WO2012012403A1 - Corn with increased yield and nitrogen utilization efficiency - Google Patents

Abstract

The subject invention relates in part to the use of insect-protected corn to modify fertility recommendations for given yield targets on any transgenic corn type.

Description

DESCRIPTION

CORN WITH INCREASED YIELD AND NITROGEN UTILIZATION EFFICIENCY

BACKGROUND OF THE INVENTION

Current fertility recommendations for corn were developed over a long period of time with traditional corn susceptible to insect attack, or with corn protected from insects via application of chemical insecticides. Subject to the price of chemical fertilizers, typical current practice is for farmers to over-saturate their fields with fertilizers to a lesser or greater degree.

FIELD OF THE INVENTION

This invention is in the field of genetically engineered plants having improved yield. BRIEF SUMMARY OF THE INVENTION

The subject invention concerns the surprising discovery that transgenic corn that produces Bacillus thuringiensis (Bt) insecticidal toxins to provide in-plant protection against feeding damage by above-ground and below-ground insect pests exhibits desirable agronomic characteristics apart from the protection against insect feeding damage.

The subject invention relates in part to the use of insect-protected transgenic corn to modify fertility recommendations for a given yield target. Specifically, it is found that transgenic corn commercially adopted as HERCULEX-XTRA; thus containing genes that encode Cry34Abl , Cry35Abl , and Cry I F insecticidal proteins, produces increased grain yield, as measured by kernel weight and number, when grown in field conditions and compared to isogenic control populations. It is additionally found that these enhanced yields are obtained with a further advantage of increased efficiency of nitrogen fertilizer utilization.

The present invention also includes methods of modulating nitrogen utilization efficiency (NUE) in a plant cell, comprising: (a) introducing into a plant cell a recombinant expression cassette comprising a Cry protein operably linked to a promoter that drives expression in a plant; and (b) culturing the plant cell under plant cell growing conditions; wherein the nitrogen uptake in the plant cell is modulated. Other methods include increasing yield in a plant, wherein such methods comprise the steps of: (a) introducing into a plant cell a construct comprising a Cry protein operably linked to a promoter functional in a plant cell, so as to yield transformed plant cells; and, (b) regenerating a transgenic plant from said transformed plant cell, wherein said Cry protein is expressed in the cells of said transgenic plant at levels sufficient to increase yield in said transgenic plant; wherein increased yield comprises enhanced root growth, increased seed size, increased seed weight, seed with increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size, nitrogen utilization or chlorophyll content.

Seeds and plants made from these methods may also be included.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 : cry34 plant-optimized gene sequence

SEQ ID NO:2: cry35 plant-optimized gene sequence

SEQ ID NO:3: truncated cry I F sequence encoding core toxin

SEQ ID NO:4: truncated / core-toxin Cry I F protein sequence

SEQ ID NO:5: native / full-length cry l F sequence

SEQ ID NO:6: native / full-length Cryl F protein sequence

SEQ ID NO:7: native cry34 sequence

SEQ ID NO:8: Cry34 protein sequence

SEQ ID NO:9: native cry35 sequence

SEQ ID NO: 10: Cry35 protein sequence

DETAILED DISCLOSURE OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the invention.

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Langenheim and Thimann, ( 1982) Botany: Plant Biology and Its Relation to Human Affairs, John Wiley; Cell Culture and Somatic Cell Genetics of Plants, vol. 1 , Vasil, ed. ( 1984); Stanier, et al., (1986) The Microbial World, 5.sup.th ed., Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant Pathology Methods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: A Laboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985); Oligonucleotide Synthesis, Gait, ed. ( 1984); Nucleic Acid Hybridization, Hames and Higgins, eds. (1984); and the series Methods in Enzymology, Colowick and Kaplan, eds, Academic Press, Inc., San Diego, Calif.

As used herein "operably linked" includes reference to a functional linkage between a first sequence, such as a promoter, and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. As used herein, the term "plant" includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants, which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from 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, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum. Plants of the invention include, but are not limited to, rice, wheat, peanut, sugarcane, sorghum, corn, cotton, soybean, vegetable, ornamental, conifer, alfalfa, spinach, tobacco, tomato, potato, sunflower, canola, barley or millet Brassica sp., safflower, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, palm, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beet, sugarcane, buckwheat, triticale, spelt, linseed, sugar cane, oil seed rape, canola, cress, Arabidopsis, cabbages, soya, pea, beans, eggplant, bell pepper, Tagetes, lettuce, Calendula, melon, pumpkin, squash and zucchini or oat plant. A particularly preferred plant is Zea mays.

