US20180327759A1 - Transgenic plants with enhanced agronomic traits

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Abstract

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US20180327759A1

Publication number: US20180327759A1
Application number: US15/732,668
Authority: US
Inventors: Mark Scott Abad; Adrian A. Lund; Terry L. Bradshaw; Barry S. Goldman; Joshua Stein
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2004-12-21
Filing date: 2017-12-12
Publication date: 2018-11-15
Also published as: WO2007044043A2; US20150089684A1; EP2562259B1; CA2875402C; EP2562259A3; CA2595171A1; AR051856A1; US20070294782A1; WO2007044043A3; EP1827079A2; EP3078749B1; AU2005337132B2; EP3078749A1; US9862959B2; EP1827079A4; CA2875402A1; AU2005337132A1; CA2595171C; EP2562259A2

This invention provides transgenic plant cells with recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. This invention also provides transgenic plants and progeny seed comprising the transgenic plant cells where the plants are selected for having an enhanced trait selected from the group of traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Also disclosed are methods for manufacturing transgenic seed and plants with enhanced traits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/121,455, filed on Sep. 8, 2014, which is a continuation of U.S. application Ser. No. 11/311,940, filed Dec. 19, 2005, which claims benefit under 35 USC § 119(e) of U.S. provisional application Ser. No. 60/638,099, filed Dec. 21, 2004, and U.S. provisional application Ser. No. 60/660,320, filed Mar. 10, 2005, each of which are herein incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy and a computer readable form (CRF) of the sequence listing, all on CD-ROMs, each containing the text of the file named “3126011US3.txt”, which is 34,689,024 bytes (measured in MS-WINDOWS) and was created on Nov. 27, 2017, are herein incorporated by reference.

INCORPORATION OF COMPUTER PROGRAM LISTING

Two copies of the Computer Program Listing (Copy 1 and Copy 2) containing folders hmmer-2.3.2 and 67pfamDir, all on CD-Rs, are incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated files for implementing the HMMer software for Pfam analysis. Folder 67pfamDir contains 67 Pfam Hidden Markov Models. Both folders were created on the CD-R on Nov. 28, 2017, having a total size of 3,153,920 bytes (measured in MS-WINDOWS).

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics and developmental biology. More specifically, the present inventions provide plant cells with recombinant DNA for providing an enhanced trait in a transgenic plant, plants comprising such cells, seed and pollen derived from such plants, methods of making and using such cells, plants, seeds and pollen.

BACKGROUND OF THE INVENTION

Transgenic plants with improved agronomic traits such as yield, environmental stress tolerance, pest resistance, herbicide tolerance, improved seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. Merely introducing recombinant DNA into a plant genome doesn't always produce a transgenic plant with an enhanced agronomic trait. Methods to select individual transgenic events from a population are required to identify those transgenic events that are characterized by the enhanced agronomic trait.

SUMMARY OF THE INVENTION

This invention employs recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic traits to the transgenic plants. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names as identified in Table 28. In more specific embodiments of the invention the protein expressed in plant cells has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ NO:84 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO:166 and homologs thereof listed in Table 2. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1.
Other aspects of the invention are specifically directed to transgenic plant cells comprising the recombinant DNA of the invention, transgenic plants comprising a plurality of such plant cells, progeny transgenic seed and transgenic pollen from such plants. Such plant cells are selected from a population of transgenic plants regenerated from plant cells transformed with recombinant DNA and that express the protein by screening transgenic plants in the population for an enhanced trait as compared to control plants that do not have said recombinant DNA, where the enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
In yet another aspect of the invention the plant cells, plants, seeds and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell. Such tolerance is especially useful not only as a advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
Yet other aspects of the invention provide transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants. In other important embodiments for practice of various aspects of the invention in Argentina the recombinant DNA is provided in plant cells derived from corn lines that that are and maintain resistance to the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.
This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA for expressing a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 28. More specifically the method comprises (a) screening a population of plants for an enhanced trait and a recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) verifying that the recombinant DNA is stably integrated in said selected plants, (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ NO:1-83; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed.
Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (h) is operably linked to DNA that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 28. The methods further comprise producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repealing the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.
Another aspect of the invention provides a method of selecting a plant comprising plant cells of the invention by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Yet another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a plant cells of this invention.
Still other aspects of this invention relate to transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G and 2A-2G are alignments of amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

As used herein a “plant cell” means a plant cell that is transformed with stably-integrated, non-natural, recombinant DNA, e.g. by Agrobacterium-mediated transformation or by baombardment using microparticles coated with recombinant DNA or other means. A plant cell of this invention can be an originally-transformed plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant DNA, or seed or pollen derived from a progeny transgenic plant.
As used herein a “transgenic plant” means a plant whose genome has been altered by the stable integration of recombinant DNA. A transgenic plant includes a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.
As used herein “recombinant DNA” means DNA which has been a genetically engineered and constructed outside of a cell including DNA containing naturally occurring DNA or cDNA or synthetic DNA.
As used herein “consensus sequence” means an artificial sequence of amino acids in a conserved region of an alignment of amino acid sequences of homologous proteins, e.g. as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
As used herein “homolog” means a protein in a group of proteins that perform the same biological function, e.g. proteins that belong to the same Pfam protein family and that provide a common enhanced trait in transgenic plants of this invention. Homologs are expressed by homologous genes. Homologous genes include naturally occurring alleles and artificially-created variants. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a polynucleotide useful in the present invention may have any base sequence that has been changed from SEQ ID NO:1 through SEQ ID NO:83 by substitution in accordance with degeneracy of the genetic code. Homologs are proteins that, when optimally aligned, have at least 60% identity, more preferably about 70% or higher, more preferably at least 80% and even more preferably at least 90% identity over the full length of a protein identified as being associated with imparting an enhanced trait when expressed in plant cells. Homologs include proteins with an amino acid sequence that has at least 90% identity to a consensus amino acid sequence of proteins and homologs disclosed herein.
Homologs are be identified by comparison of amino acid sequence, e.g. manually or by use of a computer-based tool using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A local sequence alignment program, e.g. BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal query is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal query's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. A further aspect of the invention comprises functional homolog proteins that differ in one or more amino acids from those of disclosed protein as the result of conservative amino acid substitutions, for example substitutions are among: acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; basic (positively charged) amino acids such as arginine, histidine, and lysine; neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; amino acids having aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; amino acids having aliphatic-hydroxyl side chains such as serine and threonine; amino acids having amide-containing side chains such as asparagine and glutamine; amino acids having aromatic side chains such as phenylalanine, tyrosine, and tryptophan; amino acids having basic side chains such as lysine, arginine, and histidine; amino acids having sulfur-containing side chains such as cysteine and methionine; naturally conservative amino acids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.
As used herein, “percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, for example nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100.
As used herein “Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO:84 through SEQ ID NO:166. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 27 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams for use in this invention, as more specifically disclosed below, are AAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 1 2, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60 TCP1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP, FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro 1, Givoxalase, Gotl, HATPase c, HSF DNA-bind, HSP20, HisKA, Homeobox, Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 sly, NAF, NAM, NIF, Oxidored FMN, PAS, PDL, PRA1, Peptidase C15, Peptidase S10, Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1, RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4, the databases for which are included in the appended computer listing.
As used herein “promoter” means regulatory DNA for initializing transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. is it well known that Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters that initiate transcription only in certain tissues are referred to as “tissue specific”. 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 “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions.
As used herein “operably linked” means the association of two or more DNA fragments in a DNA construct so that the function of one, e.g. protein-encoding DNA, is controlled by the other, e.g. a promoter.
As used herein “expressed” means produced, e.g. a protein is expressed in a plant cell when its cognate DNA is transcribed to mRNA that is translated to the protein.
As used herein a “control plant” means a plant that does not contain the recombinant DNA that expressed a protein that impart an enhanced trait. A control plant is to identify and select a transgenic plant that has an enhance trait. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant, i.e. devoid of recombinant DNA. A suitable control plant may in some cases be a progeny of a hemizygous transgenic plant line that is does not contain the recombinant DNA, known as a negative segregant.
As used herein an “enhanced trait” means a characteristic of a transgenic plant that includes, but is not limited to, an enhance agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In more specific aspects of this invention enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. In an important aspect of the invention the enhanced trait is enhanced yield including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
Increased yield of a transgenic plant of the present invention can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tonnes per acre, tons per acre, kilo per hectare. For example, maize yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Recombinant DNA used in this invention can also be used to provide plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. Also of interest is the generation of transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield. Such properties include enhancements in seed oil, seed molecules such as tocopherol, protein and starch, or oil particular oil components as may be manifest by an alterations in the ratios of seed components.
A subset of the nucleic molecules of this invention includes fragments of the disclosed recombinant DNA consisting of oligonucleotides of at least 15, preferably at least 16 or 17, more preferably at least 18 or 19, and even more preferably at least 20 or more, consecutive nucleotides. Such oligonucleotides are fragments of the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:83, and find use, for example as probes and primers for detection of the polynucleotides of the present invention.
In some embodiments of the present invention, a dominant negative mutant of a native gene is generated to achieve the desired effect. As used herein, “dominant negative mutant” means a mutant gene whose gene product adversely affects the normal, wild-type gene product within the same cell, usually by dimerizing (combining) with it. In cases of polymeric molecules, such as collagen, dominant negative mutations are often more deleterious than mutations causing the production of no gene product (null mutations or null alleles). SEQ ID NO: 6 and SEQ ID NO: 7 are constructed to encode agl11 protein with K-box deleted and MADs 3 protein with MAD box deleted, respectively. MADS box proteins similar to AGL11 can be considered as having three functional domains. There is an N-terminal DNA-binding domain (the MADS box), a more distal dimerization domain (the K-box) and a C-terminal domain that is usually involved in interactions with other proteins. In plants the region between the MADS box and the K-box has been shown to be important for DNA binding in some proteins and is often referred to as the I-box (Fan et al., 1997). Several different classes of dominant negative constructs are considered. Deletion or inactivation of the DNA-binding domain can create proteins that are able to dimerize with their native full length counterparts as well as other natural dimerization partners. Likewise, removal of the C-terminal domain can allow dimerization with both the native protein and it's natural dimerization partners. In both cases these types of constructs disable both the target protein and any other protein capable of interacting with the K-box.
In other embodiments of the invention a constitutively active mutant is constructed to achieve the desired effect. SEQ ID NO:3 encodes only the kinase domain from a calcium-dependent protein kinase (CDPK). CDPK1 has a domain structure similar to other calcium-dependant protein kinases in which the protein kinase domain is separated from four efhand domains by 42 amino acid “spacer” region. Calcium-dependant protein kinases are thought to be activated by a calcium-induced conformational change that results in movement of an autoinhibitory domain away from the protein kinase active site (Yokokura et al., 1995). Thus, constitutively active proteins can be made by over expressing the protein kinase domain alone.
DNA constructs are assembled using methods well known to persons of ordinary skill in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the enhanced agronomic trait. Other construct components may include additional regulatory elements, such as 5′ leasders and introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), DNA for transit or signal peptides.
Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938, which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,641,876, which discloses a rice actin promoter, U.S. Patent Application Publication 2002′0192813A1, which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/757,089, which discloses a maize chloroplast aldolase promoter, U.S. patent application Ser. No. 08/706,946, which discloses a rice glutelin promoter. U.S. patent application Ser. No. 09/757,089, which discloses a maize aldolase (FDA) promoter, and U.S. patent application Ser. No. 60/310,370, which discloses a maize nicotianamine synthase promoter, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.
In some aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), zein Z27 and glutelin1 (Russell et al. (1997) Transgenic Res. 6(2): 157-166), and peroxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol. 31(6):1205-1216), maize L3 oleosin (U.S. Pat. No. 6,433,252), globulin 1 (Belanger et al (1991) Genetics 129:863-872).
In other aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (Fischhoff et al. (1992) Plant Mol Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi el al. (2000) Plant Cell Physiol. 41(1):42-48).
Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted upstream (5′) or downstream (3′) to the coding sequence. In some instances, these 5′ enhancing elements are introns. Particularly useful as enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No. 5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase gene intron, the maize heat shock protein 70 gene intron (U.S. Pat. No. 5,593,874) and the maize shrunken 1 gene.
In other aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein 727 (Russell et al. (1997) Transgenic Res. 6(2): 157-166), globulin 1 (Belanger et al (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol. 31(6): 1205-1216).
Recombinant DNA constructs prepared in accordance with the invention will also generally include a 3′ element that typically contains a polyadenylation signal and site. Well-known 3′ elements include those from Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, ms 3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference; 3′ elements from plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in U.S. published patent application 2002/0192813 A1, incorporated herein by reference; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3), and 3′ elements from the genes within the host plant.
Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference. For description of the transit peptide region of an Arabidopsis EPSPS gene useful in the present invention, see Klee, H. J. et al (MGG (1987) 210:437-442).
Transgenic plants comprising or derived from plant cells of this invention transformed with recombinant DNA can be further enhanced with stacked traits, e.g. a crop plant having an enhanced trait resulting from expression of DNA disclosed herein in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are well-known in the art and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for imparting glyphosate tolerance; polynucleotide molecules encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in U.S. Patent Application publication 2003/0083480 A1 also for imparting glyphosate tolerance; dicamba monooxygenase disclosed in U.S. Patent Application publication 2003/0135879 A1 for imparting dicamba tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) Plant J. 6:481-489 for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance to sulfonylurea herbicides; polynucleotide molecules known as bar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for imparting glufosinate and bialaphos tolerance; polynucleotide molecules disclosed in U.S. Patent Application Publication 2003/010609 A1 for imparting N-amino methyl phosphonic acid tolerance; polynucleotide molecules disclosed in U.S. Pat. No. 6,107,549 for impartinig pyridine herbicide resistance; molecules and methods for imparting tolerance to multiple herbicides such as glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 and U.S. Patent Application Publication 2002/0112260, all of said U.S. Patents and Patent Application Publications are incorporated herein by reference. Molecules and methods for imparting insect/nematode/virus resistance is disclosed in U.S. Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent Application Publication 2003/0150017 A1, all of which are incorporated herein by reference.
In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol.
These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC 11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM 1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (vv) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
Fusion procedures usually produce viable hybrids at low frequencies, about 1×10⁻⁶to 1×10⁻⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

Plant Cell Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA are known in the art and may be used in the present invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean): U.S. Pat. No. 5,550,318 (corn): U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
In general it is useful to introduce recombinant DNA randomly, i.e. at a non-specific location, in the genome of a target plant line. In special cases it may be useful to target recombinant DNA insertion in order to achieve site-specific integration, for example to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, for example various media and recipient target cells, transformation of immature embryo cells and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein by reference.
The seeds of transgenic plants can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plants line for selection of plants having an enhanced trait. In addition to direct transformation of a plant with a recombinant DNA, transgenic plants can be prepared by crossing a first plant having a recombinant DNA with a second plant lacking the DNA. For example, recombinant DNA can be introduced into first plant line that is amenable to transformation to produce a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, e.g. enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, e.g. marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line In the practice of transformation DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the herbicides to which plants of this invention may be resistant are useful agents for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA.
Commonly used selective marker genes include those conferring resistance to antibiotics such as kananmycin and paromomycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (aroA or EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Selectable markers which provide an ability to visually identify transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.
Plant cells that survive exposure to the selective agent, or plant cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets regenerated from transformed plant cells can be transferred to plant growth mix, and hardened off, for example, in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced, for example self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its progeny seed or plants can be tested for expression of the recombinant DNA and selected for the presence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from the plant cells of this invention are grown to generate transgenic plants having an enhanced trait as compared to a control plant and produce transgenic seed and haploid pollen of this invention. Such plants with enhanced traits are identified by selection of transformed plants or progeny seed for the enhanced trait. For efficiency a selection method is designed to evaluate multiple transgenic plants (events) comprising the recombinant DNA, for example multiple plants from 2 to 20 or more transgenic events. Transgenic plants grown from transgenic seed provided herein demonstrate improved agronomic traits that contribute to increased yield or other trait that provides increased plant value, including, for example, improved seed quality. Of particular interest are plants having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
Table 1 provides a list of protein encoding DNA (“genes”) that are useful as recombinant DNA for production of transgenic plants with enhanced agronomic trait, the elements of Table 1 are described by reference to:
“PEP SEQ” which identifies an amino acid sequence from SEQ ID NO:84 to 166.
“NUC SEQ” which identifies a DNA sequence from SEQ ID NO: 1 to 83.
“Base Vector” which identifies a base plasmid used for transformation of the recombinant DNA.
“PROTEIN NAME” wvhich is a common name for protein encoded by the recombinant DNA.
“Enhanced trait” which identifies an enhanced trait which is imparted by the expression of the protein in a transgenic crop plant.
“Plasmid ID” which identifies an arbitrary name for the plant transformation plasmid comprising recombinant DNA for expressing the recombinant DNA in plant cells.

TABLE 1

PEP	NUC
SEQ	SEQ
ID	ID
NO	NO	Base Vector	PROTEIN NAME	Enhanced trait(s)	Plasmid ID

