side. of the same leaf. Experiments were performed in triplicate. Following inﬁltration the plants were grown for a further five days before leaf discs were taken 10 and freeze-dried for lipid class fractionation and quantiﬁcation analysisas described in Example 1. This analysis revealed that both DGATl and DGAT2 were oning I ’ .to increase leaf oil levels in Nicotiana benthamiana (Table 8).

Leaf tissue transformed with the 3SS:p19 construct (negative control) contained an average of 25 pg TAG/100 mg dry leaf weight. Leaf tissue transformed 15 with the 35821319 and 3SSzDGAT1 constructs (positive control)‘contained an average of 241 ug TAG/100 mg dry leaf weight. Leaf tissue transformed with the 3SS:p19 and 3SS:*DGAT2_ constructs contained an average of 551 ug TAG/100 mg dry leaf ' . .

. . The data described above demonstrates that the A. thaliana DGAT2 enzyme is 20 more active than the A. thaliana DGATl enzyme in promoting TAG accumulation in leaf tissue. Expression of the DGAT2 gene ed in 229% as much TAG accumulation in leaf tissue compared to when the TAG amount from DGATI over- expressed was set as relative 100% (Figure 9).

Transiently-transformed N. benthamiana leaf tissues expressing P19 alone 25 (control), or P19 with either AtDGATl or 2 were also used toiprepare microsomes for in vitro assays of enzyme activity. A DGAT biochemical assay was i. , med using microsomes corresponding to 50 pg protein and adding 10 nrnole [l4]C6:0-DAG and 5 nmole\acyl-COA, in 50 mM Hepes buffer, pH 7.2, containing 5 mM MgC12, and 1% BSA in a ﬁnal volume of 100 pL for each assay. The assays 30 wereiconducted at 30°C for 30 minutes. Total lipid from each assay was extracted and samples loaded on TLC plates, which were developed using a hexanezDEEzHac solvent (70:30:1 voltvolzvol). The amount of radioactivity in DAG and TAG spots was quantiﬁed by\PhosphorImage measurement. The percentage of DAG converted to TAG was calculated for each of the ome preparations. 35 some endogenous DGAT activity was ed in the N. benthamiana leaves, as the P19 control assay showed low levels of TAG production. The expression of AtDGATl yielded increased DGAT ty relative to the P19 control when the assays were supplemented with either C18:1—CoA or C18:2-COA, but not when D. substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 194 supplemented with C18z3-COA, where the levels of TAG for the P19 l and the AtDGATl were similar. However, in all of the microsomal assays when AtDGAT2 V was expressed in the leaf tissues, greater levels of DGAT activity (TAG production) were observed ed to‘ the AtDGATl microsomes. Greater levels of TAG production were observed when the imicrosomes were supplemented with either C1822-COA or C18:3-C0A ve to C18:l-COA.(Figure 10). This indicated that DGAT2 had a different substrate preference, in particular for C1823-CoA (ALA), than DGATl. 10 Example 9. Co-expression of MGAT and GPAT in transgenic seed Yang et a1. (2010) described two glycerolphosphate acyltransferases (GPAT4 and GPAT6) from A. na both having a sn-2 ence (i.e. preferentially forming sn-2 MAG rather than 'sn-l/3 MAG) and atase activity, which were able to produce sn-2 MAG from G—3-P (Figure 1). These enzymes were 15 proposed to be part of the cutin synthesis pathway. GPAT4 and GPAT6 were not expressed highly in seed tissue. Combining such a bifunctional hosphatase with a MGAT yields a novel DAG synthesis pathway using GP as a substrate that can replace or supplement the typical Kennedy Pathway for DAG synthesis in plants, V particularly in oilseeds, or other cells, which results in increased oil content, in 20 ular TAG levels. , ‘ Chimeric DNAs designated pJP3382 and pJP3383, encodingthe A. thaliana GPAT4 and GPAT6, respectively, together with the M. musculus MGAT2 for ' expression in plant seeds were made by ﬁrst inserting the entire MGAT2 coding region, contained within a SwaI fragment, into pJP3362 at the Smal site to yield ' 25 8. pJP3362 was a binary expression vector containing empty FAEl and FPl expression cassettes and a kanamycin resistance gene as a selectable marker. The A. thaliana GPAT4 was ampliﬁed from cDNA and cloned into pJP3378 at the NotI site to yield pJP33 82 in which the GPAT4 was expressed by the truncated napin promoter, .

PP], and the MGAT2 was expressed by the A. thaliana FAEl promoter. Similarly, 30 the A. na GPAT6 was ed from cDNA and cloned into pJP3378 at the ' Natl site to yield pJP3384 in which the GPAT6 was operably linked to the truncated napin promoter, FPl, and the MGAT2 was expressed by the A. thaliana FAEl promoter. VpJP3382 and pJP3383 were transformed into A. thaliana' (ecotype ia) by the ﬂoral dip method. Seeds from the treated plants were plated onto 35 media containing the antibiotic, kanamycin, to select for progeny plants (T1 plants) which were transformed. Transgenic seedlings were transferred to soil and grown in the greenhouse. Expression of the transgenes in the ping embryos ’ was determined. Transgenic plants with the highest level of expression and which show a D , Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 193 3:1 ratio for transgenicznon-transgenic plants per line, indicative of a single locus of insertion of the transgenes, are selected and grown to maturity. Seeds were obtained . from these plants (T2) which included some (which were homozygous for the transgenes. 30 to 32 (T2 plants) from each line were grown in pots of soil in a random arrangement in the greenhouse with control plants, and the lipid content, TAG content and fatty aCid compositions ofthe resultant seed was ined. The total - fatty acid content (as determined from the total FAME), in particular the TAG content of the seeds comprising both a MGAT and a GPAT4 or GPAT6 was substantially and signiﬁcantly increased by nearly 3% (absolute level) or by about 9% (relative 10 increase) over the controls, and increased relative to seeds comprising the MGAT alone or the A. thaliana DGATl alone (Figure 11). ' . p The coding region of the mouse MGAT2 gene, codon optimised for expression in plant cells, was introduced into Brassica napus together with a chimeric gene encoding Arabidopsis GPAT4. Seeds from the resultant transgenic plants were 15 harvested and some were analysed. Data from these preliminary analyses showed ility in the oil content and fatty acid composition, ly due to the plants . being grown at different times and under different environmental conditions; Seeds are planted to produce progeny plants, and progeny seeds are harvested. 20 Example 10. Testing the effect of GPAT4 and GPAT6 on MGAT-mediated TAG increase by GPAT silencing and mutation The GPAT family is large and all known members contain. two conserved domains, .a plsC acyltransferase domain and a HAD—like ase superfamily . In addition to this, A. na GPAT4-8 all contain an N—terminal region 25 homologous to a phosphoserine phosphatase domain. A. thaliana GPAT4 and ' GPAT6 both contain conserved residues that are knoWn to be al to phosphatase activity (Yang et al., 2010). _ Degenerate primers based on the conserved amino acid Sequence ‘ GDLVICPEGTTCREP (SEQ ID NO:228) were. designed to amplify fragments on N. 30 miana GPATS expressed in leaf tissue. 3' RACE will be performed using these primers and oligo-dT reverse primers on RNA isolated from N. miana leaf tissue. GPATs with atase activity (i.e. GPAT4/6-like) will be identiﬁed by their homology with the N—terminal phOsphoserine atase domain region described above. SSS-driven RNAi constructs targeting these genes will be generated 35 and transformed in A. tumefaciens strain AGLl. Similarly, a 3SS:V2 construct containing the V2 viral silencing-suppressor protein will be transformed in A. tumefaciens strain AGLlL V2 is known to suppress the native plant silencing D Substitute Sheet (Rule 26) RO/AU ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 196 mechanism to allow effective transient expression but also allow ased gene ~ silencing to function.

TAG accumulation will then be compared between _ transiently-transformed leaf samples ated with the following strain mixtures: l) 3SS:V2. (negative control); 2) 3SS:V2 + SSStMGATZ (positive control); 3) 3SS:V2 + NAi; 4) 3SS:V2 + NAi + ‘3SSzMGAT2. It is expected that the 35$:V2 + GPAT- RNAi + 3SS:MGAT2 mixture will result in less TAG accumulation than the 3SS:V2 + 3SS:MGAT2 sample due to interrupted sn-2 MAG synthesis resulting from the I I GPAT silencing. ’ 10 A similar experiment will be performed using A. thaliana and N. benthamiana 6-like sequences which are mutated to remove the conserved residues that are known to be critical to phosphatase activity (Yang et a1.) 2010). These mutated genes ’ (known collectively as GPAT4/6-delta) will then be cloned into 3SS-driven expression binary vectors and transformed in A. tumefaciens. TAG accumulation will 15 then be compared between transiently-transformed leaf samples inﬁltrated with the following strain mixtures: 1) 3SS:p19 (negative control); 2) 3SSzpl9 + 3582MGAT2 I (positive control); 3) 3SS:p19 + GPAT4/6—delta; 4) 3SS:p19+ GPAT4/6-delta + 3SS:MGAT2. It is expected that the 9 + 6-delta + AT2 mixture Willresult in less TAG accumulation than the 3SS:p19 + 35$:MGAT2 sample 20 due to interrupted sn-2 MAG synthesis resulting from the GPAT mutatiOn. Whilst the native N. benthamiana GPAT4/6—like genes will be present in this experiment it is expected that high-level expression of the GPAT4/6-delta constructs will outcompete the endogenous genes for access to the GP substrate. 25‘ Example 11. Constitutive expression of a diacylglxcerol acyltransferase and WRIl ription factor in plant cells A vector designated 358-pORE04 was made by inserting a PM fragment containing a 358 promoter'into the Sfol site of vector pORE04 after T4 DNA polymerase treatment to blunt the ends (Coutu et al., 2007). A genetic construct 30 3SSrArath—DGAT1 encoding the A. thalianq diacylglycerol ransferase DGATl (Bouvier-Nave et al., 2000) was made. Example '3 of WC 2009/129582 describes the construction of l in pXZP163. A PCR ampliﬁed fragment with KpnI and ’ EcoRV ends was made from pXZP163 and inserted into pENTRll to generate pXZP513E. The entire AtDGATl coding region of 3E contained within a '35 EcoRV fragment was inserted into 358-pORE04 at the BamHI-EcoRV site, generating pJP2078. A tic fragment, Arath-WRII, coding for the A. thaliana WRIl transcription factor (Cemac and Benning, 2004), ﬂanked by EcoRI restriction sites and codon optimized for B. napus, was synthesized. A genetic construct D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk 197 designated 3SS:Arath-WRII was made by cloning the entire coding region of Arath! WRIl, ﬂanked by EcoRI sites into 358-pORE04 at the EcoRI site generating pJP3414. Expression of the genes in N. benthamiana leaf tissue was performed according to the ent expression system as described in Example 1.

Quantiﬁcation of TAG levels of inﬁltrated N. benthamiana leaves by latroscan revealed that the combined expression of the A. thaliaria DGATl and WRIl genes resulted in 45-fold and 14.3-fold increased TAG content compared to expression of WRII and the V2 negative control respectively (Table 9). This corresponded to an ‘average and maximum observed TAG yield per leaf dry weight of 5.7 % and 6:51 % 10 respectively (Table 9 and Figure 12). The increase in leafon was not solely due to the activity of the overexpressed DGATl acyltransferase as was nt in the reduced TAG levels when WRll was left out of the combination. Furthermore, a synergistic effect was observed accounting for 48 % of the total TAG increase.

‘ Both DGATl and WRIl constructs also led to increased oleic acid levels at the 15 expense of linoleic .acid in TAG fractions of ated N. benthamiana' leaves (Table 10). These results conﬁrm recent ﬁndings by Andrianov et a1. (2010) who reported similar shiﬂs in the TAG, phospholipid and TFA lipid fractions of transgenic tobacco plants transformed with the A. thaliana DGATl ansferase. However, when DGATl and WRIl genes were co-expressed, a synergistic effect was observed on the «20 accumulation of oleic acid in the N. benthamiana leaves — this synergism accounted for an estimated at 52 % of the total oleic acid content when both genes were sed. The unexpected synergistic effects on both TAG-accumulation and oleic acid levels in transgenic N. benthamiana leaves demonstrated the potential of simultaneously up-regulating fatty acid biosynthesis and acyl uptake into non-polar 25 lipid such as TAG in vegetative tissues, two metabolic processes that are highly active in developing oilseeds.

‘ The transient expression experiment was ed except that the [’19 viral silencing suppressor as substituted for the V2 suppressor, and with careful comparison - of samples on the same leaf to avoid any leaf-to-leaf variation. For this, a chimeric 3O 9 uct for expression of the tomato bushy stunt virus P19 viral silencing suppressor‘protein (Wood et al., 2009) was separately introduced into A. tumefaciens GV3101 for co-inﬁltration. ' Quantiﬁcation of TAG levels of inﬁltrated N. benthamiana leaves by Iatroscan in this experiment revealed that the combined transient expression of the A. na 35 DGATl and WRIl genes resulted in ld increased TAG conte'ntcompared to P19 negative control (Figure 13). When compared to the expression of the DGATl and WRIl genes separately ‘on the same leaf, the combined ation increased TAG levels by 17- and 5- fold respectively. Once again, the re'ssion of both genes .

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 198 had a synergistic r than ve) effect on leaf oil accumulation with the synergistic component accounting for 73% of the total TAG increase. The greater extent of the increased TAG. content in this experiment (l41-fold) compared to the previous experiment (14.3-fold) may have been due to use of the P19 silencing suppressor rather than V2 and therefore increased gene expression from the transgenes. .

Table 11 shows the fatty acid composition of the TAG. When DGATI and WRIl genes were co-expressed in N. benthamiana, a synergistic effect was once ‘ again observed on the level of oleic aCid accumulation in the leafTAG fraction. This ' 10 increase was largely at the expense of the medium chain unsaturated fatty acids palmitic acid and c acid (Table 11). Linoleic acid was also increased which can be explained by the higher oleic acid substrate levels available to the endogenous FAD2 AlZ-desaturase. Individual sion of the DGATI and WRII genes in N. benthamiana led to intermediate changes in the TAG proﬁle without as great an 15 increase in oleic acid. In addition, but in centrast to the ﬁrst experiment, higher levels of a-linolenic acid (ALA) were detectedwhile this was ed to a lesser extent ‘ ' upon the DGATl and WRII co-expression in leaf tissue.