As used herein, "yield" may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15% typically for maize, for example), and the volume of biomass generated (for forage crops such as alfalfa, and plant root size for multiple crops). Grain moisture is measured in the grain at harvest. The adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated.

As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue preferred." A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "regulatable" promoter is a promoter, which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development. Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter, which is active under most environmental conditions, for example, the ubiquitin gene promoter Ubi 1.

Transgenic corn varieties with insect protection traits have been available for several years. Multiple events are available for control of above-ground and below-ground feeding insects. These events are transformed with genes that produce Bt proteins and are marketed under names such as HERCULEX, YIELDGARD and AGRISURE. They are widely recognized to be efficacious against insect damage and are recommended for planting by University field extension services, and by their manufacturers, in locations where insect damage is expected to adversely affect corn yields. The growing recommendations (e.g. fertilizer application rates) for all of these insect protected corn varieties are identical to those recommended for nonprotected corn varieties.

While insect-protected corn varieties such as HERCULEX-XTRA are currently planted in many corn growing regions globally, farming methods of growing them with reduced fertilizer recommendations were not previously taught or suggested and are a subject of the present invention. Fertilization rates, ranges, and amounts as described elsewhere herein and as specifically exemplified in the Examples can be used to define methods of the subject invention. Various units and rates can be used to express such rates and/or ranges - some of which are used in the Examples (e.g., pounds of nitrogen fertilizer per acre).

Nitrogen fertilizer inputs are the third most costly input (behind land and seed) in corn production, and the cost may vary greatly depending on the price of natural gas required to produce nitrogen fertilizer. Nitrogen use efficiency (NUE) represents an important target for maize (corn) breeding programs. Previous research demonstrates that genetic variability exists for NUE and its components, N uptake efficiency (NUpE) and N uti lization efficiency (NUtE). Thus, widespread adoption of the subject invention, having a result of less natural gas devoted to fertilizer production, can reduce costs for farmers as well as for consumers who use natural gas for heating and cooking.

Grain yield in corn is largely affected by the ability of the plant to take up nitrogen (N) from the soil and utilize it for growth and reproduction. Plant responses to nitrogen fertilizer are observed across a wide range of application rates. The subject invention demonstrates that grain yield and its components, kernel weight and number, are increased across a range of N application rates by the presence of the transgenic insecticidal proteins Cry34Ab l , Cry35Abl and Cry I F. These enhancements are accompanied by prolonged stay-green after flowering, and they correlate with increased N uptake efficiency before and after flowering. Further, it is surprisingly seen that the plants of the subject invention have increased grain yield per unit of plant nitrogen, both within and between N application rates. The effect is not seen with nontransgenic isogenic lines protected from insect attack by application of chemical insecticides, thus demonstrating that these benefits may be in addition to the root protection afforded by the transgenic insect control.

The subject invention stems in part from our observation that yields of genetically similar corn lines, with and without the insect resistance traits, unexpectedly respond differently to inputs, particularly nitrogen fertilizer. The insect-protected plants have less root damage from below-ground insect feeding. In addition, when damaged by root feeding insects such as corn root worm, the damaged plants regrow quicker and produce a larger root mass and have improved overall plant health. This combination of factors allows the rate of nitrogen and other fertilizers to be reduced to obtain the same amount of yield as is obtained with non insect- protected corn lines grown with higher fertility amounts.

The present invention can comprise transgenic plants that accumulate the insecticidal proteins Cry34Ab l and Cry35Abl as well as CryF l . These proteins protect the root system of maize from damage from corn rootworm feeding and facilitate improved N uptake and utilization, thereby providing increased grain yield. Fertilizer application rates can be determined from the subject disclosure. Exemplary application rates, included within the subject invention, are demonstrated. Some preferred embodiments are also further specified in the Claims. Additionally, Herculex can be used in other crop species such as canola, wheat, rice, barley and other non-legume crops.

The present invention also includes methods of increasing nitrogen uptake efficiency in a plant by administering to a plant an expression cassette containing at least one Bacillus thuringiensis insect-resistance gene which functions to improve expression of at least one insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in a plant cell. Such method can further increase in the yield of the plant. The increase in yield can include an increase in the kernel number per plant and/or an increase in the kernel mass per plant. Nitrogen utilization can also be modulated. Additionally, the increase in nitrogen can occur during the flowering and grain filling periods of development.