84	1	pMON65154	lactoylglutathione lyase	Enhanced seed protein	pMON69462
85	2	pMON72472	rab7c	Enhanced cold tolerance	pMON69456
86	3	pMON65154	CDPK kinase domain	Enhanced water use	pMON67754
				efficiency
87	4	pMON72472	SCOF-1	Enhanced water use	pMON72494
				efficiency and enhanced
				cold tolerance
88	5	pMON72472	Synechococcus sp. PCC	Increased yield, enhanced	pMON68399
			6301 Delta9 desaturase	cold tolerance and enhanced
				water use efficiency
89	6	pMON72472	Arabidopsis agl11 delta	Improved cold tolerance	pMON73765
			K-box
90	7	pMON72472	rice MADS3 delta	Enhanced cold tolerance	pMON73829
			MADS-box—L37528
91	8	pMON72472	corn MADS box	Enhanced nitrogen use	pMON73816
			protein 110	efficiency and enhance cold
				tolerance
92	9	pMON72472	Arabidopsis	Enhanced cold tolerance	pMON75305
			homeodomain
			transcription factor-
93	10	pMON72472	Arabidopsis AP2	Enhanced cold tolerance	pMON75306
			domain transcription
			factor
94	11	pMON72472	Arabidopsis GATA	Enhanced cold tolerance	pMON75309
			domain transcription
			factor
95	12	pMON72472	Arabidopsis AT-hook	Enhanced cold tolerance	pMON75312
			domain transcription
			factor-
96	13	pMON72472	rice DET1-like -	Enhanced nitrogen use	pMON80270
			BAB16336	efficiency and enhanced
				cold tolerance
97	14	pMON72472	soybean G482-like 1	Enhanced water use	pMON76342
				efficiency
98	15	pMON72472	Arabidopsis	Enhanced cold tolerance	pMON79174
			hypothetical protein
			[NM_114802]
99	16	pMON72472	corn hypothetical	Enhanced cold tolerance	pMON79413
			protein
100	17	pMON72472	soy Pra2-like protein 2	Enhanced nitrogen use	pMON75511
				efficiency
101	18	pMON72472	Agrobacterium	Enhanced cold tolerance	pMON75515
			cryptochrome-like
			protein - AE008050
102	19	pMON72472	rice SNF1-like protein	Enhanced nitrogen use	pMON80542
			9 [OsPK4] - AB011967	efficiency, enhanced water
				use efficiency, increased
				yield
103	20	pMON72472	corn SNF1-like protein 3	Enhanced water use	pMON78949
				efficiency and enhanced
				nitrogen use efficiency
104	21	pMON72472	corn SNF1-like protein 8	Enhanced cold tolerance	pMON78936
				and enhanced water use
				efficiency
105	22	pMON72472	Corn Rubisco Activase 2	Increased yield, enhanced	pMON75524
				cold tolerance and enhanced
				nitrogen use efficiency
106	23	pMON72472	NLI Interacting Isoform	Enhanced cold tolerance	pMON79163
			T1-	and increased yield
107	24	pMON72472	maize synaptobrevin-	Enhanced cold tolerance	pMON75533
			related sequnece 1 -	condition and increased
				yield
108	25	pMON72472	maize magnesium	Enhanced nitrogen use	pMON79709
			transporter mrs2-1-like	efficiency and increased
			1 sequence	yield
109	26	pMON72472	Corn Protein similar to	Enhanced water use	pMON79422
			nodulin MtN3 protein	efficiency
110	27	pMON72472	Corn glyoxalase II	Enhanced cold tolerance	pMON79425
			isozyme
111	28	pMON72472	Corn RNA 3-	Enhanced cold tolerance	pMON79718
			TERMINAL
			PHOSPHATE
			CYCLASE-LIKE
			PROTEIN
112	29	pMON72472	rice Di19 like sequence	Enhanced cold tolerance	pMON79447
113	30	pMON72472	soybean MAP kinase 6	Enhanced cold tolerance	pMON78232
			like 2 sequence
114	31	pMON72472	Ralstonia metallidurans	Enhanced cold tolerance,	pMON75980
			glutamate	and enhanced nitrogen use
			decarboxylase	efficiency
115	32	pMON72472	rice HSF5 like	Enhanced water use	pMON80489
			sequence	efficiency
116	33	pMON72472	soybean hsp17.4 like 1	Enhanced cold tolerance	pMON79697
			sequence	and enhanced water use
				efficiency
117	34	pMON72472	Corn putative	Enhanced water use	pMON78237
			pyrrolidone carboxyl	efficiency
			peptidase
118	35	pMON72472	Arabidopsis E2F	Enhanced cold tolerance	pMON80461
				enhanced nitrogen use
				efficiency
119	36	pMON72472	Arabidopsis protein	Enhanced cold tolerance	pMON78235
			phosphatase 1A
120	37	pMON72472	Arabidopsis CtpA	Enhanced cold tolerance,	pMON80452
				and enhanced water use
				efficiency
121	38	pMON74532	Arabidopsis CtpA	Increased yield
122	39	pMON72472	Corn protein similar to	Enhanced cold tolerance	pMON80500
			Arabidopsis Probable
			microsomal signal
			peptidase
123	40	pMON72472	[Oryza sativa] putative	Enhanced nitrogen use	pMON80850
			aldose reductase	efficiency
124	41	pMON72472	Zea Mays Kinase II	Increased seed protein	pMON78949
			(similar to Yeast IKS1 &
			At MRK1)
125	42	pMON72472	Fructose-1-6-	Increased yield	pMON81853
			bisphosphatase
126	43	pMON72472	soy G1928 like 1	Increased seed protein	pMON83769
127	44	pMON74532	Synechocystis sp. 6803	Increased yield	pMON78911
			Hik19
128	45	pMON72472	Synechocystis sp. 6803	Increased yield
			Hik19
129	46	pMON72472	Arabidopsis NAC	Increased yield	pMON73787
			domain transcription
			factor
130	47	pMON72472	yeast alanine	Increased yield and	pMON77895
			aminotransferase 1 -	enhanced nitrogen use
			AAB67593	efficiency
131	48	pMON72472	soybean catalase-like 1	Increased yield	pMON79152
132	49	pMON72472	corn ALG-2 interacting	Increased yield	pMON80921
			protein
133	50	pMON72472	Putative Serine	Increased yield	pMON75505
			Carboxypeptidase-
134	51	pMON72472	Putative Ankyrin Like	Increased yield	pMON80925
			Protein-
135	52	pMON72472	Putative Kinase Like	Increased yield	pMON78942
			Protein-
136	53	pMON72472	Putative Protein-	Increased yield	pMON79164
137	54	pMON72472	yeast YPR145W/asn1 -	Increased yield	pMON79653
			U40829
138	55	pMON72472	rice AtHSP17.6A like 1	Increased yield	pMON81228
			sequence
139	56	pMON72472	yeast YDL123w	Increased yield	pMON79430
140	57	pMON72472	rice 12-	Increased yield	pMON79731
			oxophytodienoate
			reductase like 1
			sequence
141	58	pMON72472	soybean MAP kinase 6	Increased yield	pMON78229
			like 3 sequence
142	59	pMON72472	Arabidopsis GAD1	Increased yield	pMON79696
143	60	pMON74532	Arabidopsis GAD1
144	61	pMON72472	soybean hsp17.4 like 4	Increased yield	pMON78240
			sequence
145	62	pMON72472	maize hsp60 like 4	Increased yield	pMON80283
			sequence
146	63	pMON72472	soy dsPTP 1	Increased yield	pMON80866
147	64	pMON72472	Yeast GLC3 Glycogen	Increased yield	pMON80292
			branching enzyme
148	65	pMON72472	Arabidopsis unknown	Increased yield	pMON82223
			protein
149	66	pMON72472	beta-D-glucosidase	Increased yield	pMON83553
150	67	pMON72472	unknown protein1	Increased yield	pMON81857
151	68	pMON72472	aldehyde oxidase	Increased yield	pMON82218
152	69	pMON72472	corn hypothetical	Improved growth under cold	pMON78227
			protein	stress
153	70	pMON72472	corn hypothetical	Improved growth under cold	pMON78904
			protein	stress
154	71	pMON72472	Arabidopsis cysteine	Increased yield	pMON78920
			proteinase inhibitor
155	72	pMON82053	Arabidopsis cysteine	Increased yield	pMON92646
			proteinase inhibitor
156	73	pMON72472	Arabidopsis	Improved growth under cold	pMON78922
			hypothetical protein	stress
157	74	pMON72472	yeast SNF1 - A26030	Improved growth under low	pMON78948
				nitrogen, drought, and/or
				cold stresses
158	75	pMON72472	soy SNF1-like protein 1	Increased yield	pMON79660
159	76	pMON72472	soy SNF-like protein 2	Enhanced nitrogen use	pMON78931
				efficiency, enhanced water
				use efficiency, increased
				yield
160	77	pMON72472	soy G1760	Increased yield and	pMON82645
				enhanced water use
				efficiency
160	77		Soy G1760	Increased yield	pMON74470
161	78	pMON72472	Rice Glyoxalase II	Increased yield	pMON79665
162	79	pMON72472	corn OsPK7-like	Enhanced nitrogen use	pMON82629
				efficiency, enhanced water
				use efficiency, increased
				yield
163	80	pMON74532	rice phyA with	Increased yield	pMON81344
			Arabidopsis phyC
			intron 1
164	81	pMON82060	rice G975 like1	Improved growth under cold
				stress
165	82		Corn Phytochrome A	Increased yield	pMON74916
166	83		Arabidopsis G1760	Increased yield	pMON73957

Selection Methods for Transgenic Plants with Enhanced Agronomic Trait

Within a population of transgenic plants regenerated from plant cells transformed with the recombinant DNA many plants that survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Selection from the population is necessary to identify one or more transgenic plant cells that can provide plants with the enhanced trait. Transgenic plants having enhanced traits are selected from populations of plants regenerated or derived from plant cells transformed as described herein by evaluating the plants in a variety of assays to detect an enhanced trait, e.g. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. These assays also may take many forms including, but not limited to, direct screening for the trait in a greenhouse or field trial or by screening for a surrogate trait. Such analyses can be directed to detecting changes in the chemical composition, biomass, physiological properties, morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics can be made on greenhouse or field grow n plants and can include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, for example assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other selection properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain may be evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality. Although the plant cells and methods of this invention can be applied to any plant cell, plant, seed or pollen, e.g. any fruit, vegetable, grass, tree or ornamental plant, the various aspects of the invention are preferably applied to corn, soybean, cotton, canola, alfalfa, wheat and rice plants. In many cases the invention is applied to corn plants that are inherently resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.
The following examples are included to demonstrate aspects of the invention, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar results without departing from the spirit and scope of the invention.

Example 1. Plant Expression Constructs

A. Plant Expression Constructs for Corn Transformation

This example illustrates the construction of plasmids for transferring recombinant DNA into plant cells which can be regenerated into transgenic plants of this invention.
Primers for PCR amplification of protein coding nucleotides of recombinant DNA were designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 1 was amplified by PCR prior to insertion into the insertion site of one of the base vectors as referenced in Table 1.
A base plant transformation vector pMON65154 was fabricated for use in preparing recombinant DNA for transformation into corn tissue using GATEWAY™ Destination plant expression vector systems (available from Invitrogen Life Technologies, Carlsbad, Calif.). With reference to the elements described in Table 3 below and SEQ ID NO:10024, pMON65154 comprises a selectable marker expression cassette and a template recombinant DNA expression cassette. The marker expression cassette comprises a CaMV 35S promoter operably linked to a gene encoding neomycin phosphotransferase II (nptII) followed by a 3′ region of an Agrobacterium tumefaciens nopaline synthase gene (nos). The template recombinant DNA expression cassette is positioned tail to tail with the marker expression cassette. The template recombinant DNA expression cassette comprises 5′ regulatory DNA including a rice actin 1 promoter, exon and intron, followed by a GATEWAY™ insertion site for recombinant DNA, followed by a 3′ region of a potato proteinase inhibitor II (pinII) gene. Once recombinant DNA has been inserted into the insertion site, the plasmid is useful for plant transformation, for example by microprojectile bombardment.

TABLE 3

FUNCTION	ELEMENT	REFERENCE

Plant gene of interest	Rice actin	1 promoter	U.S. Pat. No. 5,641,876
expression cassette	Rice actin	1 exon 1, intron 1	U.S. Pat. No. 5,641,876
	enhancer
Gene of interest	AttR1	GATEWAY ™ Cloning Technology
insertion site		Instruction Manual
	CmR gene	GATEWAY ™ Cloning Technology
		Instruction Manual
	ccdA, ccdB genes	GATEWAY ™ Cloning Technology
		Instruction Manual
	attR2	GATEWAY ™ Cloning Technology
		Instruction Manual
Plant gene of interest	Potato pinII 3′ region	An et al. (1989) Plant Cell 1: 115-122
expression cassette
Plant selectable	CaMV 35S promoter	U.S. Pat. No. 5,858,742
marker expression	nptII selectable marker	U.S. Pat. No. 5,858,742
cassette	nos 3′ region	U.S. Pat. No. 5,858,742
Maintenance in E. coli	ColE1 origin of replication
	F1 origin of replication
	Bla ampicillin resistance

A similar base vector plasmid pMON72472 (SEQ ID NO: 10025) was constructed for use in Agrobacterium-mediated methods of plant transformation similar to pMON65154 except (a) the 5′ regulatory DNA in the template recombinant DNA expression cassette was a rice actin promoter and a rice actin intron, (b) left and right T-DNA border sequences from Agrobacterium are added with the right border sequence is located 5′ to the rice actin 1 promoter and the left border sequence is located 3′ to the 35S promoter and (c) DNA is added to facilitate replication of the plasmid in both E. coli and Agrobacterium tumefaciens. The DNA added to the plasmid outside of the T-DNA border sequences includes an oriV wide host range origin of DNA replication functional in Agrobacterium, a pBR322 origin of replication functional in E. coli, and a spectinomycin/streptomycin resistance gene for selection in both E. coli and Agrobacterium.
Another base vector pMON82060 (SEQ ID NO: 10026), illustrated in Table 4, was assembled using the technology known in the art.

TABLE 4

			Coordinates
			of SEQ ID
function	name	Annotation	NO: 10026

Agro	B-AGRtu.right border	Agro right border sequence, essential for	5235-5591
transformation		transfer of T-DNA.
Gene of	P-Os.Act1	Promoter from the rice actin gene act1.	5609-7009
interest plant	L-Os.Act1	Leader (first exon) from the rice actin 1
expression		gene.
cassette	I-Os.Act1	First intron and flanking UTR exon
		sequences from the rice actin 1 gene
	T-St.Pis4	The 3′ non-translated region of the	7084-8026
		potato proteinase inhibitor II gene which
		functions to direct polyadenylation of the
		mRNA
Plant	P-CaMV.35S	CaMV 35S promoter	8075-8398
selectable	L-CaMV.35S	5′ UTR from the 35S RNA of CaMV
marker	CR-Ec.nptII-Tn5	nptII selectable marker that confers	8432-9226
expression		resistance to neomycin and kanamycin
cassette	T-AGRtu.nos	A 3′ non-translated region of the	9255-9507
		nopaline synthase gene of
		Agrobacterium tumefaciens Ti plasmid
		which functions to direct
		polyadenylation of the mRNA..
Agro	B-AGRtu.left border	Agro left border sequence, essential for	39-480
transformation		transfer of T-DNA.
Maintenance	OR-Ec.oriV-RK2	The vegetative origin of replication from	567-963
in E. coli		plasmid RK2.
	CR-Ec.rop	Coding region for repressor of primer	2472-2663
		from the ColE1 plasmid. Expression of
		this gene product interferes with primer
		binding at the origin of replication,
		keeping plasmid copy number low.
	OR-Ec.ori-ColE1	The minimal origin of replication from	3091-3679
		the E. coli plasmid ColE1.
	P-Ec.aadA-SPC/STR	promoter for Tn7 adenylyltransferase	4210-4251
		(AAD(3″))
	CR-Ec.aadA-	Coding region for Tn7	4252-5040
	SPC/STR	adenylyltransferase (AAD(3″))
		conferring spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-SPC/STR	3′ UTR from the Tn7 adenylyltransferase	5041-5098
		(AAD(3″)) gene of E. coli.

B. Plant Expression Vector for Soybean Transformation

Plasmids for use in transformation of soybean were also prepared. Elements of an exemplary common expression vector plasmid pMON74532 (SEQ ID NO: 10027) are shown in Table 5 below.

TABLE 5

Function	Element	Reference

Agro transformation	B-ARGtu.right border	Depicker, A. et al (1982)
		Mol Appl Genet 1: 561-573
Antibiotic resistance	CR-Ec.aadA-SPC/STR
Repressor of primers from the ColE1	CR-Ec.rop
plasmid
Origin of replication	OR-Ec.oriV-RK2
Agro transformation	B-ARGtu.left border	Barker, R. F. et al (1983)
		Plant Mol Biol 2: 335-350
Plant selectable marker expression	Promoter with intron and	McDowell et al. (1996)
cassette	5′UTR of Arabidopsis act 7	Plant Physiol. 111: 699-711.
	gene (AtAct7)
	5′ UTR of Arabidopsis act 7
	gene
	Intron in 5′UTR of AtAct7
	Transit peptide region of	Klee, H. J. et al (1987)
	Arabidopsis EPSPS	MGG 210: 437-442
	Synthetic CP4 coding region
	with dicot preferred codon
	usage
	A 3′ UTR of the nopaline	U.S. Pat. No. 5,858,742
	synthase gene of
	Agrobacterium tumefaciens
	Ti plasmid
Plant gene of interest expression	Promoter for 35S RNA from	U.S. Pat. No. 5,322,938
cassette	CaMV containing a
	duplication of the −90 to −350
	region
	Gene of interest insertion site
	Cotton E6 3′ end	GenBank accession
		U30508

Another base vector pMON82053 (SEQ ID NO: 10028), illustrated in Table 6, was assembled using the technology known in the art.

TABLE 6

			Coordinates of SEQ ID
Function	Name	Annotation	NO: 10028

Agro	B-AGRtu.left border	Agro left border	6144-6585
transforamtion		sequence, essential for
		transfer of T-DNA.
Plant	P-At.Act7	Promoter from the	6624-7861
selectable		arabidopsis actin 7 gene
marker	L-At.Act7	5′UTR of Arabidopsis
expression		Act7 gene
cassette	I-At.Act7	Intron from the
		Arabidopsis actin7 gene
	TS-At.ShkG-CTP2	Transit peptide region of	7864-8091
		Arabidopsis EPSPS
	CR-AGRtu.aroA-	Synthetic CP4 coding	8092-9459
	CP4.nno_At	region with dicot
		preferred codon usage.
	T-AGRtu.nos	A 3′ non-translated region	9466-9718
		of the nopaline synthase
		gene of Agrobacterium
		tumefaciens Ti plasmid
		which functions to direct
		polyadenylation of the
		mRNA.
Gene of	P-CaMV.35S-enh	Promoter for 35S RNA	1-613
interest		from CaMV containing a
expression		duplication of the −90 to −350
cassette		region.
	T-Gb.E6-3b	3′ untranslated region	688-1002
		from the fiber protein E6
		gene of sea-island cotton;
Agro	B-AGRtu.right border	Agro right border	1033-1389
transformation		sequence, essential for
		transfer of T-DNA.
Maintenance	OR-Ec.oriV-RK2	The vegetative origin of	5661-6057
in E. coli		replication from plasmid
		RK2.
	CR-Ec.rop	Coding region for	3961-4152
		repressor of primer from
		the ColE1 plasmid.
		Expression of this gene
		product interferes with
		primer binding at the
		origin of replication,
		keeping plasmid copy
		number low.
	OR-Ec.ori-ColE1	The minimal origin of	2945-3533
		replication from the E. coli
		plasmid ColE1.
	P-Ec.aadA-SPC/STR	romoter for Tn7	2373-2414
		adenylyltransferase
		(AAD(3″))
	CR-Ec.aadA-	Coding region for Tn7	1584-2372
	SPC/STR	adenylyltransferase
		(AAD(3″)) conferring
		spectinomycin and
		streptomycin resistance.
	T-Ec.aadA-SPC/STR	3′ UTR from the Tn7	1526-1583
		adenylyltransferase
		(AAD(3″)) gene of E. coli.

Protein coding segments of recombinant DNA are amplified by PCR prior to insertion into vectors at the insertion site. Primers for PCR amplification are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions.

Example 2. Corn Transformation

This example illustrates plant cell transformation methods useful in producing transgenic corn plant cells, plants, seeds and pollen of this invention and the production and identification of transgenic corn plants and seed with an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Plasmid vectors were prepared by cloning DNA identified in Table 1 in the identified base vectors for use in corn transformation of corn plant cells to produce transgenic corn plants and progeny plants, seed and pollen.
For Agrobacterium-mediated transformation of corn embryo cells corn plants of a readily transformable line (designated LH59) is grown in the greenhouse and ears harvested when the embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels on surface sterilized ears. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature. Immature maize embryo cells are inoculated with Agrobacterium shortly after excision, and incubated at room temperature with Agrobacterium for 5-20 minutes. Immature embryo plant cells are then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos are transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Transformed plant cells are recovered 6 to 8 weeks after initiation of selection.
For Agrobacterium-mediated transformation of maize callus immature embryos are cultured for approximately 8-21 days after excision to allow callus to develop. Callus is then incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration. The callus and Agrobacterium are co-cultured without selection for 3-6 days followed by selection on paromomycin for approximately 6 weeks, with biweekly transfers to fresh media, and paromomycin resistant callus identified as containing the recombinant DNA in an expression cassette.
For transformation by microprojectile bombardment immature maize embryos are isolated and cultured 3-4 days prior to bombardment. Prior to microprojectile bombardment, a suspension of gold particles is prepared onto which the desired recombinant DNA expression cassettes are precipitated. DNA is introduced into maize cells as described in U.S. Pat. Nos. 5,550,318 and 6,399,861 using the electric discharge particle acceleration gene delivery device. Following microprojectile bombardment, tissue is cultured in the dark at 27 degrees C. Additional transformation methods and materials for making transgenic plants of this invention, for example, various media and recipient target cells, transformation of immature embryos and subsequence regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
To regenerate transgenic corn plants a callus of transgenic plant cells resulting from transformation is placed on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber at 26 degrees C. followed by a mist bench before transplanting to 5 inch pots where plants are grown to maturity. The regenerated plants are self fertilized and seed is harvested for use in one or more methods to select seed, seedlings or progeny second generation transgenic plants (R2 plants) or hybrids, e.g. by selecting transgenic plants exhibiting an enhanced trait as compared to a control plant.
Transgenic corn plant cells were transformed with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants and seed of the transformed plant cells were screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as reported in Example 5.

Example 3. Soybean Transformation

This example illustrates plant transformation useful in producing the transgenic soybean plants of this invention and the production and identification of transgenic seed for transgenic soybean having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
For Agrobacterium mediated transformation, soybean seeds are germinated overnight and the meristem explants excised. The meristems and the explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette are mixed no later than 14 hours from the time of initiation of seed germination and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Trait positive shoots are harvested approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil. Additionally, a DNA construct can be transferred into the genome of a soybean cell by particle bombardment and the cell regenerated into a fertile soybean plant as described in U.S. Pat. No. 5,015,580, herein incorporated by reference.
Transgenic soybean plant cells were transformed with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants and seed of the transformed plant cells were screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as reported in Example 5.

Example 4. Homolog Identification

This example illustrates the identification of homologs of proteins encoded by the DNA identified in Table 1 which is used to provide transgenic seed and plants having enhanced agronomic traits. From the sequence of the homologs, homologous DNA sequence can be identified for preparing additional transgenic seeds and plants of this invention with enhanced agronomic traits.
An “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a polynucleotide sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; it is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database was queried using amino acid sequences provided herein as SEQ ID NO:84 through SEQ ID NO:166 using NCBI “blastp” program with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes of the polynucleotides provided herein, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database was queried using polypeptide sequences provided herein as SEQ ID NO:84 through SEQ ID NO:166 using NCBI “blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using NCBI “blastp” program with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, othervwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism.
Homologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 167 through SEQ ID NO: 10023. These relationship of proteins of SEQ ID NO:84 through 166 and homologs of SEQ ID NO:167 through 10023 is identified in Table 2. The source organism for each homolog is found in the Sequence Listing.