The observed synergistic effect of DGATI' and WRII expression on TAG biosynthesis was conﬁrmed in more detail by comparing the effect of uction 20 into N. benthamiana of both genes dually or in combination, corﬁpared to introduction of a P19 gene alone as a control, within the same leaf. This was beneﬁcial in reducing leaf to leaf variation. In addition, the number of replicates was increased to 5 and samples were pooled across different leaves from the same plant to improve the quality of the data. s are presented in Table 12. 25 Substitute Sheet (Rule 26) RO/AU I [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 199 .mddumwm 38:2 mNdv Nde mmdm univ— 3”.va odd Ndmw om.NwN 0%.de on“ dHVNO. dd dd dd Nd dd 06 dd vd Nd 53» . 858.“me RNNU dd dd dd mé. né m4 Nd Nd dd ncnudN 9o 3 ad Nd Nd dd wd —.d dd b28655 NudN dd dd dd dd dd dd Pd dd dd :v—adN dd dd .3 od od ad Nd rd dd 03mm: .32 dndNU dd N.N N.N dd dd dd .mw Né N; crumﬁcﬁrud mud—.0 N.Nw mdw ddN m.v~ méN ndp ﬁnd mdN —.mN ._ Nudwo 3 «.2 «.2 9: :8 ﬁn 323N332 o3 Zn own :utwwu ad ed od no no to to no no 88:th Né d.N m6 QN d.N d.N N.N dd m.» E taro O<H dudwo 35 m9 We 0.: N.: v.NF Md NS 3 _ do :23 «Snug dd dd dd vd Vd dd md , md md 05:83 Q9538 . huﬁdwo dd dd dd Pd rd dd pd ed pd 98 .0505 Ewan—“mm”. unpiwud— ad ad md dd dd. md Ron 33m 98 dumwo NV? v.3 adv «:8 0.8 98 .2: mNF v.3 7359mm . a d E. 0355 oiEuw <+d va— «209,12; Substltute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 200 .28 .55, E<Onnwmm 86 :l So a? 3.0 0.2 ._§nmmm a ﬁ a . % a aw." VNN mud .N> EEG o. 3.0 8.8 2.3.26 «.0; :36 moo 8.: N H as H 23m M34; 91» ”mama am>ummm , 559mm . £5 ESQ 85 m: a.“ Ed a « 3,5883 a a _ o 3.3 o omﬁ .9 o E o 8.2 532 . .x. 86 v0.0 omd .5; as, am a? an ﬂ ﬂ H 26 36 a who a Eacemﬁb :6 @5355 0<H mNd 3.6. a a 0:3: .~> EEO .o 33 mm: 332:. mod H3: $6 3.2 . mm“: beams“: :.<OQ CRUD HH<OQ 355$“me . 8.0 Emma 5:» :3 86 34 H a a a . 25mins 5 .Eé .2: .Eg .9 .o 8.8 who o o mg o 3.2 $50 ﬂ 832 ,N> .N> .N> 3 083 $389.2 Es§s§ 35:83 05 E<on .

E _ no Eugen .25» 26 :3 SA :3 m; a a a: ms w a 63 mnemtmmﬁoo 8.8 33:25 a a $3892 823» @2355 .9 o 86 8.0 when one 8.: c 2.9 00.0 de Ox? .x. “m “m ﬂ mo 28:? O<H Sud 34 Nmé 3:2:ch uEwSQép—m 2d So m3 6% who a a . a a 25.5th co aoEmanoo 88:? a um , 2&2 comma Eon Bow 9 o on; 2.3 .o 3.: 8w o 2.3 . 025 05 0:8 33m was .«o a ENQOQ SM? .3 28 QED 5325883550 N> .N> .N> omﬂo>< omwuo>< _ N 033. :manmmm 33 920 238 .065 3:0 $36 .236 séméo .235. . . m Substltute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 201 3.0 86 H um Rd 9% 2533 2.3:: 3a mg a a £9 m? 2.013 0.333 2.“ 25 a a m; mg 8632 8.32: :3 8.0 a a 2.: «3 o o :4 Rd ﬁtnv a 8.” 33m... 25:: MS a 32 o o o. 2.2%me 980 38 38 Substitﬁte Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 202 m dobcoov Emummmﬁg wogotawb : b30355 E<UQ+H§+QE Qoaao Q_a~x~ qoamo aOHVO Qoﬁoo ‘vdams ndavd_ ﬁdaod ogamdm hoam_~ ﬂoawN Qoamo ,QOﬁmd odamd <o«m_ ﬁoumog 85mm: «m2 33:8323 Haﬁz—$3 _daNd mdavaw adamd Qoawo _d%md cdan;~ ,Ndamc Qoamo mdaawm voﬁﬁw_ ~dawe Qoamo ﬂoaﬁd odaﬁd adama woavﬁ .2 E 30.:on Evian aoaoc mgﬁahav ‘mdamo Ndaad «came oaaas \mdama “sand oaahaﬁ mdﬂamgw noavﬁ 9o. 90 QC. woawo Noano Ox? mo aoEmomEoo .msﬁmnoo Sm Nuaqm ﬁwﬁwév q~am~ o5 od hvahﬁ mgamw 9c avan: caamd Noﬁhm ed ad od wean" odawd , , Eon 359mm .\ 53m .5 28 03D 585 3m 9:0 920 .2:20 2:20 2.~_.2mu20 920 gwﬁo ,_2:20 «22650 Béimnwﬂo oému :téﬁu .._._a§8 2.3.:qmuomo o&mu 9X6 lll'llllll'l Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ' 203 Table 12. Com anson ofWRIl + DGATl to ether with the sin - le enes -__— _—_ Based on the individual effects of both DGATl and WRIl genes upon expression in N. benthamiana, in the ce of merely an ve effect but the absence of any synergistic , the present inventors expected a TAG level of about 0.35 or a 35-fold increase compared to the P19 negative control. However, the uction of both genes resulted in TAG levels that were 129-fold higher than the .

P19 control. Based on these results, the present inventors estimated the additive effect and the synergistic effect on TAG accumulation as 26.9 % and 73.1 %, respectively. 10 In addition, when the fatty acid composition of the total lipid in the leaf s was analysed by GC, a synergistic effect was observed on Cl8:lA9 leVels in the TAG fraction ofN. benthamiana leaves inﬁltrated with WRll and DGATl (3 repeats each).

The data is shown in Table _1 1. _ For seed-speciﬁc expression of the WRIl + DGATl combination, Arabidopsis 15 thaliana was transformed with a binary vector construct including a chimeric DNA - having both pFAEl ::WRII and pCln2::DGAT1 genes, or, for comparison, the single genes pFAEl::WRll or pCln2::DGATl. Tl seeds were harvested from the plants.

The oil t of the seeds is determined. The seeds have an increased oil content. 20 Example 12. Constitutive expression of a monoacylglxcerol acyltransferase and WRIl transcription factor in plant cells A chimeric DNA encoding the Mus musculus MGAT2 (Cao et al., 2003; Yen and , 2003) and codon-optimised for B. napus was synthesized by Geneart. A genetic construct designated smu-MGAT2 was made by inserting the entire ' 25 coding region of 1022341_MusmuMGAT2, contained within an EcoRI fragment, into 3 at the EcoRI site, generating pJP3347. Cloning of the SSSzArath-WRII construct is described in Example 11. Transient expression in N. benthamiana leaf tissue was performed as described in Example 1. _ When the mouse MGAT2 and the A. thaliana WRll transcription vector were 30 coexpressed, average N. benthamiana leaf TAG levels were increased by 33—fold compared to the expression of WRIl alone (Table 13). In addition, the expression of the two genes resulted in a small (29 %) istic effect on the accumulation of leaf D .

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 204 TAG. The TAG level obtained with the MGAT2 gene in the presence of WRIl was 3.78 % as quantiﬁed by Iatroscan (Figure 12). The similar results ed with the animal MGAT2 and plant DGATl- acyltransferases in combination with the A. thaliana WRIl suggests that a synergistic effect might be a general enon when WRI and acyltransferases are overexpressed in non-oil accumulating vegetative I plant tissues.

. The experiment was repeated to introduce constructs for expressing T2 compared to V2+MGAT2-FWRII , such that inﬁltrated leaf samples were pooled across three leaves from the same plant, for two plants each. In total, each 10 combination therefore had 6 replicate inﬁltrations. This d a smaller standard deviation than pooling leaf samples between different plants as was done in the ﬁrst experiments. The data from this experiment is shown in Table 14. Earlier results (Table 13) were conﬁrmed. Although absolute TAG levels are different (inherent to the Benth assay and also different pooling of samples), relative se in TAG when 15 WRIl is co-expressed with V2+MGAT2 are r (2.45— and 2.65- fold).

Example 13. Constitutive expression of a monoacylglycerol acyltransferase, diacylglycerol acyltransferase and WRII transcription factor in plant cells The genes coding for the A. thaliana diacylglycerol acyltransferase DGATl, 20 the mouse ylglycerol acyltransferase MGAT2 and the A. thaliana WRIl were sed in different combinations in N. .benthamiand leaf tissue according to the transient expression system as described in Example 1. A detailed description of the different constructs can be found in Examples 11 and 12.

The combined sion of the DGATl, WRII and MGAT2 genes resulted in _ 25 an almost 3-fold further average TAG increase when compared to the expression of the latter two (Table 15). The maximum observed TAG yield ed Was 7.28 % as quantiﬁed by Iatroscan (Figure 12). Leaf TAG levels were not signiﬁcantly affected When the gene of the mouse MGAT2 acyltransferase was left out this combination.

Results described in Example 16, however, clearly demonstrated the positive effect of 30 the mouse MGAT2 on the biosynthesis of l lipids in N. benthamiana leaves when expressed in combination with WRIl, DGATl and the Sesamum m oleosin protein.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 205 .53an 3d m: 93 N a a 8.52m: 23 cam 3d ~ gummm . . , . as, .x. 8.0 2: a « 858%: c5 3.” mam . r2225 832 N56: N202 gaming x0 .55 .55» 3089; mu>m2 880 .9 .~> ~3 05mm 3353523 05 _ 3.5ng no E, . mm $328.2 c 832 x Rd o; maomtsmﬁoo a a 28:25 05 8d 02 83:55 €643.38 a no 222 388va 05 sougEEoo 025 woman we ova .2 S52 3.5200 5S, 93D .9 .9 uwﬁo>< owgo>< _ N m . Subsutute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 206 . 8.0 one 3% a ”ma , _ . . . . as :8 “mam a _ «3 $52 SE: . .55, A . .55» . .81 . 32““: 8mm ,9 Essa :3 ~e<oo 58853 an mmB 06am 2: 33:33qu . ‘ . ? so t mm .2 3o 02 328— anemia—boo 33.555 . 33. Um t NE 688:9: no oEmSnéEm 3880 Eobmbu .co . $462 no.3“ 3ch O<H 533588 53o . mo can“ .3 on. .53» 423 935. , .9 .9 owﬁo>< owSo>< H N Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 207 Additional data was obtained ﬁ‘om a further experiment where leaf samples were-pooling across leaves within same plant, 6 replicates of each. The data is shown p ' in Table 16. \ 5 Table 16. TAG content of inﬁltrated N. benthamiana leaf sam les.‘ TAG %d W t. ’m V2+MGAT2+DGAT1 1.08 d: 0.1 2.06 V2+MGAT2+DGAT1+WRII 2.22 i- 0.31 . 4 .

Example 14. Constitutive expression of a monoacylglycerol acyltransferase, diacycerol acyltransferase, WRIl transcription factor and glycerolphosphate acyltransferase in plant cells , 10 A 3SS:GPAT4 genetic construct was made by cloning the 1A. thaliana GPAT4 gene (Zheng et al., 2003) from total RNA isolated from developing siliques, followed i by insertion as an EcoRI fragment into pJP3343 resulting in pJPéS44. Other constructs are described in Examples 1] and '12. Transient expression in N. benthamiana leaftissue was med as described in Example 1. 15 Transient expression of the A. thaliana GPAT4 acyltransferase in combination with MGATZ, DGATl and WRIl led to a small se in the N. benthamiana leaf TAG content as quantiﬁed by Iatroscan (Table 17). The TAG level (5.78 %) was also found to be lower when GPAT4 was included in the inﬁltration mixture (Figure 12).

However, this ﬁnding does not rule out the hypothesis of sn2-MAG synthesis from 20 .,G3P as catalysed by the GPAT4 acyltransferase. , it ts that this catalytic step is unlikely to be rate ng in leaf tissue due to the high expression levels of the / endogenous GPAT4 gene (Li et al., 2007). Moreover, the A. thaliana GPAT8 acyltransferase ys a similar expression proﬁle as GPAT4 and has been shown to exhibit an pping function (Li et al., 2007). In developing seeds the expression 25 levels of GPAT4 and GPAT8 are low. As a result, coexpression of GPAT4 in a seed _ , context might be crucial to ensure sufﬁcient an-MAG substrate for a heterologous expressed MGAT acyltransferase.

Substitute Sheet ‘ ‘ . (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 208 .Eﬂunnmmm .S<o2”mmm m; mg H a Rd 5: ”9&2 £3, ..

Ragga , o3 VS a a :4". 2:“ beams: "N56: ago: 2%: E50 EEG $2 .53» .55 u§§s§ “9 4559 .9 4,559 . 3380.2 . 28:? 3.0 Ed u a 3+ . Sum a 232 3&0”me .852 .E<oa . oi .

Ea . . .5. 5.53588 .53» 4:5 2.3. 533D SEED ﬁg E<oa .9 ESE , Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 209 onal data was obtained from a ﬁirther experiment where leaf samples were pooling across leaves within same plant, 6 replicates of each. The data is shown ' " ‘ in Table 18. 5 Table 18. TAG content of inﬁltrated N. benthamiana leaf 831110168. ' » ‘ Gene combination TAG % d wei t ’ " T2+DGAT1 1.54 :i: 0.36 1.01" V2+MGAT2+DGAT1+GPAT4 1.56 i 0.18 Example 15. Constitutive expression of a monoacylglycerol acyltransferase, diacycerol acyltransferase, WRIl transcription factor and AGPase-hpRNAi silencing construct in plant cells . ' 10 A DNA fragment corresponding to nucleotides 595 to 1187. of the mRNA encoding the Nicotiana tabacum AGPase small subunit (DQ399915) (Kwak et 'al., 2007) was synthesized. The 593 bp 1118501_NtAGP fragment wasﬁrst cut with Ncol, treated with DNA polymerase I large (Klenow) fragment to te 5' blunt ends and ﬁnally digested with Xhol. Similarly, the pENTRl l-NCOI entry vector was 15 ﬁrst digested with BamHI, treated with DNA rase I large (Klenow) fragment and cut with Xhol. Ligation of the 1118501_NtAGP insert into pENTRll-NCOI generated the pENTRl l-NCOI-NtAGP entry clone. LR recombination between the ' pENTRll-NCOI—NtAGP entry clone and the pHELLSGATE12 destination vector generated pTV35, a binary .vector containing the NtAGPase RNAi cassette under the 20 control of the 358 promoter. Other ucts are described in es 11 and 12.

Transientkexpression in N. benthamiana leaf tissue was performed as described in Example 1.

Expression of- the N. tabacum AGPase silencing construct together with the genes coding for MGAT2 and WRI resulted in a 1.7-fold increase in leaf TAG levels 25 as quantiﬁed by Iatroscan (Table 19). In the absence of the MGAT2 acyltransferase TAG levels dropped almost 3-fold. Thereforethe ed TAG increase cannot be attributed solely to the silencing of the endogenous N. miana AGPase gene.

