PLANTS WITH ENHANCED YIELD AND METHODS OF CONSTRUCTION

This application claims the benefit of U.S. Provisional Application No. 62/144,727, filed on Apr. 8, 2015, 62/145,757 filed on Apr. 10, 2015 and 62/190,281 filed on Jul. 9, 2015. The entire teachings of the above application(s) are incorporated herein by reference.

This invention was made in part with government support under Grant Number DE-AR0000201 from the United States DOE, ARPA-e PETRO program. The government has certain rights in this invention.

The world faces a major challenge in the next 35 years to meet the increased demands for food production to feed a growing global population which is expected to reach 9 billion by the year 2050 (Food and Agriculture Organization of the United Nations (2009), How to Feed the World in 2050, (http://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How_to_Feed_the_World_in_2050.pdf). The added population in combination with increased demand for improved diet, in particular increased animal protein, linked to improving living standards in developing countries requires food output to increase by up to 70% in this time period. The added population and concomitant land use changes for new living space and infrastructure, alternative uses for crops such as biofuels and biobased products and changing weather patterns makes achieving this a very challenging goal. Crop productivity is limited by numerous factors, one factor being the relative inefficiency of photochemical conversion of light energy to fixed carbon during photosynthesis and another the loss of fixed carbon by photorespiration and/or other metabolic pathways having enzymes catalyzing decarboxylation reactions. The invention can be applied to any crop however it is particularly useful for major agricultural crops used for animal feed or for direct human consumption. These crops including corn, wheat, rice, barley, oats, millet, sorghum, cassava, sugarbeets, potatoes and oilseeds such as Brassica, soybean, sunflower, safflower, camelina, that are primarily grown and harvested for seed production. Current crop production relies primarily on crop species that were bred by conventional means for improved seed yield which was improved by continuous incremental changes over many years. Over this period any step changes in yield were typically enabled by new technologies such as the advent of nitrogen fertilizers, dwarf wheat varieties, dwarf rice, hybrids such as corn with “hybrid vigor”, and more recently improved agronomic practices such as increased density of seed planting enabled largely by transgenic input traits including herbicide resistance and pesticide resistance. Thus, there is a need for transgenic plants with enhanced carbon capture systems to improve crop yield and/or seed yield. Given the inherent complexity of plant metabolism and the fact that plants have evolved to balance inputs with growth and reproduction, it is highly likely that achieving step changes in crop yield will require new technology approaches. One such new technology with the potential to enable step changes in crop yield is based on the science of metabolic engineering.

Over the last twenty years metabolic engineering primarily of microbial systems to improve and/or introduce entirely new metabolic pathways to increase carbon utilization or make entirely new products based on multiple enzymatic steps has advanced enormously. This technology has already demonstrated some success in plants. There are multiple known existing prokaryotic carbon fixation pathways (Fuchs, G., Alternative Pathways of Carbon Dioxide Fixation: Insights into the Early Evolution of Life? Annual Review of Microbiology, 2011, 65, 631; Bar-Even, A., E. Noor, and R. Milo, A survey of carbon fixation pathways through a quantitative lens. J Exp Bot, 2012, 63, 2325) as well as synthetic pathways based primarily on prokaryotic enzymes (Mainguet, S. E., L. S. Gronenberg, S. S. Wong, and J. C. Liao, A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli. Metab Eng, 2013, 19, 116; US 2014/0150135; WO2014210587, Bar-Even, A., E. Noor, N. E. Lewis, and R. Milo, Design and analysis of synthetic carbon fixation pathways. Proc Natl Acad Sci USA, 2010, 107, 8889) that may be applicable for supplementing or replacing the Calvin Benson cycle in land plants. It is however very uncertain that engineering of these novel carbon fixation pathways into land plants can be successfully accomplished as noted by Bar-Even, A., E. Noor, N. E. Lewis, and R. Milo, Design and analysis of synthetic carbon fixation pathways. Proc Natl Acad Sci USA, 2010, 107, 8889) in the abstract of their publication: “Although implementing such alternative cycles presents daunting challenges related to expression levels, activity, stability, localization, and regulation, we believe our findings suggest exciting avanues of exploration—”. This is in part due to the extreme environments in which the microbes having these pathways exist such that the microbial enzymes available may not function in plants or function well within the temperature range at which plants can be grown. Other challenging factors include the tightly controlled balance of metabolic intermediates, the availability of enzyme cofactors such as the types and levels of redox cofactors and energy in the form of ATP and subcellular compartmentation in plant cells and tissues all add additional complexity. Furthermore, the normal development of plants from seed to mature plant to seed production and senescence and the shifting flow of plant metabolism in different plant tissues represents a further challenge to successfully using microbial carbon fixation systems to enhance crop yield or the yield of specific crop targets such as seed in particular.

The present disclosure relates to methods of using novel metabolic pathways having enzymes catalyzing carboxylation reactions and/or enzymes using NADPH or NADH as a cofactor to enhance the yield of desirable crop traits including increased biomass yield, increased seed yield, increased oil content in seed, increased protein content in seed, increased starch content in seed, or increased sucrose content in stalks or seed. These modifications can be combined with other traits including herbicide resistance, pest resistance, nitrogen use efficiency, heat tolerance, drought tolerance and water use efficiency or industrial traits such as polyhydroxyalkanoate polymers or modified oil compositions. The invention is particularly relevant to reducing economic costs to farmers and increasing food production.

Disclosed herein are transgenic plants and seeds of transgenic plants selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s). The metabolic pathways and enzymatic steps, which are the subject of the disclosed invention are shown in FIGS. 1-2.

In a first embodiment of the disclosed invention, the transgenic plant comprises one or more transgenes encoding two, three, four, five, six, seven, eight or more enzymes selected from the group: an oxygen tolerant pyruvate oxidoreductase (Por); pyruvate carboxylase (Pyc); malate synthase (AceB), malate dehydrogenase (Mdh); malate thiokinase (SucC and SucD), malyl-CoA Lyase (Mcl) and isocitrate lyase (Icl) wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a second embodiment of the disclosed invention, the transgenic plant comprises one or more transgenes encoding an oxygen tolerant pyruvate oxidoreductase (Por) and a pyruvate carboxylase (Pyc) wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a third embodiment of the disclosed invention, the transgenic plant comprises two or more transgenes encoding an oxygen tolerant pyruvate oxidoreductase (Por), a pyruvate carboxylase (Pyc), and a malate synthase (AceB), wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a fourth embodiment of the disclosed invention, the transgenic plant comprises five or more transgenes encoding an oxygen tolerant pyruvate oxidoreductase (Por), a pyruvate carboxylase (Pyc), a malate synthase (AceB), malate thiokinase (SucC, SucD), and a malyl-CoA Lyase (Mcl), wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a fifth embodiment of the disclosed invention, the transgenic plant comprises six or more transgenes encoding an oxygen tolerant pyruvate oxidoreductase (Por), a pyruvate carboxylase (Pyc), a malate synthase (AceB), malate thiokinase (SucC, SucD), a malyl-CoA Lyase (Mcl), and a malate dehydrogenase (Mdh), wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a sixth embodiment of the disclosed invention, the transgenic plant comprises seven or more transgenes encoding an oxygen tolerant pyruvate oxidoreductase (Por), a pyruvate carboxylase (Pyc), a malate synthase (AceB), malate thiokinase (SucC, SucD), a Malyl-CoA Lyase (Mcl), a malate dehydrogenase (Mdh), and an isocitrate lyase (Id), wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a seventh embodiment of the disclosed invention, the transgenic plant comprises two or more transgenes encoding an oxygen tolerant pyruvate oxidoreductase (Por), a malate synthase (AceB), and one or more transgenes encoding a pyruvate carboxylase (Pyc), malate thiokinase (SucC, SucD), a Malyl-CoA Lyase (Mel), a malate dehydrogenase (Mdh), and an isocitrate lyase (Id), wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a eighth embodiment of the disclosed invention, the transgenic plant of embodiments one through seven further comprises an additional one or more transgenes encoding one or more additional enzymes selected from the group: NADP-malate dehydrogenase (NADP-Mdh); fumarate hydratase (FumC); NADH-dependent fumarate reductase (FRDg); aconitase hydratase 1 (AcnA); ATP-citrate lyase A-1 (AclA-1); and ATP-citrate lyase subunit B2 (AclB-2), wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a ninth embodiment of the disclosed invention, the transgenic plant of embodiments one through seven further comprises an additional one or more transgenes encoding an NADP-malate dehydrogenase (NADP-Mdh) enzyme or the transgenes used in embodiments one through seven are expressed in a plant which has been modified through precise genome engineering to increase the expression of an existing plant gene encoding NADP-malate dehydrogenase (NADP-Mdh) enzyme activity.

In a tenth embodiment of the disclosed invention, the transgenic plant comprises one or more transgenes encoding an NADP-malate dehydrogenase (NADP-Mdh) enzyme or is a plant which has been modified through precise genome engineering to increase the expression of an existing plant gene encoding NADP-malate dehydrogenase (NADP-Mdh) enzyme activity wherein the transgenic plant or plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a first aspect of embodiments one through ten, the heterologous enzymes expressed from the transgenes are targeted to the plastids of the plant wherein the transgenic plant is selected on the basis of having a higher yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In a second aspect of embodiments one through ten, the expression of the transgene(s) is under the control of one or more seed specific promoter(s) and the heterologous enzymes expressed from the transgenes are targeted to the plastids of the plant wherein the transgenic plant is selected on the basis of having a higher seed yield in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In an aspect of embodiments one through ten including the first and second aspects, the transgenic plant is selected on the basis of having a yield increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 60%, or at least 80%, at least 90%, at least 100% at least 120% or higher in comparison with a corresponding plant that is not expressing the heterologous enzyme(s).

In an aspect of embodiments one through ten including the first and second aspects, the transgenic plant is selected on the basis of having a seed yield increase at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50% or higher in comparison with a corresponding plant that is not expressing the heterologous enzyme(s). In an embodiment, the transgenic plant has a seed oil content at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% or higher than the oil content of a wild type plant of the same species.

In an additional embodiment of the invention, the transgenic plants of all of the previous embodiments and aspects include additional transgenes encoding a bicarbonate transporter localized to the chloroplast membrane to increase the level of bicarbonate and/or carbon dioxide available for use by carbon fixation enzymes within the disclosed metabolic pathways.

In an embodiment, a method of producing a transformed plant having enhanced yield comprises transforming a plant cell with the disclosed transgenes; growing a plant from the plant cell until the plant produces seed; and selecting seeds from a plant in which yield is enhanced in comparison with a corresponding plant that is not expressing the heterologous enzyme(s) is disclosed. In an additional embodiment describe methods and genetic constructs are described that minimize the number of transgenes, or transgenes plus modifications to the expression of genes present naturally in the plant, to achieve the yield change outcomes.

In an embodiment methods, metabolic pathways, enzymes, and crops for enhancing the yield of food crops to support the future needs of the growing world population are disclosed.

In an embodiment the transgenic plants of all of the previous embodiments and aspects include additional transgenes encoding input traits such as herbicide or pesticide tolerance, insect resistance, drought tolerance, stress tolerance, nitrogen and water use efficiency or additional enzymes or traits to further increase yield. For example, the disclosed constructs may also contain expression cassettes for one or more transgenes encoding enzymes or other proteins for enhancing the availability of substrates for the disclosed metabolic pathways and enzymes. These include for example enzymes capable of increasing photosynthesis, increasing carbon flow through the Calvin cycle in photosynthesis, and/or increasing regeneration of ribulose 1,5-bisphosphate, the acceptor molecule in the Calvin cycle that upon fixation of CO₂is converted to two molecules of 3-phosphoglycerate, the key intermediate for acetyl-CoA production.

Candidate enzymes include but are not limited to sedoheptulose 1,7-bisphosphatase (SBPase, EC 3.1.3.37), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a bi-functional enzyme encoding both SBPase and FBPase activities, transketolase (EC 2.2.1.1), and aldolase (EC 4.1.2.13). SBPase, transketolase, and aldolase activities have been shown to have an impact on the control of carbon fixed by the Calvin cycle (Raines, 2003, Photosynthesis Research, 75, 1-10) which could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Such enzymes have been introduced into plants to enhance the flux of carbon to acetyl-CoA (U.S. Pat. Appl. Publ. (2012), US 2012/0060413), a key intermediate in the disclosed pathways (FIGS. 1, 2, 12, 14, and 18). Vectors expressing transcription factors, such as those described in patent application WO 2014/100289, can be combined with the vectors described in the invention, including vectors pMBXS1022, pMBXS1023, pMBXS919, pMBXS1056, pMBXS1057, pMBXS1058, pMBXS1059 and pMBXS1060, to further enhance yield.