The Bacillus thuringiensis insect-resistance gene used can include a Cry protein. The insect-resistance gene can be selected from the group consisting of, e.g., a cry34 gene, a cry35 gene, a cry I F gene, and a cry3A gene. The Bacillus thuringiensis insect-resistance gene can comprise, e.g., Cry34Ab, Cry35Ab, and/or Cry I F. Sequences of the relevant proteins and genes of HERCULEX products are readily determinable. Unless otherwise indicated herein, the Cry I F protein and gene are as described in U.S. Patent No. 6,218, 188 (preferably the truncated, plant- optimized version described therein), and the Cry34/35 genes and proteins are as described in U.S. Patent No. 6,340,593 (preferably the 149B 1 genes and proteins). Other genes and proteins that can be used according to the subject invention are also known in the art. See e.g. U.S. Patent Nos. 7, 179.965; 7,524,810; 7,939,651 ; 6,893,872; and 6,900,371. The Crickmore et al. website of the official Bacillus thuringiensis nomenclature committee is also well-known in the art and provides links to many, publically available Cry protein and gene sequences. Truncated and/or core-toxin fragments of Cry 1 F, for example, can be used, as is known in the art. Variants thereof are included. The official nomenclature committee uses boundaries of at least 45% identity (e.g. Cry l F), 78% identity (e.g. Cryl Fa), and 95% protein sequence identity (Cry l Fal ) as primary, secondary, and tertiary ranks, respectively. Such boundaries, as well as 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, and/or 99% sequence identity (with an exemplified or suggested protein or gene sequence) can be used according to the subject invention in some embodiments.

Either monocotyledonous plants or dicotyledonous plants may be used in the present invention. The present invention also includes method of growing transgenic corn plants having an increased yield by using a reduced amount of fertilizer, wherein the transgenic corn is insect resistant due to expression of an insect-resistance gene, and wherein the reduced amount of fertilizer is relative to fertilizer recommended for use on non-transgenic corn, wherein said non- transgenic corn is optionally protected by granular chemical insecticide to control rootworms.

The increase in year can include an increase in the kernel number per plant or can include an increase in the kernel mass per plant. Such transgenic corn plants can yield corn comparable to corn yield from said non-transgenic corn grown using said recommended amounts of fertilizer.

The fertilizer used can be a nitrogenous fertilizer. The nitrogenous fertilizer can be applied at any rate of less than 150 pounds per acre. Alternatively the nitrogenous fertilizer can be applied at a rate of less than 50, 60, 70, 75, 80, 90, 100, 1 10, 120, 125, 130, 140 pounds per acre. ^" In general, it may be preferable to have some nitrogenous fertilizer added to a crop. The nitrogenous fertilizer can be applied to a field after planting said corn plants in the field but prior to emergence.

The present invention also includes methods of increasing yield of monocotyledonous plants or dicotyledonous plants due to nitrogen utilization.

Likewise, by means of the present invention, other agronomic genes can be expressed in plants of the present invention. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with cloned resistance genes to engineer plants that are resistant to specific pathogen strains. See, for example, Jones et al., Science 266:789 ( 1994) (cloning of the tomato Cf- 9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262: 1432 ( 1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78: 1089 ( 1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

B. A gene conferring resistance to a pest, such as soybean cyst nematode. See e.g., PCT Application WO 96/30517; PCT Application WO 93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection, Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793 ( 1987) (nucleotide sequence of rice cysteine proteinase inhibitor); Huub et al., Plant Molec. Biol. 21 :985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani et al., Biosci. Biotech. Biochem. 57: 1243 ( 1993) (nucleotide sequence of Streptomyces nitrosporeus alpha-amylase . inhibitor); and U.S. Pat. No. 5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344:458 ( 1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone. H. An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269:9 ( 1994) (expression cloning yields DNA coding for insect diuretic hormone receptor); and Pratt et al., Biochem. Biophys. Res. Comm. 163: 1243 ( 1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 1 16: 165 ( 1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.

. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691 ( 1993), who teach the nucleotide sequence of a cDNA encoding tobacco hornworm chitinase; and Kawalleck et al., Plant Molec. Biol. 21 :673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see the disclosure by Botella et al., Plant Molec. Biol. 24:757 ( 1994), of nucleotide sequences for mung bean calmodulin cDNA clones; and Griess et al., Plant Physiol. 104: 1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO 95/16776 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance).

N. A membrane permease, a channel former or a channel blocker. For example, see the disclosure of Jaynes et al., Plant Sci. 89:43 ( 1993), of heterologous expression of a cecropin-.beta., lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. rev. Phytopathol. 28:451 ( 1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366:469 ( 1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo alpha- 1 ,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-alpha- 1 ,4-D-galacturonase. See Lamb et al., Bio/Technology 10: 1436 ( 1992). The cloning and characterization of a gene which encodes a bean endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 ( 1992). s. A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease. 2. Genes that Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee et al., EMBO J. 7: 1241 (1988); and Miki et al., Theon. Appl. Genet. 80:449 ( 1990), respectively.

B. Glyphosate (resistance conferred by, e.g., mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate acetyl transferase (GAT) genes, respectively), other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes), See, for example, U.S. Pat. No. 4,940,835 to Shah, et al. and U.S. Pat. No. 6,248,876 to Barry et. al., which disclose nucleotide sequences of forms of EPSPs which can confer glyphosate resistance to a plant. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. European patent application No. 0 333 033 to umada et al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a PAT gene is provided in European application No. 0 242 246 to Leemans et al., DeGreef et al., Bio/Technology 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for PAT activity. Exemplary of genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop are the Acc l -S l , Acc l -S2 and Acc l -S3 genes described by Marshall et al., Theon. Appl. Genet. 83:435 ( 1992). GAT genes capable of conferring glyphosate resistance are described in WO 2005012515 to Castle et. al. Genes conferring resistance to 2,4-D, fop and pyridyloxy auxin herbicides are described in WO 2005107437 and U.S. patent application Ser. No. 1 1/587,893, both assigned to Dow AgroSciences LLC.

C. An herbicide that inhibits photosynthesis, such as a triazine (psbA and l s+ genes) or a benzonitrile (nitrilase gene). Przibila et al., Plant Cell 3: 169 (1991 ), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441 , and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al., Biochem. J. 285: 173 ( 1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant. See nultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992).

B. Decreased phytate content- 1 ) Introduction of a phytase-encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see Van Hartingsveldt et al., Gene 127:87 ( 1993), for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene. 2) A gene could be introduced that reduced phytate content. In maize for example, this could be accomplished by cloning and then reintroducing DNA associated with the single allele which is responsible for maize mutants characterized by low levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al., J. Bacteol. 170:810 ( 1988) (nucleotide sequence of Streptococcus mutants fructosyltransferase gene); Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene); Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacillus lichenifonnis alpha-amylase); Elliot et al., Plant Molec. Biol. 21 :515 (1993) (nucleotide sequences of tomato invertase genes); Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directed mutagenesis of barley alpha-amylase gene); and Fisher et al., Plant Physiol. 102: 1045 (1993) (maize endosperm starch branching enzyme II).

D. Abiotic Stress Tolerance which includes resistance to non-biological sources of stress conferred by traits such as nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance cold, and salt resistance. Genes that affect abiotic stress resistance (including but not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, and salt resistance or tolerance) and increased yield under stress.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Unless specifically indicated or implied, the terms "a", "an", and "the" signify "at least one" as used herein.

The present invention is explained in greater detail in the Examples that follow. These examples are intended as illustrative of the invention and are not to be taken are limiting thereof. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Corn hybrids derived from the IBMRIL population (Lee, M; Sharopova, N.; Beavis, W. D.; Grant, D.; att, M.; Blair, D.; and Arnel Hallauer, A. (2002) Expanding the genetic map of maize with the intermated B73 x Mo 17 (IBM) population. Plant Molec. Biol. 48:453-461 ) were crossed to HERCULEX-XTRA and non-HERCULEX-XTRA isogenic testers. One hundred female inbreds were used in this experiment. These inbreds consisted of 99 recombinant inbred lines from the intermated B73 x Mo l 7 population (IBMRI Ls) and one Dow AgroSciences proprietary inbred (QASM7). The genotypes were consistent between 2008 and 2009 with the exception of M0329 which was grown only in 2008 and

M0379 which was grown only in 2009. Furthermore, the hybrids formed from the parental inbred lines, B73 and Mo 1 7, were only grown in 2009. The I BMRILs were chosen based on previous data obtained in 2006 and 2007, and were selected to minimize any confounding effects due to differences in maturity. Each of the female lines was crossed to both DASV8 and its near isogenic l ine, DASV8XT, which contains the HERCULEX-XTRA traits, to create a total of 200 hybrids.