Surprisingly, tuting MGAT2. for the A. thaliana DGATI did not alter TAG levels in inﬁltrated N: benthamiana leaves in combination with the N. tabacum 30 AGPase silencing construct. Silencing of the N. miana AGPase therefore s to have a ent metabolic effect On MGAT and DGAT acyltransferases. A ' similar difference is also observed in the maximum observed TAG levels with WRII and the AGPase silencing construct in combination with MGAT2 or DGATl yielding 6.16 % and 5.51 % leaf oil respectively (Figure 12).

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 210 JF<OQHmmm 32%: 05am .NH<OEUmmm 93 a; So 2: a a no a a 28m of ., . :2 85 . ﬁm>nmmm . gaming—:8 _ a .

HEB «03 oEm$néEm vogom‘mgb. u Mad w: . .x., So no ﬁ a a O<H 8.... 8m Re woman— 362895 552 ﬁg: .Eéa orﬁ omﬁo>< cams @038 N .32 . xo .55» manomobﬂ ﬁg .5; . 35:52:23 ,9 .9 Ezmﬁéﬁg 8:00 Ezmﬁbmﬁg .9 %£o< 3 . _ a vows—83 a=5382 3 mo? , c MS and ad 3.32 382%.: .x. a a a O<H 92 a: . a? . 5 33.555 V £32, M<de£éwmm0<nmmm . 3&3 28826 04:. can $32 , E<oa 025 \ we .3 Godwin—Eco Smaummm .55, .SE 2sz .SE ozn 3.580 .9 “9. .N> owﬁo>< _ Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 211 pression of WRI] and MGAT in combination with AGPase silencing is particularly promising to increase oil yields in starch accumulating tissues. Examples include tubers such as for potatoes, and the endosperm of cereals, potentially leading to cereals with increased grain oil content (Barthole et al., 2011). Although N. tabacum andN. miana AGPase genes are likely to bear signiﬁcantsequence identity, it is likely that a N. benthamia/na AGPase-hpRNAi uct will further elevate TAG yields due to improved silencing efﬁciency.

Additional data was obtained from a further experiment where leaf samples 2 were pooled across leaves within same plant, 6 ates of each. The data is shown '10 in Tables 19 and 20.

Table 20. TAG content of inﬁltrated N. benthamiana leaf samles V2+MGAT2+DGAT1+WRII+Oleosin 1.93 :t 0.18 V2+MGAT2+DGAT1+WR11+Oleosin+AGPase- 2.19 :1: 0.19 h_.RNAi ' .

Exam le 16. Constitutive ex n of a monoac l l cerol ac ltransferase diac cerol ac ltransferase WRIl transcri tion factor and an oleosin rotein in 15 plant cells A‘ pRShl binary vector containing the gene coding for the S. indicum seed oleosin (Scott et a1., 2010) under the control of the 358 promotor was provided by Dr. .

N. Roberts earch Limited, New Zealand). Other constructs are described in Examples 11 )and 12. Transient expression in N. benthamiana leaf tissue was 20 performed as described in Example 1.

When the sesame oleosin protein was expressed together with the A. thaliana WRI transcription factor and M. us MGAT2 acyltransferase,‘ TAG levels in N. benthamiana leaves as quantiﬁed by Iatroscan were found to be 2.2-fold higher (Table 21). No signiﬁéantchanges in the leaf TAG fatty acid proﬁles were detected (Table ' 25 22). A small increase in TAG was also observed when the A. thdliana DGATl acyltransferase was ed. COmpared to the V2 negativeControl, the combined eXpression ofWRIl, DGATl and the sesame oleosin protein resulted in a 3-fold TAG increase and a maximum observed TAG IeVel of 7.72 % (Table 21 and Figure 12).

LeafTAG levels were further elevated by a factor of 2.5 upon including the MGAT2 30 acyltransferase. This ponded to an average of15.7 % and a m observed of 18.8 % TAG on a dry weight basis. The additional se in leaf TAG when D J Substitute Sheet (Rule 26) RO/AU \l [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 212 p MGAT2 was included clearly trates the positive effect of this acyltransferase on the biosynthesis and accumulation of l lipids in transgenic leaf tissues.

. The ment was repeated with the combination of genes for expressing V2 ' and V2+MGAT2+DGAT1+WRIl+Oleosin, tested in different N. benthamiana plants, with samples pooled acress leaves from the same plant and with 12 replicate inﬁltrations for each. The data is shown in Table 23.

‘ Replicate samples were also pooled across leaves from same p1'ant,with 6 repeats for each inﬁltration: The data is shown in Tables 24 and 25.

Although inﬁltration of N. miana leaves resulted in increased levels of 10 leaf oil (TAG), 'no signiﬁcant" increase in the total lipid content was detected, suggesting that a redistribution of fatty acids from different lipids pools into TAG Was occurring. In contrast, when the MGAT2 gene was coexpressed with the DGATl, WRIl and oleosin genes, total lipids were increased 2.21-fold, demonstrating a net' increase in the synthesis of leaf lipids. 15.

Example 17. Constitutive expression of a monoacylglycerol acyltransferase, glycerol acyltransferase, WRIl transcription factor and a FAD2-hpRNAi ing construct in plant cells .

, A N. benthamiana FAD2 RNAi cassette under the control of a 358 promotor 20 was obtained by LR recombination. into the pHELLSGATES destination vector to te vector pFN033. Other ucts are described in Examples 11 and 12.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 213 .Eénumwm "Neézummm o: 33 as :.o 8.3 ME mom a a a a . a . cam a: N3 8; v2 ”33 a: gum: . , ‘ . 5.5» . . . 85583“ ed o: R; . v: 93 o? a ﬂ a a , a N3.“ n. #2 and 86 “33 ”in RM v3 @535: “~56: .S<oz .

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So on 8.32.5. a: ”3 8.0 o: «no «3 mg 3mm: ESQ a «2.2 a a m a .« a a o Q}: 533$. .9 8.0 $2 on” 8.0 m3 N3 3.0 :32 . . 55 . 4,559 33882.. $23 36 85 3.? 2.3 86 x: 8.0 £3 one 26 £8 Gmmooﬁo a a. a a um a « a 28:95 .9 No.0 Ed 3.3 mg 8.2 8.2 aid 3: 3m Gd :dao: who . go 3an .Emoﬂonmmm E<oo .53, 36 85 NE 2; «no Bus :5 «2 ”S can. N2 85 a; a a a a a a a a a a 35 nod «3 $2 one a; 2% 85 mm: £22. .9 on.” 8.32; 85 Oz: 235%? ~ .NN “and 4.559%? Boa 33m 9:0 ELHED 920 ELHSD oumﬁo Sﬁwﬂo :qﬁwHU N535 2.2..2mnwﬁo onomo. OHNND ouvmo I'll-Ill Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk . 215 Table 23. TAG content of inﬁltrated N. benthamiana leaf samles.

T_AG°...weitM __.__o.19io.os __18.74 V2+MGAT2+DGAT1+WRIl+Oleosin 3.56 i 0.86‘ Table 24. TAG content of inﬁltrated N. benthamiana leaf sam-les. ' 5 Table 25. Total fatt acid content of inﬁltrated N. benthamiana leaf sam. les.

“W 1 V2+MGAT2 3 28 :l: o 33 T2+DGAT1+WRII+Oleosin, 6 88 :t 0 37 The genes coding for the mouse monoacylglycerol acyltransferase MGATZ, A. thaliana‘ diacylglycerol acyltransferase DGATl, A. na WM] and a N. benthamiana FAD2 A12-fatty acid desaturase n RNAi construct (Wood et al., 10 manuscript in preparation) were expressed in combination in N. miana leaf tissue using the ent expression system as described in Example 1.

Similar changes were ed in the fatty acid compositions of TAG, polar lipids and TFA of N benthamiana leaves inﬁltrated with WRIl, MGATZ, DGATl and the Fad2 silencing contruct (Tables 26—28). In all three lipid fractions, oleic acid ‘15 levels were further increased and reached. almost 20 % in polar lipids, 40 % in TFA and more than 55 % in TAG. This increase came mostly at the expense of linoleic acid reﬂecting the ing effect on the endoplasmic reticulum FAD2 A12- desaturase. Leaf TAG alSo contained less olenic acid while levels in TFA and ' polar lipids Were unaffected. 20 When these experiments were repeated and the fatty acid compositions determined for TAG, polar lipids and total lipids, the results (Table 29) were consistent with the. ﬁrst experiment. " Substitute Sheet - (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 2‘16 .mhiuﬁummm 22%;- Nam fax/”m9” HEB JE<OQ h E .Zg 2.386 NE N; H :.3 36 .26 :3 a So 3.0 36 bacomwcg. 5136 3:1”? a :5 H a. a a a .N> 35. Ga SS Rd :2 $5 as 85 05mm: .32 ~H<OD u§§uﬁ§e .2252 33:82 .EMB NS a: 8.0 a; m3 go omd 3.0 a H So a 8.0 36 8.0 um a a a a a .H .H .N> 25 8% Ra imam ”do 23 mNdN Eﬁ m? «.3 35 Ed 8 ‘ SEE mo 8&on 28 .w<zmﬁ-ma<mnmm3§ 3.0 93 3: 3.: NS 2.2 a a 93 a H a 55 :maummmhﬁénum a ~> wmﬂo 33 , _ o H2 3.3 o can 83 ammo 05 .3 Eon 2.. 58m 238 920 236 3:0 23:0 .236 Sago m_.~_.o<mnw_0 .058 £38 38 OHVNO Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 217' aN>Hmwm HEB a—ab Ezmﬁ.wm<mh§<oo biommnab 53: 253 J23 :5 So 85 who 8d N3 mm; Ed u 86 a .4“ a a a a « a .32 g. o 32 an 84 32 So 8w 8.3 3o o o :3 anuwsuﬁtg 323382 Ego 3823b .362 .Engwnﬁnmﬂ .53» 2.0.13 Ed 93 Ed o3 So MS cm; 26 25 2.0 50% a a a a a a a a a .9 3.2 a; mod $2 Ed 3.2 “2.3 m3 .o :5 Ed @328“ 3E: c5 “Eon mo 55%? 8589 omo .§<oanmmm 832.0. mac :5 NS was 3.0 x: 25 2.9 « a u u a a H a Boa 9 8.2 03 “in NE mac 3.2 $30 mod o o 86 _ 33m EN £20252 28 03D 32:0 066 236 36 a; 23w 2.23 5a 8 6 8 2.2.236 38 :158 38 38 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 218 .N>Nwmm 5E €2m$8§ BEBE“: ESQ bucommﬁb $52 03mm: .59» Nod mg 8.9 85 «3 «2 4m a 86% 35 86 a 86 :3 86 H on: a a 3.8 H a a $9” H H .32 “~> 3.6 mg n_.v who 3+ 8o 8.0 26 8.0 u§§uﬁ§e 33:82 HH<OQ 8.8sz .352 So 8+ 91 88% .M<ZMQ:-NQ<mHmmm in? So No.0 86 «mm a 85 a So 8.0 8.0 8o H a % 2.2. H a a 8.8 mm: a mtmm a a t .9, Rd 383$ vmd m? $5 3o £8. 0.3 26 «in: was 38 mo :Mkrummm 8an mod a: So 3o Fe 86 as .CQOQmem VS . a H a .26 a «3 aod w a % a H H a N> 8.2 Boa 8.0 8N 3». 2.: 28 3.: 8.8 .35 o 3.0 wmd beam .3 How 93.; .N,H<Ozum ban 2:30 one—U 9:65 cums oinwa 22:9 2.2520 2.~_.a<muw_U 058 238 38 38 Substitute Sheet ' (Rule 26) RO/AU' [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk ed set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk . $53:. w; ﬁ . _ 9% 8.385 Seaman.. .2332 OEOHWmA: ~H<OQ+NH<OE+N>. ‘ . $92:.. g 3.393 mmdﬁﬁuv <OQ+NH<O§+N> . 26 a mmdﬂ “ma E 2.2 OD+~H<O§+N> 36 $3.

Q+NH<OE+N> +=M>9+HH<O m2 E820, « 36 $633 3:0 33:0 .

S.n h.e 8 2 wk0A/n [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 220 50.0 0N6 H." ﬂ 3.0 hm o wwwo mmd H um ﬁnd “Nd ~OHNNU OHVNO Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 221 Example 18. Expression of Mus musculus MGATI and MGAT2 in Nicotiana bentliamiana cells by stable transformation Constitutive expression in N. benthamiana ' The enzyme ty of the MI musculus MGATl and MGAT2 was demonstrated in Nicotiana benthamiana. The chimeric vectors smu-MGAT’1 and 3SSzMusmu-MGAT2 were introduced, into A. tumefaciens strain AGLl via standard electroporation procedure and grown on solid LB media supplemented with ' . kanamycin (50 mg/L) and icin (25 mg/L) and incubated at 28°C o days.

A single colony wasused to initiate fresh culture. Following 48 hours culturing with vigorous aeration, the cells were collected by fugation at 2,000x g and the supernatant were removed. The cells were resuspended in a new solution containing ‘ 50 % LB and 50 % Ms medium at the density of 0D600 =o.5. Leaf samples of Nicotiana benthamiqna plants grown asceptically in vitro were excised and cut into square sections around 0.5-1 cm2 in size with a sharp scalpel while immersed in the A. tumefaciens solution. The wounded N. miana leaf pieces submerged in A., tumefaciens were allowed to stand at room temperature for 10 min prior to being blotted dry on a sterile ﬁlter paper and transferred onto MS plates without supplement. Following a co-cultivation period of two days at 24°C, the explants were washed three times with sterile liquid MS medium, and ﬁnally blot dry with sterile ﬁlter paper and placed on the selective agar-solidified MS medium supplemented with 1.0. mg/L aminopurine (BAP), 0.25 mg/L acetic acid (IAA), 50 mg/L kanamycin and 250 mg/L cefotaxime and incubated at 24°C for two weeks to allow for shoot development from the transformed N. ‘benthamiana leaf discs. ‘To establish in vitra transgenic plants, healthy green shoots were cut off and transferred onto a new _ 200 mL tissue e pots containing agar—solidiﬁed MS medium supplemented with 25 [lg/L IAA and 50 mg/L kanamycin and 250 mg/L cefotaxime.

Expression of the MGATl and MGAT2 transgenes was determined by Real- Time PCR. Highly-expressing lines were selected and their seed harvested. This seed ' was planted directly onto soil and the ating population of seedlings harvested after four weeks. Highly-expressing events were selected and seed produced by these d out directly onto soil to result in a segregating population of 30 seedlings.

After three weeks leaf discs were taken from each seedling for DNA extraction and subsequent PCR to determine which lines were transgenic and which were null for the transgene. The population was then harvested with the entire aerial tissue from each seedling cleaned of soil and freeze-dried. The dry weight of each sample was recorded and total lipids isolated. The TAG in these total lipid samples was quantiﬁed by TLC-FID and the ratio of TAG to an internal rd (DAGE) in each Dple determined (Figure 14). The e level of TAG in the transgenic seedlings Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 222 I of 3SSzMusmu-MGAT2 line 3347-19 was found to be 4.1-fold higher than the average level of TAG in the null seedlings. The event with the largest increase in TAG had 73-fold higher TAG than the average of the null events.