Transgenes encoding proteins involved in the transport of bicarbonate in cyanobacterial and algal systems can be added to increase the availability of CO₂for the Calvin cycle and for the carboxylation enzymes present in the metabolic pathways disclosed herein. Suitable bicarbonate transporter genes can be obtained from cyanobacteria and algal species. A novel bicarbonate transporter from Chlamydomonas reinhardtii (CCP1) that significantly increases plant yield has recently been described (pending PCT Application No. PCT/US2014/072347, “Plants with enhanced photosynthesis and methods of manufacture thereof”, incorporated herein by reference in its entirety, and in pending U.S. Provisional Patent Application No. 62/291,341, “Transgenic land plants comprising a bicarbonate transporter protein of an edible eukaryotic algae”)) and would be particularly useful for the purposes of the disclosed invention. An example embodiment of a suitable bicarbonate transporter transgene is the bicarbonate transporter from Chlamydomonas reinhardtii (CCP1) of SEQ ID NO: 6 (NCBI Genbank NCBI Reference Sequence: XM 001692145.1, Chlamydomonas reinhardtii strain CC-503 cw92 mt+, available at http://www.ncbi.nlm.nih.gov/nuccore/XM_001692145.1) that encodes a protein of SEQ ID NO: 7 (Low-CO2-inducible chloroplast envelope protein, available at Source: http://www.uniprot.org/uniprot/A8IT08). SEQ ID NO:6 is shown in FIG. 25; SEQ ID NO: 7 is shown in FIG. 26.

Other suitable examples of bicarbonate transporter genes are provided in Table 11 shown in FIGS. 24A through 24D, and in Table 12, shown in FIG. 28A and FIG. 28B.

The transgenic plants of embodiments 1-10 may have additional transgenes that provide resistance to one or more herbicides seleceted from, but not limited to, the following group: glyphosate, 2,4-D, 2,4-D choline, Liberty Link, Dicambia, glufosinate, mesotrione, isoxaflutole, tembotrione, pyroxasulfone, fluthiacet-methyl, atrazine, triazines, metolachlor, imazethapyr, fomesafen, metribuzin, and bicyclopyrone.

Vectors expressing genes encoding enzymes to metabolize glyoxylate in the plastid, including plastid targeted glyoxylate carboligase and/or plastid targeted tartronic semialdehyde reductase, can be combined with the vectors described in the invention to further enhance the efficiency of the yield traits disclosed in this invention. Glyoxylate is a key intermediate in the disclosed pathways (FIG. 1) as well as a key product in the disclosed minimum gene sets to increase seed yield (FIGS. 1, 14, and 18). Plastid targeted glyoxylate carboligase would convert glyoxylate to tartronic semialdehyde and plastid targeted tartronic semialdehyde reductase would convert tartronic semialdehyde to glycerate. Both of these metabolites may be more readily metabolized within the plastid. Previous researchers have shown that heterologous expression of plastid targeted glyoxylate dehydrogenase (also known as glyoxylate reductase), glyoxylate carboligase, and tartonic semialdehyde reductase to convert glyoxylate formed during photorespiration to glycerate increases photosynthesis and biomass production in Arabidopsis thaliana (Kebeish, R. 2007, 25, 593-599). Synthetic CO₂fixation pathways to produce glyoxylate in plants have also been described (US 2014/0150135) and can be combined with the enzyme systems descrived herein

Alternatively, endogenous plastid localized glyoxylate reductase activity can be increased through promoter replacement, precise genome engineering, or heterologous expression of the transgene to increase the conversion of glyoxylate to glycolate. Previous researchers have suggested that cytosolic and plastid localized glyoxylate reductases in Arabidopsis detoxify the glyoxylate and/or contribute to redox balance (Allan et al., 2009, Biochem. J., 423, 15-22).

In an embodiment, transgenic plants are generated which express the gene and enzyme combinations disclosed herein and are grown through a number of planting cyles to generate homozygous lines and screened for increased seed yield and/or increased seed oil content and those lines having significantly higher seed yield and/or increased seed oil content are selected.

In a method embodiments, metabolic pathways, enzymes and crops for enhancing the yield of food crops to support the future needs of the growing world population are disclosed.

The disclosed invention describes the in planta expression of combinations of transgenes encoding multiple enzymes in complex metabolic pathways resulting in step changes in crop yield. An exemplary seed crop was engineered to increase its yield. Unexpected step changes in yield have been obtained by engineering seed specific expression of novel combinations of enzymes to increase carbon fixation. Furthermore, the targeting of this yield increase to the harvested product of interest was demonstrated in the embodiment of the oilseed crop, where the product of interest is the seed.

Herein there is described the introduction of multiple transgenes, encoding novel metabolic pathways having enzymes catalyzing carboxylation reactions and/or enzymes using NADPH or NADH as a cofactor, into crops, screening the resulting transgenic crop lines produced for increased yield and selecting those transgenic lines having higher yield. In particular, by using plant promoters active in the developing seed and targeting each of the enzymes introduced by the transgenes to the plastids in the plant cells step changes had been demonstrated in seed yield and increased oil content. Although described and demonstrated with large numbers of transgenes, it will be obvious to those skilled in the art that it is routine experimentation to define the minimum gene sets essential to achieve the yield change outcomes demonstrated and provide the simplest system possible to facilitate regulatory approval for large scale planting.

A description of example embodiments of the invention follows.

For the purposes of the invention, “plant” refers to all genera and species of higher and lower plants of the Plant Kingdom. The term includes the mature plants, seeds, shoots and seedlings, and parts, propagation material, plant organ tissue, protoplasts, callus and other cultures, for example cell cultures, derived from them, and all other species of groups of plant cells giving functional or structural units. Mature plants refers to plants at any developmental stage beyond the seedling. Seedling refers to a young, immature-plant at an early developmental stage.

“Plant” encompasses all annual and perennial monocotyldedonous or dicotyledonous plants and includes by way of example, but not by limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Populus, Camelina, Beta, Solanum, and Carthamus.

Preferred plants are those from the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Poaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceae, Umbelliferae.

The invention can particularly be applied advantageously to monocotyledonous or dicotyledonous plant organisms. Preferred dicotyledonous plants are selected in particular from the dicotyledonous crop plants such as, for example, Asteraceae such as sunflower, tagetes or calendula and others; Compositae, especially the genus Lactuca, very particularly the species sativa (lettuce) and others; Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other cabbages; and the genus Arabidopsis, very particularly the species thaliana, and cress or canola and others; Cucurbitaceae such as melon, pumpkin/squash or zucchini and others; Leguminosae, particularly the genus Glycine, very particularly the species max (soybean), soya, and alfalfa, pea, beans or peanut and others; Rubiaceae, preferably the subclass Lamiidae such as, for example Coffea arabica or Coffea liberica (coffee bush) and others; Solanaceae, particularly the genus Lycopersicon, very particularly the species esculentum (tomato), the genus Solanum, very particularly the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, very particularly the genus annuum (pepper) and tobacco or paprika and others; Sterculiaceae, preferably the subclass Dilleniidae such as, for example, Theobroma cacao (cacao bush) and others; Theaceae, preferably the subclass Dilleniidae such as, for example, Camellia sinensis or Thea sinensis (tea shrub) and others; Umbelliferae, particularly the genus Daucus (very particularly the species carota (carrot)) and Apium (very particularly the species graveolens dulce (celery)) and others; and linseed, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various tree, nut and grapevine species, in particular banana and kiwi fruit. Preferred moncotyledonous plants include maize, rice, wheat, sugarcane, sorghum, oats and barley.

In some cases preferred crops are used for food production for animals, humans or both.

In some cases, the entire crop is used, for example by animal consumption directly in the field, or harvested after the growing season and used or processed in which case it is desirable to increase the yield of the entire plant biomass. In this case, the transgenes should be expressed in the green tissue of the plant using for example constitutive or leaf-specific promoters and the enzymes encoded by the transgenes to the plastids, in particular the chloroplasts of the plants. Examples of these types of crops include forage crops such as hay, alfalfa, silage corn etc.

In other cases the seed is the most valuable part of the plant harvested and the plant stems, stalks leaves etc. are left in the field. Examples of this include the majority of the major food crops including maize (corn), wheat, oats, barley, soybean, millet, sorghum, potato, pulses, beans, tomatoes, oilseeds, etc. In the case of plants used for the harvesting of seed, it is desirable to increase the yield of the seed without necessarily increasing the yield of the other parts of the plant to maximize the use of agronomic inputs such as fertilizer, water etc for the production of the seed. This can be achieved as described in the disclosed invention by using seed specific or silique specific promoters to control the expression of the transgenes in the developing seed and targeting the enzymes expressed from the transgenes to the plastid of the seed using plastid targeting signals as is well known in the art.

Of particular interest for transformation are plants, which are oilseed plants. In oilseed plants of interest the oil is accumulated in the seed and can account for greater than 10%, greater than 15%, greater than 18%, greater than 25%, greater than 35%, greater than 50% by weight of the weight of dry seed. Oil crops encompass by way of example: Borago officinalis (borage); Camelina (false flax); Brassica species such as B. campestris, B. napus, B. rapa, B. carinata (mustard, oilseed rape or turnip rape); Cannabis sativa (hemp); Carthamus tinctorius (safflower); Cocos nucifera (coconut); Crambe abyssinica (crambe); Cuphea species (Cuphea species yield fatty acids of medium chain length, in particular for industrial applications); Elaeis guinensis (African oil palm); Elaeis oleifera (American oil palm); Glycine max (soybean); Gossypium hirsutum (American cotton); Gossypium barbadense (Egyptian cotton); Gossypium herbaceum (Asian cotton); Helianthus annuus (sunflower); Jatropha curcas (jatropha); Linum usitatissimum (linseed or flax); Oenothera biennis (evening primrose); Olea europaea (olive); Oryza sativa (rice); Ricinus communis (castor); Sesamum indicum (sesame); Thlaspi caerulescens (pennycress); Triticum species (wheat); Zea mays (maize), and various nut species such as, for example, walnut or almond.

Camelina species, commonly known as false flax, are native to Mediterranean regions of Europe and Asia and seem to be particularly adapted to cold semiarid climate zones (steppes and prairies). The species Camelina sativa was historically cultivated as an oilseed crop to produce vegetable oil and animal feed. It has been introduced to the high plain regions of Canada and parts of the United States as an industrial oilseed crop. In addition to being useful as an industrial oilseed crop, Camelina is a very useful model system for developing new tools and transgenic approaches to enhancing the yield of crops in general and for enhancing the yield of seed and seed oil in particular. Demonstrated transgene encoded enzyme combinations and improvements in Camelina can then be deployed in major oilseed crops including Brassica species including B. napus (canola), B. rapa, B. juncea, B. carinata, crambe, soybean, sunflower, safflower, oil palm, flax, cotton. The disclosed invention can be used to increase the yield of any crop.

Metabolic Enzymes and Genes Encoding them Useful for Practising the Invention

Metabolic pathways and the enzymes useful for practicing the disclosed invention are illustrated in FIG. 1 and FIG. 2 which show the enzymatic reactions catalyzed by these enzymes. A list of exemplary enzymes and genes encoding them are shown in Table 1.

It is well known in the art that alternative genes encoding these metabolic enzymes can be identified based on nucleotide and or protein sequence homology and either isolated from their species of origin or constructed by DNA synthesis techniques. Metabolic enzyme includes metabolic enzymes homologous to the enzymes listed in Table 1 so long as the metabolic enzyme can catalyze the same enzymatic reaction shown in either FIG. 1 or FIG. 2. “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the sequences being compared. Falling within this generic term are the terms “ortholog” meaning a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species, and “paralog” meaning a functionally similar sequence when considered within the same species. Paralogs present in the same species or orthologs of the gene in other species can readily be identified without undue experimentation, by molecular biological techniques well known in the art.

As used herein, “percent homology” of two amino acid sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci., U.S.A. 87: 2264-2268. Such an algorithm is incorporated into the NBLAST and) XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, word length 12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters are typically used. (See http://www.ncbi.nlm.nih.gov)

In addition, polynucleotides that are substantially identical to a polynucleotide encoding any of the metabolic enzymes listed in Table 1 are included. By “substantially identical” is meant a polypeptide or polynucleotide having a sequence that is at least about 85%, specifically about 90%, and more specifically about 95% or more identical to the sequence of the reference amino acid or nucleic acid sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, or specifically at least about 20 amino acids, more specifically at least about 25 amino acids, and most specifically at least about 35 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, specifically at least about 60 nucleotides, more specifically at least about 75 nucleotides, and most specifically at least about 110 nucleotides. Typically, homologous sequences can be confirmed by hybridization, wherein hybridization under stringent conditions. Using the stringent hybridization [i.e., washing the nucleic acid fragments twice where each wash is at room temperature for 30 minutes with 2× sodium chloride and sodium citrate buffer (2×SSC buffer; 300 mM sodium chloride and 30 mM sodium citrate, pH 7.0) and 0.1% sodium dodecyl sulfate (SDS); followed by washing one time at 50° C. for 30 minutes with 2×SCC and 0.1% SDS; and then washing two times where each wash is at room temperature for 10 minutes with 2×SSC], homologous sequences can be identified comprising at most about 25 to about 30% base pair mismatches, or about 15 to about 25% base pair mismatches, or about 5 to about 15% base pair mismatches.