A split-block design with three replications was used in which N rate and female parent were the whole plot treatments. Male parent was included as a spl it-plot within each N rate/female parent subplot. Plots were planted on May 7, 2008 and May 21 , 2009 on the University of Illinois Cruse Farm in Champaign, IL. Al l plots received an in~furrow application of chlorpyrifos (Lorsban 1 5G) at a rate of 1 .3 lbs a. i. per acre in 2008 and tefluthrin (Force 3G) at a rate of 0.099 lbs a. i. per acre in 2009. N was applied as ammonium sulfate ((NF^ SC ) in a diffuse band after emergence and incorporated between V2 and V3 plant growth stage. The N rates used in the study were 0 and 225 lbs per acre. Each experimental unit consisted of two rows spaced 2.5 feet apart. The rows were 1 5 feet long in 2008 and 1 7.5 feet long in 2009. Plots were thinned to an approximate density of 32,000 plants acre. A t flowering, four (2008) or five (2009) representative plants were sampled and weighed. A shredded aliquot was dried to constant weight and ground in a Wiley mil l to pass through a 20 mesh screen. Dried, ground stover samples were analyzed for total N concentration using a combustion technique (NA2000 N~Protein, Fisons Instruments, San Carlos, CA). A similar sampl ing approach was used at the R6 plant growth stage except that the ears were removed, dried, and shelled to al low for calculation of per-plant grain weight at physiological maturity. At harvest, al l plants within a single row of the two row plot were harvested. A subsample of the grain was analyzed for protein concentration using near-infrared transmittance spectroscopy (Foss 1241 NIT grain analyzer; FOSS, Eden Prairie, MN). Three-hundred kernels from each plot were counted using an electronic seed counter and weighed to obtain an estimate of individual kernel weight.

The phenotypic data were analyzed using the MIXED procedure of SAS. Nitrogen rate, female parent, and male parent were treated as fixed effects whi le repl ication was considered random.

Genetic utilization (GU) is defined as grain weight (kg) per unit of plant N (kg) under nonfertilized conditions, and has units of kg/kg plantN. Nitrogen Use Efficiency (NUE) was calculated as the ability to produce grain (kg) per unit of added fertilizer N (kg) and has units of kg/kgN. Nitrogen Uptake Efficiency (NUpE) was calculated as the difference in plant nitrogen content at high and low N application levels, divided by the difference in the applied levels of N. This is a measure of the efficiency of fertilizer N uptake into the plant per unit of fertilizer N (kg) and has units of kg plantN/kgN. Nitrogen Utilization Efficiency (NUtE) is defined as the grain weight (kg) per unit of N taken up by plant and has units of kg/kg plantN

The fields in which these hybrids were grown were treated with chemical pesticides at rates recommended by the manufacturer to control corn rootworms. Thus control of root damage was not dependent upon efficacy of the Cry34Abl and Cry35Ab l HERCULEX-XTRA genes. The data summarized in Table 1 show that when averaged across the 99 IBMRIL female parents, in both 2008 and 2009, the hybrids with the HERCULEX-XTRA trait (DASV8XT) had significantly better Nitrogen Uptake Efficiency (NUpE) than did the hybrids which did not contain the HERCULEX-XTRA genes.

The values of Nitrogen Use Efficiency (NUE) and Nitrogen Utilization Efficiency (NUtE) were found not to correlate with the presence/absence of the HERCULEX-XTRA traits in 2008 and 2009 (Table 1 )

Table 1. N use components of corn lines having the H ERCU LEX-XTRA traits (DASV8XT) compared to isogenic lines without the traits (DASV8) at Champaign, I L in 2008 and 2009, at

0 DASV8 49.5a 67.4a

0 DA SV8XT 46. b 66.0a

225 DASV8 12.9a 0.32a 40.7a 30.5a 0.56a 55.4a

225 DASV8XT 14.0b 0.35b 42.4a 29.2b 0.60b 49.3b

Means within a column followed by the same letter are not significantly different at P <0.05.