Constitutive expression in A. thaliana The enzyme activity of the M. musculus MGATl and MGAT2 was demonstrated in A. thaliana. The chimeric vectors 3SSzMusmu-MGAT1 and . 3SSzMusmu-MGAT2 along with the empty vector control pORE04 were transformed in A. thaliana by the ﬂoral dip method and seed from primary transformants selected on kanamycin media. The T2 seed from these T1 plants was harvested and TFA of the seeds from each plant determined-(Figure 1-5). The e mg TFA/g seed was found to be 139:13 for the control pORE04 lines with median 136.0, 152:14 for the 35$:MGAT1 lines with median 155.1 and 15:11 for the AT2 lines with median 154.7. This represented an e TFA increase cOmpared to the control of 9.7 % for 3SS:MGAT1 and 12.1 % for 3SS;MGAT2. ' Example 19. Additional genes I ' Further increasesin oil ‘ Additional genes are tested alongside the combinations described above to determine whether further _oi1 increases can be achieved. These include the following Arabidopsis genes: AT4G02280, Sucrose synthase SUS3; ATZG36190, Invertase CWINV4; AT3G13790, Invertase ‘ ; 800, Glucose 6 phosphatezphosphate translocator GPTZ; ATSG33320, Phosphoenolpyruvate transporter PPTl; AT4G15530, Pyruvate orthophosphate Vdikinase Plastid-PPDK; 920, Pyruvate kinase pPK—Bl. The genes coding for these enzymes are synthesised and cloned into the constitutive binary expression vector 3 as EcoRI fragments for testing in N. benthamiana.

When a number of genes were added to the combination of WRII, DGATl, MGAT2 and oleosin and expressed in N. benthamiana , no additional increase in the level ofTAG was observed, namely for: safﬂower PDAT, Arabidopsis thaliana PDATl, Arabidopsis thaliana DGATZ, Arabidopsis thaliana in, peanut oleosin, Arabidopsis thaliaria haemoglobin 2, Homo sapiens iPLAh, Arabidopsis thaliana GPAT4, E. cali G3P ogenase, yeast G3P dehydrogenase. castor LPAAT2, Arabidopsis na beta-fructoﬁiranosidase (ATBFRUCTI, NM_112232), Arabidopsis na beta-fructoﬁiranosidase (cwINV4, NM_129177 ), indicating that none of these enzyme activities were rate ng in N. benthamiana leaves when expressed transiently. This does not indicate that they will have no effect in stably- Dformed plants, such as in seed, or in other organisms.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 223 Further additional genes are tested for additive or synergistic oil increase ty. These include the following opsis thaliana gene models or their encoded proteins, and homologues from other species, which are grouped by putative on and have previously been shown to be upregulated in tissues with increased oil content. Genes/proteins involved in sucrose ation: ATlG73370, AT3G43190, AT4G02280, AT5620830, ATSG37180, AT5G49190, AT2G36190, 784, ATSGI3790, AT3G526’00. Genes/proteins involved in the oxidative pentose ate pathway: AT3GZ7300, 760, AT1G09420, AT1G24280, 110, ATSG35790, AT3GOZ360, ATSG41670, ATlG64190, AT2G45290, 750, AT1612230, AT5G13420, ATlGl3700, ATSGZ4410, AT5G24420, AT5G24400, AT1663290u 850, AT5G61410v ATlG71100, AT2601290, AT3G04790, AT5G44520w AT4G26270, AT4G29220, AT4G32840, 810, ATSGS6630, AT2G22480, ATSG61580, AT1G18270,’ATZG36460_, AT3GSZ930, AT4G2653'0, AT2G01140, AT2G21330, AT4G38970, AT3GSS440, ATZG21170.

’ Genes/proteins involved in glycolysis: AT1G13440, AT3G04120, AT1G16300, ATlG7§530, 550, AT3G45090, AT5G60760, ATlGS6l90, AT3GlZ780, 450, ATIGO9780, AT3608590, AT3G30841, AT4GO9520, AT1622170, ATlG78050, ATZG36530, AT1G74030. Genes/proteins which function as plastid transporters: AT1661800, AT5G16150, AT5G33320, ATSG46110, AT4GlSS30, ATZG36580, AT3G52990, AT3655650, AT3GSS810, AT4G26390, AT5G08570, AT5G56350, AT5G63680, 440, AT3G22960, AT3G49160, AT5652920.

Genes/proteins involved in malate and pyruvate metabolism: AT1G04410, ATSG43330, AT5G5_6720, ATIGS3240, AT3GlSOZO, AT2622780» 660, AT3G47520, 330, AT2G19900, AT5G11670, AT5625880, AT2613560, AT4G00570.

Constructs are prepared which e sequences encoding these candidate proteins, which are inﬁltrated into N. benthamiana leaves as in previous experiments, and the fatty acid content and composition analysed. Genes, which aid in increasing non-polar lipid-content are ed with the other genes as described above, principally those encoding MGAT, Wril, DGATl and an Oleosin, and used to ' transform plant cells. i Increases in unusual fatt acids Additional genes are tested alongside the combinations described above to determine whether increases in unusual fatty acids can be achieved. These include the ing genes (provided are the GenBank Accession Nos.) which are grouped by putative function and homologues frOm other species. Delta-12 acetylenases DCOO769, CAA76158, AAO38036, AAO38032; Delta-12 conjugases AAG42259, Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 224 60, 74; Delta-12 desaturases, P46313, ABSl87l6, AASS7577, AAL61825, AAF04093, AAF04094; Delta-12 epoxygenases XP__001840127, .

CAA76156, AAR23815; Delta-lZ’hydroxylases ACF237O70, 55, ABQOlltSS, AAC49010; and Delta-12 P450 s such as AF406732.

Constructs are prepared which include sequences encoding these candidate proteins, which are inﬁltrated into N. benthamiana leaves as in previous experiments, and the fatty acid content and composition analysed. The nucleotide sequences of the coding regions may be codon—optimised for the host species of st. Genes which aid in sing unusual fatty acid' content are combined with the other genes as _ described above, principally those encoding MGAT, WRIl, DGATI and an Oleosin, ‘ and used to transform plant cells.

K Example 20. Stable transformation of plants including Nicotiana tabacum with combinations of oil increase genes .

An existing binary expression , +11ABGBEC (US Provisional Patent Application No. 61/660392), which contained a double er-region 35$ promoter expressing the NPTII kanamycin resistance gene and three gene expression cassettes, was used as a starting vector to prepare several contiucts each containing a ation of genes for sstable transformation of plants. This vector was modiﬁed by exchanging the expressed genes with oil increase genes, as follows. +l lABGBEC was ﬁrst modiﬁed by'inserting an -interrupted sesame oleosin gene, ﬂanked by Natl sites, from the vector pRShl-PSPl into the pORE04+l lABGBEC Noll sites to generate pJP3500 pJP3500 was then modiﬁed by inserting a codon-optimised DNA fragment encoding the A. thaliana WRLl gene into the EcoRI sites to generate 1 pJP3501 was further modiﬁed by inserting a DNA fragment encoding the type A. thaliana DGATI coding region, ﬂanked by AsiSI sites, into the AsiSI sites to generate pJP3502 (SEQ ID NO:409). A ﬁnal . modiﬁcation was made by inserting another expression cassette, consisting of a double enhancer-region 358 promoter expressing a coding region encoding the M. musculus MGAT2, as a Stul-Zral fragment into the SfoI site of pJP3 502 to te pJP3503 (SEQ ID ). The MGAT2 expression cassette was excised from pJP3347 at the StuI + ZraI sites. pJP3502 and pJP3503 were both used to stably transform N. tabacum as described below. By these constructions, pJP3502 contained the A. thaliana WRLl and DGATl coding'regions drivenby the A. thaliana Rubisco small subunit promoter (SSU) » and double enhancer-region 358 promoter, respectively, as well as, a SSUtsesame oleosin cassette. The T-DNA region of this construct is shown schematically in Figure 16. The vector pJP3503 additionally Gained the e3SSzzMGAT2 cassette. This construct is shown schematically in Figure .

' Substitute Sheet (Rule 26)'RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ,225 17. The nucleotide sequence of the T-DNA region of the construct pJP3503 is given as SEQ ID NO:412.

Stable ormation ofNicotiana tabacum with combinations of genes The binary vectors pJP3502 and pJP3503 were separately introduced into the A. tumefdciens strain AGLl by a standard electroporation procedure. Transformed cells were selected and grown on LB-agar supplemented with kanamycin (50 mg/l) and rifampicin (25 mg/l) and incubated at 28°C for two days. A single colony Of each i was used to initiate fresh cultures in LB broth. Following 48 hours incubation with vigorous aeration, the cells "were ted by centrifugation at 2,000 g and the supernatant was removed. The cells were resuspended at the density of OD600 =0.5 .in fresh medium ting of 50% LB and 50% MS medium.

, Leaf samples of N. tabacum cultivar W38 grown asceptically in vitro were excised with a'scalpel and cut into pieces of about 0.5-1 cm2 in size while immersed in the A. tumefaciens sions. The cut leaf pieces were left in the A. tumefaciens suspensions at room temperature for 15 s prior to being blotted dry on a sterile ﬁlter paper and transferred onto MS plates without antibiotic supplement. Followmg ’ a co-cultivation period of two days at 24°C, the explants were washed three times with sterile, liquid MS medium, then blotted dry with sterile ﬁlter paper and placed on the selective MS agar supplemented with 1.0 mg/L benzylaminopurine (BAP), 0.5 mg/L indoleacetic acid (1AA), 100 mg/L kanamycin and 200 myL cefotaxime. The plates were incubated at 24°C for two weeks to allow for shoot development from the I transformed N. tabacum leaf pieces.

To establish rooted transgenic plants in vitro, healthy green shoots were cut off and transferred to. MS agar medium supplemented with 25 rig/L IAA, .100 mg/L kanamycin and 200 mg/L cefotaxime. After roOts had developed, individual plants were erred to soil and grown in the glasshouse. Leaf samples were harvested at different stages of plant pment including before and during ﬂowering. Total fatty acids, polar lipids and TAG were quantiﬁed and their fatty acid proﬁles determined by TLC/GC as described in Example 1. ' ' n Analysis of pJP3503 transformants Fer the transformation with pJP3503 (“4-gene construct”), leaf samples of ' about 1 cm2 were taken from 30 y ormants prior to ﬂower buds forming and TAG levels in the samples were quantiﬁedby Iatroscan. Seven plants were _ ed for further analysis, of which ﬁve displaying sed leaf oil levels and two ' exhibiting oil. levels essentially the same as wild-type plants. Freeze-dried leaf Dues from these plants were analysed for total lipid content and TAG content and Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk 226 fatty acid composition by TLC and GC. Transformed plants numbered ‘4 and 29 were found to have considerably increased levels of leaf oil compared to the wild type, while plant number 21 exhibited the lowest TAG levels at essentially wild-type levels (Table 30). Plants ed 11, 15 and 27 had intermediate levels of leaf oil. Oleic acid levels in TAG were found to be inversely correlated to the TAG , consistent with the "results of the r transient expression experiments in N. benthamiana.

, . In the transformed plants numbered 4 and 29, leaf oil content (as a percentage of dry ) was found to increase considerably at the time of ﬂowering (Table 31).

From the data in Table 31, the increase was at least 1.7- and ld for plants 4 and 29, tively. No such change was observed for plant 21 which had TAG levels similar to the wild-type control. Oleic acid levels in the TAG ﬁ'actions isolated from each sample followed a similar pattern. This fatty acid accumulated up to 22.1% of the fatty acid in TAG from plants‘4 and 29, a l7-18—fold increase compared to plant , 21 and the wild-type. The increase in? oleic acid was accompanied by.increased linoleic acid and palmitic acid levels while a-linolenic acid levels dropped 8-fold compared to in plant 21 and the wild-type control. Unlike TAG, polar lipid levels decreased slightly at the ﬂowering stage in the three lines (Table 32). Changes in C18 monounsaturated and polyunsaturated fatty acid levels in the polar lipid fractions of the three lines were similar to the shifts in their'TAG composition although the. changes in oleic acid and linoleic acid were less marked. Signiﬁcant increases in total leaf lipids were observed for lines 4 and 29 during ﬂowering with levels reaching more than 10% of dry weight (Table 33). Total leaf lipid levels in plant 21 before and during ﬂowering were similar to levels observedin wild-type plants at similar stages s 33 and 35). Changes in the total lipid fatty acid composition of all three plants were r to the respective TAG fatty acid compositions. Leaf oil in plant 4 during seed setting was found to be further elevated at the onset of leaf chlor'osis. The highest leaf TAG levels detected at this stage ponded to a 65-fold increase compared to similar aged leaves in plant 21 during seed setting and a 130-fold increase compared to similar leaves of ng wild-type plants (Table 34; Figure 18). ' .

. The increased TAG in this plant ded with elevated oleic acid levels.

Unlike plant 4, leaf TAG levels in the other two primary transformants and wild-type tobacco did not increase, or only marginally increased, after ﬂowering and during chlorosis. The lower leaves of plants 4 and 29 exhibited reduced TAG levels upon senescence. In all plants, linoleic acid levels dropped while a-linolenic acid levels were sed with progressng leaf age.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 227 Consistent with the increased TAG levels, total lipid levels in leaves of plants 4' and 29 during seed setting were further elevated compared to similar leaves of both plants during ﬂowering (Tables 33 and 35). The highest total lipid level detected in plant 4 on a dry weight basis was 15.8 %, lent to a 7.6— and 11.2-fold increase ed to similar leaves of plant 21 and wild-type plants, respectively. Whilst the fatty acid composition of total lipid in' the leaves of the wild type plant and plant 21 were similar, signiﬁcant ences were observed-in plants 4 and 29. These changes ed those found. in TAG of both y transformants.