The term metabolic enzymes includes polynucleotides that encode the enzyme activities listed in Table 1 including polypeptides or full-length proteins that contain substitutions, insertions, or deletions into the polypeptide backbone. Related polypeptides are aligned with the metabolic enzymes listed in Table 1 by assigning degrees of homology to various deletions, substitutions and other modifications. Homology can be determined along the entire polypeptide or polynucleotide, or along subsets of contiguous residues. The percent identity is the percentage of amino acids or nucleotides that are identical when the two sequences are compared. The percent similarity is the percentage of amino acids or nucleotides that are chemically similar when the two sequences are compared. Metabolic enzymes and homologous polypeptides are preferably greater than or equal to about 75%, preferably greater than or equal to about 80%, more preferably greater than or equal to about 90% or most preferably greater than or equal to about 95% identical.

A homologous polypeptide may be produced, for example, by conventional site-directed mutagenesis of polynucleotides (which is one avenue for routinely identifying residues of the molecule that are functionally important or not), by random mutation, by chemical synthesis, or by chemical or enzymatic cleavage of the polypeptides. In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide that is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria.

In some cases, for example the Pyruvate oxidoreductase enzyme it is desirable to use an enzyme which retains its enzymatic activity in the presence of oxygen for example, from D. africanus (Pieulle, L., V. Magro, and E. C. Hatchikian, Isolation and analysis of the gene encoding the pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability. J Bacteriol, 1997, 179, 5684; Vita, N., E. C. Hatchikian, M. Nouailler, A. Dolla, and L. Pieulle, Disulfide Bond-Dependent Mechanism of Protection against Oxidative Stress in Pyruvate-Ferredoxin Oxidoreductase of Anaerobic Desulfovibrio Bacteria. Biochemistry, 2008, 47, 957). Preferably the metabolic enzymes and the genes encoding them are not obtained from mammalian, specifically human species, or from organisms which are known pathogens.

Unless otherwise indicated, the disclosure encompasses all conventional techniques of plant transformation, plant breeding, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, 2001; Current Protocols in Molecular Biology, F. M. Ausubel et al. eds., 1987; Plant Breeding: Principles and Prospects, M. D. Hayward et al., 1993; Current Protocols in Protein Science, Coligan et al., eds., 1995, (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach, M. J. MacPherson, B. D. Hames and G. R. Taylor eds., 1995.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, 2001 (Oxford University Press), The Encyclopedia of Molecular Biology, Kendrew et al., eds., 1999 (Wiley-Interscience) and Molecular Biology and Biotechnology, a Comprehensive Desk Reference, Robert A. Meyers, ed., 1995 (VCH Publishers, Inc), Current Protocols In Molecular Biology, F. M. Ausubel et al., eds., 1987 (Green Publishing), Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, 2001.

A number of terms used herein are defined and clarified in the following section. As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors described herein can be expression vectors.

As used herein, an “expression vector” is a vector that includes one or more expression control sequences. As used herein, an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.

As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid (e.g., a vector) into a cell by a number of techniques known in the art.

“Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers.

The term “plant” is used in its broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and photosynthetic green algae (e.g., Chlamydomonas reinhardtii). It also refers to a plurality of plant cells that is largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.

The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, inflorescences, anthers, pollen, ovaries, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.

The term “plant part” as used herein refers to a plant structure, a plant organ, a plant tissue or a plant cell.

A “non-naturally occurring plant” refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants created through genetic engineering.

The term “plant cell” refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

The term plastid refers to a subcellular organelle of the plant and includes chloropolasts and plastids in developing seed. The term “plant cell culture” refers to cultures of plant units such as, for example, protoplasts, cells and cell clusters in a liquid medium or on a solid medium, cells in plant tissues and organs, microspores and pollen, pollen tubes, anthers, ovules, embryo sacs, zygotes and embryos at various stages of development. The term “plant material” refers to leaves, stems, roots, inflorescences and flowers or flower parts, fruits, pollen, anthers, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

A “plant organ” refers to a distinct and visibly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, inflorescence, spikelet, floret, seed or embryo.

The term “non-transgenic plant” refers to a plant that has not been genetically engineered with heterologous nucleic acids. These non-transgenic plants can be the test or control plant when comparisons are made, including wild-type plants.

A “corresponding non-transgenic plant” refers to the plant prior to the introduction of heterologous nucleic acids. This plant can be the test plant or control plant, including wild type plants.

A “trait” refers to morphological, physiological, biochemical and physical characteristics or other distinguishing feature of a plant or a plant part or a cell or plant material. The term “trait modification” refers to a detectable change in a characteristic of a plant or a plant part or a plant cell induced by the expression of a polynucleotide or a polypeptide of the invention compared to a plant not expressing them, such as a wild type plant. Some trait modifications can be evaluated quantitatively, such as content of different metabolites, proteins, pigments, lignin, vitamins, starch, sucrose, glucose, fatty acids and other storage compounds, seed size and number, organ size and weight, total plant biomass, yield of seed and yield of genetically engineered products.

Physical plant characteristics that can be modified include cell development (such as the number of trichomes), fruit and seed size and number, yields and size of plant parts such as stems, leaves and roots, the stability of the seeds during storage, characteristics of the seed pod (e.g., susceptibility to shattering), root hair length and quantity, internode distances, or the quality of seed coat. Plant growth characteristics that can be modified include growth rate, germination rate of seeds, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, rate of nitrogen uptake, biomass or transpiration characteristics, as well as plant architecture characteristics such as apical dominance, branching patterns, number of organs, organ identity, organ shape or size. The ability to improve plant yield, plant seed yield, and plant seed oil content would be of great economic advantage to farmers worldwide and would allow for increased food production necessary to meet the demands of the growing global population.

Described herein are methods of producing a transgenic plant, plant tissue, seed, or plant cell, wherein said plant, plant tissue, seed or plant cell comprises incorporated in the genome of said plant, plant tissue, seed, or plant cell: one or more polynucleotides encoding one or more transgenes encoding metabolic pathway enzymes, heterologous to the plant with DNA sequences to enable their expression or in the case of a metabolic enzyme native to that plant its increased expression or the cellular location of that enzyme. In some cases alternative regulatory sequences, homologous or heterologous to the plant can be inserted in front of a native plant gene to alter the expression of a plant enzyme and/or alter the cellular location in which the plant enzyme is functionally active. The term transgene refers to a recombinant polynucleotide or nucleic acid that comprises a coding sequence encoding a protein or RNA molecule. The transgenes encoding the specific enzymes illustrated in FIG. 1 and FIG. 2 are operatively linked to the regulatory elements necessary for expression in the plant and in some cases, targeting of the expressed enzymes to subcellular organelles such as the plastid, and inserted into a vector adapted for expression in a plant cell as illustrated in FIGS. 3, 4, 5, 6, and 7. Suitable vectors for plant expression include T-DNA vectors. Alternatively, DNA fragments containing the transgene and the necessary regulatory elements for expression of the transgene can be excised from a plasmid and delivered to the plant cell using microprojectile bombardment-mediated methods. “Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. Combinations of heterologous and homologous enzymes shown in FIGS. 1 and 2 and listed in Table 1 are also suitable for practicing the invention. For example, plants express a Malate dehydrogenase enzyme in plastids of developing seeds (Beeler, S., H. C. Liu, M. Stadler, T. Schreier, S. Eicke, W. L. Lue, E. Truernit, S. C. Zeeman, J. Chen, and O. Kotting, Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis. Plant Physiol, 2014, 164, 1175).

It was found that incorporation of combinations of genes encoding subsets of the metabolic enzymes listed in Table 1 increased the yield of the plant as determined by measuring the weight of the transgenic plant or measuring the weight of the seed produced by the transgenic plant and comparing it to a transgenic plant or plant seed containing vector sequences without the transgenes encoding the metabolic anzymes. For example, increases in the yield of seed up to two times or higher than plants not having the metabolic enzymes expressed are shown in the examples herein. In some cases, in addition to the increase in seed yield, the oil content of those seed are measurably higher.

DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes into plants. As used herein, “transgenic” refers to an organism in which a nucleic acid fragment containing a heterologous nucleotide sequence has been introduced. The transgenes in the transgenic organism are preferably stable and inheritable. The heterologous nucleic acid fragment may or may not be integrated into the host genome.

Several plant transformation vector options are available, including those described in Gene Transfer to Plants, 1995, Potrykus et al., eds., Springer-Verlag Berlin Heidelberg New York, Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins, 1996, Owen et al., eds., John Wiley & Sons Ltd. England, and Methods in Plant Molecular Biology: A Laboratory Course Manual, 1995, Maliga et al., eds., Cold Spring Laboratory Press, New York. Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of 5′ and 3′ regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene.

Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA sequence and include vectors such as pBIN19. Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001, as well as the binary vector pCIB 10 and hygromycin selection derivatives thereof. (See, for example, U.S. Pat. No. 5,639,949).

Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences are utilized in addition to vectors such as the ones described above which contain T-DNA sequences. The choice of vector for transformation techniques that do not rely on Agrobacterium depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non-Agrobacterium transformation include pCIB3064, pSOG 19, and pSOG35. (See, for example, U.S. Pat. No. 5,639,949). Alternatively, DNA fragments containing the transgene and the necessary regulatory elements for expression of the transgene can be excised from a plasmid and delivered to the plant cell using microprojectile bombardment-mediated methods.

Engineered minichromosomes can also be used to express one or more genes in plant cells. Cloned telomeric repeats introduced into cells may truncate the distal portion of a chromosome by the formation of a new telomere at the integration site. Using this method, a vector for gene transfer can be prepared by trimming off the arms of a natural plant chromosome and adding an insertion site for large inserts (Yu et al., 2006, Proc. Natl. Acad. Sci. USA 103: 17331-17336; Yu et al., 2007, Proc. Natl. Acad. Sci. USA 104: 8924-8929).

An alternative approach to chromosome engineering in plants involves in vivo assembly of autonomous plant minichromosomes (Carlson et al., 2007, PLoS Genet. 3: 1965-74). Plant cells can be transformed with centromeric sequences and screened for plants that have assembled autonomous chromosomes de novo. Useful constructs combine a selectable marker gene with genomic DNA fragments containing centromeric satellite and retroelement sequences and/or other repeats.

Another approach useful to the described invention is Engineered Trait Loci (“ETL”) technology (U.S. Pat. No. 6,077,697; US 2006/0143732). This system targets DNA to a heterochromatic region of plant chromosomes, such as the pericentric heterochromatin, in the short arm of acrocentric chromosomes. Targeting sequences may include ribosomal DNA (rDNA) or lambda phage DNA. The pericentric rDNA region supports stable insertion, low recombination, and high levels of gene expression. This technology is also useful for stacking of multiple traits in a plant (US 2006/0246586).

Zinc-finger nucleases (ZFNs) are also useful for practicing the invention in that they allow double strand DNA cleavage at specific sites in plant chromosomes such that targeted gene insertion or deletion can be performed (Shukla et al., 2009, Nature 459: 437-441; Townsend et al., 2009, Nature 459: 442-445).

The CRISPR/Cas9 system (Sander, J. D. and Joung, J. K., Nature Biotechnology, published online Mar. 2, 2014; doi; 10.1038/nbt.2842) is particularly useful for editing plant genomes to modulate the expression of homologous genes encoding enzymes, for example the NADP-specific malate dehydrogenase enzyme found naturally in the plant cell plastids useful for practicing the disclosed invention. Several examples of the use of this technology to edit the genomes of plants have now been reported (Belhaj et al. Plant Methods 2013, 9:39).