TABLE 2

PEP
SEQ
ID
NO:	homolog SEQ ID NOs

84:	4274	4007	7537	1472	2465	1788	1873	8538	2486	2101	2090
	3705
	513	7264	6280	4902	2624	8820	1614	5907	8247	2717	4147
	5559
	1631	7278	6566	6687	2116	9018	192	2002	5150	322	6314
	6458
	6281	1285	7292	4226	4543	2496	9903	1478	554	5383	7751
	2484
	4954	7695	5821	6271	3339	443	8542	1561	2321	5876	6877
	3452
	2879	3497	2097	4257	7449	7281	3708	4513	2001	4425	9319
	4133
	6686	2146	9698	1036	2026	1292	5566	181	6951	9794	2439
	2621
	5202	878	8081	1392	1950	9999	4392	2121	7824	2367	5102
	6717
	1541	9444	7051	529	4096	602	8266
85:	1163	3954	9565	5913	8096	1310	3871	3019	2926	1456	2770
	4461
	2570	5099	7946	3700	9665	1600	7270	7312	6531	9978	8803
	8920
	4917	6067	6352	6902	2025	2516	4213	9446	8483	5404	2213
	4311
	3724	9926	9599	3835	727	8396	190	3701	7478	706	4038
	7149
	5413	1538	8094	9467	7385	7520	7275	3299	3658
86:	2511	2513	7067	7055	5647	9608	9399	4420	9867	4564	2527
	7769
	2323	347	6509	2052	5258	4504	5363	3847	329	7133	1751
	3243
	8135	4767	5558	2719	6177	6161	6180	1606	3066	514	7725
	4747
	2868	3953	3995	9218	8245	1471	1050	4602	9788	5705	1043
87:	7338	2565	1372	619	8819	7803	7216	9263	8478	7286	2051
	8010
	4629	2569	8521	7659	6081	6080	2727	1944	5731	7616	8198
	8166
	6312	9586	2010	7801	4694	4265	3928	9925	1675	6099	5725
	1040
	5933	270	4135	6356	8593	7015	3351	9045	5105	9655	3874
	5951
	2184	7921	9476	3408	7095	1214	9077	3211	7050	7106	4788
	3534
	3093	7715
88:	9004	8450	3918	3721	516	8506	8664	3458	6365	2464	1564
	4322
	7760	3673	7547	2603	8146	1755	7919	4542	436	2278	4913
	2453
	9651	2319	3659	678	4640	3600	4171	1156	1807	5765	6619
	2992
	354	8233	2386	9454	9453	8837	1238	6971	7874	6538	8258
	1371
	1609	3120	3437	8825	7158	5623	1313	7335	6137	3691	8239
	415
	7580	5147	8818	6282	4612	543	6639	9686	7662	7683	7682
	7664
	5278	5260	8016	2558	2566	2530	9515	5921	8962	3892	7174
	6793
	6936	6938	8284	5225	9323	2932	4932	5328	6697	6602	5109
	9625
	1876	7435	7758	1719	662	6913	4095	5563	4919	8188	6804
	360
	9790	7742	7745	2584	8776	8004	862	6690	8757	5193	6618
	9595
	225	4815	5192	1055	4061	4017	9781	9955	8231	1254	5944
	4087
	8234	319	1180	4631	9258	6546	699	3498	866	588	189
	4577
	1244	5332	3952	9818	2700	9827	6958	7167	3762	9259	3504
	4434
	4968	3204	8580	1077	5275	9915	1474	9160	1653	2701	1637
	5350
	5299	1843	4178	10018	7040	2894	2821	5624	680	9370	7560
	4573
	2144	6813	6722	3689	6721	3373	8902	6656	8928	8462	1336
	9106
	6956	220	3196	221	6118	4162	7171	9683	3870	4094	8343
	8342
	213	904	6335	8822	2482	3806	1766	2692	9038	384	9008
	9007
	3843	2040	7982	9001	9002	4781	606	4807	4852	5595	1509
	1018
	8100	8751	6816	9347	8346
89:	9376	7987	7994	8028	8008	9732	7256	7258	6605	6606	7280
	3139
	9607	7439	6711	9237	9236	6585	1956	1982	1979	7291	7290
	1200
	8901	7293	7295	7315	7297	7279	6624	6601	6622	6584	6583
	6387
	7341	9612	264	284	3163	7321	7324	3947	6330	6620	6600
	6598
	6675	9922	4296	3753	4899	6243	4253	4128	8555	2069	9831
	3105
	6073	6074	6075	7571	9157	3157	7568	8071	7565	5977	4467
	7244
	1849	7820	7235	3250	6000	1058	6372	9381	7369	4775	4109
	609
	9998	9373	6579	6559	6561	206	8729	1166	4317	1863	5710
	381
	2162	4270	6680	6925	752	2268	4086	394	378	3059	6262
	9338
	2659	8145	6646	9005	4294	6457	6451	8075	8093	3747	3558
	7476
	7475	7317	1709	6558	3764	3280	6580	1845
90:	446	4338	4342	9732	4169	7256	7258	7260	6605	6606	7280
	9607
	3131	3148	6585	4388	3940	177	7243	7291	7290	1200	1231
	1207
	6265	7295	7293	7315	7297	7279	7792	7788	6624	6601	6622
	6583
	6584	6891	7253	7277	7262	7272	7271	7324	7321	6598	6620
	6600
	9922	7623	6675	9831	2069	3105	6073	6075	6074	7571	9157
	7107
	7110	7108	7109	7244	1849	7235	3250	176	179	3357	1058
	6798
	5204	9410	1816	5206	6794	1553	6056	6795	6372	9585	9587
	7819
	383	510	175	2068	2865	2823	450	9643	1555	1636	3418
	2608
	701	6147	6165	1470	6578	9307	4775	3206	4245	7525	8255
	2331
	9277	9305	9278	9274	6559	6579	6561	8729	7851	2561	7850
	2208
	1166	9701	670	6640	8134	5364	3904	6039	1320	6481	5993
	3541
	207	8345	8326	4696	7508	1266	2160	2162	1270	3958	4250
	6887
	3932	4270	5104	6680	8202	6925	1088	8251	1466	8249	9677
	3579
	7756	2119	4273	1011	245	3713	9490	5245	4735	2153	854
	4214
	5025	6660	5001	4086	4099	4077	7241	7237	3059	6262	2659
	820
	6646	9005	4294	6457	6451	2869	2767	7746	1732	1738	1734
	1740
	1617	6714	8105	7317	7318	1709	1731	6558	7716	7971	7968
	1210
	1248	925	6580	1247	1273	1274	3369	2586	3664
91:	7402	7398	3583	3592	4729	6500	6496	2752	215	7280	3139
	3136
	7439	7440	7424	9236	9237	3124	3128	3938	177	1956	1982
	1979
	7291	7290	4868	6436	7355	7315	7297	7279	794	7341	7272
	7262
	290	304	289	291	288	7324	7321	3947	6330	6661	6658
	4296
	3753	8555	1943	8457	3157	7565	7568	7088	5977	4467	3998
	3185
	3181	3152	3155	7534	7516	7244	1847	7442	7468	6000	8494
	375
	1057	9773	6372	7369	957	2685	3508	3586	3087	8866	5749
	1377
	2317	723	4586	2111	4703	4413	3687	8541	9052	1793	6037
	2164
	2626	6824	5207	3238	7618	9974	9973	1815	1813	206	4540
	2230
	6716	5597	2206	2232	2200	5599	1187	3435	9672	9040	9039
	2881
	7223	2377	2292	7024	6395	4348	4880	744	8064	5301	8492
	8447
	4312	4317	8472	1863	7956	6694	8324	7358	381	5710	3028
	2182
	7020	3016	531	5699	9003	7798	7421	2299	787	1905	3641
	1934
	1893	1889	7496	5036	7100	3584	3055	2018	2043	2042	7345
	1084
	1086	8473	8350	4908	4934	8469	8477	8098	8215	6937	8496
	4319
	8499	8500	1741	5722	1844	1930	2265	2272	605	4099	4086
	5667
	7379	7378	6262	9338	4236	9976	9969	9972	819	9011	9012
	4937
	4956	2236	6461	5469	8073	6550	3501	2047	2133	7747	6304
	1321
	7230	5154	5153	7376	7373	7476	7490	6862	7512	7475	7562
	7538
	7536	7559	212	200	2238	3159	8122	1753	435	442	2337
	3280
	3274	8476	5502	6810	2934	4335	3380	8421	1125	1368
92:	4117	4611	3810	2575	6435	3730	689	8501	7519	5065	7840
	1174
	8079	3550	8678	8411	9895	1790	4481	2401	2373	1224
93:	3314	5148	1284	2180	7766	9728	8528	6328	7621	9726	1134
	7569
	7567	5040	7727	8537	7497	8041	1784	8956	8953	3029	5087
	7642
	2764	1736	6023	8126	164	8296	4997	6279	3822	9989	6339
	5750
	4196	2427	7030	8232	2440	5671	7115	6494	3608
94:	370	4530	5496	2209	8764	5878	2102	3133	4500
95:	7835	7905	4016	3657	6757	2948	2947	3710	3091	4869	8309
	6256
	3302	1832	7091	9509	2972	4439	667	3396	7147
96:	7080	6259	8950	8331	6582	1510	8054	801	3389	4111	2614
	4255
	5795	2476	10023	6565	3866	1300	6088	7775	6346	9576	8029
	6701
	1803	4281	816	2806	396	2594	3089
97:	6736	292	4996	5983	8898	6886	8845	1312	9032	1586	9964
	9971
	6476	455	357	6653	3756	4574	3311	920	3295	3443	9601
	6906
	5214	5215	5216	5217	7960	7945	7009	8847	4936	6359	5296
	6571
	2233	3288	8736	5420	4531	9662	4891	7545	7948	7980	2201
	9853
	8153	4861	8591	1746	7233	8693	3252	8690	709	5069	9505
98:	9658	9420	3779	6609	3006	2471	3567	4983	434	3565	10008
	8348
	5728	8033	8302	5426	8854	6705	4160	4243	9780	9744	8244
	4469
	317	152	522	6171	3571	3894	8512	2442	9642	6255	3980
	3725
99:	9060	9635	3511	3575	1890	1852	7041	4217	2648	156	2833
	8456
	7232	8503	5709	8742	6107	9824	9121	1142	886	9105	8210
	2910
100:	9526	1911	5622	9279	2726	1935	4608	7672	9536	4429	260
	4235
	356	5588	4995	2426	5771	4203	1942	6086	3697	2322	9359
	1534
	9550	1034	8012	7732	7017	1448
101:	4840	6812	777	7939	1059	6995	5721	4881	5741	9047	566
	3050
	8123	2590	8060	4040	6354	2078	2081	3304	2079	3305	2734
	1841
	9901	9621	1060	6385	3676	2300	1773	9674	4263	7267	9920
	7962
	1042	6416	8813	2382	4599	5670	3931	4207	2985	400	5678
	2687
	7693	5971	2534	4262	9246	1286	9506	3796	9450	271	6880
	5182
	6830	3290	2631	572	3716	9890	9594	9073	7979	8835	7060
	503
	4838	6333	2562	8179	3698	4825	8783	2604	673	2632	5822
	3084
	9095	9913	3912	9850	7102	7121	4583	5132	3859	3618	4979
	1024
	4655	9239	1027	3466	8177	1137	6704	1902	3898	6320	3990
	8921
	9789	7780	1867	720	7797	8984	8792	6677	2787	6486	4974
	7096
	6414	1120	4261	4688	9633	7118	1304	7906	4639	8137	6317
	6512
	5194	7524	3719	8879	5892	6719	10015	2432	5524	5347	4620
	7838
	2349	3742	3336	8156	4407	751	9048	1574	8745	9064	5982
	3677
	3335	8828	2296	6016	2954	2083	7531	750	6967	8907	4754
	7306
	9325	8831	8687	6604	8124	953	5357	6150	4106	7305	448
	7370
	2848	4299	6468	9430	4333	9395	6953	5311	6493	5514	1883
	2876
	4693	645	4187	4174	955	2372	7506	9290	4134	4176	2986
	9835
	4353	3004	4350	4916	2653	9517	5416	2953	2619	8038	8443
	5903
	8092	4082	5327	1767	1761	1781	1763	9578	9787	3277	4719
	7767
	4559	3285	7164	1444	6028	2320	6608	1684	1706	1687	1710
	7111
	7099	5737	6814	6811	386	1976	9411	7973	2663	5521	9734
	8257
	5695	2667	1484	5781	1479	2298	9281	9280	9689	502	6909
	4165
	426	2941	8935	9201	1089	5055	7239	831	1203	9814	9688
	385
	7326	6723	7768	5838	2982	242	2577	3232	9055	8305	5794
	7974
	6114	3340	1764	5723	5940	5567	5093	9792	1635	7645	4958
	6863
	2987	9330	3245	9438	4298	534	3316	5125	6755	4873	1065
	6730
	6904	3603	5948	6903	9042	2384	3197	1325	9053	5820	971
	3970
	7014	4645	2517	5757	7712	3115	3116	3113	6008	3494	9096
	9270
	8942	5369	3727	5382	6563	7718	4888	3393	9383	834
102:	4624	1430	1961	1964	5156	999	3374	6267	4084	9627	4383
	1556
	1098	783	6842	7112	7177	7189	4841	9547	456	3255	2850
	8504
	1846	9224	7752	1367	8373	3039	6783	3653	4153	6429	9457
	8323
	4252	6669	202	162	8940	1117	956	5660	7829	5669	4893
	5253
	9367	6594	4400	6788
103:	4624	9456	5156	999	6267	3374	4084	5394	6413	7397	8169
	6610
	9718	7876	4383	1098	1556	783	7112	3279	6197	9663	6252
	7970
	7356	4596	1390	5051	5754	7566	980	5947	6353	2721	2365
	3317
	2005	5483	8362	7498	6681	1616	4872	1367	6742	1167	8605
	7077
	3202	8267	3674	8623	7344	6893	869	8068	1122	1124	3653
	4153
	6429	9457	8101	8228	1091	8323	5427	3242	9678	7829	5669
	6594
	4893	157	2141	2249	205	7148	4801	1348	5789	371	9957
	3281
	2933	6102	9219	4400	7744
104:	4624	9456	5156	999	6267	4084	5394	6413	7397	8169	9718
	6610
	7876	1098	4383	1556	783	5129	3279	6197	7970	4596	7356
	1390
	5051	5754	7566	5947	980	6353	2721	2365	3317	7189	7177
	456
	1367	6742	1167	3202	7077	8605	8623	8267	3368	7344	6893
	3674
	6783	8068	9341	3935	4153	6429	9457	8101	3987	9140	3035
	238
	8323	5427	3242	7829	5669	6594	4893	157	2141	205	2249
	7148
	4801	4054	1348	5789	9493	8525	371	9957	3281	2933	6102
	9219
	4400	6788	9027
105:	7812	882	3025	2739	5708	3306	1560	7612	5164	8369	4656
	9015
	2778	2824	4488	9482	4490	7160	1576	8990	3445	9496	1081
	8581
	8589	9860	6511	559	8419	7407	6212	6227	9129	4498	7269
	8072
	9605	1531	7089	1967	8562	9391	1385	7893	1070	3042	8370
	8206
	1811	5662	5196	4406	4389	5144	4681	4683	4661	7698	3800
	6641
	7414	4547	9756	5711	5714	4033	4037	4039	4035	948
106:	9402	8595	2366	8382	1968	3420	6998	1544	1165	8998	1193
	5810
	8051	1252	8171
107:	1291	7911	2740	6326	1417	8943	7052	3303	9661	5223	7511
	5891
	5111	6321	4259	4424	4358	9191	2891	8424	8353	2521	6427
	1960
	355	5300	5739	2460	3831	6920	9695	6754	4501	2964	4399
	7640
	8431	7924	3977	1557	2027	3409	1651	8086	9083	8265	313
	1774
	2339	1155	8549	234	6176	5862	3417	4736	6612	2434
108:	3765	171	1106	382	6195	8056	5268	5113	472	8844	4982
	666
	8778	6706	7739	2918	9074	3017	4218	9363	9914	5967	7796
	9712
	5449	5635	9520
109:	1826	2705	9393	2425	1213	7660	9089	9054	4327	3778	303
	4458
	9486	6741	5431	6169	3381	1431	930	1853	2573	4821	8454
	5435
	1573
110:	5969	7138	7828	3577	489	5529	6737	9681	6534	1722	8881
	4557
	8810	4325	7209	9670	5063	2588	3570	2468	3921	9958	9301
	5466
	3869	3523	1591	5729	9783	2404	7123	10012	1898	2921	1361
	2199
	6079	4823	6562	3582	4198	2988	1673	9091	6045	745	7254
	4887
	977	564	9761	790	3944	608	1196	5222	966	8036	326
	1477
	2535	9234	1206	4824	2506	5853	1778	6765	919	8702	4812
	5353
	1322	3733	5817	3053	4591	5238	4483	786	852	3201	1588
	7489
	5700	2914	5503	2660	2500	2582	8351	2766	9436	4685	9940
	4443
	8414	1785	1349	417	5680	3107	1447	4089	3425	2643	8291
	7197
	5541	6047	4613	2239	7734	4497	881	7549	2390	7637	6418
	5266
	5855	5325	7607	3119	859	3220	1382	4268	240	5654	3739
	8059
	4397	806	301	2748	4529	838	9166	769	7471	8997	2099
	1829
	3560	8685	3440	4648	6234	642	8422	3978	1239	3217	755
	3790
	2682	1051	5269	8390	6076	676	6647	1805	8971	7955	788
	2675
	161	8217	9631	9610	2898	569	3240	6155	3722	2713	9746
	5523
	6768	5103	4879	6805	6194	5775	7593	7966	1217	1068	592
	5777
	2301	9364	7176	1747	7810
111:	1848	7581	8716	1381	3763	9600	464	830	1454	2697	1946
	3371
	3331	5569	5388	8095	1812	8144	5798	9118	4416	2526	6791
	6506
	2295	1757	521	807	6077	9190	8758	1157	2605	5928	4987
	256
	8104	3453	5802	9866	374	6761
112:	3041	1135	1343	4097	5330	4575	1412	1253
113:	7726	7410	8023	8039	8037	3318	7855	3370	1661	7688	5627
	4943
	8955	8914	4862	1500	344	3426	5227	3502	293	9110	3083
	2887
	9766	1619	6856	2546	6874	6190	6923	5100	4621	9470	8754
	9389
	6376	6441	4422	9154	345	457	6292	571	1061	3909	4751
	2793
	5101	4673	4878	3491	6065	7011	7632	5390	4909	8328	5391
	6022
	9063	1804	3189	3803	880	2123	2931	6650	7789	2392	3478
	2204
	1188	1993	6419	5463	1704	1718	4102	6349	4776	8793	1919
	7221
	1154	8756	3192	1603	8648	1347	1692	9684	3073	3212	8125
	1563
	5012	2143	2731	10006	5240	4915	7389	1729	7639	6319	844
	2263
	6343	891	3352	590	593	578	591	8035	6049	6731	7592
	1145
	7686	9079	4699	5323	4303	1000	579	340	9696	539	7817
	7814
	352	6724	1917	3594	8858	8853	8855	1866	2559	5673
114:	9538	6322	7202	4360	8216	2187	2190	2706	8864	7631	4191
	753
	4188	3875	4002	8728	5819	8780	4946	5823	5429	9409	4797
	1914
	9292	4470	1705	8384	2457	7603	5603	8655	376	7135	3249
	3241
	5239	1064	1066	7518	6038	6924	3213	3207	5845	2936	6683
	5372
	10011	9763	9153	4074	4666	7043	8312	4569	1296	3499	3846
	5324
	5997	7365	3321	2810	4489	5162	9846	1282	8791	5848	5696
	6367
	5854	3923	6828	9284	2736	6767	4015	4001	7119	7128	6819
	6637
	6746	5893	8436	3198	954	9398	4215	6750	1094	429	6157
	7446
	5377	6108	7283	2695	597	5092	227	5082	143	142	4345
	4731
	5584	4024	1933	9022	1446	8586	6315	9886	8896	1441	3322
	2485
	9887	1859	4473	4598	1725	1723	1711	1727	4684	1789	3375
	2128
	1830	7859	7156	4538	461	5632	4722	4804	449	7646	4227
	5060
	4568	5604	6193	2360	3728	5736	1183	4672	3037	9327	688
	466
	1275	1707	2012	6397	463	3789	2075	6517	6083	2533	2036
	726
	6533	899	8318	6219	508	312	5191	1874	4144	704	762
	1810
	4409	6251	5115	4914	526	3447	1665	9116	2732	5462	7997
	1995
	8681	1540	3384	9404	2304	4761	3200	9823	622	1354	5145
	9265
	1685	8769	8873	8475	1862	5428	3807	1585	1457	9535	7023
	7207
	8840	3150	7215	4107	3879	4554	1912	9215	1955	2217	4314
	6198
	6851	7248	3714	1842	6726	3068	1234	1314	8417	5801	6952
	7120
	2118	643	7187	7053	7671	9298	4633	4795	2872	634	6275
	3390
	5246
115:	6527	5534	7630	1822	3812	8830	7349	8173	8230	6454	823
	5742
	7635	2370	6734	3441	7401	1825	7723	1776	8502	4376	9849
	8505
	9918	1246	5086	9880	5477	1168	6035	263	6347	7450	4783
	8460
	3576	1626	1550	6648	3860	6467	9774	1264	5066	3347	9869
	4031
	1128	9691	6162	7227	3529	3184	3182	6301	6712
116:	6340	2512	9223	8511	1411	9838	4614	2313	8017	3726	7853
	4136
	7242	5908	4839	3629	2463	8002	685	2572	5679	9641	5049
	6231
	3777	9346	570	2598	5766	4493	1856	3171	633	3079	3519
	6914
	6119	4782	9549	944	6394	1818	3916	7353	2995	5902	2095
	747
	9917	9933	4752	4734	4779	493	3829	5900	7925	2928	2157
	1988
	7313	4921	4689	9543	1857	4528	407	9514	9214	3665	3506
	2973
	3045	4875	6374	6390	4895	6392	6393	8200	4551	6433	5790
	2532
	6707	8937	4894	7719	7722	6196	4546	7019	6334	5718	2693
	8868
	8337	7624	8084	5368	1792	1809	5308	5291	3604	2470	4720
	1363
	2244	6555	1691	5954	5228	5208	5224	3448	5362	9613	5230
	7162
	8616	2935	5756	4391	4390	4378	9775	1461	914	4939	6668
	2178
	4494	4491	4492	4447	4446	4398	4401	4423	3968	4462	6837
	7793
	8451	922	9304	4070	7852	1526	1527	2223	387	246	1554
	8977
	6547	3865	1386	684	8322	252	248	249	4690	2214	7084
	6873
	328	9533	8682	4926	4698	7268	2344	9907	6963	6033	7577
	297
	5424	3515	8897	9306	2854	9203	7342	949	6487	8468	2389
	9019
	6360	377	9321	7871	1683	1290	4183	3397	3219	4855	4853
	7564
	8066	2391	2364	2341	2308	2336	2359	2312	2355	2291	8674
	8676
	2309	2334	2338	2293	6361	5880	4164	2896	8031	6630	5425
	7499
	1442
117:	3877	5727	4241	1513	5672	903	2629	7240	1570	3522	6209
	6399
	7453	4618	848	9822	1978	8047	7126	9241	3754	6401	6838
	8863
	8632	7454	5580	8522	7895
118:	3495	5400	9889	6676	3537	2811	5961	7327	7081	3672	180
	3487
	729	9474	1384	3951	5271	717	7942	6235	5455	4990	9372
	1754
	1073	6133	7304	7521
119:	9634	7978	4066	4065	2009	5886	5171	8727	7846	9806	2815
	4941
	1225	8445	4436	2407	3775	5616	4453	5778	6515	6090	4925
	4646
	5370	6739	7765	4381	8172	1108	1839	7443	330	7806	7804
	1251
	7085	1192	1195	8607	6203	9771	698	8705	5590	5598	5592
	5596
	3601	9117	2765	3559	343	7898	8931	3693	3692	1611	1610
	365
	5981	1171	764	5611	5444	5881	8797	7530	2017	8983	2997
	2930
	6738	5270	1786	9245	3759	1888	4980	6122	5354	3372	9297
	5120
120:	9228	4471	4904	8878	2071	9785	822	6100	8130	8957	6961
	1787
	9828	6974	5058	6048	2058	1334	8704	4992	625	3328	4222
	2494
	8453	5397	4969	4450	4318	2859	1370	1074	323	8974	4426