Intriguingly, leaves of plants 4 and 29 before and dining seed setting were characterized by a glossy surface, providing a ypic change that Can serve as a . phenotype that is easily scored visually, which could aid the timing of harvest for maximal oil content. , _ ‘ In summary, leaves of plants 4 and 29 rapidly accumulated TAG during ng up till seed setting. At the latter stage, the majority of leaves exhibited TAG levels n 7 % and 13 % on a dry weight basis, compared to 0.1% - 0.2% for line 21. These observed TAG levels and total lipid levels far exceed the levels achieved by Andrianov et al., (2010) who reported up to a maximum of 5.8% and 6.8%" TAG in leaves of N. tabacum upon constitutive expression of the A. thaliana DGATl and inducible expression of the A. thaliang LECZ genes. ' Table 30. Percentage TAG (% weight of leaf dry weight) and oleic acid levels (% of total fatty acids) in the TAG isolated from leaves of selected primary tobacco plants transformed with pJP3503 Plant No. %TAG (DW) % C183”~ Development stage _ i ' of «lant Wild {p8 . 2.3 Budding Wlld tre 1.3 Flowerin ; b: 0.05 1.5 Buddin 1.29 010.2 Buddin; ’NNNt—v— Urn-d 0.21 7.4 . Flowering 0.23 4.5 No buds I—a No buds - \O\) 0.) DJ Buddin_ ' 11 U! 10.4 No buds I Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 228 0838 .8898 an woaaa vﬁawea 0.0 do EN 08028 80059005 0.0 0.0 0.0 253 odaao H H HNA H H00 Qﬁanem voaﬁs ﬁdacm qoﬂmd quwo H N0 N0 ”.mN 0 H ~.0Hh.0 8:0 m-~ 000 00 0 «dams odaro qﬁaﬁam odawo womwm odﬁao .ﬁdanm wgaﬁaﬁ odawm ooamwm mdaw0_ odanﬁ Qoaﬂd 00H50 0.0 H 089-003 2830060 «:05 m0 ‘ 890 0000qu an 0.0 0.0 wan—h H 02280. a H 0.0 odﬂVo moaowﬂ odaqo odwwd ﬁdﬁwo odavm Ndaw; odﬁeo noamdﬁ teamsm odﬂwo goaﬁd 3 235 00HN.0 8 _.0 05 O<H H . 00 390096 , 0208088 98 5.532% m. ﬂ m N. .._. 0 mu 00 m0 0m 0N MA 0 haw mdm 0 0 O m0 030% 0; 800 H mHH NOH 2: 3 0.0 Eon H H one 0.0m m: ~N. 2 3 00HOA 0.0 30¢ 05 0005300 5.0 H n.0H 0.0H 0.0H 0.0 9:39.» H 05.80 NMON 0.2 04 InnIEIE! m0 0.0 C0 05 .002 HA wdH a 0.0 .«o Roman H 0.0H 0.0 a 3; H 3325 «:2.— VON 0.0m n893.3 0.0 g S 0.0HN.0 H ”5.530: 0 £90 . 0. £32 605 vacuum , y 30: 0:3 2. N v m m.N 00 0.0m 0:06 m0 “.0 N0 N O<H O 0 . .nm 030,—. 35D 3380:. ouv—U cue—U .2ﬁ0uu 2—"20 2.220"qu 930 «(EEO 02:30 aﬁunwa 2.2.2Q0HU 930 52.8 .2228 2.242930 930 0NVNU .

Subsutute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 229 mama 3093 8039 .5898 3823 Eat 3020ng 253 Wm mo HR: mo mconugov mwaz 83a ESE“; . we was cocmmomﬁoo owSmSm “5.833% 0.5 Eon 2a 33m 559% 98 San Gamma? Ev .mctogo¢ maE mo MES—u 3??» was £9 80.39 £95: Em: .83th .chuoBom 520m £39 Samoa E 3 «w .Nm o—Auhvogoumﬁn ~53 8* :<n.cNU Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 230 . ,conuoﬁcab E 3.0 Emma 06 od H H H m _ oond Nd v.0 8038 80¢ 828— 2.13 06H odHﬁo o.o od H H E 04 m.o md 33: :38 u mo .8383 0.0 3 ﬁo 06 {036388 $2 Eggs—m H H H H m6 No m; m6 Wm mo Bug owﬁgm . . baa 05 SHE. ﬁo 3 “.0 od . was 8a . H a H H o.“ _ m . o m6 o Gamma? 550% Ev «an waE o o ! mo $5.532“ od od H H H £395 g N. 0 md m6 u A5 3E6 332 USN 5.3.55 2809 271.." ed ed od 0.0 Ba: vacuum H a H H 3on 3:.“ «6 3 Nd N6 .mm £333 23%5 358:.x. cueNU aiguo anN0 9qu Substltute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 231 mo ,mcuoaoc . anon III1N md llm.wm mo .82“: .mSﬁOm 38 cows UV ﬁr Nd d.mm E! No mag“.

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XL 382. :83 coaﬂuﬁ £32 0838 359 O Md wd ‘mﬁ vd md ('1 {N m; _d EMNV mdm md O Nd Hd 0.83 528 O<H 3 @8023 a 838% mowSm .«52 .3” 33m 39 a So 035.and 353 amou— . 9:0 930 .ZWSU 2:30 Baxﬁmueﬁo mama .32 b?» “a ”a H a Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 232 .3? we 832 come baeoue ‘ matoaom 5% wESw no 8E2 ea: ,mctoaoa 33% 283 “a 85838 .ommEoﬁo ES @0832“ 55 9a .mommmm. 32:: Emma mag 5T5 Bum be :2? cab Ia 032:8 Ems; $51 § matsomua: 8032 o 2% Sana can @9028 ﬁo 0.: S 50¢ Boa 09:: . :83 .mm >99 was 33m and 0.: an»: haw—Ex. 9:0 930 «ZHHEU 335.3 ”a.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 233 Analysis of tobacco plants ormed with 2]P3502 For the transformation with pJP3502 (“3 gene construct”), the sucrose level in the MS agar medium was reduced to half the standard level'pntil sufficient calli were established, which aided the recovery of ormants expressing WRIl. Forty-one , primarytransformants were obtained from the transformation with pJP3502 and transferred to the greenhouse. Leaf samples of different age were collected at either ng or seed setting stages (Table 36). The plants look ypically normal except for three transformants, originating from the same callus in the transformation procedure and therefore likely to be from the same transformation event, which were 10 slightly r and displayed a glossy leaf phenotype similar to that observed for plant 4 with pJP3 503 (above) but less in .

Leaf disk samples of primarytransformants were harveSted during'ﬂowering and TAG was quantiﬁed visualized by iodine ng after TLC. Selected transgenic plants displaying increased TAG levels ed to thelwild type controls 'were 15 further analyzed in more detail by TLC and GC. The highest TAG level in young green leaves was detected in line 8.1 and corresponded to 8.3 % TAG on a dry weight basis or an approximate 83-fold increase compared to wild type leaves of the same age (Table 36). Yellow-green leaves typically contained a higher oil content compared to younger green leaves with maximum TAG levels observed in line 14.] 20 (17.3 % TAG on a, dry weight basis). Total lipid content and fatty acid composition I V of total lipid in the leaves was also quantitated (Table 37).

Seed (Tl seed) was ted‘from the primary transforrnants at seed maturity and some were sown to prOduce‘ Tl plants. These plants were predicted to be segregating for the transgene and therefore some null segregants were expected in the 25 T1 populations, which could serve as appropriate negative controls in addition to known wild—type plants which were grown at the same time and under the same conditions. 51 T1 plants, deriVed from primary transformant 14.1 which had a single- copy T-DNA insertion, which were 6-8 weeks of age and 10-25 cm in height were analysed together with ’12 wild-type plants. The plants appeared phenotypically 30 normal, green and healthy, and did not appear smaller than the corresponding wild— type plants. Leaf samples of about 1 cm diameter were taken ﬁom fully expanded green leaves. 30 of the T1 plants showed elevated TAG levels in the leaves, of which 8 plants showed high levels of TAG, about double the level of TAG ed to the primary transformant 14.1 at the same stage of plant development. These latter plants 35 are likely to be homozygous for the transgenes. The level of TAG and the TAG fatty acid ition in leaves of selected Tl plants were measured by g lipid isolated from about 5 mg dry weight of leaf tissue onto each TLC lane, the data is own in Table 38.

. Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 234 8238 c8023. Nﬂ > N». do SM do o.o do do v.2 do woN dNN 2 do o.o o.o Na Hz“ :2 Na u. do do EN do 3 do do SN 3 nmm 3 N do do o 3 md Sow d3 > no. do 2% o.o o.o do do 3 o.o dom 3H .N.N o.o o.o o.o 3 HA $32 d8 u. dm do N.MN do 3 do w 3N 3 mm. mo S do do o o.o 50 5on 2: > N do «on o.o 3 do Yo.m 3 do «:3 33 o.o o.o o.o o.o mo md Eob . «:3 u. m do E: No N.N‘ do dm .3 N mom EN Na do do o mo «.0 336$ Ned > ed do mNN do 3 do 3. m5 No nmm N2 ,N.N o.o do o.o 3 HA Oar—L Ni m m.N do doN no 3 no 3. d2 o.o 3m 3; dN no o. o N ad mo 2056388 d3 > d: do 3N o.o 3 do do MEN 3 2N o.o o.N o.o do o.o 3 02—” d3 m o... No NoN do S do 3 32 3 mom do «N do do o 3 HA. 34 n. 3 do NN do N do Ne MIN, do MEN o.o N do o o 3 o.o Boo . CC b3.“ N2 s as do EN, do 3 do o.o do 2m doN mN do do o.o 3 NA 82R: 98 N2 m o.o do o.oN do 3 do o dad: N mNm do N do do o, Na md. 9363. d2 > o.o . «.5 do o.oN do 3 do m.m in do d: NoN 3 o.o do o.o 5.0 330:0» be d8 8 u. No do ,oNN do 3 do o.o, dmm 3 wN o.o 3 mo o o do o.o mo>o2 .32 mo do > NN do 3m do 3 do o.o Ni mo o.Nm NS .2 do do o.o 3 HA. 30:?» 323m Emmoiﬁo 83% do u. m.» do 3N no 3 o.o m.m 2: Na mom «.2 N do do o Na md ABC . Eco mdN > m.N do o.oN do o.o do dN o.o mo 3m m: m do .do o 3 md 20>“; a? mam—39:8 manages Biemﬁo .292: 5w; 0449 A . to ESQ 6m 2 mdeE we N2 2.342 Bo magnum 2nd 353 2: ommﬂm we.» 38 38 38 238 38 38 238 N238 NdooNumU mdeoomaG 2.8 33 34238 38 onNNu 9ku b2, 88 u a m Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk with transformed plants tobacco selected from leaves yellow Cl8z3n3 DW fiom ted C18:2 lipid H total C18:l "'3 K0 of H composition NH me ".

H acid fatty (DGAT1+WRIl+Oleosin) . ”2'9"! HHH and ’5 o lipid Total 37. 502 Dble pJP3 (Rule 26) RO/AU 236 a H 0839 3823 88m mo>m2 Ihm m6 1m m4 Nd N; NA ﬁn TN gum 09 In 0') VI M N N NA 82m 3 68m HiQ.E.E.iiiE.iHIBIHIElEHI .

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.Nommmm V m XL g . £32 5MB E oém E E 0’2. .wm wagommﬁwb «Ssh £23. ! E I Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 237 Genetic constructs suitable for transformation of monocotyledonous plants are made by exchanging the Arath—SSU promoters in 2 and pJP3503 for promoters "more active in monocots. Suitable promoters include constitutive viral promoters from monocot viruses or promoters that have demonstrated to function in a transgenic context in monocot species (e.g., the maize Ubi er described by Christensen et al., 1996). Similarly, the CaMV-3SS promoters in pJP3502 and pJP3503 are exchanged for promoters that are more active in monocot species. These constructs are transformed into wheat, barley and maize using rd methods. 10 Miscanthus species Genetic constructs for Miscanthus species transformation .are made by exchanging the Arath-SSU promoters in pJP3502 and p1P3503 for promoters more active in 'Miscanthus. Suitable promoters include constitutive viral promoters, a ubiquitin prbmoter (Christensen et al., 1996) or ers that have demonstrated to 15 function in a transgenic t in Miscanthus. Similarly, the CaMV—3SS prombters in plP3502 and pJP3503 are exchanged for ers that are more active in Miscanthus. New constructs are transformed in Miscanthus by a microprojectile— mediated method r to that described by Wang et al., 201 l. 20 Switchgass (Panicum virgatum) ' / Genetic constructs for switchgrass transformation are made by ging the Arath-SSU promoters in .pJP3502 and pJP3503 for promoters more active in switchgrass. Suitable promoters include constitutive viral promoters or ers that have demonstrated to function in a transgenic context in switchgrass (e.g., Mann et 25 al., 2011). Similarly, the SS ' promoters in pJP3502 and pJP3503 are exchanged for promoters that are more active in switchgrass. New ucts are transformed in grass by an Agrobacterium-mediated method similar to that ~ described by Chen et al., 2010 and Ramamoorthy and Kumar, 2012. 30 Sugarcane c constructs for sugarcane transformation are made by exchanging the Arath—SSU promoters in pJP3502 and pJP3503 for promoters more active in sugarcane. le promoters include constitutive viral promoters or promoters that _ have demonstrated to function in a transgenic context in sugarcane (e.g., the maize . 35 ,Ubi promoter described by Christensen et al., 1996). Similarly, the CaMV-3SS ’ promoters in plP3502 and pJP3503 are exchanged for promoters that are more active in sugarcane. New constructs are ormed in sugarcane by a microprojectile— aiated method similar to that described by Bower et al., 1996.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 238 Elephant grass Genetic constructs for Pennisetum purpureum transformation are made by exchanging the Arath~SSU promoters in pJP3502 and 3 for promoters more active in elephant grass. Suitable promoters include constitutive viral promoters or I promoters that have demonstrated to function in a transgenic context in Pennisetum species such as P. glaucum like (e.g. the maize Ubi promoter described by Christensen et al., 1996). Similarly, the CaMV-3SS promoters in 2 and 3 are exchanged for ers that are more active in Pennisetum species; 10 New constructs are transformed in P. purpureum. by a microprojectile—mediated method similar to that described by Girgi et al., 2002.

Lolium Genetic constructs for Lolium perenne and other Lolium species 15 transformation are made by exchanging the Arath—SSU promoters in pJP3502 and 3 for ers more active in ryegrass. Suitable promoters include constitutive viral promoters or ers that have demonstrated to function in a transgenic context in Lolium species (e.g. the maize Ubi promoter described by . Christensen et al., 1996).. Similarly, the CaMV-3SS ers in pJP3502 and 20 pJP3503 are ged for promoters that are more active in Pennisetum species.

New constructs are transformed in Lolium perenne by a silicon carbide-mediated method similar to that bed by Dalton et al., 2002 or an Agrobacterium-mediated I I method similar to that described by Bettany et al., 2003. pJP3502 and pJP3503 are d to seed-speciﬁc expression genetic 25 constructs by exchanging the CaMV-SSS and Arath-SSU promoters (except the selectable marker cassette) with seed-speciﬁc promotersactive in the target species. 923% Genetic constructs for Brassica napus transformation are made by exchanging 30 the CaMV-3SS and SSU promoters in pJP3502 and pJP3503 for'promoters more active in canola. Suitable promoters include promoters that have previously ' been demonstrated to function in a transgenic context in Brassica napus (e.g., the A. thaliana FAEl promoter, 'Brassica napus napin promoter, Linum usitatz‘ssimum Vconlininl and conlinin2 promoters). New constructs are transformed in B. napus as 35 previously described.