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U U.S. Pat. No. 4,945,050; Tomes et al. (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. Biotechnology 6:923-926 (1988)). Also see Weissinger et al. Ann. Rev. Genet. 22:421-477 (1988); Sanford et al. Particulate Science and Technology 5:27-37 (1987) (onion); Christou et al. Plant Physiol. 87:671-674 (1988) (soybean); McCabe et al. (1988) BioTechnology 6:923-926 (soybean); Finer and McMullen In Vitro Cell Dev. Biol. 27P:175-182 (1991) (soybean); Singh et al. Theor. Appl. Genet. 96:319-324 (1998)(soybean); Dafta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988) (maize); Klein et al. Biotechnology 6:559-563 (1988) (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. Plant Physiol. 91:440-444 (1988) (maize); Fromm et al. Biotechnology 8:833-839 (1990) (maize); Hooykaas-Van Slogteren et al. Nature 311:763-764 (1984); Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. Proc. Natl. Acad. Sci. USA 84:5345-5349 (1987) (Liliaceae); De Wet et al. in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (1985) (pollen); Kaeppler et al. Plant Cell Reports 9:415-418 (1990) and Kaeppler et al. Theor. Appl. Genet. 84:560-566 (1992) (whisker-mediated transformation); D'Halluin et al. Plant Cell 4:1495-1505 (1992) (electroporation); Li et al. Plant Cell Reports 12:250-255 (1993) and Christou and Ford Annals of Botany 75:407-413 (1995) (rice); Osjoda et al. Nature Biotechnology 14:745-750 (1996) (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference in their entirety. References for protoplast transformation and/or gene gun for Agrisoma technology are described in WO 2010/037209. Methods for transforming plant protoplasts are available including transformation using polyethylene glycol (PEG), electroporation, and calcium phosphate precipitation (see for example Potrykus et al., 1985, Mol. Gen. Genet., 199, 183-188; Potrykus et al., 1985, Plant Molecular Biology Reporter, 3, 117-128), Methods for plant regeneration from protoplasts have also been described [Evans et al., in Handbook of Plant Cell Culture, Vol 1, (Macmillan Publishing Co., New York, 1983); Vasil, I K in Cell Culture and Somatic Cell Genetics (Academic, Orlando, 1984)].

Methods for transformation of plastids such as chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation may be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase (McBride et al., Proc. Natl. Acad. Sci. USA, 1994, 91:7301-7305) or by use of an integrase, such as the phiC31 phage site-specific integrase, to target the gene insertion to a previously inserted phage attachment site (Lutz et al., Plant J, 2004, 37, 906-13). Plastid transformation vectors can be designed such that the transgenes are expressed from a promoter sequence that has been inserted with the transgene during the plastid transformation process or, alternatively, from an endogenous plastidial promoter such that an extension of an existing plastidial operon is achieved (Herz et al., Transgenic Research, 2005, 14, 969-982). An alternative method for plastid transformation as described in WO 2010/061186 wherein RNA produced in the nucleus of a plant cell can be targeted to the plastid genome can also be used to practice the disclosed invention. Inducible gene expression from the plastid genome using a synthetic riboswitch has also been reported (Verhounig et al. (2010), Proc Natl Acad Sci USA 107: 6204-6209). Methods for designing plastid transformation vectors are described by Lutz et al. (Lutz et al., Plant Physiol, 2007, 145, 1201-10).

Recombinase technologies which are useful for producing the disclosed transgenic plants include the cre-lox, FLP/FRT and Gin systems. Methods by which these technologies can be used for the purpose described herein are described for example in (U.S. Pat. No. 5,527,695; Dale And Ow, 1991, Proc. Natl. Acad. Sci. USA 88: 10558-10562; Medberry et al., 1995, Nucleic Acids Res. 23: 485-490).

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.

Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome are described in US 2010/0229256 A1 to Somleva & Ali and US 2012/0060413 to Somleva et al.

The transformed cells are grown into plants in accordance with conventional techniques. See, for example, McCormick et al., 1986, Plant Cell Rep. 5: 81-84. These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.

Procedures for in planta transformation can be simple. Tissue culture manipulations and possible somaclonal variations are avoided and only a short time is required to obtain transgenic plants. However, the frequency of transformants in the progeny of such inoculated plants is relatively low and variable. At present, there are very few species that can be routinely transformed in the absence of a tissue culture-based regeneration system. Stable Arabidopsis transformants can be obtained by several in planta methods including vacuum infiltration (Clough & Bent, 1998, The Plant J. 16: 735-743), transformation of germinating seeds (Feldmann & Marks, 1987, Mol. Gen. Genet. 208: 1-9), floral dip (Clough and Bent, 1998, Plant J. 16: 735-743), and floral spray (Chung et al., 2000, Transgenic Res. 9: 471-476). Other plants that have successfully been transformed by in planta methods include rapeseed and radish (vacuum infiltration, Ian and Hong, 2001, Transgenic Res., 10: 363-371; Desfeux et al., 2000, Plant Physiol. 123: 895-904), Medicago truncatula (vacuum infiltration, Trieu et al., 2000, Plant J. 22: 531-541), camelina (floral dip, WO/2009/117555 to Nguyen et al.), and wheat (floral dip, Zale et al., 2009, Plant Cell Rep. 28: 903-913). In planta methods have also been used for transformation of germ cells in maize (pollen, Wang et al. 2001, Acta Botanica Sin., 43, 275-279; Zhang et al., 2005, Euphytica, 144, 11-22; pistils, Chumakov et al. 2006, Russian J. Genetics, 42, 893-897; Mamontova et al. 2010, Russian J. Genetics, 46, 501-504) and Sorghum (pollen, Wang et al. 2007, Biotechnol. Appl. Biochem., 48, 79-83).

Following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.

The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al. Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.

In some scenarios, it may be advantageous to insert a multi-gene pathway into the plant by crossing of lines containing portions of the pathway to produce hybrid plants in which the entire pathway has been reconstructed. This is especially the case when high levels of product in a seed compromises the ability of the seed to germinate or the resulting seedling to survive under normal soil growth conditions. Hybrid lines can be created by crossing a line containing one or more genes with a line containing the other gene(s) needed to complete a biosynthetic pathway. Use of lines that possess cytoplasmic male sterility (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52) with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently. Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina.

Transgenic plants can be produced using conventional techniques to express any genes of interest in plants or plant cells (Methods in Molecular Biology, 2005, vol. 286, Transgenic Plants: Methods and Protocols, Pena L., ed., Humana Press, Inc. Totowa, N.J.; Shyamkumar Barampuram and Zhanyuan J. Zhang, Recent Advances in Plant Transformation, in James A. Birchler (ed.), Plant Chromosome Engineering: Methods and Protocols, Methods in Molecular Biology, vol. 701, © Springer Science+Business Media). Typically, gene transfer, or transformation, is carried out using explants capable of regeneration to produce complete, fertile plants. Generally, a DNA or an RNA molecule to be introduced into the organism is part of a transformation vector. A large number of such vector systems known in the art may be used, such as plasmids. The components of the expression system can be modified, e.g., to increase expression of the introduced nucleic acids. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Expression systems known in the art may be used to transform virtually any plant cell under suitable conditions. A transgene comprising a DNA molecule encoding a gene of interest is preferably stably transformed and integrated into the genome of the host cells. Transformed cells are preferably regenerated into whole fertile plants. Detailed description of transformation techniques are within the knowledge of those skilled in the art.

Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, 1989, Science 244: 1293-1299). In one embodiment, promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plants and algae. In a preferred embodiment, promoters are selected from those that are known to provide high levels of expression in monocots.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize 1n2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophlic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 promoter which is activated by salicylic acid. Other chemical-regulated promoters include steroid-responsive promoters [see, for example, the glucocorticoid-inducible promoter (Schena et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10421-10425; McNellis et al., 1998, Plant J. 14:247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al., 1991, Mol. Gen. Genet. 227: 229-237; U.S. Pat. Nos. 5,814,618 and 5,789,156, herein incorporated by reference in their entirety).

A three-component osmotically inducible expression system suitable for plant metabolic engineering has recently been reported (Feng et al., 2011, PLoS ONE 6: 1-9).

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050, the core CaMV 35S promoter (Odell et al., 1985, Nature 313: 810-812), rice actin (McElroy et al., 1990, Plant Cell 2: 163-171), ubiquitin (Christensen et al., 1989, Plant Mol. Biol. 12: 619-632; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689), pEMU (Last et al., 1991, Theor. Appl. Genet. 81: 581-588), MAS (Velten et al., 1984, EMBO J. 3: 2723-2730), and ALS promoter (U.S. Pat. No. 5,659,026). Other constitutive promoters are described in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

“Tissue-preferred” promoters can be used to target gene expression within a particular tissue. Compared to chemically inducible systems, developmentally and spatially regulated stimuli are less dependent on penetration of external factors into plant sells. Tissue-preferred promoters include those described by Van Ex et al., 2009, Plant Cell Rep. 28: 1509-1520; Yamamoto et al., 1997, Plant J. 12: 255-265; Kawamata et al., 1997, Plant Cell Physiol. 38: 792-803; Hansen et al., 1997, Mol. Gen. Genet. 254: 337-343; Russell et al., 199), Transgenic Res. 6: 157-168; Rinehart et al., 1996, Plant Physiol. 112: 1331-1341; Van Camp et al., 1996, Plant Physiol. 112: 525-535; Canevascini et al., 1996, Plant Physiol. 112: 513-524; Yamamoto et al., 1994, Plant Cell Physiol. 35: 773-778; Lam, 1994, Results Probl. Cell Differ. 20: 181-196, Orozco et al., 1993, Plant Mol. Biol. 23: 1129-1138; Matsuoka et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590, and Guevara-Garcia et al., 1993, Plant J. 4: 495-505. Such promoters can be modified, if necessary, for weak expression.

“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al., 1989, BioEssays 10: 108-113, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19B1 (maize 19 kDa zein), milps (myo-inositol-1-phosphate synthase), and celA (cellulose synthase). Gamma-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, and globulin 1. The stage specific developmental promoter of the late embryogenesis abundant protein gene LEA has successfully been used to drive a recombination system for excision-mediated expression of a lethal gene at late embryogenesis stages in the seed terminator technology (U.S. Pat. No. 5,723,765 to Oliver et al.).

Leaf-specific promoters are known in the art. See, for example, WO/2011/041499 and U.S. Patent No 2011/0179511 A1 to Thilmony et al.; Yamamoto et al., 1997, Plant J. 12: 255-265; Kwon et al., 1994, Plant Physiol. 105: 357-367; Yamamoto et al., 1994, Plant Cell Physiol. 35: 773-778; Gotor et al., 1993, Plant J. 3: 509-518; Orozco et al., 1993, Plant Mol. Biol. 23: 1129-1138, and Matsuoka et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590.

Certain embodiments use transgenic plants or plant cells having multi-gene expression constructs harboring more than one promoter. The promoters can be the same or different.

Any of the described promoters can be used to control the expression of one or more of the genes of the invention, their homologs and/or orthologs as well as any other genes of interest in a defined spatiotemporal manner.

Nucleic acid sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter active in plants. The expression cassettes may also include any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes.

A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and the correct polyadenylation of the transcripts. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tm1 terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These are used in both monocotyledonous and dicotyledonous plants.

The coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (Perlak et al., 1991, Proc. Natl. Acad. Sci. USA 88: 3324 and Koziel et al., 1993, Biotechnology 11: 194-200).

Individual plants within a population of transgenic plants that express a recombinant gene(s) may have different levels of gene expression. The variable gene expression is due to multiple factors including multiple copies of the recombinant gene, chromatin effects, and gene suppression. Accordingly, a phenotype of the transgenic plant may be measured as a percentage of individual plants within a population. The yield of a plant can be measured simply by weighing. The yield of seed from a plant can also be determined by weighing.

The present inventors have transformed plants with recombinant DNA molecules that encode heterologous metabolic enzymes in the nuclear genome. The expressed recombinant metabolic enzymes are transported into the plastid compartments of the plant cells. Transgenic plants and plant cells expressing the recombinant metabolic enzymes are selected on the basis of having higher yield of total biomass or seed compared to wild type plants of the same species not comprising the recombinant metabolic enzymes. The transgenic plants also show increased seed yield compared to wild type plants of the same species not comprising the recombinant heterologous enzymes. In some cases the transgenic plants show increased seed yield and higher oil content as compared to wild type plants of the same species not comprising the recombinant heterologous enzymes.

A recombinant DNA construct including a plant-expressible gene or other DNA of interest is inserted into the genome of a plant by a suitable method. Suitable methods include, for example, Agrobacterium tumefaciens-mediated DNA transfer, direct DNA transfer, liposome-mediated DNA transfer, electroporation, co-cultivation, diffusion, particle bombardment, microinjection, gene gun, calcium phosphate coprecipitation, viral vectors, and other techniques. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert DNA constructs into plant cells. A transgenic plant can be produced by selection of transformed seeds or by selection of transformed plant cells and subsequent regeneration.

In one embodiment, the transgenic plants are grown (e.g., on soil) and harvested. In one embodiment, above ground tissue is harvested separately from below ground tissue. Suitable above ground tissues include shoots, stems, leaves, flowers, grain, and seed. Exemplary below ground tissues include tubers, roots, and root hairs. In one embodiment, whole plants are harvested and the above ground tissue is subsequently separated from the below ground tissue.