	6663
	5231	4130	5715	6857	4963	5687	5392	8795	5871	8912	6299
	1801
	2634	2636	1802	3896	9268	8278	3174	7026	7220	8074	8650
	9102
	9287	7583	7097	8408	7371	7667	195	5536	2730	3535	2115
	4088
	1078	5734	6232	6400	5785	9041	5946	5806	533	6762	5167
	3509
	7720	6425	1775	5393	9808	2671	5316	1712	9504	4010	5818
	4041
	4667	2542	3854	9371	235	4204	6216	4774	1599	2661	3975
	8489
	1947	6860	3465	9213	3323	6144	9350	1311	6868	1029	5543
	7654
	5689	9138	4901	3646	4370	5447	3568	9049	191	9432	7500
	3216
	1937	1287	2215	3057	4331	2431	3662	2335	4680	9924	9464
	9069
	2446	1136	9899	6634	3821	7491	5743	3818	5952	7694	5008
	7044
	427	2844	993	3332	3542	9555	7509	2655	9733	5930	9935
	2885
	3456	4859	5366	9953	703	1152	8430	4437	7699	8295	1423
	695
	8683	1317	3715	6834	613	3411	1147	6674	3578	9109	2999
	7881
	8241	6528	3430	2831	9772	7685	4897	7480	601	7250	9392
	2656
	8567	4984	3736	9512	9647	5430	4593	748	6576	8827	3346
	9892
	4300	3364	7086	6670	4459	7543	3063	3463	5232	5575	9624
	1514
	1882	4732	5860	3475	5168	6684	6581	6437	8357	2423	4876
	2589
	4866	4870	2803	6735	8307	4675	4142	798	2816	5545	867
	2253
	5522	3595	2944	1158	840	7251	6225	8152	6818	7384	8440
	8966
	5155	6030	7494	5408	8341	7914	7909	1602	8527	9112	2828
	2829
	5360	4922	4427	459	9254	1278	7587	10010	5614	5712	6699
	1041
	6085	1953	5422	1038	1533	8211	5116	4737	7492	9123	1237
	3782
	5218	8775	8058	7078	9592	1095	9142	325	368	4520	6553
	5378
	6134	8299	1309	2520	7542	9412	7800	6452	5355	3976	9725
	7636
	7309	525	9834	4503	5283	870	5203
121:	9228	4471	4904	8878	2071	9785	822	6100	8130	8957	6961
	1787
	9828	6974	5058	6048	2058	1334	8704	4992	625	3328	4222
	2494
	8453	5397	4969	4450	4318	2859	1370	1074	323	8974	4426
	6663
	5231	4130	5715	6857	4963	5687	5392	8795	5871	8912	6299
	1801
	2634	2636	1802	3896	9268	8278	3174	7026	7220	8074	8650
	9102
	9287	7583	7097	8408	7371	7667	195	5536	2730	3535	2115
	4088
	1078	5734	6232	6400	5785	9041	5946	5806	533	6762	5167
	3509
	7720	6425	1775	5393	9808	2671	5316	1712	9504	4010	5818
	4041
	4667	2542	3854	9371	235	4204	6216	4774	1599	2661	3975
	8489
	1947	6860	3465	9213	3323	6144	9350	1311	6868	1029	5543
	7654
	5689	9138	4901	3646	4370	5447	3568	9049	191	9432	7500
	3216
	1937	1287	2215	3057	4331	2431	3662	2335	4680	9924	9464
	9069
	2446	1136	9899	6634	3821	7491	5743	3818	5952	7694	5008
	7044
	427	2844	993	3332	3542	9555	7509	2655	9733	5930	9935
	2885
	3456	4859	5366	9953	703	1152	8430	4437	7699	8295	1423
	695
	8683	1317	3715	6834	613	3411	1147	6674	3578	9109	2999
	7881
	8241	6528	3430	2831	9772	7685	4897	7480	601	7250	9392
	2656
	8567	4984	3736	9512	9647	5430	4593	748	6576	8827	3346
	9892
	4300	3364	7086	6670	4459	7543	3063	3463	5232	5575	9624
	1514
	1882	4732	5860	3475	5168	6684	6581	6437	8357	2423	4876
	2589
	4866	4870	2803	6735	8307	4675	4142	798	2816	5545	867
	2253
	5522	3595	2944	1158	840	7251	6225	8152	6818	7384	8440
	8966
	5155	6030	7494	5408	8341	7914	7909	1602	8527	9112	2828
	2829
	5360	4922	4427	459	9254	1278	7587	10010	5614	5712	6699
	1041
	6085	1953	5422	1038	1533	8211	5116	4737	7492	9123	1237
	3782
	5218	8775	8058	7078	9592	1095	9142	325	368	4520	6553
	5378
	6134	8299	1309	2520	7542	9412	7800	6452	5355	3976	9725
	7636
	7309	525	9834	4503	5283	870	5203
122:	5149	8274	1269	5690	5410	6173	3556	3468	4438	1208	5663
	545
	7791	167	8526	3554	3890	5517	2633	5799	7332	3855
123:	6613	2955	6069	4371	9778	2358	6094	6089	6526	8516	5547
	6071
	3313	6112	4034	2957	2978	5525	5509	5531	5528	5561	5607
	5578
	5565	5581	5579	5526	5533	8275	9649	3706	6832	6350	5088
	1150
	1439	5916	5281	2689	4704	6036	7641	3679	9051	6455	6836
	861
	3525	9267	551	1047	9231	6871	6525	6523	3862	6179	7206
	2549
	3992	178	3349	2003	9082	2354	3011	3013	6910	9731	555
	1033
	8344	6990	7131	5177	8976	9092	1601	1598	1608	8048	8418
	3451
	7002	4523	8340	5474	2917	7702	4505	7759	6992	6991	4145
	9590
	9714	3423	9656	7573	3644	350	6892	6889	403	423	9597
	10004
	8938	1952	9076	2084	3308	849	8160	1899	2114	8649	3702
	4565
	6473	4071	5833	2038	3309	9303	3587	3472	9061	6685	194
	646
	6121	5506	2376	9559	2417	3376	3360	1834	9759	2899	2901
	6629
	5631	7670	8613	2962	9803	2743	2363	6848	9146	8108	8882
	1697
	9779	3960	9830	4022	7740	4346	4615	3012	2583	803	5888
	8151
	1450	8428	6588	5909	3963	6185	5814	1539	274	3076	10003
	10005
	1221	1737	4288	6470	8686	2601	6469	5276	2937	6200	5380
	172
	7523	9242	1590	266	973	7743	1032	9357	5552	9716	3259
	6628
	3263	4120	3258	2581	3260	4555	5197	1295	7028	5131	9343
	8641
	5953	2790	4533	3684	6756	8425	7620	9044	4484	4442	6589
	7205
	2544	5999	5110	7690	3707	6257	8989	5519	4451	9747	4199
	8386
	3597	3792	4276	797	2990	3048	5601	359	7576	1657	3740
	8176
	9455	612	694	7192	9414	7194	254	581	2378	2380	1235
	7600
	1272	3830	6792	5403	3464	3261	6428	8573	8600	9589	5505
	5333
	2150	9945	536	8952	6290	8741	9349	7929	6110	6101	6554
	6551
	7005	6556	2195	2194	9934	6053	5550	6091	6097	1294	4304
	6796
	5401	3015	3974	9921	2789	8083	8082	6710	8905	7514	576
	3319
	5564	4590	9666	515	6745	5138	5139	2130	2645	6228	3562
	8634
	9178	6536	4361	3337	9699	8491	7757	4905	3257	7611	6743
	9014
	3439	6120	1783	1850	8136	4851	661	800	6654	2866	1759
	7551
	2690	4141	6430	654	527	7495	5412	8061	2455	8022	4435
	4155
	1201	7714	5423	7689	7485	7469	7473	7463	7348	5512	575
	5548
	7021	6070	5585	9708	3749
124:	7858	8376	2905	7510	6066	7834	594	9979	7103	3214	2892
	4020
125:	1965	1299	9851	8509	7263	1130	8178	2737	8180	3769	8759
	9805
	1981	9059	8530	1473	9765	4269	4292	1925	7809	4290	9845
	314
	7374	231	4410	7211	4323	7033	6382	6485	4356	7873	9342
	5560
	5071	3031	305	8559	247	1595	5021	4580	1249	8659	3872
	6504
	5803	5906	8242	4525	8657	8070	598	5089	283	2903	2720
	7170
	2274	8111	2729	7063	1409	2646	5774	5385	2760	5834	8876
	7082
	3856	4842	5034	1645	7857	1744	9722	3900	2635	3326	6930
	444
	5800	4668	3489	3366	3363	6548	1067	6568	3580	9755	462
	1517
	3282	5254	7231	5879	5640	9730	8264	6866	8271	8219	8982
	8518
	7330	5152	6006	362	6587	3857	8189	1021	4928	6146	7366
	1017
	4985	6287	269	9285	8103	6140	8900	8461	8042	7513	1530
	8908
	9187	7436	5320	7656	1494	1750	9428	8182	5863	1567	4944
	9088
	4138	3394	3493	2812	5989	8558	2028	7289	1771	1452	9932
	5027
	5309	2398	5097	8009	201	2519	8192	5965	8374	8356	6799
	7965
	6539	3530	4701	563	8194	7382	410	5432	2428	265	5697
	6229
	331	2681	8269	4012	7866	7729	1913	2904	4157	3917
126:	5740	333	3685	6489	6369	4339	7931	3455	392	7908	5602
	1268
	5664	6024	5557	2057	1003	8161	4098	8684	6875	9916	6781
	936
	9710	7735	5417	7273	887	5812
127:	5856	1578	7377	3621	5827	3671	2110	9481	4233	1104	8163
	9262
	4971	2839	3787	5613	3078	2259	8139	5793	3459	5929	2801
	2151
	8722	3469	2286	3100	5637	6002	1085	7774	3391	1250	5804
	1639
	512	660	4885	7730	3038	9471	9992	825	7282	9937	2080
	2108
	8747	7188	1529	5986	8046	6381	1331	2403	209	1777	8557
	1506
	2579	8482	3343	2553	565	3342	3401	6464	5117	8449	3528
	7042
	1092	4056	4605	3546	5074	4182	6213	3686	8439	7265	9894
	2923
	4307	3005	7934	287	3138	6664	6013	4832	4468	3283	6236
	9800
	7555	7668	2445	8349	8224	8032	6172	7963	7938	7860	7910
	7940
	7907	7451	8523	7548	8917	4291	4210	5628	7633	8801	2658
	5498
	6844	9034	992	6878	638	5185	3614	3001	2430	5371	2297
	3099
	3329	2769	5307	818	5470	1002	3141	1414	9309	8679	8656
	5433
	8459	4544	7977	2596	4036	6311	8615	7375	4284	7400	2159
	1895
	6521	4711	1896	2666	9809	1537	3569	6829	6890	8347	5244
	2023
	9596	210	8696	3632	3636	7413	7411	3341	5996	9252	6570
	6106
	2550	827	9397	7998	5434	6501	421	6820	4792	8862	2587
	1686
	5844	8143	2650	9719	2952	7764	7310	7350	4419	7343	4953
	2281
	6922	998	732	4907	3656	3861	7773	1362	540	9872	4889
	4717
	5358	4967	3442	9026	1186	1545	3768	3848	397	1733	7778
	3230
	3231	2907	5201	4679	4662	2132	7944	9893	9351	9176	7697
	4264
	4814	2142	7990	9856	9426	9361	4582	1406	2310	8164	8601
	8738
	3162	3793	5143	9494	8400	2046	2045	4978	5677	7136	2474
	5014
	5007	5017	5011	5639	3867	5691	5713	4168	4184	2860	1543
	9881
	2070	8772	9552	6943	577	8594	2433	2435	6495	9544	4137
	1833
	9946	6276	6143	6141	5825	6671	5735	8848	7146	7144	4623
	5738
	184	9876	7225	9462	9460	9465	5910	2383	4800	5870	5869
	273
	9875	9873	222	9840	4786	981	3797	1770	4201	7394	3143
	9906
	1220	6221	4682	4697	6465	7159	6643	3631	3660	3640	3639
	3638
	3635	3154	3650	3082	9499	1198	8043	8063	9598	7101	1871
	1197
	4535	6057	7181	5995	6202	934	5123	5106	5576	5128	6688
	6678
	4536	952	3461	5374	6774	9942	1230	6306	3483	5047	5450
	4652
	9013	4964	7678	5914	3945	5931	5883	3330	584	7843	7842
	7848
	7868	7847	4173	8628	8577	8303	8804	4589
128:	5856	1578	7377	3621	5827	3671	6513	2110	9481	4233	1104
	8163
	9262	4971	2839	3787	5613	3078	2259	8139	5793	3459	5929
	2801
	2151	8722	3469	2286	3100	5637	6002	1085	7774	3391	1250
	5804
	1639	512	660	4885	7730	3038	9471	9992	825	7282	9937
	2080
	2108	8747	7188	1529	5986	8046	6381	1331	2403	209	1777
	8557
	1506	2579	8482	3343	2553	565	3342	3401	3108	8272	3784
	4267
	6464	5117	8449	3528	7042	1092	4056	4605	3546	5074	4182
	6213
	3686	8439	7265	9894	2923	4307	3005	7934	287	3138	6664
	6013
	4832	4468	3283	6236	9800	7555	7668	2445	8349	8224	8032
	6172
	2670	7963	7938	7860	7910	7940	7907	7451	8523	7548	8917
	4291
	4210	5628	7633	8801	2658	5498	6844	9034	992	6878	638
	5185
	3614	3001	2430	5371	2297	3099	3329	2769	5307	8420	818
	5470
	1002	3141	1414	9309	8679	8656	5433	8459	4544	7977	2596
	4036
	6311	8615	7375	4284	7400	2159	1895	6521	4711	1896	2666
	9809
	1537	3569	6829	6890	8347	5244	2023	9596	210	8696	3632
	3636
	7413	7411	3341	5996	9252	6570	6106	2550	827	9397	7998
	5434
	6501	421	6820	4792	8862	2587	1686	5844	8143	2650	9719
	2952
	7764	7310	7350	4419	7343	4953	2281	6922	998	732	7563
	4907
	3656	3861	7773	1362	540	9872	4889	4717	5358	4967	3442
	9026
	1186	1545	3768	3848	397	1733	7778	3230	3231	2907	5201
	4679
	4662	2132	7944	9893	9351	9176	7697	4264	4814	2142	7990
	9856
	9426	9361	4582	1406	2310	8164	8601	8738	3162	3793	5143
	9494
	8400	2046	2045	4978	5677	7136	2474	5014	5007	5017	5011
	5639
	3867	5691	5713	4168	4184	2860	1543	9881	2070	8772	9552
	6943
	577	8594	2433	2435	6495	9544	4137	1833	9946	6276	6143
	6141
	251	5336	5825	6671	5735	8848	7146	7144	4623	5738	184
	9876
	7225	9462	9460	9465	5910	2383	4800	5870	5869	273	9875
	9873
	222	9840	4786	981	3797	1770	4201	7394	3143	9906	1220
	6221
	4682	4697	6465	5600	7159	6643	3631	3660	3640	3639	3638
	3635
	3154	3650	3082	9499	1198	8043	8063	9598	7101	1871	1197
	4535
	6057	7181	5995	6202	934	5123	5106	5576	5128	4166	6688
	6678
	4536	952	9386	3367	3461	5374	6774	9942	1230	6306	3483
	5047
	5450	4652	9013	4964	7678	5914	3945	5931	5883	3330	584
	7843
	7842	7848	7868	7847	4173	8628	8577	8303	8804	4589
129:	6283	2094	3062	2271	6254	9056	4151	3773	1819	5768	1674
	7328
	5634	9770	3114	9282	6309	9704	845	5395	7738	5398	1796
	5098
	1232	5972
130:	7784	5453	9584	6859	9085	5136	9525	2342	2327	2326	8227
	3072
	6540	5884	9046	3774	9221	8368	4647	6152	6616	4140	5610
	267
	1654	9864	3834	9194	5858	3771	2074	3054	970	1835	1677
	7614
	6954	3403	6800	1641	7395	5591	3386	9950	8611	856	1858
	1520
	473	8885	6011	9711	9825	9859	3111	5119	1429	1523	3561
	1945
	9938	5482	3348	2385	6226	3194	3839	9243	3008	4170	6662
	1892
	2971	766	6573	409	5075	4890	9646	5026	9188	942	940
	7917
	938	7915	6978	1566	4359	5107	6149	1921	9205	558	2593
	9767
	3663	3485	4395	5451	1327	3271	725	793	4006	2369	465
	3590
	858	4660	8466	5760	453	2677	3069	3964	3914	4301	3064
	3301
	7884	2946	9593	1301	1216	9620	2994	2405	3203	6375	2147
	4373
	9796	8212	4251	5659	3187	9170	2662	2694	5304	4050	4069
	4047
	6861	9115	2858	481	4119	7615	7762	10016	6239	337	338
	8599
	8597	929	927	924	363	7717	204	5991	3852	6318	1176
	1179
	2056	2441	8533	628	2053	8174	5619	1515	5080	7386	6250
	7883
	2826	5083	1164	9070	1162	1160	1438	1436	913	3235	596
	3680
	4588	4570	6183	6184	8547	367	1416	380	1891	3924	5262
	5013
	4687	5467	8364	7479	6919	5452	6845	2870	2993	3956	4175
	1153
	7594	1878	2488	9668	3669	9251	5263	7422	8183	5174	6764
	8181
	404	2913	6297	604	812	8190	9618	3480	3278	2591	5960
	1324
	7168	7056	8115	5520	9207	8128	218	1127	3477	3670	3125
	8175
	7877	1752	2733	1141	4208	4347	1178	1655	4864	9388	7195
	1831
	7319	2809	9983	8576	8209	6211	5985	7193	7172	3476	6946
	8553
	8127	336	9078	7210	7452	842	6296	7259	8099	262	7208
	5730
	3224	5151	9273	3752	2509	4744	9275	8065	3772	8089	1647
	761
	393	9365	3811	2436	2699	9248	2055	5846	4705	4287	1594
	2863
	4479	6327	416	3939	1110	8556	8259	1688	3628	1083	9741
	6541
	4025	4046	4026	815	2691	8354	2970	2976	8363	5782	6532
	6520
	7608	3545	6249	10017	8969	170	7412	2402	1726	2886	6760
	2893
	8941	8926	1099	8899	5043	8842	9093	9097	9094	3237	997
	5732
	6302	4820	483	599	3492	485	9120	4799	5221	6286	3496
	9084
	5010	8262	3704	5443	4626	583	9310	4048	8575	3981	3979
	7967
	9931	8221	203	193	9874	470	4105	864	6260	2222	791
	2909
	2949	8495	479	5872	216	3623	3236	9418	3607	4308	7455
	3010
	9192	1583	9348	5873	796	9882	8903	2724	9328	3934	3514
	7540
	6894	8289	10013	8967	7832	9868	5251	8336	6242	6691	6785
	2443
	1097	7889	7093	6508	4232	2438	2247	9065	6246	6168	6186
	621
	1149	9707	2641	3609	2735	7025	420	1824	7038	9975	5137
	8767
	7252	6126	3695	478	4249	9959	6269	8608	5706	951	3884
	316
	6858	4028	4644	7046	1823	1357	6802	7127	9616	4121	3047
	7634
	1870	302	4118	2842	652	6907	9815	9812	3365	9764	4844
	4849
	4847	4846	2929	6567	8044	7445	2818	8838	8841	7861	8986
	5665
	8545	9143	6751	6789	9944
131:	5504	7201	9080	4524	6201	3840	4320	2523	2135	7287	7299
	9617
	6355	4519	632	4321	7887	8869	366	8507	2280	8201	3170
	697
	696	7153	6491	8338	8389	8397	8381	8377	8380	8358	8393
	8391
	8394	8313	8352	8415	8317	8334	8333	8361	8314	8315	8332
	8335
	8375	5831	5829	7224	1326	8514	946	7226	2939	327	649
	1493
	3809	4297	4080	2845	1795	5346	6667	309	1854	1140	7837
	2545
	7199	1133	1129	1151	1115	1146	1985	1959	1957	1983	3307
	8045
	3741	3761	3758	6087	9379	4826	6544	675	538	8707	5857
	9832
	8154	8796	1629	5286	8321	7217	824	2711	9035	9036	3030
	975
	7674	6483	7661	8936	1875	5973	4043	1380	1475	4930	2958
	7261
	1499	3014	1118	4597	9776	4045	9394	1075	5052	3239	6900
	4517
	7736	1105	9534	2745	1713	8191	6041	1894	9169	2207	9058
	9057
	7606	9010	6901	6139	8120	7458	1100	8906	6027	419	4516
	1121
	1840	5685	742	3398	4315	7584	5975	7854	6034	580	672
	3555
	7613	5650	1836	8040	3428	8666	2077	1708	437	4402	3169
	7183
	6217	6054	7882	665	3473	2638	2784	674	8719	5127	7087
	5042
	9757	8027	395	2919	511	6695	7456	560	3549	8964	286
	3470
	9697	8561	2708	6753	9472	2611	7750	7001	342	3233	3234
	5990
	4867	5918	321	9956	2156	5769	6864	320	6627	7213	7184
	9216
	544	4092	5302	6529	1682	1907	4691	5608	1063	7337	2808
	9500
	1975	9163	2361	4923	6408	6445	7405	1974	5389	9491	3969
	6957
	8378	3026	3751	5198	8427	8438	8584	8587	5030	5340	9031
	5970
	3246	5935	5841	7679	391	4480	451	4813	6214	3457	8155
	8236
	9366	710	8960	3718	4721	7862	3936	8627	1552	3825	1633
	6948
	4863	7763	8131	5038	335	6404	1109	253	5375	991	1827
	1503
	2479	2503	6313	4724	4816	6673	9553	2085	9222	9687	2686
	3888
	5409	2127	5964	9572	681	3989	6809	9602	5922	9591	2756
	2758
	6689	8560	1663	5987	4502	5912	8708	8737	8713	1575	6507
	4057
	2862	469	1869	7285	8781	7390	1437	3602	9071	9713	2412
	2922
	1209	5744	5692	1026	9522	5688	3097	9739	9900	7071	7359
	7822
	4049	8760	349	690	2499	2502	2505	2498	1337	2791	5897
	5606
	4991	3385	3598	9168	5609	9723	9452	5114	1422	542	9513
	1289
	3327	6439	1584	8412	4773	1330	5478	8055	9066	3902	8510
	1970
	3043	422	425	4014	7799	6230	2105	6258	6010	9413	8660
	2628
	6270	6876	4757	1582	1581	5586	9313	2411	3471	2774	9575
	4886
	4883	4884	1215	785	4052	9911	8024	9256	3819	2592	4122
	4055
	2104	8273	8711	3776	4609	5894	5414	5418	5415	5642	996
	5943
	6109	4051	4053	8715	8689	8709	8717	8692	8688	5287	2817
	9609
	7733	9086	6128	4075	7772	6277	6273	4787	3051	8442	8712
	4124
	2000	4725	1483	9000	9477	2674	278	7578	3218	1900	1350
	6241
	1920	6638	5305	6621	9702	4686	7782	6159	7362	5339	1623
	8116
	1283	7783	3123	3850	5188	8534	6749	6062	7680	4280	6471
	5968
	5335	9529	7785	6059	4403	9104	1212	9615	6740	3591	2050
	2501
	802	9315	8981	1486	9161	2203	821	6989	8360	5767	501
	5220
	5243	5219	1306	2318	1308	1569	3903	7296	1023	5411	7922
	10000
	8193	5033	7826	8112	1031	480	9434	8638	2548	6644	6659
	926
	1281	3863	6885	5018	4324	8240	5645	1451	1428	9255	5020
	4604
	4477	2089	4986	5016	4449	4981	4472	4466	7926	3449	7930
	5796
	7352	8479	4487	8945	8107	3619	1039	5716	943	5762	4636
	6158
	553	6272	3897	8815	1257	974	2294	2276	804	846	2807
	7643
	2248	9745	506	6040	557	2227	6288	6293	4240	257	8598
	6115
	7125	4527	7301	5367	5048	2504	3901	5615	6160	7427	9645
132:	7833	5889	3007	8109	5915	2022	6345	3652	3805	4242	6959
	5248
	6384	1580	8887	6294	1739	9842	6700	3666	2480	3620	7472
	9016
	9795	276	1758	5135	1256	3574	7061	4918	7154	1916	2076
	9159
	3416	4625	1865	1670	9896	6240	8603	8205	6782	8883	877