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 239 ‘ Soybean {Glycine max! 'A genetic construct is made by cloning the PspOMI fragment from a sised DNA nt having the nucleotide sequence shown in SEQ ID NO:415 (Soybean synergy insert; Figure 19A) into a binary vector such as pORE04 at the Nail site. This. fragment contains Arath-WRII sed by a Arath-FAEI promoter, Arath~DGATl expressed by a Linus-Cn12 promoter, Musmu-MGAT2 expressed by Linus-Cull and Arath-GPAT4 sed by Linus-Cull. A further genetic construct is made by exchanging the GPAT coding region for an oleosin coding region. ‘A further c construct 'is made by ng the MGAT expression cassette. 10 A genetic construct, pJP3569 (Figure. 21), was generated by cloning the Sbfl- PstI fragment from the DNA molecule having the nucleotide sequence shown in SEQ ID NO:415 into the PstI site of pORE04. This construct contained (i) a coding region encoding the A. thaliana WRIl transcription factor, codon optimised for G. max expression, and expressed from the G. max kunitz n inhibitor 3 (Glyma-K'I‘i3) 15 er, (ii) a coding region encoding the Umbelopsis ramanm‘ana DGATZA (codon optimised as described by Lardizabal et al., 2008) and expressed from the G. max alpha-subunit beta-conglycinin (Glyma-b-conglycinin) promoter and (iii) a coding region encoding the M. us MGAT2, codon optimised for G. max expression. A second genetic construct, pJP3570, was generated by cloning the Sbjl-Swal fragment 20 of the DNA molecule having the tide sequence shown in SEQ ID NO:415 into pORE04 at the EcoRV-Pstl sites to yield a binary vector containing genes sing the A. thaliana WRIl transcription factor and U. ramanniana DGATZA enzyme.

Similarly, a third genetic uct, pJP3571, was generated by cloning the 'AsiSI nt of the DNA molecule having the nucleotide sequence shown in SEQ ID 25 NO:415 into the AsiSI site of pORE04 to yield a binary vector containing a gene encoding the U. ramanniana DGATZA enzyme. A fourth genetic construct, pJP3 572, was generated by cloning the NotI fragment of the DNA molecule having the nucleotide sequence shown in SEQ ID NO:4l 5 into pORE04 at the Not] site to yield a binary vector containing a.gene expressing the A. na WRII transcription factor. 3O A ﬁfth genetic construct, pJP3573, was generated by cloning the SwaI fragment of the DNA molecule having the nucleotide sequence shown in SEQ ID N02415 into pOREO4 at the EcoRV site to yield a binary vector containing the gene encoding M.

I musculus MGAT2.

A sixth genetic construct, pJP3580, is generated by replacing the M. musculus 35 MGAT2 with the Sesamum indicum oleosin gene.

Each of these six constructs are used to transform soybean, using the methods as described in Example 6. Transgenic plants produced by the transformation with Db of the constructs, particularly pJP3569, produce seeds with increased oil content.

Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 240 Sugarbeet ‘ I The vectors pJP3502 and 3 (see above) as used for the transformation of tobacco are used to transform plants of sugarbeet (Beta ‘vulgaris) by Agrobacterium—mediated transformation as described by Lindsey & Gallois (1990).

The plants produce greatly increased levels of TAG in their leaves, similar in extent to the tobacco plants produced as described above. Transgenic sugarbeet plants are harvested while the leaves are still green or preferably green/yellow just prior to beginning of senescence or early in that pmental process, i.e., and while the 10 sugar content of the beets is at a high level and aﬁer allowing lation ofTAG in the leaves. This allows the production of dual-purpose sugarbeets which are suitable for production of both sugar from the beets and lipid from the leaves; the lipid may be converted directly to biodiesel ﬁiel by crushing the leaves and centrifu'gation of the. resultant material to Separate the oil fraction, or the direct productibn of hydrocarbons 15 by pyrolosis of the leaf material.

. Promoters that are active in the root (tuber) of sugarbeet are also used to s transgenes in the tuber.

Example 21. Stable transformation of Solanum sum- with oil increase .‘20 genes pJP3502, the intermediate binary expression vector described in the previous e, was modiﬁed by ﬁrst excising one SSU promoter by ,AscI+NcoI digestion and replacing it with the potato. B33 promoter ﬂanked by AscI and NcoI to generate pJP3504. The SSU promoter in pJP3504 along with a fragment of the A. thaliana 25 WRLI. gene was ed at the PspOMI sites by a potato B33 promoter with the sMe A. na WRLl gene fragment ﬂanked by No‘tI-PspOMI to generate 6.

The pJP3347 was added to pJP3506 as described in the above example to generate pJP3507. This construct is shown schematically in Figure 20. Its sequence is given in SEQ ID NO:4l3. The construct is used to orm potato (Solanum tuberosum) to 30 increase oil content in tubers.

Exam le 22. GPAT-MGAT fusion enz mes The enzyme activity of GPAT-MGAT enZyme fusions‘is tested to determine A ‘ whether this would increase the accessibility of the GPAT-produced MAG for MGAT 35 activity. A suitable linker region was ﬁrst synthesised and cloned into a cloning vector. This linker contained suitable sites for g the N-terminal (EcoRI—Zral) ' and C-terminal coding regions (NdeI-Smal or NdeI—Pstl).

D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 241 atttaaatgcggccgcgaattcgtcgattgaggacgtccctactagacctgctggacctcctcctgctacttactacgattctct cgctgtgcatatggtcagtcatgcccgggcctgcaggcggccgcatttaaat (SEQ ID NO:414) A GPAT4-MGAT2 fusion (GPAT4 N-terminus and MGATZ C-terminus) was made by ﬁrst cloning a DNA fragment encoding the A. thaliana GPAT4, ﬂanked by MfeI and ZraI sites and without a C-terminal stop codon, into the EcoRI—Zral sites. i The DNA fragment encoding the M. musculus MGAT2, ﬂanked by NdeI-PstI sites, was then cloned into the NdeI-Pstl sites to generate a single GPAT4—MGAT2. coding sequence. The ﬁised coding sequence was then cloned as 3 Nail fragment into pYESZ 10 to generate pYESZ::GPAT4—MGAT2 and the constitutive binary sion vector ' pJP3343 to generate pJP3343zzGPAT4-MGAT2.

' Similarly, a MGAT2-GPAT4 fusion (MGAT2 inus and GPAT4 C- terminus) was made by first cloning the DNA ﬁagment encoding M; musculus MGAT2, ﬂanked by EcoRI and ZraI sites without a C-terminal stop codon, into the 15 EcoRI-Zral sites. The DNA fragment encoding the A. thaliana GPAT4, ﬂanked by NdeI—Pstl sites, was then cloned into the NdeI-Pstl sites to generate a single MGAT2- GPAT4 coding sequence.’ The fused. coding sequence was then cloned as a NotI ' ' fragment into pYESZ to generate pYESZ::MGAT2-GPAT4 and the constitutive binary expression vector pJP3343 to generate pJP3343§2MGAT2—GPAT4. ' 20 The yeast sion vectors are tested in yeast S. cerevisiae and the binary . s are tested in N. benthamiana and ed for oil content and ition with single-coding region controls. le 23. Discoveﬂ of novel WRLI seg'uences '25 Three novel WRLl sequences are cloned into pJP3343 and other suitable binary constitutive expression vectors and tested in N. benthamiana. These include the genes ng Sorbi-WRLI (from Sorghum bicolor; SEQ ID NO:334), Lupan- WRLl (from Lupinus angustifolius; SEQ ID N02335.) and Ricco-WRLI (from Ricinus communis; SEQ ID NO:336). These constructs are tested in comparison with 30 the Arabidopsis WRIl-encoding gene in the N. benthamiana leaf assay. _ As an initial step in the ure, a partial cDNA fragment ponding to the WRLl was identiﬁed in the developing seed EST database of Lupinus angustifolius (NA-080818_Platel4f06.bl, SEQ ID N02277). A full-length cDNA (SEQ ID N02278) was subsequently red by ming 5’- and 3’- RACE PCR 35 using nested primers and cDNAs isolated from developing seeds of Lupinus angustifolius. The full length cDNA was l729_bp long, including a 1284 bp protein , coding sequence ng a predicted polypeptide of 428 amino acids (SEQ ID D337). The entire coding region of the full length lupin WRLl cDNA was then Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk ation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk ation] jyk Unmarked set by jyk 24?.

' PCR ampliﬁed using forward and reverse primers which both incorporated EcoRI restriction sites to facilitate the cloning into the pJP3343 vector under the control of a 358 promoter in the sense orientation. A. tumefaciens strain AGLl, harbouring the pJP3343-LuangWRLl was ated in N. miana leave s as described'in Example l. Leaf discs transiently expressing the pJP3343-LuangWRL1 were then harvested and analysed for oil content. ‘ Example 24. Silencing of the CGI-58 homologue in N. m James et a1. (2010) have reported that the ing of the A. thaliana CGI-58. 10 homologue ed in up to 10-fold TAG accumulation in leaves, mainly as lipid droplets in the cytosol. Galactolipid levels were also found to be higher, whereas levels of most major phospholipid species ed unchanged. Interestingly, TAG levels in seeds were unaffected and, unlike other TAG degradation mutants, no negative effect on seed germination was observed. 15 Three full length and two partial transcripts were found in the N. benthamiana transcriptome showing gy to the A. thaliana CGI-58 gene. A 434 bp region present in all ﬁve transcripts was ampliﬁed from N. benthamiana isolated leaf RNA and cloned via LR cloning (Gateway) into the pHELLSGATEIZ destination vector.

The resulting expression vector designated pTV46 encodes a hairpin RNA ) 20 molecule for reducing expression of the tobacco gene encoding the homologue of CG1-58 and was used to transform N. tabacum as bed in Example 1, yielding 52 primary transformants. ' Primary transformants displaying increased TAG levels in their vegetative - tissues are crossed with homozygous lines described in Example 20. 25 Example 25. Silencing of the N. tabrzcum ADP-glucose osphogylase (AGPase) small subunit Sanjaya et a1. (2011) demonstrated that silencing of the AGPase small subunit in combination with WRI over-expression further increases TAG accumulation in A. 30 thaliana seedlings while starch levels were reduced. An AGPase small subunit has been cloned from ﬂower buds (Kwak et al., 2007). The deduced amino acid sequence Showed 87 % identity with the A. Ithaliana AGPase. A 593 bp fragment was synthesized and cloned into pHELLSGATElZ via LR cloning (Gateway) resulting in the binary vector pTV35ﬂ Transformation of N. tabacum was done as described in 35 Example 1 and yielded 43 primary transformants. , ‘ Primary transformants displaying a reduction in total leaf starch levels are crossed with homozygous lines described in Examples 20 and 21. In on, D Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 243 primary transformants are crossed with homozygous lines that are the result of a. crossing of the lines described in 20 and 21.

Example 26. Production and use of constructs for gene combinations including an inducible promoter Further genetic constructs are made using an inducible promoter system to drive expression of at least one of the genes in the combinations of genes as described above, particularly in pJP3503 and pJP3502. In the modiﬁed constructs, the WRIl gene is expressed .by an inducible promoter such as the Aspergilus niger ach lo promoter in the presence of an expressed Aspergilus niger alcR gene. atively, a IDGAT is eXpressed using an inducible promoter. This is advantageous when maximal TAG lation is not desirable at all times during development. An inducible promoter system or a pmentally-controlled promoter system, preferably to drive the ription factor such as WRIl, allows the induction of the 15 high TAG phenotype at an appropriate time during development, and the uent I accumulation ofTAG to high levels.

TAG can be further sed by the co-expression of transcription factors including embryogenic transcription factors such as LEC2 or BABY BOOM (BBM, Srinivasan et a1., 2007). These are expressed under control of ble promoters are -20 described above and super-transfonned on transgenic lines or co-transformed with W111 and DGAT. 0 is generated by_ g a MAR spacer as a Aatll fragment into the Aatll site of pOREO4. pJP359l is generated by cloning a second MAR spacer as an KpnI fragment into the KpnI site of pJP3590. pJP3592 is generated by cloning the 25 AsiSI—Smal fragment of the DNA molecule having the nucleotide sequence shown in SEQ ID NO:416 (12ABFJYC_pJP3569_insert; Figure 193) into the AsiSI-EcoRV sites of pJP3591. pJP3596 is generated by cloning a Pstl-ﬂanked inducible sion cassette containing the ach er expressing the M. musculus MGAT2 and a e max lectin polyadenylation signal into an introduced Sbﬂ site in 30 pJP3592. Hygromycin-resistant versions of both pJP3592 and pJP3596 (pJP3598 and pJP3597, respectively) are generated by replacing the NPTII selectable marker gene with the HPH ﬂanked gene at the FseI-Ascl sites. ' ' These constructs are used to orm the same plantspecies as described in Example 20. Expression from the inducible promoter is increased by ent with 35 the inducer of the transgenic plants after they have grown substantially, so that they accumulate increased levels of TAG. These constructs are also super—transformed in stably transformed constructs already ning an'oil-increase construct including Dthree-gene or four-gene TDNA region (SEQ ID NO:4ll and SEQ ID NO:412, Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 244 respectively). Alternatively, the gene expression cassettes from the three-gene and four-gene constructs are cloned into the Natl sites of 7 and 15m598 to yield a combined constitutive and inducible vector system for high fatty acid and TAG synthesis, lation and e. _ In addition to other inducible promoters, an alternative is that gene expression can be temporally and spatially restricted by using promoters that are only active during. specific developmental s or ‘in specific tissues. nous chemically inducible ers. ' ‘ are also used to limit expression to ic developmental windows.

It will be appreciated by persons skilled in the art that numerous ions ‘10 and/or modiﬁcations may be made to the invention as shown in the speciﬁc - embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in alllrespects as illustrative and not restrictive.

, The present application claims priority from US 61/580590 ﬁled 27 December 15 2011 and US 61/718,563 ﬁled 25 October 2012, the entire contents of both of.which g . are incorporated herein by reference. ._ All publications discussed and/or referenced herein are incorporated herein in V their entirety. g Any discussion of documents, acts, materials, devices, articles or the like 20 which has been included in the present speciﬁcation is solely for the purpose of providing a context for the t invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the ﬁeld relevant to the present invention as it existed before the priority date of each claim of this application“ 25 Substitute Sheet (Rule 26) RO/AU [Annotation] jyk None set by jyk [Annotation] jyk ionNone set by jyk [Annotation] jyk Unmarked set by jyk ation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk 245 REFERENCES Abdullah et al. (1986) Biotech. 4:1087.

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SubstitLite Sheet (Rule 26) RO/AU

Claims

1. A plant or part thereof, or alga, comprising two or more exogenous polynucleotides and an increased level of one or more non—polar lipid(s) relative to a corresponding plant or a part thereof, or alga, lacking the two or more exogenous polynucleotides, wherein the two or more exogenous polynucleotides se a ﬁrst exogenous polynucleotide which encodes a first fatty acid acyltransferase polypeptide and one or more further ous polynucleotide(s) encoding one or both of the following: (i) an RNA molecule which inhibits sion of a gene encoding a polypeptide involved in the degradation of lipid and/or which reduces lipid content such as a lipase, or (ii) a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a plant or part thereof, or alga, wherein a vegetative part of the plant has a total non—polar lipid t of at least about 7% (w/W dry ), and wherein, if the transcription factor polypeptide of (ii) is present, the plant or part f, or alga, comprises the RNA molecule of (i).