Genetic constructs may encode a selectable marker to enable selection of transformation events. There are many methods that have been described for the selection of transformed plants [for review see (Miki et al., Journal of Biotechnology, 2004, 107, 193-232) and references incorporated within]. Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptII (U.S. Pat. No. 5,034,322, U.S. Pat. No. 5,530,196), hygromycin resistance gene (U.S. Pat. No. 5,668,298, Waldron et al., (1985), Plant Mol Biol, 5:103-108; Zhijian et al., (1995), Plant Sci, 108:219-227), the bar gene encoding resistance to phosphinothricin (U.S. Pat. No. 5,276,268), the expression of aminoglycoside 3″-adenyltransferase (aadA) to confer spectinomycin resistance (U.S. Pat. No. 5,073,675), the use of inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Pat. No. 4,535,060) and methods for producing glyphosate tolerant plants (U.S. Pat. No. 5,463,175; U.S. Pat. No. 7,045,684). Other suitable selectable markers include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al., (1983), EMBO J, 2:987-992), methotrexate (Herrera Estrella et al., (1983), Nature, 303:209-213; Meijer et al, (1991), Plant Mol Biol, 16:807-820); streptomycin (Jones et al., (1987), Mol Gen Genet, 210:86-91); bleomycin (Hille et al., (1990), Plant Mol Biol, 7:171-176); sulfonamide (Guerineau et al., (1990), Plant Mol Biol, 15:127-136); bromoxynil (Stalker et al., (1988), Science, 242:419-423); glyphosate (Shaw et al., (1986), Science, 233:478-481); phosphinothricin (DeBlock et al., (1987), EMBO J. 6:2513-2518).

Methods of plant selection that do not use antibiotics or herbicides as a selective agent have been previously described and include expression of glucosamine-6-phosphate deaminase to inactive glucosamine in plant selection medium (U.S. Pat. No. 6,444,878) and a positive/negative system that utilizes D-amino acids (Erikson et al., Nat Biotechnol, 2004, 22, 455-8). European Patent Publication No. EP 0 530 129 A1 describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. U.S. Pat. No. 5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants.

Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293). Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., 1987, EMBO J. 6: 3901-3907; U.S. Pat. No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Sci. 20: 448-455; Pan et al., 1996, Plant Physiol. 112: 893-900).

Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Matz et al. (1999), Nat Biotechnol 17: 969-73). An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein.

Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al. (2002), Nat Biotech 20: 87-90), the blue fluorescent protein, the cyan fluorescent protein, and the green fluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis and Vierstra (1998), Plant Molecular Biology 36: 521-528). A summary of fluorescent proteins can be found in Tzfira et al. (Tzfira et al. (2005), Plant Molecular Biology 57: 503-516) and Verkhusha and Lukyanov (Verkhusha, V. V. and K. A. Lukyanov (2004), Nat Biotech 22: 289-296) whose references are incorporated in entirety. Improved versions of many of the fluorescent proteins have been made for various applications. Use of the improved versions of these proteins or the use of combinations of these proteins for selection of transformants will be obvious to those skilled in the art.

For plastid transformation constructs, a preferred selectable marker is the spectinomycin-resistant allele of the plastid 16S ribosomal RNA gene (Staub J M, Maliga P, Plant Cell 4: 39-45 (1992); Svab Z, Hajdukiewicz P, Maliga P, Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990)). Selectable markers that have since been successfully used in plastid transformation include the bacterial aadA gene that encodes aminoglycoside 3′-adenyltransferase (AadA) conferring spectinomycin and streptomycin resistance (Svab et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 913-917), nptII that encodes aminoglycoside phosphotransferase for selection on kanamycin (Carrer H, Hockenberry T N, Svab Z, Maliga P., Mol. Gen. Genet. 241: 49-56 (1993); Lutz K A, et al., Plant J. 37: 906-913 (2004); Lutz K A, et al., Plant Physiol. 145: 1201-1210 (2007)), aphA6, another aminoglycoside phosphotransferase (Huang F-C, et al, Mol. Genet. Genomics 268: 19-27 (2002)), and chloramphenicol acetyltransferase (Li, W., et al. (2010), Plant Mol Biol, DOI 10.1007/s11103-010-9678-4). Another selection scheme has been reported that uses a chimeric betaine aldehyde dehydrogenase gene (BADH) capable of converting toxic betaine aldehyde to nontoxic glycine betaine (Daniell H, et al., Curr. Genet. 39: 109-116 (2001)).

Plastid targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. Plant Mol. Biol. 30:769-780 (1996); Schnell et al. J. Biol. Chem. 266(5):3335-3342 (1991)); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. J. Bioenerg. Biomemb. 22(6):789-810 (1990)); tryptophan synthase (Zhao et al. J. Biol. Chem. 270(11):6081-6087 (1995)); plastocyanin (Lawrence et al. J. Biol. Chem. 272(33):20357-20363 (1997)); chorismate synthase (Schmidt et al. J. Biol. Chem. 268(36):27447-27457 (1993)); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. J. Biol. Chem. 263:14996-14999 (1988)). See also Von Heijne et al. Plant Mol. Biol. Rep. 9:104-126 (1991); Clark et al. J. Biol. Chem. 264:17544-17550 (1989); Della-Cioppa et al. Plant Physiol. 84:965-968 (1987); Romer et al. Biochem. Biophys. Res. Commun. 196:1414-1421 (1993); and Shah et al. Science 233:478-481 (1986). Alternative plastid targeting signals have also been described in the following: US 2008/0263728; Miras, S. et al. (2002), J Biol Chem 277(49): 47770-8; Miras, S. et al. (2007), J Biol Chem 282: 29482-29492.

The engineered plants for increased yield may have stacked input traits that include herbicide resistance and insect tolerance, for example a plant that is tolerant to the herbicide glyphosate and that produces the Bacillus thuringiensis (BT) toxin. Glyphosate is a herbicide that prevents the production of aromatic amino acids in plants by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase). The overexpression of EPSP synthase in a crop of interest allows the application of glyphosate as a weed killer without killing the genetically engineered plant (Suh, et al., J. M Plant Mol. Biol. 1993, 22, 195-205). BT toxin is a protein that is lethal to many insects providing the plant that produces it protection against pests (Barton, et al. Plant Physiol. 1987, 85, 1103-1109). Other useful herbicide tolerance traits include but are not limited to tolereance to Dicamba by expression of the dicamba monoxygenase gene (Behrens et al, 2007, Science, 316, 1185), tolerance to 2,4-D and 2,4-D choline by expression of a bacterial aad-1 gene that encodes for an aryloxyalkanoate dioxygenase enzyme (Wright et al., Proceedings of the National Academy of Sciences, 2010, 107, 20240), glufosinate tolerance by expression of the bialophos resistance gene (bar) or the pat gene encoding the enzyme phosphinotricin acetyl transferase (Droge et al., Planta, 1992, 187, 142), as well as genes encoding a modified 4-hydroxyphenylpyruvate dioxygenase (HPPD) that provides tolerance to the herbicides mesotrione, isoxaflutole, and tembotrione. (Siehl et al., Plant Physiol, 2014, 166, 1162). The invention is further illustrated by the following non-limiting examples. Any variations in the exemplified compositions and methods that occur to the skilled artisan are intended to fall within the scope of the present invention.

Plasmids pMBXS918, pMBXS919, pMBXS994, pMBXS1022, pMBXS1023, and pMBXS1024, are derivatives of pCAMBIA binary vectors (Centre for Application of Molecular Biology to International Agriculture, Canberra, Australia) and were constructed using conventional molecular biology and cloning techniques. The transgenes encoded by these plasmids are listed in Table 2. The enzyme activities, substrates, and metabolic pathways are shown in FIGS. 1 and 2.

Maps illustrating the metabolic enzyme encoding genes and plant expression elements for directing their expression in plants in the plasmid vectors pMBXS918, pMBXS919, pMBXS1022, pMBXS1023, and pMBXS1024 are shown in FIGS. 3-7. Plasmid vectors pMBXS1022 and pMBXS994 have the same metabolic enzymes but differ in the expression of green fluorescent protein (GFP) for visual selection of transformants. pMBXS1022 has two expression cassettes for GFP, one seed specific and one constitutive, whereas pMBXS994 has only a constitutive expression cassette for GFP.

To construct gene expression cassettes for metabolic pathway enzymes, a DNA sequence encoding a plastid signal peptide was fused to the N-terminus of each gene to direct the encoded protein to the plastid. The plastid signal peptide consisted of DNA encoding the signal peptide from the ribulose-1,5-bisphosphatase carboxylase (Rubisco) small subunit from Pisum sativum, including the first 24 amino acids of the mature protein (Cashmore, Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase, in Genetic Engineering of Plants, T. Kosuge, Meredith, C. P. & Hollaender, A., Editor. 1983, Plenum: New York. p. 29). A three amino acid linker containing an Xba I restriction site allowed direct fusion of the desired transgene to the plastid signal peptide (Kourtz et al., 2005, Plant Biotechnol. J., 2005, 3, 435). Each plastid targeting signal modified gene was placed between the seed specific promoter and corresponding 3′-termination sequence from the soya bean oleosin isoform A gene (Rowley and Herman, Biochim. Biophys. Acta, 1997, 1345, 1) to form the seed specific expression cassettes. Seed specific expression cassettes were cloned into pCAMBIA vectors using conventional cloning techniques. The nucleotide sequence of the complete vectors pMBXS918, pMBXS919, pMBXS1022, pMBXS1023, and pMBXS1024 are shown in FIGS. 19-23.

In preparation for plant transformation experiments, seeds of Camelina sativa germplasm 10CS0043 (abbreviated WT43, obtained from Agriculture and Agri-Food Canada) were sown directly into 4 inch pots filled with soil in the greenhouse. Growth conditions were maintained at 24° C. during the day and 18° C. during the night. Plants were grown until flowering. Plants with a number of unopened flower buds were used in ‘floral dip’ transformations.

Agrobacterium strain GV3101 (pMP90) was transformed with the construct of interest using electroporation. A single colony of GV3101 (pMP90) containing the construct of interest was obtained from a freshly streaked plate and was inoculated into 5 mL LB medium. After overnight growth at 28° C., 2 mL of culture was transferred to a 500-mL flask containing 300 mL of LB and incubated overnight at 28° C. Cells were pelleted by centrifugation (6,000 rpm, 20 min), and diluted to an OD600 of ˜0.8 with infiltration medium containing 5% sucrose and 0.05% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, Tex., USA). Camelina plants were transformed by “floral dip” using transformation constructs as follows. Pots containing plants at the flowering stage were placed inside a 460 mm height vacuum desiccator (Bel-Art, Pequannock, N.J., USA). Inflorescences were immersed into the Agrobacterium inoculum contained in a 500-ml beaker. A vacuum (85 kPa) was applied and held for 5 min. Plants were removed from the desiccator and were covered with plastic bags in the dark for 24 h at room temperature. Plants were removed from the bags and returned to normal growth conditions within the greenhouse for seed formation.

To identify Camelina seeds expressing GFP, fully mature seeds were harvested from transformed plants and dried for 2 days in an oven with mechanical convection set at 22° C. GFP expressing seeds were visualized by fluorescent microscopy using a Nikon AZ100 microscope with a eGFP filter (Excitation bandpass 470/40, Emission Bandpass 525/50). For plasmids pMBXS919 and pMBXS918, the presence of a bar gene on the T-DNA allowed selection of transformants by spraying a solution of 400 mg/L of the herbicide Liberty (active ingredient 15% glufosinate-ammonium).

Transgenic plant lines produced using the different plasmid vectors and vector combinations are shown in Table 3 together with the analysis of the yield of the T2 generation seed from each line.

Seed weight yield was determined by harvesting all of the mature seeds from a plant and drying them in an oven with mechanical convection set at 22° C. for two days. The weight of the entire harvested seed was recorded. Total seed oil content and oil fatty acid profile were determined using published procedures for preparation of fatty acid methyl esters (Li et al., Phytochemistry, 67, 904) with some modifications. Briefly, 25-30 mg of mature seeds were placed in 13×100 mm screw-cap test tubes. To each tube, 1.5 mL of 2.5% (v/v) sulfuric acid in methanol (w/ 0.01% w/v BHT), 400 μL toluene, and 500 μg of a triheptadecanoin (Nu-Chek Prep, Elysian, Minn.) solution (10 mg/mL in toluene) as internal standard were added. Tubes were purged with nitrogen, capped, and heated at 90° C. for 1 h. Upon cooling, 1 mL of 1 M sodium chloride and 1 mL of heptane were added to each tube. Following mixing and centrifugation, the heptane layer containing fatty acid methyl esters was analyzed with an Agilent 7890A gas chromatograph with a 30 m×0.25 mm (inner diameter) INNOWax column (Agilent) and flame ionization detection. The oven temperature was programmed from 185° C. (1 min hold) to 235° C. (1 min hold) at a rate of 10° C./min (11 min total run time), and the front inlet pressure was 35.8 psi of He. The oil content (% of seed weight) was determined by comparison of the detector response from seed-derived fatty acid methyl esters relative to methyl heptadecanoate from the triheptadecanoin internal standard. Transgenic lines produced with either of plasmids pMBXS994 or pMBSX1022 not only had significantly higher seed yield but in addition the seed oil content was increased by up to 25% as compared to the control plants.