	7470
	7190
133:	3767	1936	390	6879	5949	8320	4341	1797	9923	5720	7626
	5638
	8294	5250	5783	4649	3513	3058	519	7034	4635	7649	7684
	4411
	7300	4860	7598	9786	3415	4337	4344	7610	3729	4393	7627
	1303
	9821	5057	520	6928	3675	494	8993	2837	7701	2841	8011
	6082
	6341	495	7302	7878	8508	7303	9857	4723	3748	5707	6773
	7432
	918	3886	8446	2456	7372	1768	3467	6125	5805	4521	2163
	2258
	9581	4275	4278	4277	6407	5172	4628	2564	4139	2574
134:	9820	3178	3325	9629	4845	7949	7988	6657	1236	4592	310
	4063
	1123	2925	2783	7441	3361	4193	7477	4906	2980	4000
135:	4310	4445	1549	1551	3520	6747	1048	9936	8536	4394	3173
	8330
	2117	6095	9293	4238	4200	4617	3566	9898	2444
136:	7669	3429	8564	5459	5927	2547	8365	5562	7856
137:	7203	1791	7073	4234	972	3070	439	9332	8118	4541	3482
	648
	537	5289	5540	490	702	664	1008	6187	7074	6233	6163
	4384
	8150	5035	1644	4408	6682	3350	8610	3596	230	3531	3948
	898
	219	1056	4256	8367	3221	4110	4671	5460	2049	9396	2900
	2969
	9748	389	9212	1864	5652	2316	3193	8423	7844	243	7890
	1403
	2437	6939	332	5108	1139	3836	4791	3688	3868	5811	9804
	1305
	7891	2082	8288	5255	7561	8519	5295	7387	5694	5274	9863
	6912
	8622	3682	5241	8843	9768	4465	5859	1838	5905	1199	524
	2161
	2578	9604	1383	9340	2346	616	1159	7597	8811	4810	945
	968
	5461	5898	1202	5573	8859	3581	6206	6103	4456	3135	1772
	2420
	1542	9260	6174	562	1605	1656	6955	6926	6358	441	9249
	2800
	8387	3191	334	2847	6264	872	687	8724	8761	549	567
	582
	507	5130	8455	3399	4068	2609	6331	5656	4441	2510	2332
	3481
	4081	9524	6696	6218	4369	4945	4309	4977	405	6983	6870
	7927
	4526	2262	7867	4653	2920	5839	2357	3696	2951	3527	3462
	1263
	1881	5247	9669	6111	9269	6592	4622	5763	8052	4584	5303
	9133
	9742	1799	5298	5348	2353	408	6603	1498	4948	1468	6569
	4552
	4474	6215	9167	8635	3889	3190	6852	6001	2924	6593	1769
	9650
	8304	5072	3090	6124	6966	6156	5792	7585	9548	4237	4190
	6153
	7245	9753	4877	3298	3746	1328	6557	8823	8653	2152	353
	424
	5626	9311	7214	4534	4537	8243	2229	1536	1559	3540	3539
	2772
	294	1511	1204	9644	1227	1332	6205	8680	6524	9632	2875
	1173
	7336	9927	8110	3755	9682	7367	8630	2454	3973	5134	3040
	8782
	8372	199	1015	2599	6284	2131	9652	9977	4718	2747	2315
	1977
	4229	5568	2490	4404	3624	3794	1205	6459	5436	5438	3000
	2998
	223	1445	2176	3648	3647	614	9919	3503	1333	4230	285
	6261
	2409	4607	8222	3883	8049	9508	7003	3315	8218	5212	1954
	7696
	7141	6117	3021	5587	3606	7591	1054	3882	9819	548	2096
	4432
	2507	2406	4146	3563	603	7104	9676	3832	5518	4567	1994
	7175
	3096	6776	7786	6338	186	8520	5752	630	8740	5658	5674
	2763
	8877	5701	6518	1260	8398	8918	7622	8077	2861	9735	8643
	5686
	6827	5648	4664	950	1885	9164	668	528	656	6068	9043
	8808
	4152	8817	4150	5780	5776	9902	5830	5073	324	5617	5625
	7408
	5329	6642	318	7647	4115
138:	8968	2512	9223	8511	3726	6931	2463	9346	570	2598	2374
	2120
	6914	6119	944	6394	1818	7353	9429	747	3887	9917	9933
	4752
	2216	1923	9626	493	2928	3665	4875	4912	6403	6405	6433
	8937
	4874	9507	1293	1302	4892	6196	7019	2623	2620	9448	9929
	3532
	198	7447	1262	9816	2942	214	5895	8132	6833	2470	6044
	1360
	3864	3354	5078	3712	3622	2243	7841	7704	7058	8513	4248
	5756
	1329	1650	915	3098	4511	4507	6837	7793	8451	8617	4070
	7852
	4387	2840	9705	7098	9546	9283	7821	9334	1355	8515	4515
	3849
	1103	4638	7596	1101	6492	7805	4585	2129	3813	3165	6012
	2459
	4855	3219	4853	7564	4163	2896	6630
139:	3997	2703	1918	5761	6918	6142	9908	5187	2508
140:	9075	587	5843	3210	5471	8034	8884	3547	8432	5657	433
	3460
	5473	789	471	3122	7813	7075	3837	3743	8256	348	3077
	5593
	5832	987	3146	9721	5493	5966	5508	2938	8924	7628	2462
	6274
	719	8951	9226	7284	2916	4116	8624	4496	2668	9288	1814
	5032
	893	5334	3572	6043	1404	6379	5861	9837	8946	1512	2796
	2246
	1405	9622	7329	4715	1148	4231	4695	5190	4903	7517	1440
	3444
	9782	8850	2260	9801	3324	6632	2884	9126	3044	2991	3786
	8888
	7132	3383	9017	8248	1660	7652	3827	8250	7599	5788	2798
	8406
	3056	2529	8973	1049	1082	6220	6984	9090	4858	8488	484
	6596
	1558	7383	8535	6808	9568	6266	168	406	1425	5748	6969
	6702
	9912	3294	9324	3291	6801	9081	1476	5724	8405	372	4330
	5141
	3067	7461	6014	3410	4643	4606	2961	6780	414	3074	4632
	6130
	1316	1319	2004	3438	2134	9758	1126	3244	3215	7234	722
	1394
	2679	7238	7007	4659	1903	7605	5015	9769	9693	6982	2630
	6050
	1465	244	4085	1276	4159	5963	9264	2927	6093	8298	3359
	8697
	8399	1481	4594	5950	5882	5282	6872	2524	9738	9706	6151
	4349
	498	4669	9149	5901	9185	7875	169	183	6175	4512	8771
	174
	6208	768	8720	1460	770	4495	4692	724	901	6744	6651
	7901
	1649	7047	5090	8755	5784	5815	9826	5681	1076	7590	1185
	7406
	3024	8988	7169	4476	2458	561	241	259	1191	7004	1219
	6720
	5056	7916	5849	2343	2984	6652	2915	261	4127	1948	6310
	8213
	7655	6289	6015	5992	713	737	9023	1906	3484	3994	4180
	6042
	4627	4179	6063	1342	4478	1760	735	7308	1211	211	2016
	3413
	2761	8403	9671	3223	2610	6373	2912	3345	6189	373	3613
	430
	7399	7985	4778	509	3557	3379	5229	629	3927	7993	8030
	7991
	505	9441	8091	635	8392	3382	9291	9299	2607	712	4192
	7064
	4220	5454	5175	8836	7428	1630	2531	4634	7795	3052	1659
	532
	8922	6060	8806	1643	1046	6432	530	5257	6181	5009	1144
	173
	2348	7322	7083	9654	255	2956	7522	9545	9523	3395	3709
	236
	4405	187	4154	2983	3573	5555	9760	7294	9331	2109	2167
	1280
	4396	1044	3149	4750	4896	2106	3020	6679	6549	7288	8013
	9862
	3450	1045	6386	9884	896	7380	3022	5932	492	3526	9847
	7879
	5847	2718	9458	4283	4104	5361	8282	6752	1668	9638	7885
	3930
	863	7550	4244	5787	2543	2906	4959	4952	418	4994	6927
	5582
	4076	9962	2092	8524	3145	4431	2557	5249	8225	6443	9667
	4131
	2416	8413	3643	4181	7952	9690	5499	2154	2155	2177	2061
	8834
	2175	2749	523	1716	8963	3110	3009	5753	4947	9619	3694
	7995
	9473	9431	1492	7403	4539	623	9195	2989	8057	1634	5530
	3661
	4674	8260	6635	4058	3824	6199	5809	3436	2191	9530	9501
	1972
	2813	6148	8444	1808	6021	7831	5292	3950	6999	6997	5133
	6170
	5636	3521	2788	9752	9527	1910	6847	7134	4013	1102	4506
	7666
	3334	5267	2644	3985	651	5046	4819	6263	9628	3518	3516
	2113
	2098	7129	3412	3092	5544	6537	4415	841	1037	836	6545
	9865
	9685	1632	9736	9510	6775	6136	3176	721	5911	3424	7246
	5062
	3402	7541	4475	7969	8925	307	8929	8910	8913	8915	5176
	440
	399	4558	1132	5824	8138	5406	8078	847	4706	2943	2722
	9939
	8129	6474	9988	9994	6797	4455	4457	4454	1607	916	9574
	5091
	9573	9416	6129	2974	9630	9904	8114	1131	1742	7897	5186
	5618
	2362	2379	468	7173	6703	2170	9387	9362	9562	1646	5807
	3814
	9186	6763	6268	2429	902	5140	4247	885	2652	6247	3161
	3723
	6096	7818	6599	2688	7827	1622	6633	8485	6498	4882	5163
	5076
	4563	5733	9810
141:	7726	7410	8023	8039	8037	6113	3318	7855	3370	7688	1661
	4943
	5627	8955	8914	4862	1500	344	3426	5227	3502	293	9110
	3083
	2887	9766	6856	1619	2546	6874	6190	5100	6923	4621	9470
	8754
	9389	6441	6376	4422	9154	345	457	6292	571	1061	3909
	4751
	2793	5101	4673	4878	8732	3491	6065	7011	7632	5390	8328
	6022
	880	2123	2931	4044	6650	7789	2392	3478	2204	1188	4440
	1993
	6419	5463	1704	1718	4102	6349	398	4776	8793	1919	7221
	8756
	1154	3192	1603	8648	2048	1692	1347	3073	9684	3212	8125
	1563
	5012	2143	2731	10006	5240	4915	7389	1729	7639	6319	844
	2263
	6343	891	3352	590	593	578	591	8035	6049	6731	7592
	1145
	7686	9079	4699	5323	4303	1000	579	340	9696	539	7817
	7814
	352	6724	1917	8987	3594	8858	8853	8855	1866	2559	5673
	7130
142:	9538	6322	8216	2187	2190	2706	2702	8864	7631	5819	4946
	5429
	1914	9292	4470	1705	2457	8384	7603	8655	376	1064	7518
	5845
	2936	6683	9763	9153	7043	3846	5324	5162	4489	5848	6367
	5696
	5854	2736	9284	6767	4015	4001	7119	7128	6819	6746	6637
	8436
	3198	7283	2695	597	5092	4684	1789	8417	7187	643	7053
	7671
	4633	4795	6275	3390	5246	4731
143:	9538	6322	7202	114	8216	2187	2190	2706	2702	8864	7631
	4188
	753	4191	8728	4002	3875	5819	8780	4946	5429	1914	9292
	4470
	1705	2457	8384	7603	8655	376	7135	1064	1066	7518	6038
	6924
	5845	3207	2936	6683	10011	9763	9153	7043	4569	1296	3846
	5324
	5997	3321	5162	4489	9846	1282	8791	5848	6367	5696	5854
	6828
	2736	9284	6767	4015	4001	7119	7128	6819	6746	6637	8436
	3198
	9398	1094	7283	2695	597	5092	227	5082	4684	1789	3375
	6456
	8602	2128	5736	1183	4672	6560	688	466	3789	2075	6517
	726
	6083	2036	2533	4914	762	5115	4409	6251	704	1810	526
	1665
	7997	2732	5462	7207	8475	8840	7215	1585	1862	3807	5428
	1234
	8417	5801	6952	7120	7187	643	7053	7671	9298	4633	4795
	6275
	3390	5246	4731
144:	7151	6340	8968	2512	9223	8511	6831	1411	8429	640	9838
	8939
	4614	2313	8017	7853	3726	4136	7242	5908	4839	3629	5553
	2463
	8002	685	2572	5679	5651	9641	5049	2397	6231	3777	9346
	570
	2598	5766	4493	4464	1856	3171	4078	2374	633	615	3079
	3519
	6914	6119	4782	8753	9483	1625	9549	944	6394	1818	7353
	491
	9429	2995	10019	8167	5902	2890	636	9917	9933	4734	4779
	493
	6410	3829	1821	2762	5900	7925	2780	627	6949	2928	9995
	2157
	1988	7313	4921	9729	2461	4689	9543	3358	1857	4528	9514
	9214
	3665	3506	2973	4875	3045	6374	6390	4895	6393	6392	8200
	4665
	3446	4551	1111	6433	5790	2532	6707	8937	4894	7719	7722
	7644
	6196	4546	7019	6334	4112	2693	5718	8868	8337	7105	8401
	3479
	9883	5297	5826	5195	1694	1052	7624	8084	5368	1792	1809
	5308
	5291	3604	6611	2470	4720	1363	1360	1366	2541	2540	2244
	7448
	7845	5208	5228	5224	5362	5230	3448	7779	7781	9870	7776
	4248
	2935	5756	4391	4378	9775	4390	1461	914	4939	6837	7793
	8451
	9304	9302	922	718	4070	7852	1527	1526	8977	246	684
	3865
	6547	1386	387	2223	1554	8322	252	249	248	2214	7084
	4690
	328	6873	7325	2968	9025	8682	9533	6729	9375	1072	6897
	4926
	7268	2344	6963	9907	4698	7342	949	5424	6033	1290	7577
	297
	3515	8897	9306	2854	9019	6360	6487	2389	9203	7871	9321
	377
	1683	8468	3397	4855	3219	4853	7825	8066	2391	2312	2364
	2341
	2308	2336	2359	2355	2291	2309	8676	2334	8674	2338	2293
	6361
	5880	4164	2896	6929	5179	4595	8031	1887	6630	5425	7499
	1442
	7539	5770
145:	585	3920	765	759	2616	8355	4161	2637	7094	9183	4433
	9037
	5612	7711	708	9981	1449	535	369	3735	3293	3266	3269
	8463
	8467	8543	8464	3289	3286	3297	3287	3262	5702	5683	5698
	7139
	1676	9405	9107	8749	779	4302	6597	6771	5571	7748	6790
	5122
	8975	8959	6766	8978	8958	9114	7307	6807	8875	5118	8980
	6787
	7247	6769	6530	5704	6823	5121	6786	8954	873	8636	3808
	3804
	2021	8654	3510	3490	6003	413	2715	3140	589	7741	3717
	7864
	4809	1615	1297	3474	2657	8486	1090	4379	3610	1681	7276
	3538
	9250	5439	9541	6420	6446	6448	6453	1779	2759	4209	8839
	5759
	6316	5079	9067	7346	965	5537	4924	932	4239	5440	4550
	4553
	9891	728	6411	3757	1365	749	8359	4212	5384	5887	5885
	7619
	5344	8695	4266	8762	895	5170	9300	4177	3407	3406	1658
	4158
	3996	5386	9435	3353	2967	7334	1571	9189	1662	9905	784
	8069
	1679	4032	8480	1420	8470	2469	6970	3941	10007	1233	7595
	1223
	3392	9479	5643	4657	5675	2843	6552	8891	733	1929	6409
	4271
	1806	8106	906	5484	7989	1861	5345	3310	2776	6368	2838
	8416
	5284	3760	9836	7972	7638	6223	760	8339	639	626	4576
	7072
	6595	3817	5644	3801	500	8184	9664	182	3226	3544	2678
	5693
	8861	428	9165	4156	8484	9539	275	279	280	730	736
	739
	741	758	763	767	778	781	3505	5684	3264	2966	7474
	1093
	7069	8585	5272	2654	2683	624	5465	5356	7266	5797	3536
	1828
	547	1597	8870	6843	10022	8087	8540	2613	3377	9407	1980
	4818
	5874	7036	1298	1335	2836	7790	3404	1182	3845	3785	8290
	3075
	7048	6182	3791	4149	2282	6718	5538	679	9878	2857	8645
	5464
	7964	1621	5456	477	476	475	2742	8088	8670	6713	9308
	3421
	5326	5668	4641	9333	1369	1749	6883	4532	961	7152	6032
	5703
	8658	774	9119	7953	3512	9537	3823	4365	8395	1780	488
	2388
	3356	6398	7648	3878	3906	7124	7182	5189	746	2122	644
	8448
	9888	8140	6672	217	4167	8812	6029	3543	4023	7409	8142
	2473
	5200	772	546	1868	7354	6135	3651	9257	8149	1604	9928
	3589
	1820	6020	4900	8814	963	2723	5479	5280	7755	1762	775
	7059
	776	5842	6839	3690	6921	8787	4186	8301	3732	1119	1114
	1715
	7142	7143	714	3780	9326	6442	5485	1407	6888	4871	3002
	6383
	1161	7161	7157	7065	3783	8739	2979	4246	2649	3844	2647
	2325
	2642	743	740	734	731	738	6590	6591	4561	8433	7535
	8157
	346	499	9813	773	7165	780	8640	2945	2556	438	7219
	7179
	4482	2856	2709	3962	5211	3292	3296	3300	3802	8102	9987
	995
	4700	1720	8404	5786	3433	8639	454	7777	1004	1491	1490
	5199
	969	982	2555	3984	1007	990	5359	9982	306	8565	8574
	8570
	8551	7460	6248	8548	868	8546	8994	6342	6224	6145	6748
	1028
	8067	7339	7140	1005	2536	2771	1006	7381	6324	4286	1013
	985
	983	986	988	1012	1014	5904	6917	9390	8571	3799	9941
	1435
	1184	1700	7836	6450	6415	3826	460	4702	1613	989	3627
	7006
	3820	2775	2019	6031	3109	9844	1190	8286	2676	7249	8053
	8050
	8021	5583	1288	9211	1388	9566	9567	8578	1194	5183	5441
	5476
	9139	1941	7180	2347	7137	7145	3046	3434	2250	659	4654
	5998
	4123	2044	7609	3431	4126	7018	9353	2424	8631	5621	716
	3265
	1927	4113	5029	2741	3179	1255	1258	1259	4029	1699	1680
	4579
	1701	4581	1678	4578	6472	5994	5755	5840	9542	620	967
	8297
	8661	2254	2618	3734	388	1999	6815	6462	3737	3738	5682
146:	7116	5816	2975	2710	7572	5242	8569
147:	9980	8517	5865	3276	1782	5877	3378	2835	6460	7031	637
	7150
	5976	5053	8379	6853	5054	6835	2396	2400	10014	8794	2777
	3049
	3065	4260	1323	1393	3229	4514	518	6979	2478	4572	7815
	8710
	8714	2447	1880	1877	9531	6131	2371	9692	4518	7212	7070
	8383
	2539	447	4509	2757	8254	4486	1886	8886	647	3605	1340
	4340
	4352	2450	9960	9961	9984	5974	3654	2277	3683	4258	7625
	7629
	7807	1667	5942	4108	4951	7663	4817	9558	1497	9335	1922
	2552
	315	5475	1702	8621	1181	4759	6019	412	3885	6535	9659
	8860
	1138	3205	3227	3507	7092	2746	7090	8214	8238	1170	7996
	1169
	2580	4143	700	2066	5653	6377	9985	8119	8133	3999	8846
	9385
	1080	9220	7323	8985	611	9087	4272	4289	9437	9871	7731
	1640
	6396	5112	6732	308	6772	2651	6072	6882	6884	6840	1243
	4194
	8085	6138	5719	7574	9528	7823	6291	5169	6865	3858	9839
	7673
	5764	8979	3419	9556	4316	5574	9235	7604	6645	7218	7992
	923
	7314	7316	5655	2602	2600	1222	1226	9024	1998	8625	2107
	9020
	9749	9033	9750	9030	8706	8220	3106	3838	2032	5349	4566
	6025
	4658	4677	9841	8773	6626	8774	5666	5661	5458	6693	3788
	5620
	5178	6017	6018	2528	2525	226	888	799	2054	4027	4030
	9848
	4545	1071	5285	6911	1565	1568	3943	4377	5487	2125	9897
	6666
	9709	771	7870	5851	3208	8090	810	1624	8292	9433	3388
	3387
	467	2381	5050	1693	5019	1695	1698	1001	2950	1016	7419
	4009
	8816	941	3750	7912	5077	196	2275	5837	5836	311	7444
	4898
	9271	7544	7546	7570	4976	3166	5924	4603	3955	2015	1997
	4708
	8402	7880	3273	905	7113	6305	683	8306	839	272	8308
	7888
	4510	9798	2895	2832	2996	9727	6188	250	228	9569	6665
	2669
	3905	3967	3983	7320	7999	8000	6004	5158	6444	5159	2911
	2908
	4616	5890	9068	2210	3841	8458	3611	517	3630	3617	3634
	9484
	3248	2410	9577	3637	3616	3612	296	6502	232	3745	6421
	7737
	1535	1728	4293	6636	4642	2481	295	8618	7665	5500	6092
	6417
	9793	1756	1107	8880	7849	4355	2551	6728	6727	6725	5717
	1618
	6869	1735	2189	8582	431	6104	2137	4753	300	7068	4364
	5925
	7582	5337	8409	3270	7438	2020	5293	6300	6132	6898	6348
	6344
	6915	6364	6988	6944	6962	6402	6389	6896	6935	6431	6895
	7039
	6164	6960	6933	7037	6424	6363	7012	2698	7035	7054	7062
	2625
	8662	8725	2242	8550	2240	2235	8721	2622	2306	8620	8694
	8718
	2211	2185	2181	1669	2328	2288	8665	8698	8544	8691	2186
	2307
	2302	6323	2283	2261	8612	2269	2329	8588	8614	2303	8572
	8592
	2273	8668	2375	8563	8619	8566	2266	7707	7198	2415	8663
	7016
	7651	8590	7653	2413	2352	8644	7010	2981	6940	7029	6908
	6370
	6303	2330	2408	2350	6406	6964	6758	8583	6307	6899	7586
	6942
	5539	5516	5556	5542	5472	5535	8947	5497	7057	6987	7008
	7617
	7553	7032	6985	541	6058	6084	8637	6965	9469	7236	6698
	1730
	9006	3880	5551	1508	6825	595	3061	3766	1241	4738	3338
	9162
	2738	2754	2792	2755	8261	9240	8014	3333	3160	2615	6154
	7902
	1851	1638	6482	5095	2179	4637	7811	7808	9636	7191	7650
	2537
	5006
148:	9807	9021	3422	2554	9217	1648	8121	7920	3711	4189	445
	8186
	9777	5773	5629	8481	7331	9861	358	5468	2202	5988	3247
	6055
	4619	474	4670
149:	5381	9028	2279	884	4610	8904	4254	5875	6779	4755	8911
	5791
	5808	8596	5957	6105	7423	9570	6777	3703	6770	9316	9694
	6778
	1800	3633	2725	1518	2728	8293	197	7515	6222	817	5934
	3432
	4228	497	9852	9532	9797	2585	9286	4549	2707	6026	7816
	7886
	9885	8531	7941	6505	5939	8407	7918	9261	5926	4508	5980
	2753
	4600	6329	4072	482	3320	1189	6715	6733	4651	4185	6207
	4328
	4305	7347	7753	3966	7961	4768	6708	6614	809	8319	8165
	1612
	4463	2522	6308	9784	3982	7865	1315	9193	4380	5448	9717
	1962
	2714	2820	3593	6916	5746	1143	6972	8371	5751	5306	1696
	6855
	1022	2422	8196	6854	1087	7228	4448	5899	2617	2888	8141
	229
	2851	1359	6649	7079	9751	9877	8961	8311	6617	556	1951
	3744
	1307	9445	6692	2039	188	4412	6543	2640	4522	1318	8539
	8579
	5589	6447	3908	5896	6572	9171	7178	9244	1271	9817	9266
	7196
	7122	7937	2744	9623	3164	1855	3222	5747	3731	8388	631
	1245
	4663	3199	8554	9111	2314	2031	6332	1664	1672	8062	9272
	4562
	1229	1443	850	5294	1908	2034	3937	1627	9459	2172	3699