2. The plant or part thereof, or alga, of claim 1, wherein one or more or all of the following features apply: (i) the total fatty acid content in the non-polar lipid of the plant or part thereof, or alga, comprises at least 2% more oleic acid and/or at least 2% less palmitic acid than the lar lipid in the corresponding plant or part thereof, or alga, lacking the two or more exogenous polynucleotides, (ii) the non-polar lipid(s) comprise a fatty acid which comprises a hydroxyl group, an epoxy group, a ropane group, a double carbon—carbon bond, a triple carbon—carbon bond, conjugated double bonds, a branched chain such as a methylated or hydroxylated branched chain, or a combination of two or more thereof, or any of two, three, four, five or six of the entioned groups, bonds or branched chains, (iii) the plant or part thereof, or alga, comprises oleic acid in an fied or non-esteriﬁed form in its lipid, wherein at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), at least 60% (mol%), at least 65% (mol%) or at least 66% (mol%) of the total fatty acids in the lipid of the plant or part thereof, or alga, is oleic acid, 252 (iv) the plant or part thereof, or alga, comprises oleic acid in an esterified form in its non-polar lipid, wherein at least 20% (mol%), at least 22% (mol%), at least 30% (mol%), at least 40% (mol%), at least 50% (mol%), at least 60% (mol%), at least 65% (mol%) or at least 66% (mol%) of the total fatty acids in the non—polar lipid of the plant or part thereof, or alga, is oleic acid, (v) the total fatty acid content in the lipid of the plant or part thereof, or alga, comprises at least 2% more oleic acid and/or at least 2% less palmitic acid than the lipid in the corresponding plant or part thereof, or alga, lacking the two or more ous polynucleotides, (vi) the non—polar lipid(s) comprise a modified level of total sterols, free sterols, steroyl esters and/or l glycosides, (vii) the non—polar s) comprise waxes and/or wax esters, (viii) the plant or part thereof is one member of a population or collection of at least about 1000 such plants or parts thereof, (ix) a vegetative part of the plant has a total non—polar lipid content of at least 10%, (x) the two or more further exogenous polynucleotides encode a silencing suppressor ptide, (xi) the part of the plant is a seed, fruit, or a vegetative part of a plant such as an aerial plant part or a green part such as a leaf or stem, and (xii) the plant or part thereof, or alga, further comprises an exogenous polynucleotide encoding a polypeptide that stabilizes the one or more non-polar lipids, such as an oleosin.

3. The alga of claim 1 or claim 2, wherein the alga is selected from the group consisting of diatoms (bacillariophytes), green algae (chlorophytes), blue-green algae (cyanophytes), golden-brown algae (chrysophytes), haptophytes, brown algae and heterokont algae.

4. The plant or part thereof according to any one of claims 1 to 3, n the plant is mz‘a aculeata (macauba palm), Arabidopsis thaliana, Aracz‘m’s hypogaea (peanut), aryum murumuru (murumuru), Astrocaryum vulgare (tucuma), Atralea geraensz’s (Indaia-rateiro), a humilis (American oil palm), Alralea oleifera (andaia), Affalea phalerata (uricuri), a speciosa (babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica sp. such as Brassz‘ca carinaz‘a, ’ca juncea, Brassz'ca napobrassica, Brassz'ca napus (canola), Camelina sativa (false ﬂax), Cannabis sativa (hemp), Carthamus tinctorz’us (safﬂower), Caryocar brasiliense 253 (pequi), Cocos mucz’fera (Coconut), Crambe abySSimz'ca (Abyssinian kale), Cucumis melo (melon), Elaez's guimeemsz‘S (African palm), Glycine max (soybean), Gossypium hirsutum (cotton), Heliamz‘hus Sp. such as Helianthus ammuus wer), Hordeum vulgare (barley), Jarropha curcas (physic nut), Joammesia primceps (arara nut-tree), Lemma Sp. (duckweed) such as Lemma aequz‘moctz‘alis, Lemma disperma, Lemma ecuaa’oriemsz's, Lemma gibba (swollen ed), Lemma japom'ca, Lemma minor, Lemma , Lemma obscura, Lemma paucicostata, Lemma perpusilla, Lemma temera, Lemma trisulca, Lemma turiomifera, Lemma valdz‘vz‘ama, Lemma yumgemsz's, Licamia rigida (oiticica), Linum usilatz'ssz'mum (flax), Lupimus amgustz’folius (lupin), Mauritia ﬂexuosa (buriti palm), liama marl’pa (inaja palm), Miscam‘hus Sp. such as Miscamthus x gigamteus and MiscamthuS SimemSiS, Nicotiama Sp. co) such as Nicotiama tabacum or Nicotiama bemthamiama, OemocarpuS bacaba (bacaba-do-azeite), Oemocarpus bataua (pataua), Oemocarpus distichus (bacaba—de-leque), Oryza Sp. (rice) such as Oryza sativa and Oryza glaberrz'ma, m virgatum (switchgrass), Paraqueiba paraemsis (mari), Persea amemcama (avocado), Pomgamia pimmata (Indian beech), Populus Irichocarpa, Ricz'mus commamis (castor), Saccharum Sp. (sugarcane), Sesamum indicum (sesame), m sum (potato), Sorghum Sp. such as Sorghum bicolor, m vulgare, Theobroma grama’tforum (cupuassu), Trifolz'um Sp, Trithrz'max brasiliemsz's (Brazilian needle palm), Triticum Sp. (wheat) such as Triticum aestivum and Zea mays (corn).

5. The plant or part thereof, or alga, according to any one of claims 1 to 4, wherein the total sterol content and/or composition in the non-polar lipid is signiﬁcantly different to the total sterol content and/or composition in the non-polar lipid in the corresponding plant or part thereof, or alga, g the two or more exogenous polynucleotides.

6. The plant or part thereof, or alga, according to any one of claims 1 to 5, wherein the sed level of one or more non-polar s) is such that one or more or all of the following features apply: (i) the level is at least 0.5% greater on a weight basis than the level in a corresponding plant or part thereof, or alga, (ii) the level is at least 1% r on a relative basis than in a corresponding plant or part thereof, or alga, (iii) the total lar lipid content of the plant or part thereof, or alga, is at least 0.5% greater on a weight basis than the content of a corresponding plant or part thereof, or alga, respectively, 254 (iv) the total non-polar lipid t of the plant or part thereof, or alga, is at least 1% r on a relative basis than the content of a corresponding plant or part thereof, or alga, respectively, and (V) the level of one or more non-polar lipid(s) and/or the total non-polar lipid content of the plant or part f, or alga, is at least 0.5% greater on a weight basis and/or at least 1% greater on a relative basis than in a corresponding plant or part thereof, or alga, respectively, which is lacking the two or more ous polynucleotides and which comprises an exogenous polynucleotide encoding an Arabidopsis thaliana DGATl.

7. The plant or part f, or alga, according to any one of claims 1 to 6, which comprises: (i) a TAG, DAG, TAG and DAG, or MAG content which is at least 10% greater on a relative basis than the TAG, DAG, TAG and DAG, or MAG content of a corresponding plant or part thereof, or alga, and/or (ii) a total polyunsaturated fatty acid (PUFA) t which is increased or decreased relative to the total PUFA content of a corresponding plant or part thereof, or alga.

8. The plant or part thereof, or alga, according to any one of claims 1 to 7, wherein the total non-polar lipid content, or the one or more non-polar lipids, and/or the level of the oleic acid or a PUFA in the plant or part thereof, or alga, is determinable by analysis by using gas chromatography of fatty acid methyl esters obtained from the plant or part thereof, or alga.

9. The plant or part thereof, or alga, according to any one of claims 1 to 8, wherein the fatty acid acyltransferase polypeptide has diacylglycerol acyltransferase (DGAT) ty, monoacylglycerol acyltransferase (MGAT) activity, or glycerolphosphate acyltransferase (GPAT) activity.

10. The plant or part thereof, or alga, according to any one of claims 1 to 9, n one or both of the following features apply: (i) the polypeptide involved in the degradation of lipid and/or which reduces lipid content is CGi58 polypeptide or SUGAR-DEPENDENTl triacylglycerol lipase, and 255 (ii) the transcription factor polypeptide is Wrinkled l (WRIl) transcription factor, Leafy Cotyledon 1 (Lecl) transcription , Leafy Cotyledon 2 (LEC2) transcription factor, Fus3 transcription factor, ABI3 transcription factor, Dof4 transcription factor, BABY BOOM (BBM) transcription factor or Dofll ription factor.

ll. The plant or part thereof, or alga, according to any one of claims 1 to 10, wherein the two or more exogenous polynucleotides encode: (i) a Wrinkled l (WRIl) transcription factor and a DGAT, (ii) a WRIl transcription factor and a DGAT and an oleosin, (iii) a WRIl transcription factor, a DGAT, a MGAT and an oleosin, (iv) a MGAT and a glycerol—3-phosphate acyltransferase (GPAT), (v) a MGAT and a DGAT, (vi) a MGAT, a GPAT and a DGAT, (vii) a WRll transcription factor and a MGAT, (viii) a WRIl transcription factor, a DGAT and a MGAT, (ix) a WRIl transcription factor, a DGAT, a MGAT, an oleosin and a GPAT, and (x) optionally, a silencing suppressor polypeptide.

12. The plant or part thereof, or alga, of claim 11, wherein (i) the GPAT also has phosphatase activity to produce MAG, such as a polypeptide having an amino acid ce ofArabidopsis GPAT4 or GPAT6, and/0r (ii) the DGAT is a DGATl or a DGAT2, and/or (iii) the MGAT is an MGATI or an MGATZ.

13. The plant or part thereof, or alga, according to any one of claims 1 to 12, n the plant or part thereof, or alga, ses: i) a ﬁrst exogenous polynucleotide ng a DGAT and a second exogenous polynucleotide encoding a WRII, ii) a ﬁrst ous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRll, and a third exogenous polynucleotide encoding an oleosin, iii) a first exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRII, a third exogenous polynucleotide encoding an n, and a fourth exogenous polynucleotide encoding an MGAT, 256 iv) a ﬁrst exogenous polynucleotide encoding a DGAT a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding LEC2 or BBM, v) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRll, a third exogenous polynucleotide encoding an n, a fourth ous polynucleotide encoding an MGAT, and a ﬁfth exogenous polynucleotide encoding LEC2 or BBM, vi) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRll, a third exogenous polynucleotide ng an oleosin, and a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, Vii) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRll, a third exogenous polynucleotide encoding an n, a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth exogenous polynucleotide encoding LEC2 or BBM, viii) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRII, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth exogenous polynucleotide encoding an MGAT, ix) a ﬁrst exogenous polynucleotide encoding a DGAT a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide ng an RNA le which inhibits sion of a gene encoding a lipase such as a CGiS 8 polypeptide, a ﬁfth exogenous polynucleotide ng an MGAT, and a sixth exogenous polynucleotide encoding LEC2 or BBM.

14. The plant or part thereof of claim 13, wherein one or more of the following features apply i) the exogenous polynucleotides encoding the DGAT and oleosin are operably linked to a constitutive promoter, or a er active in green s of a plant at least before and up until ﬂowering, which is capable of directing expression of the polynucleotides in the plant or part f, ii) the exogenous polynucleotide encoding WRIl, and RNA le which ts expression of a gene encoding a lipase such as a CGi58 polypeptide, is 257 ly linked to a constitutive promoter, a promoter active in green tissues of a plant at least before and up until flowering, or an inducible er, which is capable of directing expression of the polynucleotides in the plant or part thereof, and iii) the exogenous polynucleotides encoding LEC2, BBM and/or MGAT2 are operably linked to an inducible promoter which is capable of directing expression of the cleotides in the plant or part thereof.

15. The plant or part thereof according to any one of claims 1 to 14, n the part thereof is a vegetative plant part that ses: (i) a total non-polar lipid content of at least 11%, at least 12%, at least about 13%, at least 14%, or at least 15% (w/w dry weight or seed weight), and/or (ii) a total TAG content of at least 7%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least 17% (w/w dry weight or seed weight).

16. The plant or part thereof according to any one of claims 1 to 15, which comprises a first exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an MGAT, and a fourth exogenous polynucleotide encoding an oleosin, wherein the vegetative part of the plant has one or more or all of the following features: i) a total lipid content of at least 8%, ii) at least a 3 fold higher total lipid content in the vegetative part of the plant to a corresponding vegetative part of a plant lacking the exogenous polynucleotides, iii) a total TAG content of at least 5% (% weight of dry weight or seed ), iv) at least a 40 fold higher total TAG content relative to a ponding vegetative part of a plant lacking the exogenous polynucleotides, v) oleic acid comprises at least 15% (% weight of dry weight or seed weight) of the fatty acids in TAG, vi) at least a 10 fold higher level of oleic acid in TAG relative to a corresponding vegetative part of a plant lacking the exogenous polynucleotides, vii) palmitic acid ses at least 20% (% weight) of the fatty acids in TAG, viii) at least a 1.5 fold higher level of palmitic acid in TAG ve to a corresponding vegetative part of a plant lacking the exogenous polynucleotides, ix) linoleic acid comprises at least 22% (% weight) of the fatty acids in TAG, x) or—linolenic acid comprises less than 20% (% weight) of the fatty acids in TAG, and 258 xi) at least a 5 fold lower level of Ot-lihOlGI’liC acid in TAG relative to a corresponding vegetative part of a plant lacking the exogenous polynucleotides.

17. The plant or part thereof, or alga, according to any one of claims 1 to 16, wherein the first fatty acid acyltransferase polypeptide is a DGAT, such as a DGATl, and wherein the transcription factor polypeptide is a WRIl transcription factor, and wherein the plant or part thereof, or alga, further comprises an exogenous polynucleotide encoding n, and wherein the plant or part thereof, or alga, has one or more or all of the ing features: (i) a total TAG content of at least 10%, at least 12.5%, at least 15% or at least 17% (% weight of dry weight), (ii) at least a 40 fold, at least a 50 fold, at least a 60 fold, or at least a 70 fold, or at least a 100 fold, higher total TAG content in the plant or part thereof, or alga, relative to a ponding plant or part thereof, or alga, lacking the ous polynucleotides, (iii) oleic acid comprises at least 19%, at least 22%, or at least 25% (% weight) of the fatty acids in TAG, (iv) at least a 10 fold, at least a 15 fold, at least a 17 fold, or at least a 19 fold, higher level of oleic acid in TAG in the plant or part thereof, or alga, relative to a corresponding plant or part thereof, or alga, lacking the ous polynucleotides, (v) palmitic acid comprises at least 20%, at least 25%, or at least 28% (% weight) of the fatty acids in TAG, (vi) at least a 1.25 fold higher level of palmitic acid in TAG in the plant or part f, or alga, relative to a corresponding plant or part thereof, or alga, lacking the exogenous polynucleotides, (vii) linoleic acid comprises at least 15%, or at least 20%, (% weight) of the fatty acids in TAG, (viii) oc—linolenic acid comprises less than 15%, less than 11% or less than 8% (% weight) of the fatty acids in TAG, and (ix) at least a 5 fold, or at least an 8 fold, lower level of olenic acid in TAG in the plant or part thereof, or alga, relative to a corresponding plant or part thereof, or alga, lacking the exogenous polynucleotides.