Numeric ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes the degree of error associated with measurement of the particular quantity).

Two single colonies of GV3101 (pMP90), each carrying a specific construct required for co-transformation, were obtained from freshly streaked plates and inoculated into two separate vials containing 5 mL LB medium. After overnight growth at 28° C., 2 mL of culture of each construct was transferred to separate 500-mL flask containing 300 mL of LB and incubated overnight at 28° C. Cells were pelleted by centrifugation (4,000 rpm, 20 min), and diluted to an OD600 of ˜0.8 with infiltration medium containing 5% sucrose. The cultures, each carrying a specific construct, were mixed 1:1 by volume and 0.05% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, Tex., USA) was added. Camelina plants were transformed by “floral dip” using transformation constructs as described above.

To identify co-transformed lines, GFP expressing seeds were visualized by fluorescent microscopy using a Nikon AZ100 microscope with an eGFP filter (Excitation bandpass 470/40, Emission Bandpass 525/50) and planted in soil. The presence of the bar gene on the T-DNA of pMBXS918 or pMBXS919 constructs allowed selction of co-transformants by spraying a solution of 400 mg/L of the herbicide Liberty (active ingredient 15% glufosinate-ammonium) on plantlets obtained from GFP expressing seeds.

Co-transformations of select plasmid combinations were performed and transgenic plants isolated. T1 plants were grown in a greenhouse to produce T2 seed. Seed yield from select lines is shown in Table 4.

T2 seeds were sown and eight individual T2 plants from lines transformed with constructs for high yield were grown in the greenhouse in a randomized complete block design. T3 seed was harvested and seed weight was recorded for the individual plants. Average T3 seed yield was calculated from 8 T2 plants per line and compared to yield data of 7 plants containing empty vector pMBXO12 (Table 5).

Multiple individual plants within a line showed significantly increased yield. The highest seed yield was obtained with a plant from line 15-0406 that was co-transformed with plasmids pMBXS919 and pMBXS1024. The seed yield of this plant was 276% of the average of the vector control line JS11 and produced 13.63 grams of seed from a single plant under greenhouse growth conditions.

The weight of 100 seeds from the highest yielding co-transformed lines within Table 5 was determined. Seeds from these lines were larger as determined by the increase in 100 seed weight (Table 6). The largest seeds were from a plant co-transformed with pMBXS919/p1024 which contained an average 100 seed weight of 141.53 mg, significantly higher (132%) than the control line JS11 that contained an average 100 seed weight of 106.64 mg. The T3 seed yield and 100 seed weight were used to estimate the total number of seeds per plant. Results from these calculations show that the highest yielding plants produced both heavier seeds and more seeds per plant. The approach and methods described in this example can be used to screen for and select the highest yielding lines for commercial production.

The length and width of individual seeds from the highest yielding control line (JS11) and the highest yielding transgenic lines were also measured (Table 7) showing a small increase in seed size.

The oil content of lines was measured for each of the plants described in Table 5 using the procedures described earlier. The oil content of replicate plants from an individual event is shown in FIG. 8. The total oil per plant, calculated by multiplying the % oil content of the seed with the seed yield per plant, is shown in FIG. 9. Substantial increases in total oil content per plant were observed in some lines, with the highest value obtained with plant 15-0382 (FIG. 9).

Protein content in bulk Camelina seeds was measured using the American Oil Chemists Society (AOCS, Urbana, Ill., USA) Ba 4e-93 method (Generic Combustion Method for Determination of Crude Protein). Select lines were chosen based on their total oil content per plant (FIG. 9). Seed protein content (% seed weight) remained relatively stable despite the increases in seed oil content (% seed weight) (Table 8). The higher seed yield for many of the lines (Table 5) translated into more total protein per plant (FIG. 10).

It is well known in the art that it is desirable for plant breeding and regulatory approval purposes to reduce the number of transgenes in a line for commercial development to the minimum set while still achieving the desired outcome, which in the case of this invention is higher plant yield and/or higher plant seed yield and/or higher seed oil content. Having unequivocally demonstrated the achievement of significantly higher yield in the transgenic plants containing the different sets of metabolic enzymes alone and in combination, it is routine to now proceed to determine the optimum yield increase with the minimum set of genes. For this reason a series of additional plasmid vectors are constructed encoding the metabolic enzyme combinations as shown in Table 9. By transforming camelina as described above and determining the change in seed yield as compared to a vector control and to the highest yielding lines in Tables 3, 4, and 5, it will be routine experimentation to achieve the desired outcome. Alternate combinations of transgenes that can be used to improve seed and/or seed oil yield are listed in Table 9.

In a preferred embodiment, genes sets in plasmids pMBXS1056, pMBXS1057, pMBXS1058, pMBXS1059, and pMBXS1060 are transformed into Camelina (Table 10). In one embodiment, a recA− strain of Agrobacterium, such as AGL1 [Lazo, G et al., Biotechnology 9, 963-967 (1991)], is used to increase plasmid stability during the cultivation of the Agrobacterium stock for transformation.

Vector pMBXS1056 (FIG. 11) contains expression cassettes for pyruvate carboxylase (Pyc) and pyruvate oxidioredcutase (Por) to enable conversion of 1 molecule of CO2, 1 molecule of HCO3-, and 1 acetyl-CoA to 1 molecule of oxaloacetate (FIG. 12a). In the presence of endogenous plant malate dehydrogenase activity (FIG. 12b), oxaloacetate can be converted to S-malate. This plasmid was transformed into Agrobacterium strain GV3101 (pMP90) and used to vacuum infiltrate Camelina. Transgenic T1 seeds were identified by their GFP fluorescence. T1 seeds of 40 lines were planted in soil in a greenhouse to produce T2 seeds.

Construct pMBXS1058 (FIG. 13) contains expression cassettes for pyruvate carboxylase (Pyc), pyruvate oxidoredcutase (Por), and malate synthase (AceB). In the presence of endogenous plant malate dehydrogenase activity, this construct is designed to enable the conversion of 1 molecule of CO₂and 1 molecule of HCO₃⁻ to 1 molecule of glyoxylate (FIG. 14a). This plasmid was transformed into Agrobacterium strain GV3101 (pMP90) and used to vacuum infiltrate Camelina. Transgenic T1 seeds were identified by their GFP fluorescence. T1 seeds of 59 lines were planted in soil in a greenhouse to produce T2 seeds.

Construct pMBXS1059 (FIG. 15) contains expression cassettes for pyruvate oxidoredcutase (Por), pyruvate carboxylase (Pyc), malate dehydrogenase (Mdh5), and malate synthase (AceB). This construct is designed to enable the conversion of 1 molecule of CO₂and 1 molecule of HCO₃⁻ to 1 molecule of glyoxylate (FIG. 14a). This plasmid was transformed into Agrobacterium strain GV3101 (pMP90) and used to vacuum infiltrate Camelina. Transgenic T1 seeds were identified by their GFP fluorescence. T1 seeds of 21 lines were planted in soil in a greenhouse to produce T2 seed.

Construct pMBXS1060 (FIG. 16) contains expression cassettes for pyruvate oxidoredcutase (Por), pyruvate carboxylase (Pyc), malate dehydrogenase (Mdh5), malate thiokinase (SucC and SucD), and malyl-CoA lyase (Mcl). This construct is designed to enable the conversion of 1 molecule of CO₂and 1 molecule of HCO₃⁻ to 1 molecule of glyoxylate (FIG. 14b). This plasmid was transformed into Agrobacterium strain AGL1 and used to vacuum infiltrate Camelina. Transgenic T1 seeds were identified by their GFP fluorescence.

Construct pMBXS1057 (FIG. 17) contains expression cassettes for pyruvate oxidoredcutase (Por), pyruvate carboxylase (Pyc), malate thiokinase (SucC and SucD), and malyl-CoA lyase (Mcl). In the presence of endogenous plant malate dehydrogenase activity, this construct is designed to enable the conversion of 1 molecule of CO₂and 1 molecule of HCO₃⁻ to 1 molecule of glyoxylate (FIG. 14b). This plasmid was transformed into Agrobacterium strain GV3101 (pMP90) and used to vacuum infiltrate Camelina. Transgenic T1 seeds were identified by their GFP fluorescence. T1 seeds of 50 lines were planted in soil in a greenhouse to produce T2 seed.

Alternative constructs can be constructed to convert 1 molecule of pyruvate and 1 molecule of HCO₃⁻ to 1 molecule of acetyl-CoA and 1 molecule of glyoxylate as shown in FIG. 18. These constructs can contain either endogenous or transgene encoded malate dehydrogenase activity.

Examples 1-6 describe novel sets of transgenes to increase seed and/or seed oil yield. These methods can be further enhanced by co-expression of a bicarbonate transporter localized to the chloroplast envelope since diffusion of CO₂across plastid membranes is considered to be a significant limiting factor of photosynthesis (Tholen & Zhu, 2011, Plant Physiol. 156, 90-105). The bicarbonate transporter would increase the supply of bicarbonate (HCO₃⁻) available to pyruvate carboxylase and, in the presence of a carbonic anhydrase as well as increase the supply of CO₂to the pyruvate oxidoreductase reactions described in Examples 1-6. Carbonic anhydrases are known to be present in chloroplasts to allow the interconversion of bicarbonate and carbon dioxide as shown below:

[CO₂+H₂O←→HCO₃⁻+H⁺]

Bicarbonate transporters from cyanobacteria can be modified with a targeting signal to direct the protein to the chloroplast envelope.

In a preferred embodiment, bicarbonate transporters from green algae that possess chloroplasts and whose bicarbonate transporters already localize to a chloroplast envelope can be used.

In another embodiment, the bicarbonate transporter is encoded by the CCP1 gene [Accession No. XM_001692145.1] (SEQ ID NO:6) as described in WO2015103074, incorporated herein by reference in its entirety.

In yet another embodiment, the bicarbonate transporter is expressed under a seed specific or constitutive promoter.

In an alternative embodiment, the bicarbonate transporter is expressed under a seed specific or constitutive promoter and does not localize to the plastid, such as the CCP1 gene [Accession No. XM_001692145.1] (SEQ ID NO:6) as described in pending U.S. Provisional Patent Application No. 62/291,341.

An expression cassette including a seed specific promoter sequence operably linked to a heterologous nucleotide sequence encoding a bicarbonate transporter can be co-transformed with constructs selected from those described in Examples 1-6. In the case of a cyanobacterial bicarbonate transporter, the transgene nucleotide sequence is modified with a sequence that will direct the bicarbonate transporter to the plastid envelope.

Constructs described in Examples 1-7 can be transformed into other crops to increase seed yield.

Transformation of Brassica napus, Brassica carinata, and Brassica juncea.
Transformation of Brassica carinata

Brassica carinata can be transformed using a previously described floral dip method (Shiv et al., 2008, Journal of Plant Biochemistry and Biotechnology 17, 1-4). Briefly constructs of interest are transformed into Agrobacterium strain GV3101 and cells are grown in liquid medium. Cells are harvested and resuspended in a transformation medium consisting of ½ MS salts, 5% sucrose, and 0.05% Silwet L-77. Brassica carinata plants are grown in a greenhouse until inflorescences develop and approximately 25% of their flowers are opened. Plasmids used for transformation of Brassica carinata are modified to encode the biapholos resistance selectable marker. Plants are submerged in a prepared solution of Agrobacterium containing the modified pMBXS1022, pMBXS919, or a mixture of Agrobacterium strains containing pMBXS1022 and pMBXS919, for approximately 1 minute, and covered for 24 hours. Plants are returned to the greenhouse and allowed to set seed. Transformed seeds are screened by picking DsRed seeds under the appropriate wavelength of light as described above. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield and/or increased seed oil content are selected. Transformation of Brassica napus

Brassica seeds are surface sterilized in 10% commercial bleach (Javex, Colgate-Palmolive) for 30 min with gentle shaking. The seeds are washed three times in sterile distilled water and placed in germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% (w/v) sucrose and 0.7% (w/v) phytagar, pH 5.8 at a density of 20 per plate and maintained at 24° C. an a 16 h light/8 h dark photoperiod at a light intensity of 60-80 μEm⁻²s⁻¹for 4-5 days.

Construct pMBXS1023 is modified to add the neomycin tranferase (nptII) selectable marker. Constructs pMBXS919 or pMBXS1023 are introduced into Agrobacterium tumefacians strain EHA101 (Hood et. al., 1986, J. Bacteriol. 168: 1291-1301) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct are grown in 5 ml of minimal medium supplemented with appropriate antibiotics for 48 hr at 28° C. One ml of bacterial suspension was pelleted by centrifugation for 1 min in a microfuge. The pellet was resuspended in 1 ml minimal medium.