	2324
	2220	9564	1596	6052	7728	3284	4062	4060	4351	3833	3795
	5442
	5446	8168	9551	2627	9679	6542	2567	8609	9122	6380	7657
	3272
	8965	4079	5096	9540	5633	4911	5173	3094	1532	1748	2145
	6278
	3256	4148	7431	3922	3919	5419	5331	5059	1467	921	7229
	4285
	2140	2571	573	994	3993	6950	1469	3500	2877	5376	7404
	6849
	8018	5094	1374	4042	4962	1459	1463	4601	9948	610	4064
	379
	2475	2960	6166	8246	7298	5379	9417	7830	9137	5938	5923
	4827
	5937	4780	9811	7357	5234	8731	7904	258	4100	7487	233
	1079
	8789	8798	607	2673	2477	9419	3588	2672	6167	6245	7975
	5342
	5322	1030	9660	917	2188	3645	2878	3625	6803	653	4021
	2136
	6522	2665	1990	6623	452	5945	9639	1516	7426	8934	6867
	5941
	339	1901	2174	1572	6009	586
150:	6947	9930	8552	4707	600	458	9423	3655	2834	5726	3667
	9648
	650	9611	5396	7913	8923	5649	3615	4630	7483	9614	7700
	1010
	1924	6440	9247	7839	4961	6973	7163	7602	4129	574	3851
	6285
	618	5577	8277
151:	2606	2063	2060	496	3946	10020	7076	2024	5495	6475	9700
152:	9658	9420	3779	6609	3006	2471	3567	4983	98	979	3486
	5549
	3725	6098	7872	2112	434	3565	10008	5728	8348	8033	5426
	8854
	9642	6255	3980
153:	9658	9420	3779	6609	3006	2471	3567	4983	3486	98	979
	5549
	434	3565	10008	8348	5728	8302	8033	6705	8244	9642	6255
154:	9424	4676	2340	5445	8252	8604	8158	6499	3027	1765	10009
	2963
	4716	5352	5351	5277	6850	7155	4678	8208	8329	6993	2100
	9833
	2421	9791	7936	7900	9072	9799	4367	6826	6514	4091	4090
	2751
	4093	3033	9312	9554	6607	4421	1703	4418	1817	7022	4357
	4059
	9296	9910	3071	2940	1860	7771	402	2073	9498	928	1909
	9653
	9497	2696	8498	3036	2414	4777	3899	6192	9909	3770	4485
	2467
	2419	9451	2712	837	8434	2138	6351	9108	4836	5481	2124
	2418
	6981	7274	5184	5081	1671	9295	3599	1020	4224	5554	5572
	5570
	6391	3961
155:	9424	4676	2340	5445	8252	8604	8158	6499	3027	1765	6510
	3427
	10009	2963	4716	5352	5351	5277	6850	7155	4678	8208	8329
	6993
	2100	9833	6994	9657	6996	9854	3668	8199	5365	7802	9029
	2421
	9791	7936	7900	9072	9799	4367	6826	6514	4091	4090	2751
	4093
	3033	9312	9554	6607	4421	1703	4418	1817	7022	4357	4059
	9296
	9910	3071	2940	1860	2684	782	9276	7425	9991	2492	1884
	2794
	8529	5265	8007	7771	402	3842	2073	9498	928	1909	9653
	9497
	2696	8498	3036	2414	4777	3899	6192	9909	3770	4485	2467
	2419
	9451	2712	837	8434	2138	6351	9108	4836	5481	2124	2418
	6981
	7274	5184	5081	1714	3228	2139	6846	7947	2977	5641	1671
	9295
	3599	1020	4224	5554	5572	5570	6391	3961
156:	9060	9635	3511	1890	1852	7041	8456	8503	5709	7232	9824
	9121
	1142	9105	3524	886	99	2910	9802	8893	9050	6116	8210
	2648
157:	4624	1430	1961	1964	9456	5156	5394	7397	8169	7589	3279
	4306
	7970	5051	6353	5373	7529	7189	7177	9720	9724	9737	8325
	9743
	8310	9762	5483	4841	9547	456	7027	4073	865	3681	4125
	2487
	9754	9715	5852	7311	8287	9858	4556	4386	5237	4843	8283
	6968
	3988	5146	2030	3167	10021	7754	401	6336	1019	9986	7351
	9843
	1519	5919	6463	2568	4326	3251	7257	411	8280	2612	4172
	677
	5236	6337	6941	1939	1846	3268	5264	2126	2850	1413	2595
	4865
	5338	8606	8629	3585	9954	2395	3255	1592	8504	2091	3454
	8148
	2103	6516	7601	9113	5955	8362	7498	6681	8435	682	1616
	4872
	3003	3881	9673	5402	10002	9368	1367	5310	4499	5630	4811
	486
	6466	4114	6893	1167	504	159	7344	2897	9561	6709	3828
	8373
	5779	5405	8890	3368	7467	1794	5605	3202	7077	3414	8605
	8267
	8441	6977	6210	3177	3674	9141	1069	1345	8623	3926	7892
	5387
	663	1904	869	6784	3039	6783	5341	9603	7943	1496	1495
	8633
	7703	6564	8195	4295	754	7579	7687	9829	1122	3935	9341
	8268
	4211	3564	2805	8263	792	1124	756	2716	7333	7222	2014
	2356
	3127	3653	7950	4153	8867	6429	9457	6434	8734	6905	2165
	4571
	2072	6064	4650	5252	3798	3915	1666	7185	7706	3720	883
	3254
	4920	8452	5962	7186	1091	8162	8228	843	9140	2345	3987
	7200
	2814	6078	9492	3816	4354	6881	7705	1724	2597	102	4828
	238
	552	5676	1642	4417	5646	3678	6325	4282	2664	3035	9503
	7204
	6422	6586	1652	3175	8323	2399	2394	5427	4430	6378	8097
	2704
	4444	9173	9557	8568	6575	1240	268	5166	550	2093	5835
	8235
	3242	9062	7503	1242	6669	202	4252	3942	1837	5772	7013
	3949
	2387	1872	4132	239	6362	3853	5399	5290	361	1690	6817
	1339
	1096	1489	224	351	1620	7863	9449	6123	8366	6051	2183
	1721
	9238	2680	3626	3344	1116	2750	6932	7588	7744	103	3267
	5492
	104	9588	6655	8671	8426	5594	1218	7709	9637	2882	3986
	162
	8940	7045	1364	3112	2088	6478	9289	3965	1117	5979	9329
	2311
	956	6806	9680	5660	8285	1398	5343	6127	7466	4784	3032
	5850
	5920	9197	3649	8744	8970	8852	1984	5669	7829	6594	8237
	1348
	5789	371	9957	3281	2933	6102	9219	4400	6788	4414
158:	2959	3815	4004	3362	3533	6295	3034	6178	1228	1025	5085
159:	4624	1430	1961	1964	9456	5156	999	3374	6267	1053	4084
	9627
	5394	6413	7397	8169	9718	6610	7876	4383	1098	1556	783
	6842
	7112	9675	3279	6197	9663	4306	7970	4596	7356	1390	5051
	7566
	5754	6353	2721	2365	7177	7189	4841	9547	3255	2850	8504
	1846
	9224	7752	6742	3039	8268	3564	4153	6429	9457	102	4828
	7200
	4354	6881	8323	5427	6669	4252	162	8940	1117	5660	956
	7829
	5669	6594	4893	5253	9367	5124	9369	5546	157	2141	205
	2249
	7148	8237	1348	5789	9493	371	9957	3281	2933	6102	9219
	4400
	6788	9027	8373
160:	7398	4338	6484	6449	9732	6500	6496	6480	6477	4169	7260
	6605
	6606	7280	8487	9607	7439	9236	9237	3131	6585	177	7291
	7290
	4868	7293	7295	7297	7315	7279	8159	6601	6624	6622	6584
	6583
	6388	6387	7341	6891	7253	7321	7324	3947	4452	6330	6620
	6598
	6600	3753	4899	6243	9355	3157	7108	7107	5977	4467	3998
	3181
	3185	3152	3155	7534	1849	7442	7235	3250	8494	176	3357
	7369
	8673	9585	9587	8866	6046	9103	4775	9951	9949	9943	9947
	6579
	6559	6561	6574	1261	8729	1166	9701	8492	8447	8472	4317
	1863
	7956	8326	3400	1267	4696	7761	1266	1270	5036	7100	6366
	6519
	6371	3055	3488	6680	1088	8473	8469	3126	8098	5813	8496
	4319
	8499	8500	1717	1741	4313	9009	854	6660	5001	4099	4086
	4077
	7241	8185	3059	3060	8187	2158	8207	5667	8147	7379	6262
	9338
	8145	820	6646	9005	2236	5469	8075	8093	3501	7746	1321
	6007
	3747	1915	5153	7373	3558	7476	212	200	2238	1617	6714
	8105
	7317	7318	6558	7716	3764	7971	7968	3275	6580	8476	3023
161:	7166	6423	5969	7138	7828	3577	489	5529	6737	9681	6534
	1722
	8881	8779	4557	8810	4325	7209	9670	5063	2588	3570	2468
	3921
	9958	9301	5466	911	3869	3523	1591	9783	2404	237	7123
	6479
	10012	1898	2921	1361	341	8532	2199	6079	2205	4823	6562
	3582
	4198	5437	2988	1673	7066	5126	9091	6045	745	4216	7254
	5161
	4887	977	5407	564	9761	790	1009	3944	8437	608	1196
	5222
	966	8036	1477	2535	9234	4824	5288	2506	5853	2880	1778
	3225
	6765	919	4375	8702	3209	4812	1562	833	5353	1322	3733
	5817
	6945	3053	7049	900	4591	5238	5868	4483	786	3201	1588
	7489
	5700	2914	5503	2660	2500	6191	2902	2582	8948	8351	185
	2766
	6841	9436	9478	4685	9940	658	4443	1344	8414	1785	1349
	8856
	417	5680	984	3107	1447	4089	3425	2643	3781	3118	4548
	7197
	5541	6047	4613	7933	2239	7734	4497	9378	4202	881	8999
	7549
	2390	7637	795	6418	6577	5266	3253	5855	6759	5325	7607
	3119
	859	2333	3220	1382	4560	4268	240	5654	3739	8059	4397
	806
	686	8170	301	2748	9480	4529	838	9166	769	1265	1112
	8997
	2099	1408	1829	3560	8685	3440	4648	6234	8422	642	3978
	3217
	755	1239	3790	2682	1051	5269	8390	6253	6076	676	6647
	1805
	8971	7955	788	9502	2675	9610	2898	569	3240	110	7810
	487
	9253	6155	3722	2713	9746	5523	8080	6768	5103	4879	6805
	9475
	7593	364	7966	1217	1068	592	5777	568	2301	3933	1747
162:	4624	9456	5156	999	3374	1053	4084	9627	5394	6413	2639
	7397
	8169	6610	9718	4383	1556	1098	783	6842	7112	9675	3279
	6197
	4306	7970	4596	7356	5051	7566	5754	6353	2721	2365	7177
	7189
	9724	9547	456	3255	7752	3039	6783	8268	3564	4153	6429
	9457
	102	4828	7200	6881	4354	8323	4252	6669	202	7829	5669
	5253
	9367	4893	6594	157	2141	205	2249	7148	1348	5789	9493
	371
	9957	3281	2933	6102	9219	4400	6788	1117	5660	956
163:	3151	8646	4019	4005	282	299	4018	4831	2576	4850	3134
	9124
	9125	9174	9175	9209	9210	9127	9128	9134	7493	2483	4101
	9518
	9521	907	7896	937	1505	8471	8474	8465	5165	5181	5180
	7708
	7710	5235	5233	4103	8785	8786	8790	8788	8723	8703	8733
	8735
	8748	3085	8752	8765	8763	8770	8784	1378	1402	8799	8800
	8805
	8802	8824	8821	8826	9314	9317	9320	9322	9318	9345	9344
	9352
	9354	9384	9400	9358	9356	9374	9380	9377	9382	9406	9408
	9421
	9425	9422	9427	9439	9440	9442	9447	9443	9461	9466	9463
	9485
	9468	9487	9488	9489	9495	9511	9963	9965	9970	9990	9968
	9966
	9996	10001	9997	8766	8768	8647	8651	8652	8667	8699	1432
	1504
	1455	1502	805	7528	5864	5866	2489	9147	9148	281	9206
	9208
	9225	9227	2563	9229	9230	715	2008	2011	1387	1389	9100
	9101
	9130	9131	7770	7787	9150	9151	4834	9179	9180	4837	3153
	2065
	2067	2086	2087	4460	3913	3911	3103	1391	1395	2491	1928
	1926
	1973	4008	4003	8746	8743	8833	8829	9993	1940	1963	1992
	2006
	1932	1938	5044	933	939	7558	4219	808	908	811	813
	7526
	7481	828	4221	826	814	931	829	910	851	832	7505
	853
	835	7429	7486	7391	7388	7364	7527	7417	7433	7465	964
	875
	7363	874	876	7361	7360	909	4223	7457	7462	879	7459
	4206
	7482	4733	7464	7507	7557	889	7392	935	7504	7393	7532
	7488
	4205	7554	7437	7533	960	7415	894	892	897	5532	959
	890
	7552	4942	3959	3972	3957	3991	3971	5758	5070	2448	2449
	2452
	2466	2493	2495	2497	2560	7368	7501	7556	4726	4728	1966
	9177
	1397	2514	2538	711	958	1035	7575	7434	855	871	7484
	857
	860	962	7502	7420	7416	5068	4938	1433	7986	8001	1480
	8005
	1501	1528	1373	3095	4225	4730	7396	1277	1279	947	7932
	7935
	2285	2287	4374	4910	4927	4929	4931	4933	4935	9181	9182
	1485
	6986	3088	1522	1338	1356	1353	1427	1426	1487	1525	1351
	1399
	1521	1524	1507	1488	2472	1358	1376	1401	1464	3104	4949
	4950
	4955	4957	4960	4988	4989	4993	4998	4999	5022	5023	5024
	5028
	5031	5486	5488	5489	5490	5494	5507	5510	5513	5515	5511
	5527
	3101	7954	7958	7957	7899	7903	4372	4385	7928	7959	7976
	7951
	7981	7984	8019	8006	8025	8020	4368	4366	4343	4363	4382
	7894
	8026	4334	4332	4822	8003	7923	7983	4336	3180	3158	3129
	2245
	3086	3137	2290	3183	3186	3188	8700	8701	2393	2368	3117
	4739
	4972	4763	4766	4772	4806	2241	2289	2305	2270	2264	2267
	2284
	2212	2237	6357	4975	4756	9294	8327	5259	5256	5867	4794
	8493
	8497	1931	3144	3142	3121	1113	5209	7676	7658	7675	3548
	3551
	4329	8490	9196	9198	4848	9152	9155	9145	9144	9135	9136
	9204
	8385	2228	2226	2231	2234	3552	3553	1589	7721	9640	2515
	2518
	277	3910	3018	6615	3355	6975	6980	6976	7713	7000	3517
	4790
	4802	4830	3405	2451	4011	4833	5828	4712	4805	4771	4746
	5318
	4713	4793	4835	5319	5317	4742	4764	1421	1424	9606	6625
	1969
	1971	1434	8672	8669	3130	2013	2029	9516	8726	8730	9336
	9339
	9337	4829	6934	1743	5745	1958	7869	6426	1986	1987	7340
	5037
	4785	4789	912	3876	3873	3891	3102	8807	8626	8642	3132
	4854
	9232	9233	9202	9132	1418	1419	9199	9200	9184	1628	1172
	1175
	1177	9360	4765	5061	671	657	669	655	5314	5067	8113
	8117
	978	8270	165	7255	8809	298	1989	1991	4796	4808	6244
	6488
	6490	6503	4743	3312	6822	4067	5279	9156	9158	5205	3147
	3168
	3172	4856	3156	1548	1798	1546	208	8944	8933	8949	8276
	8279
	8281	8203	8204	6005	8253	8992	8995	8996	8972	8991	9519
	8197
	5917	5978	8223	5984	5936	8316	5956	5958	8226	8229	8300
	9098
	9099	9560	9580	9579	9582	9583	8919	7418	7794	1587	1579
	7692
	5273	1593	5261	4710	4279	1547	6497	3895	3893	3907	3081
	4195
	5210	4758	4197	8832	8916	8851	8857	8894	8892	8871	8865
	8849
	8872	8889	8874	8927	8932	8930	8895	8909	4770	7681	7691
	5226
	5213	5157	5160	1375	1400	1458	1462	1482	2037	2041	9172
	5321
	1396	1415	4714	4741	4966	4970	5000	5002	4745	5003	4760
	4762
	5005	4769	4798	5064	1379	1352	1341	1453	3929	3925	3080
	8750
	7430	9401	9403	2007	2062	2059	2064	4857	8675	8677	4940
	4965
	4973	5004	5039	5041	5045	705	707	691	693	692	2033
	2035
	4740	4709	4749	4748	4803	5312	5313	5315	1745	9855	6821
164:	6237	6238	7766	2180	5501	6061	617	1996	7115	6328	7621
	9726
	1897	1879	6204	5480	6298	8537	6412	5959	1784	7642	8015
	1736
	9952	8126	432	3822	4362	5750	5671	7677
165:	3151	1577	8646	4019	4005	282	299	4018	4831	2576	4850
	3134
	9124	9125	9174	9175	9209	9210	9127	9128	9134	7493	2483
	9518
	9521	907	7896	937	1505	2252	2804	2782	2779	2822	2224
	2251
	2149	2256	2846	2781	2257	2255	2855	2852	2797	2785	2221
	2802
	2873	2849	2786	2819	2795	2799	2148	2198	2192	2168	2225
	2218
	2197	2867	2864	2825	2196	2193	2171	2169	2830	2219	2173
	2166
	2853	2827	2889	2871	2874	8471	8474	8465	5165	5181	5180
	7708
	7710	5235	5233	4103	8785	8786	8790	8788	8723	8703	8733
	8735
	8748	3085	8752	8765	8763	8770	8784	1378	1402	8799	8800
	8805
	8802	8824	8821	8826	9314	9317	9320	9322	9318	9345	9344
	9352
	9354	9384	9400	9358	9356	9374	9380	9377	9382	9406	9408
	9421
	9425	9422	9427	9439	9440	9442	9447	9443	9461	9466	9463
	9485
	9468	9487	9488	9489	9495	9511	9963	9965	9970	9990	9968
	9966
	9996	10001	9997	8766	8768	8647	8651	8652	8667	8699	1432
	1504
	1455	1502	805	7528	5864	5866	2489	9147	9148	281	3195
	2773
	9206	9208	9225	9227	2563	9229	9230	715	2008	2011	1387
	1389
	9100	9101	9130	9131	7770	7787	9150	9151	4834	9179	9180
	4837
	3153	2065	2067	2086	2087	4460	3913	3911	3103	1391	1395
	2491
	1928	1926	1973	4008	4003	8746	8743	8833	8829	9993	1940
	1963
	1992	2006	1932	1938	5044	933	939	7558	4219	808	908
	811
	813	7526	7481	828	4221	826	814	931	829	910	851
	832
	7505	853	835	7429	7486	7391	7388	7364	7527	7417	7433
	7465
	964	875	7363	874	876	7361	7360	909	4223	7457	7462
	879
	7459	4206	7482	4733	7464	7507	7557	889	7392	935	7504
	7393
	7532	7488	4205	7554	7437	7533	960	7415	894	892	897
	5532
	959	890	7552	4942	3959	3972	3957	3991	3971	5758	5070
	2448
	2449	2452	2466	2493	2495	2497	2560	7368	7501	7556	4726
	4728
	1966	9177	1397	2514	2538	711	958	1035	7575	7434	855
	871
	7484	860	857	962	7502	7420	7416	5068	4938	1433	7986
	8001
	1480	8005	1501	1528	1373	3095	4225	4730	7396	1277	1279
	947
	7932	7935	2285	2287	4374	4910	4927	4929	4931	4933	4935
	9181
	9182	1485	6986	976	3088	1522	1338	1356	1353	1427	1426
	1487
	1525	1351	1399	1521	1524	1507	1488	2472	1358	1376	1401
	1464
	3104	4949	4950	4955	4957	4960	4988	4989	4993	4998	4999
	5022
	5023	5024	5028	5031	5486	5488	5489	5490	5494	5507	5510
	5513
	5515	5511	5527	3101	7954	7958	7957	7899	7903	4372	4385
	7928
	7959	7976	7951	7981	7984	8019	8006	8025	8020	4368	4366
	4343
	4363	4382	7894	8026	4334	4332	4822	7117	8003	7923	7983
	4336
	3180	3158	3129	2245	3086	3137	2290	3183	3186	3188	8700
	8701
	2393	2368	3117	4739	4972	4763	4766	4772	4806	2241	2289
	2305
	2270	2264	2267	2284	2212	2237	6357	4975	4756	9294	8327
	5259
	5256	5867	4794	8493	8497	3144	3142	1113	7676	7658	7675
	3548
	3551	4329	8490	9196	9198	4848	9152	9155	9145	9144	9135
	9136
	9204	8385	2226	2228	2231	2234	3552	3553	7721	9640	2515
	2518
	277	3910	3018	6615	3355	6975	6980	6976	7713	7000	3517
	4790
	4802	4830	3405	2451	4011	4833	5828	4712	4805	4771	4746
	5318
	4713	4793	4835	5319	5317	4742	4764	1421	1424	9606	6625
	1969
	1971	1434	8672	8669	3130	2013	2029	9516	8726	8730	9336
	9339
	9337	6631	4829	6934	1743	5745	1958	7869	6426	1986	1987
	7340
	5037	4785	4789	912	3876	3873	3891	3102	8807	8626	8642
	3132
	4854	9232	9233	9202	9132	1418	1419	9199	9200	9184	1628
	1172
	1175	1177	9360	4765	9855	163	5084	6821	5061	671	657
	669
	655	5314	5067	8113	8117	298	1989	1991	4796	4808	6244
	6488
	6490	6503	4743	3312	6822	4067	5279	9156	9158	5205	3147
	3168
	4856	3156	1548	1798	1546	208	8944	8933	8949	8276	8279
	8281
	8203	6005	8253	8992	8995	8996	8972	8991	9519	8197	5917
	5978
	8223	5984	5936	8316	5956	5958	8226	8229	8300	9098	9099
	9560
	9563	9580	9579	9582	9583	8919	7418	7794	1587	1579	7692
	5273
	1593	5261	4710	4279	1547	3895	3893	3907	3081	4195	5210
	4758
	4197	4727	8832	8916	8851	8857	8894	8892	8871	8865	8849
	8872
	8889	8874	8927	8932	8930	8895	8909	4770	1062	7681	7691
	5213
	5226	5157	5160	1375	1400	1458	1462	1482	2037	2041	9172
	5321
	1396	1415	4714	4741	4966	4970	5000	5002	4745	5003	4760
	4762
	5005	4769	4798	5064	7114	1379	1352	1341	1453	3929	3925
	3080
	8750	7430	9401	9403	2007	2059	2062	2064	4857	8675	8677
	4940
	4965	4973	5004	5039	5041	5045	705	707	691	693	692
	2033
	2035	4740	4709	4749	4748	4803	5312	5313	5315	1745	8270
166:	7398	6484	6449	4729	6500	6496	6480	6477	4169	7256	7258
	2351
	6605	6606	7280	9607	7439	9571	9415	6711	9236	9237	6585
	177
	1949	7291	7290	4868	6438	6265	7293	7295	7297	7315	7279
	7792
	7788	8159	6601	6622	6624	6584	6583	6388	7341	7253	7277
	7321
	7324	3947	6620	6598	6600	4296	3753	4899	6243	4253	4128
	9355
	8071	7568	7534	7244	7442	7468	7820	7235	3250	8494	176
	6372
	7369	4428	8866	4775	7525	8255	5421	9967	6579	6559	6561
	2768
	1166	9701	4083	9703	8447	4312	8472	4317	1863	7956	3400
	757
	3642	5036	7100	3055	3488	6680	4587	8473	8469	3126	8477
	8098
	5813	9879	2965	9740	5142	8499	4319	8500	1717	8410	2883
	1410
	641	854	6660	5001	4099	4086	7241	4077	7237	8777	3059
	8185
	5491	3060	5667	7379	6262	9338	4236	6646	2236	8075	8093
	8076
	2767	7746	7749	1321	6007	7724	7373	7376	3558	7476	1732
	1734
	1740	1738	212	200	6714	7317	7318	1689	1709	6558	7716
	3764
	435	7971	7968	6580	8476	5457	1346