18. The plant or part thereof, or alga according to any one of claims 1 to 17, which comprises (i) one or more introduced mutations in a gene which encodes an endogenous enzyme of the plant or part thereof, or alga, and/or 259 (ii) an exogenous polynucleotide which down-regulates the tion and/or activity of an endogenous enzyme of the plant or part thereof, or alga, wherein each endogenous enzyme is selected from the group ting of a DGAT, an sn-l glycerolphosphate acyltransferase (sn-l GPAT), a l~acyl-glycerol-3—phosphate acyltransferase ), an acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), a atidic acid phosphatase (PAP), an enzyme involved in starch biosynthesis such as (ADP)-glucose pyrophosphcrylase (AGPase), a fatty acid desaturase such as at A12 fatty acid desaturase (FADZ), a polypeptide involved in the degradation of lipid and/or which reduces lipid content such as a lipase such as a CGi58 polypeptide, or a combination of two or more thereof.

19. The plant or part thereof, or alga, of claim 18, n the exogenous polynucleotide which down-regulates the tion and/or activity of an endogenous enzyme of the plant or part thereof, or alga, is selected from the group consisting of an antisense polynucleotide, a sense polynucleotide, a catalytic cleotide, a microRNA, a polynucleotide which encodes a polypeptide which binds the endogenous enzyme, a double stranded RNA molecule and a processed RNA molecule derived therefrom.

20. The plant or part thereof, or alga, ing to any one of claims 1 to 19, wherein the non-polar lipid (i) comprises TAG, DAG, TAG and DAG, or MAG, and (ii) comprises a specific PUFA which is EDA, ARA, SDA, ETA, EPA, DPA or DHA, the speciﬁc PUFA being present at a level of at least 1% of the total fatty acids in the non-polar lipid, or a combination of two of more thereof, or (iii) comprises a fatty acid which is present at a level of at least 1% of the total fatty acids in the non-polar lipid and which comprises a hydroxyl group, an epoxy group, a cyclopropane group, a double carbon-carbon bond, a triple carbon-carbon bond, conjugated double bonds, a branched chain such as a methylated or ylated branched chain, or a combination of two or more thereof, or any of two, three, four, ﬁve or six of the aforementioned groups, bonds or branched .

21. A recombinant plant cell comprising two or more exogenous polynucleotides and an increased level of one or more non-polar lipid(s) relative to a corresponding cell lacking the two or more exogenous polynucleotides, wherein the two or more exogenous polynucleotides comprise a first exogenous polynucleotide which encodes a 260 ﬁrst fatty acid acyltransferase polypeptide and one or more r exogenous polynucleotide(s) encoding one or more of the following: (i) an RNA molecule which inhibits expression of a gene encoding a polypeptide involved in the degradation of lipid and/or which s lipid content such as a lipase, (ii) or transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a plant cell, (iii) a second fatty acid acyltransferase polypeptide, or (iv) an RNA molecule which inhibits expression of a gene ng a polypeptide involved in starch biosynthesis such as a AGPase polypeptide, n the cell has a total non—polar lipid content of at least 7% (w/w dry weight), and wherein, if the transcription factor polypeptide of (ii) is present, the plant or part thereof, or alga, comprises the RNA le of (i).

22. The cell of claim 21 comprising one of more features deﬁned in any one of claims 2 to 19.

23. A process for obtaining the cell of claim 21 or claim 22, the process comprising the steps of: i) introducing into a cell two or more exogenous polynucleotides, ii) expressing the two or more exogenous polynucleotides in the cell or a progeny cell thereof, iii) analysing the lipid content of the cell or progeny cell, and iv) selecting a cell or progeny cell having an increased level of one or more non— polar lipids relative to a corresponding cell or progeny cell lacking the exogenous polynucleotides, wherein the two or more exogenous polynucleotides encode i) a Wrinkled l (WRIl) transcription factor and a DGAT, ii) a WRll transcription factor and a DGAT and an Oleosin, iii) a WRll transcription , a DGAT, a MGAT and an Oleosin, iv) a MGAT and a glycerol—3-phosphate acyltransferase (GPAT), v) a MGAT and a DGAT, vi) a MGAT, a GPAT and a DGAT, vii) a WRIl transcription factor and a MGAT, viii) a WRIl transcription , a DGAT and a MGAT, ix) a WRll transcription factor, a DGAT, a MGAT, an Oleosin and a GPAT, 261 and x) optionally, a silencing suppressor polypeptide, wherein each exogenous polynucleotide is operably linked to a promoter that is capable of directing expression of the exogenous polynucleotide in the cell or y cell.

24. The s of claim 23, wherein the selected cell or progeny cell comprises: i) a ﬁrst exogenous polynucleotide encoding a DGATl and a second exogenous polynucleotide encoding a WRII, ii) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRIl, and a third exogenous polynucleotide encoding an oleosin, iii) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an n, and a fourth exogenous polynucleotide encoding an MGAT, iv) a ﬁrst exogenous polynucleotide ng a DGAT, a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an oleosin, and a fourth exogenous polynucleotide encoding LEC2 or BBM, v) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous cleotide encoding a WRIl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide encoding an MGAT, and a ﬁfth exogenous polynucleotide encoding LEC2 or BBM, Vi) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an oleosin, and a fourth ous polynucleotide encoding an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, vii) a ﬁrst exogenous cleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRIl, a third exogenous cleotide encoding an oleosin, a fourth exogenous polynucleotide ng an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth exogenous polynucleotide encoding LEC2 or BBM, viii) a ﬁrst exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRll, a third exogenous polynucleotide encoding an oleosin, a fourth ous polynucleotide encoding an RNA molecule which inhibits sion of a gene encoding a lipase such as a CGi58 polypeptide, and a ﬁfth exogenous polynucleotide encoding an MGAT, 262 ix) a first exogenous polynucleotide encoding a DGAT, a second exogenous polynucleotide encoding a WRIl, a third exogenous polynucleotide encoding an oleosin, a fourth exogenous polynucleotide ng an RNA molecule which inhibits expression of a gene encoding a lipase such as a CGi58 polypeptide, a ﬁfth exogenous polynucleotide encoding an MGAT, and a sixth ous cleotide encoding LEC2 or BBM.

25. The process of claim 23 or claim 24, wherein the two or more exogenous polynucleotides are stably integrated into the genome of the cell or progeny cell.

26. The process of claim 25, further comprising the step of regenerating a transgenic plant from the cell or progeny cell comprising the two or more exogenous polynucleotides.

27. The process of claim 26, wherein the step of regenerating a transgenic plant is performed prior to the step of expressing the two or more exogenous polynucleotides in the cell or a y cell thereof, and/or prior to the step of ing the lipid t of the cell or progeny cell, and/or prior to the step of selecting the cell or progeny cell having an increased level of one or more lar lipids.

28. The process of claim 26 or claim 27 which further comprises a step of obtaining seed or a y plant from the transgenic plant, wherein the seed or progeny plant comprises the two or more exogenous polynucleotides.

29. The process according to any one of claims 23 to 28, wherein the selected cell or regenerated plant therefrom, or a vegetative plant part or seed of the regenerated plant, has one or more of the features as deﬁned in any one of claims 1 to 20.

30. Use of a first polynucleotide encoding a fatty acid acyltransferase polypeptide, together with a further one or more polynucleotides encoding one or more of the following: (i) an RNA molecule which inhibits expression of a gene encoding a polypeptide involved in the ation of lipid and/or which reduces lipid t such as a lipase, (ii) a transcription factor polypeptide that increases the expression of one or more glycolytic or fatty acid biosynthetic genes in a plant or part thereof, or alga, 263 (iii) a second fatty acid acyltransferase polypeptide, or (iv) an RNA molecule which inhibits sion of a gene encoding a polypeptide involved in starch biosynthesis such as a AGPase polypeptide, for producing a enic plant or part f, or alga, having an enhanced ability to produce one or more non-polar lipids relative to a corresponding plant or part thereof, or alga, lacking the polynucleotide(s), wherein the polynucleotide(s) are ous to the plant or part thereof, or alga, and are each operably linked to a promoter which is capable of directing expression of the polynucleotide(s) in the transgenic plant or part thereof, or alga, wherein a vegetative part of the plant has a total non—polar lipid content of at least about 7% (w/W dry weight), and wherein, if the transcription factor polypeptide of (ii) is present, the plant or part f, or alga, comprises the RNA molecule of (i).

31. The use of claim 30, wherein the cell, or the plant or part thereof, or alga, comprises one or more of the features defined in claims 1 to 20.

32. A process for ing seed, the process comprising: i) growing the plant according to any one of claims 1 to 20; and ii) harvesting seed from the plant.

33. The process of claim 32, which comprises growing a population of at least about 1000 plants, each being a plant according to any one of claims 1 to 20, and ting seed from the population of plants.

34. Use of the plant or part thereof, or alga, according to any one of claims 1 to 20 or the cell of claim 21 or claim 22 for the manufacture of an industrial product.

35. The use of claim 34, wherein the industrial product is a arbon product such as fatty acid esters, fatty acid methyl esters and/or a fatty acid ethyl , an alkane such as methane, ethane or a longer-chain alkane, a mixture of longer chain alkanes, an alkene, a biofuel, or carbon monoxide and/or hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon de, hydrogen and biochar.

36. A process for producing an industrial product, the process comprising the steps of: 264 (i) ing a plant or part thereof, or alga, according to any one of claims 1 to 20, and (ii) optionally, ally processing the plant or part thereof, or alga, of step (i), and (iii) converting at least some of the lipid in the plant or part thereof, or alg,a of step (i), or in the processed plant or part thereof, or alga, obtained by step (ii), to the industrial product by applying heat, chemical, or enzymatic means, or any combination thereof, to the lipid, in situ in the plant or part thereof, or alga, of step (i), or in the processed plant or part thereof, or alga, obtained by step (ii), and (iv) recovering the rial product, thereby producing the industrial product.

37. The process of claim 36, wherein the step of physically processing the plant or part thereof comprises one or more of rolling, ng, crushing or grinding the plant or part thereof.

38. The process of claim 36 or claim 37, further comprising steps of: (a) extracting at least some of the non-polar lipid content of the plant or part thereof, or alga, as non-polar lipid, and (b) recovering the extracted non-polar lipid, wherein steps (a) and (b) are performed prior to the step of converting at least some of the lipid in the plant or part f, or alga, to the industrial product.

39. The process of claim 38, wherein the extracted lar lipid comprises triacylglycerols, wherein the triacylglycerols comprise at least 90% of the extracted lipid.

40. The process according to any one of claims 36 to 39, wherein the rial t is a hydrocarbon product such as fatty acid esters, an alkane such as methane, ethane or a longer—chain , a mixture of longer chain alkanes, an alkene, a biofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen and biochar.

41, A process for producing extracted lipid, the process comprising the steps of: (i) obtaining a plant or part thereof, or alga, according to any one of claims 1 to 20, 265 (ii) extracting lipid from the plant or part thereof, or alga, and (iii) recovering the extracted lipid.

42. The process of claim 41 which comprises one or more of , rolling, pressing, ng or grinding the plant or part thereof, and/or purifying the extracted lipid.

43. The process of claim 41 or claim 42 which uses an organic t in the extraction process to extract the oil.

44. The process according to any one of claims 41 to 43 which comprises recovering the extracted lipid or oil by collecting it in a container and/or one or more of degumming, deodorising, decolourising, drying, fractionating the extracted lipid or oil, removing at least some waxes and/or wax esters from the extracted lipid or oil, or analysing the fatty acid composition of the extracted lipid or oil.

45. The process according to any one of claims 41 to 44, in which the volume of the ted lipid or oil is at least 1 litre.

46. The process according to any one of claims 41 to 45, wherein one or more or all of the following features apply: (i) the extracted lipid or oil comprises triacylglycerols, wherein the triacylglycerols comprise at least 90% of the extracted lipid or oil, (ii) the extracted lipid or oil comprises free sterols, steroyl esters, steroyl glycosides, waxes or wax esters, or any ation thereof, and (iii) the total sterol content and/or composition in the ted lipid or oil is signiﬁcantly different to the sterol t and/or composition in the extracted lipid or oil produced from a corresponding plant or part thereof, or alga.

47. The process according to any one of claims 41 to 46, wherein the process further comprises converting the extracted lipid or oil to an industrial product.

48. The process of claim 47, n the industrial product is a hydrocarbon product such as fatty acid esters, an alkane such as methane, ethane or a -chain alkane, a mixture of longer chain alkanes, an alkene, a biofuel, carbon monoxide and/or 266 hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen and biochar.

49. The process of any one of claims 36 to 48, n the plant part is an aerial plant part or a green plant part such as a plant leaf or stem.

50. The process of any one of claims 36 to 49, further comprising a step of harvesting the plant or part thereof, or by a process comprising ﬁltration, fugation, sedimentation, ﬂotation or ﬂocculation of algae such as by ing pH of the medium.

51. A process for ing fuel, the process comprising: (i) reacting lipid recovered or extracted from the plant or part thereof, or alga, according to any one of claims 1 to 20 with an alcohol, optionally, in the presence of a catalyst, to produce alkyl esters, and (ii) optionally, blending the alkyl esters with eum based fuel.

52. The process of claim 51, wherein the alkyl esters are methyl esters.

53. A process for producing a synthetic diesel fuel, the s comprising: (i) converting the lipid in the plant or part thereof, or alga, according to any one of claims 1 to 20 to a syngas by gasification, and (ii) converting the syngas to a biofuel using a metal catalyst or a microbial catalyst.

54. A process for producing a biofuel, the process comprising converting the lipid in the plant or part f, or alga, according to any one of claims 1 to 20, or the cell of claim 21 or claim 22, to bio-oil by pyrolysis, a bioalcohol by tation, or a biogas by gasiﬁcation or anaerobic digestion.

55. A process for producing a feedstuff, the process comprising admixing the plant or part thereof, or alga, of any one of claims 1 to 20, or the cell of claim 21 or claim 22, or an extract or portion thereof, with at least one other food ient.

56. An industrial product, an extracted lipid, a fuel or a biofuel produced by the process of any one of claims 36 to 55. 267 57. Feedstuffs, cosmetics or chemicals comprising the plant or part thereof, or alga, according to any one of claims 1 to 20 or the cell of claim 21 or claim 22. 58. A process for feeding an animal comprising providing an animal with the plant or part thereof, or alga, ing to any one of claims 1 to 20, or the feedstuff of claim

57.