For transformation, cotyledons are excised from 4 or in some cases 5 day old seedlings so that they included ˜2 mm of petiole at the base. Individual cotyledons with the cut surface of their petioles are immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of ˜2 mm in co-cultivation medium, MS medium with 3% (w/v) sucrose and 0.7% phytagar and enriched with 20 μM benzyladenine. For co-transformation of pMBXS1023 and pMBXS919 constructs, the bacterial suspension for immersion contains a mixture of two Agrobacterium strains containing either pMBXS1023 or pMBXS919. The inoculated cotyledons are plated at a density of 10 per plate and incubated under the same growth conditions for 48 h. After co-cultivation, the cotyledons are transferred to regeneration medium comprising MS medium supplemented with 3% sucrose, 20 μM benzyladenine, 0.7% (w/v) phytagar, pH 5.8, 300 mg/L timentin, 20 mg/L Kanamycin and 2.5 mg/L Glufosinate ammonium. After 2-3 weeks regenerant shoots obtained are cut and maintained on “shoot elongation” medium (MS medium containing, 3% sucrose, 300 mg/L timentin, 0.7% (w/v) phytagar, 300 mg/L timentin, 20 mg/L kanamycin and 2.5 mg/L Glufosinate ammonium, pH 5.8) in Magenta jars. The elongated shoots are transferred to “rooting” medium comprising MS medium, 3% sucrose, 2 mg/L indole butyric acid, 0.7% phytagar, 500 mg/L carbenicillin, 20 mg/L kanamycin and 5 mg/L Glufosinate ammonium. After roots emerge, plantlets are transferred to potting mix (Redi Earth, W.R. Grace and Co.). The plants are maintained in a misting chamber (75% relative humidity) under the same growth conditions. Plants are allowed to self pollinate to produce seeds. T1 plantlets are screened by germinating seeds on kanamycin-supplemented medium and subsequently spraying a solution of 400 mg/L of the herbicide Liberty as described above.

Brassica napus can also be transformed using the floral dip procedure described by Shiv et al. (Shiv et al., 2008, Journal of Plant Biochemistry and Biotechnology 17, 1-4) as described above for Brassica carinata. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield and/or increased seed oil content are selected.

Transformation of Brassica juncea

Brassica juncea can be transformed using hypocotyl explants according to the methods described by Barfield and Pua (Barfield and Pua, Plant Cell Reports, 10, 308-314) or Pandian et al. (Pandian, et al., 2006, Plant Molecular Biology Reporter 24: 103a-103i) as follows:

B. juncea seeds are sterilized 2 min in 70% (v/v) ethanol and washed for 20 min in 25% commercial bleach (10 g/L hypochlorite). Seeds are rinsed 3× in sterile water. Surface-sterilized seeds are plated on germination medium (1×MS salts, 1×MS vitamins, 30 g/L sucrose, 500 mg/L MES. pH 5.5) and kept in the cold room for 2 days. Seeds are incubated for 4-6 days at 24° C. under low light (20 μm m⁻¹s⁻¹). Hypocotyl segments are excised and rinsed in 50 mL of callus induction medium (1×MS salts, 1×B5 vitamins, 30 g/L sucrose, 500 mg/L MES, 1.0 mg/L 2,4-D, 1.0 mg/L kinetin pH 5.8) for 30 min without agitation. This procedure is repeated but with agitation on orbital shaker (˜140 g) for 48 h at 24° C. in low light (10 μm⁻¹s⁻¹).

Agrobacterium can be prepared as follows: Cells of Agrobacterium strain AGL1 (Lazo, G. et al. (1991), Biotechnology, 9: 963-967) containing pMBXS1023 or pMBXS919 are grown in 5 mL of LB medium with appropriate antibiotic at 28° C. for 2 days. The 5 mL culture is transferred to 250 mL flask with 45 mL of LB and cultured for 4 h at 28° C. Cells are pelleted and resuspended in BM medium (lx MS salts, 1×B5 vitamins, 30 g/L sucrose, 500 mg/L MES, pH 5.8). The optical density at 600 nm is adjusted to 0.2 with BM medium and used for inoculation.

Explants are cocultivated with Agrobacterium containing pMBXS1023, pMBXS919, or a mixture of pMBXS1023 and pMBXS919, for 20 min after which time the Agrobacterium suspension is removed. Hypocotyl explants are washed once in callus induction medium after which cocultivation proceeds for 48 h with gentle shaking on orbital shaker. After several washes in CIM, explants are transferred to selective shoot-inducing medium (500 mg/L AgNO2, 0.4 mg/L zeatin riboside, 2.0 mg/L benzylamino purine, 0.01 mg/L GA, 200 mg/L Timentin, appropriate selection agent and 8 g/L agar added to basal medium) plates for regeneration at 24° C. Root formation is induced on root-inducing medium (0.5×MS salts, 0.5×B5 vitamins, 10 g/L sucrose, 500 mg/L MES, 0.1 mg/L indole-3-butyric acid, 200 mg/L Timentin, appropriate selection agent and 8 g/L agar, pH 5.8).

Plantlets are removed from agar, gently washed, and transferred to potting soil in pots. Plants are grown in a humid environment for a week and then transferred to the greenhouse. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield and/or increased seed oil content are selected.

The binary vectors provided in the invention can be used for Agrobacterium-mediated transformation of maize following a previously described procedure (Frame et al., 2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 185-199, Humana Press). For maize transformation, the visual GFP marker described in pMBXS1023 can be changed to a selectable marker that imparts resistance to glyphosate, such as the CP4 gene.

Plant Material:

Plants grown in a greenhouse are used as an explant source. Ears are harvested 9-13 d after pollination and surface sterilized with 80% ethanol.

Explant Isolation, Infection and Co-Cultivation:

Immature zygotic embryos (1.2-2.0 mm) are aseptically dissected from individual kernels and incubated in A. tumefaciens strain EHA101 culture (grown in 5 ml N6 medium supplemented with 100 μM acetosyringone for stimulation of the bacterial vir genes for 2-5 h prior to transformation) at room temperature for 5 min. The infected embryos are transferred scutellum side up on to a co-cultivation medium (N6 agar-solidified medium containing 300 mg/l cysteine, 5 μM silver nitrate and 100 μM acetosyringone) and incubated at 20° C., in the dark for 3 d. Embryos are transferred to N6 resting medium containing 100 mg/l cefotaxime, 100 mg/l vancomycin and 5 μM silver nitrate and incubated at 28° C., in the dark for 7 d.

Callus Selection:

All embryos are transferred on to the first selection medium (the resting medium described above supplemented with 1.5 mg/l bialaphos for selection of pMBXS919 and glyphosate for selection of pMBXS1023) and incubated at 28° C., in the dark for 2 weeks followed by subculture on a selection medium containing glyphosate and 3 mg/l bialaphos. Proliferating pieces of callus are propagated and maintained by subculture on the same medium every 2 weeks.

Plant Regeneration and Selection:

Herbicide resistant embryogenic callus lines are transferred on to regeneration medium I (MS basal medium supplemented with 60 g/l sucrose, glyphosate, 1.5 mg/l bialaphos and 100 mg/l cefotaxime and solidified with 3 g/l Gelrite) and incubated at 25° C., in the dark for 2 to 3 weeks. Mature embryos formed during this period are transferred on to regeneration medium II (the same as regeneration medium I with 3 mg/l bialaphos) for germination in the light (25° C., 80-100 μE/m²/s light intensity, 16/8-h photoperiod). Regenerated plants are ready for transfer to soil within 10-14 days. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield are selected.

The vectors provided in the invention can be used for sorghum transformation following a previously described procedure (Zhao, 2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 233-244, Humana Press). For sorghum transformation, the visual GFP marker described in pMBXS1023 can be changed to a selectable marker that imparts resistance to phosphinothricin (PPT), such as the pat gene encoding phosphinotricin acetyl transferase. For biomass sorghum or energy sorghum, seed specific promoters in pMBXS1023 and pMBXS919 should be replaced with promoters active in biomass, such as the cab-m5 promoter of the chlorophyll a/b-binding protein in maize (Sullivan et al., Mol Gen Genet, 1989, 215, 431; Becker et al., Plant Mol Biol, 1992, 20, 49).

Plant Material:

Plants grown under greenhouse, growth chamber or field conditions are used as an explant source. Immature panicles are harvested 9-12 d post pollination and individual kernels are surface sterilized with 50% bleach for 30 min followed by three washes with sterile distilled water.

Explant Isolation, Infection and Co-Cultivation:

Immature zygotic embryos (1-1.5 mm) are aseptically dissected from individual kernels and incubated in A. tumefaciens strain LBA4404 suspension in PHI-I liquid medium (MS basal medium supplemented with 1 g/l casamino acids, 1.5 mg/l 2,4-D, 68.5 g/l sucrose, 36 g/l glucose and 100 μM acetosyringone) at room temperature for 5 min. The infected embryos are transferred with embryonic axis down on to a co-cultivation PHI-T medium (agar-solidified modified PHI-I medium containing 2.0 mg/l 2,4-D, 20 g/l sucrose, 10 g/l glucose, 0.5 g/l MES, 0.7 g/l proline, 10 mg/l ascorbic acid and 100 μM acetosyringone) and incubated at 25° C., in the dark for 3 d. For resting, embryos are transferred to the same medium (without acetosyringone) supplemented with 100 mg/l carbenicillin and incubated at 28° C., in the dark for 4 d.

Callus Selection:

Embryos are transferred on to the first selection medium PHI-U (PHI-T medium described above supplemented with 1.5 mg/l 2,4-D, 100 mg/l carbenicillin and 5 mg/l PPT without glucose and acetosyringone) and incubated at 28° C., in the dark for 2 weeks followed by subculture on a selection medium containing 10 mg/l PPT. Proliferating pieces of callus are propagated and maintained by subculture on the same medium every 2 weeks for the remainder of the callus selection process of 10 weeks.

Plant Regeneration and Selection:

Herbicide-resistant callus is transferred on to regeneration medium I (PHI-U medium supplemented with 0.5 mg/l kinetin) and incubated at 28° C., in the dark for 2 to 3 weeks for callus growth and embryo development. Cultures are transferred on to regeneration medium II (MS basal medium with 0.5 mg/l zeatin, 700 mg/l proline, 60 g/l sucrose and 100 mg/l carbenicillin) for shoot formation (28° C., in the dark). After 2-3 weeks, shoots are transferred on to a rooting medium (regeneration II medium supplemented with 20 g/l sucrose, 0.5 mg/l NAA and 0.5 mg/l IBA) and grown at 25° C., 270 μE/m²/s light intensity with a 16/8-h photoperiod. When the regenerated plants are 8-10 cm tall, they can be transferred to soil and grown under greenhouse conditions. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield are selected.

The vectors pMBXS1023 and/or pMBXS919 provided in the invention can be used for transformation of barley as described by Tingay et al., 1997, Plant J. 11: 1369-1376. For barley transformation, the visual GFP marker described in pMBXS1023 can be changed to a selectable marker that imparts resistance to hygromycin, such as the hygromycin phosphotranferase gene.

Plant Material:

Plants of the spring cultivar Golden Promise are grown under greenhouse or growth chamber conditions at 18° C. with a 16/8 hours photoperiod. Spikes are harvested when the zygotic embryos are 1.5-2.5 mm in length. Developing caryopses are sterilized with sodium hypochlorite (15% w/v chlorine) for 10 min and rinsed four times with sterile water.

Immature zygotic embryos are aseptically dissected from individual kernels and after removal of the embryonic axes are placed scutellum side up on a callus induction medium (Gelrite-solidified MS basal medium containing 30 g/l maltose, 1.0 g/l casein hydrolysate, 0.69 g/l proline and 2.5 mg/L dicamba. Embryos are incubated at 24° C. in the dark during subsequent culture. One day after isolation, the embryos are incubated in A. tumefaciens strain AGL1 culture (grown from a single colony in MG/L medium) followed by a transfer on to the medium described above.

After co-cultivation for 2-3 d, embryos are transferred on to the callus induction medium supplemented with 50 mg/L hygromycin, 3 mg/l bialaphos and 150 mg/l Timentin. Cultures are selected for about 2 months with transfers to a fresh selection medium every 2 weeks.

Bialaphos and hygromycin-resistant embryogenic callus lines are transferred to a Phytagel-solidified regeneration medium containing 1 mg/l BA, 50 mg/L hygromycin, and 3 mg/l bialaphos for selection of transgenic plants and grown at 24° C. under fluorescent lights with a 16/8 h photoperiod. For root development, regenerated plants are transferred to a hormone-free callus induction medium supplemented with 50 mg/L hygromycin and 1 mg/l bialaphos. After development of a root system, plants are transferred to soil and grown in a greenhouse or a growth chamber under the conditions described above. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield are selected.