Example 5. Selection of Transgenic Plants with Enhanced Agronomic Trait(s)

This example illustrates identification of plant cells of the invention by screening derived plants and seeds for enhanced trait. Transgenic corn seed and plants with recombinant DNA identified in Table 1 were prepared by plant cells transformed with DNA that was stably integrated into the genome of the corn cell. The transgenic seed, plantlets and progeny plants were selected using the methods that measure Transgenic corn plant cells were transformed with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants and seed of the transformed plant cells were screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as compared to control plants.

A. Selection for Enhanced Nitrogen Use Efficiency

The physiological efficacy of transgenic corn plants (tested as hybrids) can be tested for nitrogen use efficiency (NUE) traits in a high-throughput nitrogen (N) selection method. The collected data are compared to the measurements from wildtype controls using a statistical model to determine if the changes are due to the transgene. Raw data were analyzed by SAS software. Results shown herein are the comparison of transgenic plants relative to the wildtype controls.

(1) Media Preparation for Planting a NUE Protocol

Planting materials used: Metro Mix 200 (vendor: Hummert) Cat. #10-0325, Scotts Micro Max Nutrients (vendor: Hummert) Cat. #07-6330, OS 4⅓″×3⅞″ pots (vendor: Hummert) Cat. #16-1415, OS trays (vendor: Hummert) Cat. #16-1515, Hoagland's macronutrients solution. Plastic 5″ stakes (vendor: Hummert) yellow Cat. #49-1569, white Cat. #49-1505, Labels with numbers indicating material contained in pots. Fill 500 pots to rim with Metro Mix 200 to a weight of ˜140 g/pot. Pots are filled uniformly by using a balancer. Add 0.4 g of Micro Max nutrients to each pot. Stir ingredients with spatula to a depth of 3 inches while preventing material loss.

(2) Planting a NUE Selection in the Greenhouse

(a) Seed Germination—
Each pot is lightly altered twice using reverse osmosis purified water. The first watering is scheduled to occur just before planting; and the second watering, after the seed has been planted in the pot. Ten Seeds of each entry (1 seed per pot) are planted to select eight healthy uniform seedlings. Additional wild type controls are planted for use as border rows. Alternatively, 15 seeds of each entry (1 seed per pot) are planted to select 12 healthy uniform seedlings (this larger number of plantings is used for the second, or confirmation, planting). Place pots on each of the 12 shelves in the Conviron growth chamber for seven days. This is done to allow more uniform germination and early seedling growth. The following growth chamber settings are 25° C./day and 22° C./night, 14 hours light and ten hours dark, humidity ˜80%, and light intensity ˜350 μmol/m²/s (at pot level). Watering is done via capillary matting similar to greenhouse benches with duration of ten minutes three times a day.
(b) Seedling Transfer—
After seven days, the best eight or 12 seedlings for the first or confirmation pass runs, respectively, are chosen and transferred to greenhouse benches. The pots are spaced eight inches apart (center to center) and are positioned on the benches using the spacing patterns printed on the capillary matting. The Vattex matting creates a 384-position grid, randomizing all range, row combinations. Additional pots of controls are placed along the outside of the experimental block to reduce border effects.
Plants are allowed to grow for 28 days under the low N run or for 23 days under the high N run. The macronutrients are dispensed in the form of a macronutrient solution (see composition below) containing precise amounts of N added (2 mM NH₄NO₃for limiting N selection and 20 mM NH₄NO₃for high N selection runs). Each pot is manually dispensed 100 ml of nutrient solution three times a week on alternate days starting at eight and ten days after planting for high N and low N runs, respectively. On the day of nutrient application, two 20 min waterings at 05:00 and 13:00 are skipped. The vattex matting should be changed every third run to avoid N accumulation and buildup of root matter. Table 7 shows the amount of nutrients in the nutrient solution for either the low or high nitrogen selection.

TABLE 7

	2 mM NH₄NO₃	20 mM NH₄NO₃(high
	(Low Nitrogen Growth	Nitrogen Growth
	Condition, Low N)	Condition, High N)
Nutrient Stock	mL/L	mL/L

1M NH₄N0₃	2	20
1M KH₂PO₄	0.5	0.5
1M MgSO₄•7H₂O	2	2
1M CaCl₂	2.5	2.5
1M K₂SO₄	1	1

Note:
Adjust pH to 5.6 with HCl or KOH

(c) Harvest Measurements and Data Collection—
After 28 days of plant growth for low N runs and 23 days of plant growth for high N runs, the following measurements are taken (phenocodes in parentheses): total shoot fresh mass (g) (SFM) measured by Sartorius electronic balance, V6 leaf chlorophyll measured by Minolta SPAD meter (relative units) (LC), V6 leaf area (cm²) (LA) measured by a Li-Cor leaf area meter, V6 leaf fresh mass (g) (LFM) measured by Sartorius electronic balance, and V6 leaf dry mass (g) (LDM) measured by Sartorius electronic balance. Raw data were analyzed by SAS software. Results shown are the comparison of transgenic plants relative to the wildtype controls.
To take a leaf reading, samples were excised from the V6 leaf. Since chlorophyll meter readings of corn leaves are affected by the part of the leaf and the position of the leaf on the plant that is sampled, SPAD meter readings were done on leaf six of the plants. Three measurements per leaf were taken, of which the first reading was taken from a point one-half the distance between the leaf tip and the collar and halfway from the leaf margin to the midrib while two were taken toward the leaf tip. The measurements were restricted in the area from ½ to ¾ of the total length of the leaf (from the base) with approximately equal spacing between them. The average of the three measurements was taken from the SPAD machine.
Leaf fresh mass is recorded for an excised V6 leaf, the leaf is placed into a paper bag. The paper bags containing the leaves are then placed into a forced air oven at 80° C. for 3 days. After 3 days, the paper bags are removed from the oven and the leaf dry mass measurements are taken.
From the collected data, two derived measurements are made: (1) Leaf chlorophyll area (LCA), which is a product of V6 relative chlorophyll content and its leaf area (relative units). Leaf chlorophyll area=leaf chlorophyll X leaf area. This parameter gives an indication of the spread of chlorophyll over the entire leaf area; (2) specific leaf area (LSA) is calculated as the ratio of V6 leaf area to its dry mass (cm²/g dry mass), a parameter also recognized as a measure of NUE. The data are shown in Table 8.