"Genetic Utilization (GU). Defined as grain weight (kg) per unit of plant N (kg) under nonfertilized conditions. Units are kg/kgplantN.

""N Use Efficiency (NUE) Defined as ability to produce grain (kg) per unit of added fertilizer N (kg). Units are kg/kgN.

"N Uptake Efficiency (NUpE). Defined as efficiency of fertilizer N uptake into the plant (kg) per unit of fertilizer N

¾>). Units are kgplantN/kgN.

Utilliizzaattiicon Efficiency (NUtE). Defined as grain weight (kg) per unit of N taken up by plant (kg). Units are kg kgplantN.

The increased Nitrogen Uptake Efficiency seen with plants having the HERCULEX- XTRA traits was reflected in increased plant N content at the R6 physiological state (Table 2) in both 2008 and 2009. Table 2. Effect of N level and presence of the HERCULEX-XTRA trait on N content

Means within a column followed by the same letter are not significantly different at P <0.05.

Further, the HERCULEX-XTRA containing hybrids had significantly better yield traits than did the hybrids which did not contain the HERCULEX-XTRA genes (Table 3). Yield was increased based on both an increase in kernel number per plant and an increase in kernel mass Improved yield correlated with higher N content at the R6 stage in both years of the study. The data Table 2 and Table 3 taken together demonstrate that improved yield correlated with higher N content at the R6 stage. Thus, one skilled in the field of crop physiology will realize that the HERCULEX-XTRA traits are effective in causing an increase in N uptake during the flowering and grain filling periods on development. Table 3. Grain yield and yield components of corn lines having the HERCULEX-XTRA traits (DASV8XT) compared to isogenic lines without the traits (DASV8) at Champaign,

IL in 2008 and 2009 at two N a lication rates.

Means within a column followed by the same letter are not significantly different at P≤0.05.

Ma et al. (Ma, B.L.; Meloche, F.; and Wei, L: (2009) Agronomic assessment ofBt trait and seed or soil-applied insecticides on the control of corn rootworm and yield. Field Crops Research. 1 1 1 ^th edition: 189- 196) showed little to no yield effect of Cry3Bb l events (active against corn rootworms) in a commercial hybrid compared with an isogenic non-transgenic hybrid treated with Force 3G insecticide to control corn rootworms. Likewise, Vigna (Vigna, M.M. (2008) Comparison of the effect of corn rootworm technology and seed-applied insecticide (clothianidin) to nitrogen status in corn. MA Thesis, Iowa State University, Ames) found no difference in yield or N content of transgenic hybrids containing either Cry3Bb l or

Cry34Abl +Cry35Ab l (event DAS59122-7) with isogenic non-transgenic controls treated with Poncho 1250. Thus, the HERCULEX-XTRA transgenes may have broader effects than simply limiting rootworm damage.

Increasing nitrogen uptake efficiency and yield by production of HERCULEX-XTRA proteins in plants could have a dramatic effect on agriculture if also effective in crop species such as canola, wheat, rice, barley and other non-legume crops.

Table 4. Analysis of variance results for grain yield and N use traits measured in 2008. Trait N rat Female N rate x female Male par nt N rate x mala Female x male N rate x femala x mala

-

- -

-

Table 5. Analysis of variance results for grain yield and N use traits measured in 2009.

Source of Variation

Trait N rate Femala N rate x female Male parent N rate x male Female x male N rate x female x male

Rl whole shoot biomass 0.0200 « 0.0001 n.s." « 0.0001 n.s. 0.0153 n.s.

Rl whole shoot N content 0.0014 0.0117 0.0831 « 0.0001 « 0.0001 0.0905 n.s.

R6 grain weight 0.0023 « 0.0001 0.0001 0.0015 0.0017 0.0870 0.0489

R6 whole shoot biomass 0.0012 < 0.0001 0.0007 « 0.0001 0.0598 0.0013 0.0178

R6 total N content 0.0003 < 0.0001 0.0001 « 0.0001 0.0004 < 0.0001 « 0.0001

Harvest Index 0.0057 « 0.0001 « 0.0001 0.1091 « 0.0001 ' n.s. n.s.

Nitrogen harvest index 0.0513 0.0002 < 0.0001 n.s. « 0.0O01 n.s. n.s.

Grain protein concentration 0.0005 « 0.0001 « 0.0001 « 0.0001 n.s. 0.0329 n.s.