The binary vectors provided in the invention can be used for Agrobacterium-mediated transformation of rice following a previously described procedure (Herve and Kayano, 2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 213-222, Humana Press). For transformation of rice, the visual GFP marker described in pMBXS1023 can be changed to a selectable marker such as hygromycin appropriate for rice transformation.

Plant Material:

Mature seeds from japonica rice varieties grown in a greenhouse are used as an explant source.

Culture Transformation and Selection:

Dehusked seeds are surface sterilized with 70% ethanol for 1 min and 3% sodium hypochlorite for 30 min followed by six washes with sterile distilled water. Seeds are plated embryo side up on an induction medium (Gelrite-solidified N6 basal medium supplemented with 300 mg/l casamino acids, 2.88 g/l proline, 30 g/l sucrose and 2 mg/l 2,4-D) and incubated at 32° C., under continuous light for 5 d. Germinated seeds with swelling of the scutellum are infected with A. tumefaciens strain LBA4404 (culture from 3-d-old plates resuspended in N6 medium supplemented with 100 acetosyringone, 68.5 g/l sucrose and 36 g/l glucose) at room temperature for 2 min followed by transfer on to a co-cultivation medium (N6 Gelrite-solidified medium containing 300 mg/l casamino acids, 30 g/l sucrose, 10 g/l glucose, 2 mg/l 2,4-D and 100 acetosyringone) and incubation at 25° C., in the dark for 3 d.

For selection of transformed embryogenic tissues, whole seedlings washed with 250 mg/l cephotaxine are transferred on to N6 agar-solidified medium containing 300 mg/l casamino acids, 2.88 g/l proline, 30 g/l sucrose, 2 mg/l 2,4-D, 100 mg/l cefotaxime, 100 mg/l vancomycin and 35 mg/l G418 disulfate). Cultures are incubated at 32° C., under continuous light for 2-3 weeks.

Plant Regeneration and Selection:

Resistant proliferating calluses are transferred on to agar-solidified N6 medium containing 300 mg/l casamino acids, 500 mg/l proline, 30 g/l sucrose, 1 mg/l NAA, 5 mg/l ABA, 2 mg/l kinetin, 100 mg/l cefotaxime, 100 mg/l vancomycin and 20 mg/l G418 disulfate. After one week of growth at 32° C., under continuous light, the surviving calluses are transferred on to MS medium (solidified with 10 g/l agarose) supplemented with 2 g/l casamino acids, 30 g/l sucrose, 30 g/l sorbitol, 0.02 mg/l NAA, 2 mg/l kinetin, 100 mg/l cefotaxime, 100 mg/l vancomycin and 20 mg/l G418 disulfate and incubated under the same conditions for another week followed by a transfer on to the same medium with 7 g/l agarose. After 2 weeks, the emerging shoots are transferred on to Gelrite-solidified MS hormone-free medium containing 30 g/l sucrose and grown under continuous light for 1-2 weeks to promote shoot and root development. When the regenerated plants are 8-10 cm tall, they can be transferred to soil and grown under greenhouse conditions. After about 10-16 weeks, transgenic seeds are harvested.

Indica rice varieties are transformed with Agrobacterium following a similar procedure (Datta and Datta, 2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 201-212, Humana Press).

Following transformation transgenic lines are screened and plants having increased plant yield and/or increased seed yield are selected.

For transformation of sugarcane the visual GFP marker described in pMBXS1023 can be changed to a selectable marker appropriate for sugarcane transformation, such as the npt gene. Transformation of sugarcane via biolistics follows a previously described protocol (Taparia et al., 2012, In Vitro Cell. Dev. Biol. 48: 15-22))

Plant Material:

Greenhouse-grown plants with 6-8 visible nodes are used as an explant source. Tops are collected and surface sterilized with 70% ethanol. The outermost leaves are removed under aseptic conditions and immature leaf whorl cross sections (about 2 mm) are cut from the region 1-10 cm above the apical node.

Culture Initiation, Transformation and Selection:

The isolated leaf sections are cultured on MS basal media supplemented with 20 g/l sucrose, 1.86 mg/l p-chlorophenoxyacetic acid (CPA), 1.86 mg/l NAA and 0.09 mg/l BA at 28° C., under 30 μmol/m²/s light intensity and a 16/8-h photoperiod for 7 d. Embryogenic cultures are subcultured to fresh medium and used for transformation.

For microprojectile bombardment, leaf disks are plated on the culture initiation medium supplemented with 0.4 M sorbitol 4 hours before gene transfer. Plasmid DNA (200 ng) containing plasmids pMBXS919 and/or pMBXS1023, modified to contain the appropriate selectable marker gene, is precipitated onto 1.8 mg gold particles (0.6 μm) following a previously described procedure (Altpeter and Sandhu, 2010, Genetic transformation—biolistics, Davey & Anthony eds., pp 217-237, Wiley, Hoboken). The DNA (10 ng per shot) is delivered to the explants by a PDS-1000 Biolistc particle delivery system (Biorad) using 1100-psi rupture disk, 26.5 mmHg chamber vacuum and a shelf distance of 6 cm. pressure). The bombarded expants are transferred to the culture initiation medium described above and incubated for 4 days.

For selection, cultures are transferred on to the initiation medium supplemented with 30 mg/l geneticin and incubated for 10 d followed by another selection cycle under the same conditions.

Plant Regeneration and Selection:

Cultures are transferred on to the selection medium described above without CPA and grown at 28° C., under 100 μmol/m²/s light intensity with a 16/8-h photoperiod. Leaf disks with small shoots (about 0.5 cm) are plated on a hormone-free medium with 30 mg/l geneticin for shoot growth and root development. Prior to transfer to soil, roots of regenerated plants can be dipped into a commercially available root promoting powder. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased sugar production are selected.

The gene constructs provided in the invention can be used for wheat transformation by microprojectile bombardment following a previously described protocol (Weeks et al., 1993, Plant Physiol. 102: 1077-1084). For transformation of wheat, the visual GFP marker described in pMBXS1023 can be changed to a selectable marker appropriate for wheat transformation, such as the hygromycin phosphotransferase (hph) and phosphomannose isomerase (pmi) genes imparting resistance to hygromycin and mannose, respectively.

Plant Material:

Plants from the spring wheat cultivar Bobwhite are grown at 18-20° C. day and 14-16° C. night temperatures under a 16 h photoperiod. Spikes are collected 10-12 weeks after sowing (12-16 days post anthesis). Individual caryopses at the early-medium milk stage are sterilized with 70% ethanol for 5 min and 20% sodium hypochlorite for 15 min followed by three washes with sterile water.

Culture Initiation, Transformation and Selection:

Immature zygotic embryos (0.5-1.5 mm) are dissected under aseptic conditions, placed scutellum side up on a culture induction medium (Phytagel-solidified MS medium containing 20 g/l sucrose and 1.5 mg/l 2,4-D) and incubated at 27° C., in the light (43 μmol/m²/s) for 3-5 d.

For microprojectile bombardment, embryo-derived calluses are plated on the culture initiation medium supplemented with 0.4 M sorbitol 4 hours before gene transfer. Plasmid DNA containing pMBXS919 and/or pMBXS1023 and the marker gene bar and hpt is precipitated onto 0.6-μm gold particles and delivered to the explants as described for sugarcane.

The bombarded expants are transferred to callus selection medium (the culture initiation medium described above containing 1-2 mg/l bialaphos and 25 mg/L hygromycin and subcultured every 2 weeks.

Plant Regeneration and Selection:

After one-two selection cycles, cultures are transferred on to MS regeneration medium supplemented with 25 mg/L hygromycin, and 2 mg/l bialaphos. For root formation, the resulting antibiotic and herbicide-resistant shoots are transferred to hormone-free half-strength MS medium. Plants with well-developed roots are transferred to soil and acclimated to lower humidity at 21° C. with a 16-h photoperiod (300 μmol/m²/s) for about 2 weeks prior to transfer to a greenhouse. Transgenic lines are screened and transgenic lines having increased plant yield and/or increased seed yield are selected.

For transformation of soybean, the visual GFP marker described in pMBXS1023 can be changed to a selectable marker appropriate for soybean transformation, such as a gene imparting resistance to hygromycin. Agrobacterium-mediated transformation of soybean following a previously described procedure (Ko et al., 2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 397-405, Humana Press).

Plant Material:

Immature seeds from soybean plants grown under greenhouse or field conditions are used as an explant source. Young pods are harvested and surface sterilized with 70% 2-propanol for 30 sec and 25% Clorox for 20 min followed by three washes with sterile distilled water.

Culture Transformation and Selection:

Under aseptic conditions, immature seeds are removed from the pods and the cotyledons are separated from the seed coat followed by incubation in A. tumefaciens culture (grown from a single colony at 28° C., overnight) in co-cultivation medium (MS salts and B5 vitamins) supplemented with 30 g/l sucrose, 40 mg/l 2,4-D and 40 mg/l acetosyringone for 60 min. Infected explants are plated abaxial side up on agar-solidified co-cultivation medium and incubated at 25° C., in the dark for 4 d.

For selection of transformed tissues, cotyledons washed with 500 mg/l cephotaxine are placed abaxial side up on a medium for induction of somatic embryo formation (Gelrite-solidified MS medium medium containing 30 g/l sucrose, 40 mg/l 2,4-D, 500 mg/l cefotaxime, 3 mg/L glufosinate and 10 mg/l hygromycin) and incubated at 25° C., under a 23-h photoperiod (10-20 μE/m²/s) for 2 weeks. After another two weeks of growth under the same conditions in the presence of 6 mg/L glufosinate and 25 mg/l hygromycin, the antibiotic-resistant somatic embryos are transferred on MS medium for embryo maturation supplemented with 60 g/l maltose, 500 mg/l cefotaxime, 3 mg/L glufosinate, and 10 mg/l hygromycin and grown under the same conditions for 8 weeks with 2-week subculture intervals.

Plant Regeneration and Selection:

The resulting cotyledonary stage embryos are desiccated at 25° C., under a 23-h photoperiod (60-80 μE/m²/s) for 5-7 d followed by culture on MS regeneration medium containing 30 g/l sucrose and 500 mg/l cefotaxime for 4-6 weeks for shoot and root development. When the plants are 5-10 cm tall, they are transferred to soil and grown in a greenhouse after acclimatization for 7 d. Transgenic lines are screened and plants having increased plant yield or seed yield and/or oil content are selected. Microprojectile bombardment-mediated transformation of soybean

The genes in constructs pMBSX919 or pMBXS1023 can be co-bombarded with hygromycin resistance gene via biolitics into embryogenic cultures of soybean to obtain transgenic plants. The transformation, selection, and plant regeneration protocols were described previously (Santarëm E R, J J Finer, 1999. In Vitro Cellular and Developmental Biology—Plant 35:451-455.)

Plant Material:

Immature zygotic embryos are isolated from developing pods from plants grown under greenhouse conditions or field. The cotyledons are excised and plated on MS salts and B5 vitamins, 6% sucrose, 40 mg/l 2,4-D, (pH 7.0) and 0.2% Gelrite for 3-4 weeks at 27° C. under a 16-h photoperiod (30 μE/m²/s) to induce somatic embryos.

Transformation and Selection:

Bright green, globular, proliferative embryos are transferred to MS salts and B5 vitamins, 3% sucrose, 5 mM asparagine, 20 mg/l 2,4-D (pH 5.7) and 0.2% Gelrite and are subcultured every 2-3 weeks. Embryogenic tissues are subcultured 3-5 days prior to bombardment on the same media. For bombardment, clusters of embryogenic tissues are placed in the center of 90 mm Petri dishes containing the media and co-bombarded, using bombardment apparatus, with a fragment containing genes in constructs pMBSX919 or pMBXS1023 and a fragment with the hygomycin gene precipitated on gold particles.

For selection, after one week all tissues are transferred to MS salts with B5 vitamins, 3% sucrose, 5 mM asparagine, 20 mg/l 2,4-D, 15 mg/L Hygromycin (pH 5.7) and 0.2% Gelrite for 3-4 weeks. All resistant tissues are selected and transferred to fresh media until embryos are cream-colored and ready for desiccation.

Embryo Maturation and Germination:

Clones are regenerated by first placing embryos on MS salts with B5 vitamins, 6% Maltose (pH 5.7), 0.2% Gelrite and 0.5% activated charcoal for 3-4 weeks. Embryos are desiccated in a dry Petri dish sealed with parafilm and placed on the shelf for 2-5 days and germinated on growth regulator-free MS medium. Plants are transferred to soil after optimum root and shoot formation.

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.