Yield 0.0005 « 0.0001 « 0.0001 « 0.0001 0.0007 0.0003 0.0440

Kernel weight 0.0009 « 0.0001 0.0015 « 0.0001 « 0.0001 « 0.0001 n.s.

Kernel number 0.0008 « 0.0001 0.0016 «0.0001 n.s. 0.0024 n.s.

Reproductive success 0.0016 « 0.0001 < o.oooi « 0.0001 « 0.0001 0.0060 0.0005

NUE < 0.0001 0.0003 . 0.0140 -

NUpE - « 0.0001 « 0.0001 - 0.0002 -

NUtE « 0.0001 « 0.0001 « 0.0001 -

GU « 0.0001 0.0735 n.s. -

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMS We claim:

1. A method of increasing nitrogen uptake efficiency in a plant comprising administering to a plant an expression cassette containing at least one Bacillus thuringiensis insect-resistance gene which functions to improve expression of at least one insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in a plant cell.

2. The method according to claim 1 wherein said method further comprises an increase in the yield of the plant.

3. The method according to claim 2 wherein the increase in yield comprises an increase in the kernel number per plant and/or an increase in the kernel mass per plant.

4. The method according to claim 1 further comprising culturing the plant under plant cell growing conditions wherein the nitrogen utilization is modulated.

5. The method according to claim 4 wherein said Bacillus thuringiensis insect- resistance gene encodes a Cry protein.

6. The method according to claim 5 wherein said insect-resistance gene is selected from the group consisting of a cry34 gene, a cry35 gene, a crylF gene, and a cry3A gene.

7. The method according to claim 1 wherein the Bacillus thuringiensis insect- resistance gene comprises Cry34Abl, Cry35Abl, and CrylF.

8. The method according to claim 1 wherein the increase in nitrogen occurs during the flowering and grain filling periods of development.

9. The method according to claim 1 wherein the plant is selected from the group consisting of a monocotyledonous plant and a dicotyledonous plant.

10. The method according to claim 1 wherein the plant is a monocotyledonous plant.

11. A method of growing transgenic corn plants having an increased yield by using a reduced amount of fertilizer, wherein the transgenic corn is insect resistant due to expression of an insect-resistance gene, and wherein the reduced amount of fertilizer is relative to fertilizer recommended for use on non-transgenic corn, wherein said non-transgenic corn is optionally protected by granular chemical insecticide to control rootworms.

12. The method according to claim 11 wherein said transgenic corn plants yield corn comparable to corn yield from said non-transgenic corn grown using said recommended amounts of fertilizer.

13. The method according to claim 11 wherein said fertilizer is a nitrogenous fertilizer.

14. The method according to claim 11 wherein said transgenic plant comprises a

Bacillus thuringiensis insect-resistance gene.

15. The method according to claim 14 wherein said Bacillus thuringiensis insect- resistance gene encodes a Cry protein.

16. The method according to claim 14 wherein said insect-resistance gene is selected from the group consisting of a cry34 gene, a cry35 gene, a crylF gene, and a cry3A gene.

17. The method according to claim 14 wherein the Bacillus thuringiensis insect- resistance gene comprises Cry34Abl, Cry35Abl, and CrylF.

18. The method according to claim 13 wherein said nitrogenous fertilizer is applied at a rate of less than 150 pounds per acre.

19. The method according to claim 11 wherein said transgenic corn plants are grown in a field, and said fertilizer is nitrogenous fertilizer applied to said field after planting said corn plants in said field but prior to emergence.

20. A method of modulating nitrogen utilization efficiency (NUE) in a plant cell, comprising: (a) introducing into a plant cell a recombinant expression cassette comprising a Cry protein operably linked to a promoter that drives expression in a plant; and (b) culturing the plant cell under plant cell growing conditions; wherein the nitrogen uptake in the plant cell is modulated.

21. A method for increasing yield in a plant, said method comprising the steps of: (a) introducing into a plant cell a construct comprising a Cry protein operably linked to a promoter functional in a plant cell, so as to yield transformed plant cells; and, (b) regenerating a transgenic plant from said transformed plant cell, wherein said Cry protein is expressed in the cells of said transgenic plant at levels sufficient to increase yield in said transgenic plant; wherein increased yield comprises enhanced root growth, increased seed size, increased seed weight, seed with increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size, nitrogen utilization or chlorophyll content.

22. Seed from the transgenic plant of claim 21.