TABLE 8

PEP	Leaf chlorophyll area	Leaf chlorophyll	Shoot fresh mass

SEQ

Construct

Percent

Mean of

Percent

Mean of

Percent

Mean of

ID

Event ID

change

Mean

controls

P-value

change

Mean

controls

P-value

change

Mean

controls

P-value

91	PMON73816	ZM_M37183	4	3688.43	3558.85	0.221	3	24.54	23.73	0.0722	5	48.04	45.92	0.1289
	PMON73816	ZM_M37183	15	5963.14	5180.33	0	12	31.72	28.41	0	16	48.24	41.48	1.00E-04
	PMON73816	ZM_M37183	8	4796	4439.2	0.0438	3	27.1	26.2	0.2569	23	55.2	44.8	0
	PMON73816	ZM_M37188	12	4002.73	3558.85	0	13	26.86	23.73	0	4	47.83	45.92	0.1707
	PMON73816	ZM_M37188	13	5832.79	5180.33	3.00E-04	12	31.73	28.41	0	11	46.25	41.48	0.0046
	PMON73816	ZM_M37188	−9	4037.7	4439.2 1	0.0234	−1	26	26.2	0.7492	−10	40.4	44.8	0.0144
	PMON73816	ZM_M37197	4	5375.2	5180.33	0.2694	1	28.81	28.41	0.5194	17	48.42	41.48	0
	PMON73816	ZM_M37197	21	5374.8	4439.2	0	14	29.9	26.2	0	30	58.4	44.8	0
	PMON73816	ZM_M37197	5	3733.33	3558.85	0.0996	1	24.02	23.73	0.522	5	48.42	45.92	0.0742
100	PMON75511	ZM_M44958	18.1	5065.43	4287.52	1.00E-04	13.9	29.44	25.86	0	12	44.22	39.48	0.0096
	PMON75511	ZM_M44958	7.3	8006.21	7460.91	0.0071	5.5	40.63	38.5	0.0072	0	67.53	67.56	0.9892
	PMON75511	ZM_M44961	8.2	4639.06	4287.52	0.0583	5.8	27.36	25.86	0.0449	6.7	42.13	39.48	0.1258
	PMON75511	ZM_M44961	4.7	7810.27	7460.91	0.0947	4.9	40.41	38.5	0.0195	4.9	70.87	67.56	0.1511
	PMON75511	ZM_M46591	5.1	4504.72	4287.52	0.2951	5.5	27.27	25.86	0.0734	−4.5	37.69	39.48	0.3276
	PMON75511	ZM_M46591	−4.3	7142.88	7460.91	0.1149	−1.4	37.98	38.5	0.4997	8	72.98	67.56	0.0151
	PMON75511	ZM_M46601	12.3	4813.03	4287.52	0.0117	4.7	27.07	25.86	0.1494	22.4	48.31	39.48	0
	PMON75511	ZM_M46601	7.7	8036.73	7460.91	0.0045	5	40.44	38.5	0.014	0.3	67.76	67.56	0.93
114	PMON75980	ZM_M53387	−8	3998.29	4368.22	0.0065	2	24.35 I	23.8	0.3237	−18	37.79	45.85	0
	PMON75980	ZM_M53389	-10	3323.6	3691.69	0.0189	-3	23.05	23.65	0.3551	−8	30.6	33.21	0.0804
	PMON75980	ZM_M53389	-5	4139.75	4368.22	0.1038	-2	23.42	23.8	0.4834	-10	41.22	45.85	0.0031
	PMON75980	ZM_M53390	8	4728.73	4368.22	0.0188	5	25.07	23.8	0.072	−3	44.65	45.85	0.4407
	PMON75980	ZM_M53390	10	4044.06	3691.69	0.0245	2	24.24	23.65	0.3703	9	36.29	33.21	0.0398
	PMON75980	ZM_M53392	27	4679.18	3691.69	0	10	26.06	23.065	3.00E−04	27	42.31	33.21	0
	PMON75980	ZM_M53392	2	4446.67	4368.22	0.5757	4	24.88	23.8	0.0534	3	47.36	45.85	0.3298
	PMON75980	ZM_M53396	13	4948.67	4368.22	0	7	25.37	23.8	0.0068	8	49.32	45.85	0.0259
	PMON75980	ZM_M53396	16	4271.59	3691.69	2.00E−04	4	24.7	23.65	0.109	13	37.46	33.21	0.0046
	PMON75980	ZM_M53397	1	4411.5	4368.22	0.7574	1	24.06	23.8	0.6707	−6	43.08	45.85	0.0992
	PMON75980	ZM_M53398	2	4476.43	4368.22	0.4235	7	25.36	23.8	0.0052	−6	43.12	45.85	0.0792
103	PMON78949	ZM_M63936	−2.1	4587.66	4686.12	0.4835	3.3	30.35	29.37	0.1605	−6.1	32.65	34.77	0.0457
	PMON78949	ZM_M63936	−2.1	3863.18	3946.32	0.4391	−0.6	28.37	28.55	0.7352	8.7	45.14	41.55	0.0077
	PMON78949	ZM_M63941	7.5	5037.73	4686.12	0.0128	3.9	30.51	29.37	0.1021	7.4	37.33	34.77	0.0158
	PMON78949	ZM_M63941	−1.9	3871.03	3946.32	0.4835	−2.5	27.83	28.55	0.1742	9.8	45.63	41.55	0.0036
	PMON78949	ZM_M63942	7.5	5036.21	4686.12	0.0132	6.4	31.26	29.37	0.007	9.2	37.98	34.77	0.0025
	PMON78949	ZM_M63942	13	4459.25	3946.32	0	7.6	30.73	28.55	0	9.2	45.37	41.55	0.0047
	PMON78949	ZM_M63944	4.3	4887.29	4686.12	0.1528	4.9	30.81	29.37	0.0393	−6.6	32.48	34.77	0.0306
	PMON78949	ZM_M63944	0.8	3979.53	3946.32	0.7571	0.4	28.66	28.55	0.8318	−0.9	41.17	41.55	0.7776
108	PMON79709	ZM_M51983	3	5110.49	4947.82	0.1855	6	28.18	26.59	0.0012	4	46.1	44.36	0.076
	PMON79709	ZM_M51983	2	6011.13	5906.6	0.6174	3	28.75	27.9	0.2078	16	62.26	53.53	2.00E−04
	PMON79709	ZM_M51983	0.9	5829.16	5776.02	0.7681	−0.7	30.24	30.45	0.7671	−3.1	45.46	46.92	0.4097
	PMON79709	ZM_M51985	0	5773.16	5776.02	0.988	−0.2	30.38	30.45	0.9183	−1.6	46.12	46.92	0.682
	PMON79709	ZM_M51985	7	6301.05	5906.6	0.0602	3	28.81	27.9	0.1763	16	62.11	53.53	2.00E−04
	PMON79709	ZM_M51985	6	5263.87	4947.82	0.0079	6	28.07	26.59	0.0026	3	45.48	44.36	0.2555
	PMON79709	ZM_M52025	3	5075.34	4947.82	0.2817	4	27.58	26.59	0.0415	4	46.33	44.36	0.052
	PMON79709	ZM_M52025	3.2	5959.63	5776.02	0.3087	−1.7	29.93	30.45	0.4617	1	47.38	46.92	0.7983
	PMON79709	ZM_M52025	21	7124.16	5906.6	0	14	31.74	27.9	0	20	64.48	53.53	0
	PMON79709	ZM_M52710	6	6240.85	5906.6	0.1109	10	30.6	27.9	1.00E−04	9	58.5	53.53	0.0321
	PMON79709	ZM_M52710	8	5339.8	4947.82	0.001	7	28.46	26.59	1.00E−04	3	45.82	44.36	0.1373
	PMON79709	ZM_M52710	3.8	5995.36	5776.02	0.2241	3.6	31.55	30.45	0.1214	−4.1	45	46.92	0.2779
	PMON79709	ZM_M52720	7.4	6201.46	5776.02	0.0188	5.2	32.04	30.45	0.0258	6.1	49.8	46.92	0.1242
	PMON79709	ZM_M52720	7	5280.25	4947.82	0.0053	7	28.39	26.59	2.00E−04	−5	42.31	44.36	0.0357
	PMON79709	ZM_M52720	12	6617.79	5906.6	8.00E−04	9	30.28	27.9	9.00E−04	3	55.01	53.53	0.5222
96	PMON80270	ZM_M55967	5.2	6306.34	5993.37	0.0376	4.3	30.64	29.39	0.028	7.3	54.7	50.98	0.0017
	PMON80270	ZM_M55967	6.6	5.33	5	0.0666	6.7	33.48	31.38	0.0075	6	44.75	42.21	0.0627
	PMON80270	ZM_M55968	16.6	5.83	5	0	5.7	33.17	31.38	0.0421	17.5	49.6	42.21	0
	PMON80270	ZM_M55968	−1	5930.77	5993.37	0.6873	−0.5	29.25	29.39	0.8058	7.7	54.89	50.98	0.001
	PMON80270	ZM_M55969	−4.1	5749.51	5993.37	0.1048	0.3	29.47	29.39	0.892	4.7	53.36	50.98	0.0427
	PMON80270	ZM_M55969	5	5.25	5	0.1118	4.1	32.66	31.38	0.1464	8	45.58	42.21	0.0139
	PMON80270	ZM_M55970	−2.3	5855.83	5993.37	0.3595	1.3	29.76	29.39	0.5246	4.4	53.2	50.98	0.0504
	PMON80270	ZM_M55970	2.6	5.13	5	0.4257	−2.5	30.58	31.38	0.3062	2.9	43.45	42.21	0.3616
	PMON80270	ZM_M55971	−4	5754.31	5993.37	0.1118	0.7	29.61	29.39	0.7	1.8	51.92	50.98	0.4075
	PMON80270	ZM_M55971	6	5.3	5	0.0728	4.8	32.89	31.38	0.0536	6	44.74	42.21	0.064
	PMON80270	ZM_M55972	−1	5933.48	5993.37	0.6897	−0.3	29.29	29.39	0.8631	3.6	52.81	50.98	0.1193
	PMON80270	ZM_M55972	13.8	5.69	5	0	5.1	32.99	31.38	0.0397	9.4	46.19	42.21	0.0037
	PMON80270	ZM_M56524	8	5.4	5	0.0364	5.1	32.98	31.38	0.0413	15.5	48.74	42.21	0
	PMON80270	ZM_M56524	−1.4	5908.18	5993.37	0.5702	1	29.67	29.39	0.6255	6.3	54.18	50.98	0.0067
	PMON80270	ZM_M56526	−2.7	5829.79	5993.37	0.276	−1.4	28.98	29.39	0.4744	2.5	52.23	50.98	0.2681
	PMON80270	ZM_M56526	20	6	5	0	0.5	31.54	31.38	0.8352	13.8	48.05	42.21	0
	PMON80270	ZM_M56527	1.2	6063.11	5993.37	0.6421	−0.2	29.32	29.39	0.8978	5.6	53.82	50.98	0.0126
	PMON80270	ZM_M56527	2.2	5.11	5	0.489	2.4	32.14	31.38	0.3294	4.1	43.95	42.21	0.2012
118	PMON80461	ZM_M52932	24.5	8417.13	6759.85	0	13.4	34.66	30.57	0	25.7	76.5	60.88	0
	PMON80461	ZM_M52932	6	7095.13	6713.17	0.0553	3	30.63	29.82	0.294	−1	54.058	54.73	0.653
	PMON80461	ZM_M52932	1	4877.13	4816.31	0.5834	2	29.24	28.65	0.2351	−2	30.75	31.34	0.4187
	PMON80461	ZM_M52932	−4.5	5830.38	6107.25	0.1599	−1.1	29.45	29.77	0.6468	−2.7	37.58	38.63	0.5145
	PMON80461	ZM_M52932	−9	4808.1	5269.64	0.0084	1	30.86	30.68	0.7905	2	35.8	35.13	0.4119
	PMON80461	ZM_M52932	8.2	5068.24	4686.12	0.0069	10	32.31	29.37	0	−6	32.68	34.77	0.0483
	PMON80461	ZM_M52932	14.3	4511.99	3946.32	0	6.5	30.42	28.55	5.00E−04	11	46.12	41.55	7.00E−04
	PMON80461	ZM_M53218	−14.6	5773.62	6759.85	1.00E−04	−5.6	28.87	30.57	0.0168	−16.4	50.92	60.88	1.00E−04
	PMON80461	ZM_M53218	7	7166.44	6713.17	0.0231	5	31.33	29.82	0.0501	9	59.48	54.73	0.002
	PMON80461	ZM_M53218	2	4908.21	4816.31	0.4075	3	29.55	28.65	0.072	3	32.25	31.34	0.1908
	PMON80461	ZM_M53218	−9	4808.4	5269.4	0.0085	−2	30.04	30.68	0.3563	−2	34.52	35.13	0.4641
	PMON80461	ZM_M53218	8.2	5071.81	4686.12	0.0064	6.2	31.19	29.37	0.0096	0.4	34.91	34.77	0.8893
	PMON80461	ZM_M53218	1.7	6211.2	6107.25	0.6164	−1.5	29.33	29.77	0.5225	0.9	38.97	38.63	0.8332
	PMON80461	ZM_M53218	1.1	3987.88	3946.32	0.6988	1.3	28.92	28.55	0.484	1.3	42.07	41.55	0.6981
	PMON80461	ZM_M53235	3	4955.98	4816.31	0.2084	1	28.93	28.65	0.5828	0	31.45	31.34	0.8709
	PMON80461	ZM_M53235	20.2	8122.46	6759.85	0	13.8	34.79	30.57	0	17.3	71.4	60.88	0
	PMON80461	ZM_M53235	3	6907.56	6713.17	0.3282	5	31.36	29.82	0.0447	1	55.05	54.73	0.8357
	PMON80461	ZM_M53503	2	4921.37	4816.31	0.3438	8	30.95	28.65	0	3	32.32	31.34	0.1605
	PMON80461	ZM_M53503	14.9	7763.72	6759.85	1.00E−04	10.4	33.77	30.57	0	25.9	76.63	60.88	0
	PMON80461	ZM_M53503	7	7197.24	6713.17	0.0154	6	31.54	29.82	0.0255	12	61.48	54.73	0
	PMON80461	ZM_M53504	−1	6666.94	6713.17	0.816	1	29.98	29.82	0.8413	10	60.29	54.73	6.00E−04
	PMON80461	ZM_M53504	−1	4748.6	4816.31	0.5416	−1	28.4	28.65	0.6231	−2	30.82	31.34	0.4559
	PMON80461	ZM_M53504	−15.3	5724.41	6759.85	0	−8.6	27.93	30.57	2.00E−04	−21	48.11	60.88	0
	PMON80461	ZM_M53848	2	4897.29	4816.31	0.4654	4	29.87	28.65	0.0153	−2	30.63	31.34	0.3077
	PMON80461	ZM_M53848	−15.3	5722.73	6759.85	0	−5.6	28.87	30.57	0.0168	−24.1	46.19	60.88	0
	PMON80461	ZM_M53848	3	6882.64	6713.17	0.394	7	31.86	29.82	0.008	2	56	54.73	0.4059
	PMON80461	ZM_M54282	0	4800.09	4816.31	0.8878	2	29.31	28.65	0.2011	−1	30.98	31.34	0.6261
	PMON80461	ZM_M54282	−2	6592.76	6713.17	0.5446	−2	29.35	29.82	0.5372	3	56.57	54.73	0.2552
	PMON80461	ZM_M54282	−12.7	5900.82	6759.85	7.00E−04	−4.9	29.07	30.57	0.0346	−19.8	48.83	60.88	0
	PMON80461	ZM_M54284	7	7155.9	6713.17	0.0265	5	31.2	29.82	0.0723	1	55.01	54.73	0.855
	PMON80461	ZM_M54284	19.2	8060.14	6759.85	0	9.7	33.55	30.57	0	16.4	70.88	60.88	1.00E−04
	PMON80461	ZM_M54284	5	5052.8	4816.31	0.0404	1	28.94	28.65	0.5692	3	32.14	31.34	0.2488
	PMON80461	ZM_M55266	−2.4	5962.4	6107.25	0.4616	0.2	29.81	29.77	0.9457	−6.7	36.04	38.63	0.1098
	PMON80461	ZM_M55957	5	6414.71	6107.25	0.1187	2.9	30.63	29.77	0.2128	−3.7	37.21	38.63	0.3528
	PMON80461	ZM_M56233	2.7	6270.89	6107.25	0.4056	5	31.25	29.77	0.0426	−0.7	38.38	38.63	0.8653
	PMON80461	ZM_M56728	3.8	6338.35	6107.25	0.2405	4	30.96	29.77	0.0831	−0.5	38.43	38.63	0.8911
102	PMON80542	ZM_M57107	−3.8	5766.93	5993.37	0.1461	−0.2	29.34	29.39	0.9327	6.1	54.07	50.98	0.0089
	PMON80542	ZM_M57107	14.2	5.71	5	0	1.2	31.75	31.38	0.6312	12.3	47.4	42.21	2.00E−04
	PMON80542	ZM_M57119	−8	5512.76	5993.37	0.0015	−1.1	29.08	29.39	0.5896	4.6	53.34	50.98	0.03785
	PMON80542	ZM_M57119	11.6	5.58	5	5.00E−04	5.1	32.96	31.38	0.0429	16.2	49.03	42.21	0
	PMON80542	ZM_M57120	2.6	5.13	5	0.4257	2.5	32.16	31.38	0.3138	−1.1	41.75	42.21	0.7377
	PMON80542	ZM_M57120	−3.1	5807.66	5993.37	0.2163	0.2	29.46	29.39	0.9036	0.1	51.04	50.98	0.9595
	PMON80542	ZM_M57121	−2.7	5829.33	5993.37	0.2746	1.9	29.94	29.39	0.3311	8.4	55.24	50.98	2.00E−04
	PMON80542	ZM_M57121	4.4	5.22	5	0.2467	−1.4	30.95	31.38	0.5865	9	45.99	42.21	0.0058
	PMON80542	ZM_M57122	−3.5	5785.68	5993.37	0.1669	0.4	29.5	29.39	0.8458	8.9	55.51	50.98	1.00E−04
	PMON80542	ZM_M57122	0	5	5	1	2.3	32.1	31.38	0.3537	6.8	45.07	42.21	0.0474
	PMON80542	ZM_M57124	−3	5815.15	5993.37	0.2353	−2.7	28.61	29.39	0.1694	6.6	54.33	50.98	0.0032
	PMON80542	ZM_M57124	13.4	5.67	5	2.00E−04	0.3	31.48	31.38	0.8981	13.1	47.74	42.21	1.00E−04
	PMON80542	ZM_M57131	13.3	7776.21	6866.4	0	6.5	33.54	31.48	0.0099	27.9	68.11	53.23	0
	PMON80542	ZM_M57132	−2.3	5853.25	5993.37	0.3506	−2.3	28.71	29.39	0.2306	13.2	57.73	50.98	0
	PMON80542	ZM_M57132	7.6	5.38	5	0.0174	−1.9	30.79	31.38	0.4522	7.4	45.34	42.21	0.0221
	PMON80542	ZM_M57146	0.6	6031.47	5993.37	0.7995	4.9	30.82	29.39	0.0124	−2.6	49.63	50.98	0.2347
	PMON80542	ZM_M57146	0.4	5.02	5	0.9047	7	33.58	31.38	0.0052	0.7	42.51	42.21	0.8221
123	PMON80850	ZM_M56061	−3.7	4.94	5.13	0.1027	2.8	30.43	29.6	0.2912	−1.8	44.81	45.62	0.6326
	PMON80850	ZM_M56061	−1.1	5272.7	5331.51	0.7088	0.7	28.56	28.35	0.7113	−6.3	42.33	45.16	0.0465
	PMON80850	ZM_M56062	3.5	5.31	5.13	0.1181	0.1	29.63	29.6	0.9654	6.2	48.44	45.62	0.0972
	PMON80850	ZM_M56062	4.4	5566.18	5331.51	0.1369	1.6	28.8	28.35	0.4251	9.3	49.37	45.16	0.0032
	PMON80850	ZM_M56071	−3.3	4.96	5.13	0.141	2.5	30.33	29.6	0.379	7.6	49.1	45.62	0.0407
	PMON80850	ZM_M56071	−0.5	5302.33	5331.51	0.853	−0.8	26.11	28.35	0.6697	10.7	50.01	45.16	7.00E−04
	PMON80850	ZM_M56222	−0.8	5.09	5.13	0.719	6.1	31.41	29.6	0.0211	0.9	46.01	45.62	0.8177
	PMON80850	ZM_M56222	4	5545.23	5331.51	0.1754	4.2	29.53	28.35	0.0367	−0.8	44.78	45.18	0.7867
	PMON80850	ZM_M56722	−1.8	5.05	5.13	0.4557	0.1	29.61	29.6	0.9841	−5.6	43.05	45.62	0.1295
	PMON80850	ZM_M56722	0.9	5379.37	5331.51	0.7693	2.1	28.94	28.35	0.3101	2.8	46.42	45.16	0.3906
	PMON80850	ZM_M56723	−4.1	4.92	5.13	0.0711	−1.2	29.25	29.6	0.6582	−2.9	44.28	45.62	0.4536
	PMON80850	ZM_M56723	8.3	5774.12	5331.51	0.0052	2.1	28.94	28.35	0.2947	2.6	46.33	45.16	0.4113
	PMON80850	ZM_M57056	7.2	5.51	5.13	0.0014	5	31.06	29.6	0.0623	11.5	50.85	45.62	0.0022
	PMON80850	ZM_M57056	2.6	5472.58	5331.51	0.3707	1.4	28.75	28.35	0.4782	2.1	46.11	45.16	0.5035

Nitrogen Use Field Efficacy Assay

Level I. Transgenic plants provided by the present invention are planted in field without any nitrogen source being applied. Transgenic plants and control plants are grouped by genotype and construct with controls arranged randomly within genotype blocks. Each type of transgenic plants are tested by 3 replications and across 5 locations. Nitrogen levels in the fields are analyzed in early April pre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus(P), Potassium(K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.
Level II. Transgenic plants provided by the present invention are planted in field with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 N), medium level (80 lb/ac) and high level (180 lb/ac). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied to the 0 N treatment the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants are grouped by genotype and construct with controls arranged randomly within genotype blocks. Each type of transgenic plants is tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed in early April pre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus(P), Potassium(K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.

TABLE 9

Genes increase seed yield in transgenic plants at different nitrogen levels.

	PEP SEQ					Transgenic	Control	Percent
	ID NO	Phe ID	Gene	Construct	Event	mean	Mean	change	Pvalue

I	108	PHE0001623_1734	maize	PMON79709	ZM_M51983	137.5	124.76521	10.207	0.0908
			magnesium
			transporter,
			mrs2-1-like 1
	105	PHE0001376_1468	Corn Rubisco	PMON75524	ZM_M47998	140.2	124.76521	12.3711	0.0407
			Activase 2
	130	PHE0001111_1201	Yeast alanine	PMON77895	ZM_M61017	140.3	124.76521	12.4512	0.0394
			aminotransferase

	PEP SEQ				treat-		Transgenic	Control	Percent	P-
	ID NO	Phe ID	Gene	Construct	ment	event	yield	yield	change	value

II	114	PHE0002412_2512	Ralstonia	pMON75980	High	ZM_M53398	159.7	142.45	10.801503	0.0621
			metallidurans
			glutamate
			decarboxylase
		PHE0002412_2512	Ralstonia		Low	ZM_M53398	137.125	125.14298	8.7380273	0.0263
			metallidurans
			glutamate
			decarboxylase
		PHE0002412_2512	Ralstonia		High	ZM_M53392	202.575	190.5333333	5.9443005	0.0833
			metallidurans
			glutamate
			decarboxylase
	118	PHE0002492_2592	Arabidopsis	pMON80461	High	ZM_M53218	160.6	142.45	11.30137	0.0498
			E2F
		PHE0002492_2592	Arabidopsis		High	ZM_M53848	158.675	142.45	10.225303	0.0792
			E2F
		PHE0002492_2592	Arabidopsis		Low	ZM_M53848	141.175	125.14298	11.356132	0.0031
			E2F
		PHE0002492_2592	Arabidopsis		Med	ZM_M53218	159.15	145.075	8.843858	0.0883
			E2F
	91	PHE0001017_1108	MADS box 110	pMON73816	Low	ZM_M37188	134.575	125.14298	7.008746	0.0798

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.
Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform. well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.
Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more plating seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30.000 plants/acre. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre. Surrogate indicators for yield improvement include source capacity (biomass), source output (sucrose and photosynthesis), sink components (kernel size, ear size, starch in the seed), development (light response, height, density tolerance), maturity, early flowering trait and physiological responses to high density planting, for example at 45,000 plants per acre, for example as illustrated in Table 10 and 11.

TABLE 10

Timing	Evaluation	Description	comments

V2-3	Early stand	Can be taken any time after
		germination and prior to
		removal of any plants.
Pollen shed	GDU to 50% shed	GDU to 50% plants shedding
		50% tassel.
Silking	GDU to 50% silk	GDU to 50% plants showing
		silks.
Maturity	Plant height	Height from soil surface to	10 plants per plot - Yield
		flag leaf attachment (inches).	team assistance
Maturity	Ear height	Height from soil surface to	10 plants per plot - Yield
		primary ear attachment node.	team assistance
Maturity	Leaves above ear	visual scores: erect, size,
		rolling
Maturity	Tassel size	Visual scores +/− vs. WT
Pre-Harvest	Final Stand	Final stand count prior to
		harvest, exclude tillers
Pre-Harvest	Stalk lodging	No. of stalks broken below
		the primary ear attachment.
		Exclude leaning tillers
Pre-Harvest	Root lodging	No. of stalks leaning >45°
		angle from perpendicular.
Pre-Harvest	Stay green	After physiological maturity
		and when differences among
		genotypes are evident: Scale
		1 (90-100% tissue green)-9
		(0-19% tissue green).
Harvest	Grain Yield	Grain yield/plot (Shell
		weight)

TABLE 11

Timing	Evaluation	Description

V8-V12	Chlorophyll
V12-VT	Ear leaf area
V15-15DAP	Chl fluorescence
V15-15DAP	CER
15-25 DAP	Carbohydrates	sucrose, starch
Pre-Harvest	1st internode diameter
Pre-Harvest	Base 3 internode diameter
Pre-Harvest	Ear internode diameter
Maturity	Ear traits	diameter, length, kernel
		number, kernel weight

Electron transport rates (ETR) and CO2 exchange rates (CER): ETR and CER were measured with Li6400LCF (Licor, Lincoln, Nebr.) around V9-R1 stages. Leaf chlorophyll fluorescence is a quick way to monitor the source activity and was reported to be highly correlated with CO2 assimilation under varies conditions (Photosyn Research, 37: 89-102). The youngest fully expanded leaf or 2 leaves above the ear leaf was measured with actinic light 1500 (with 10% blue light) micromol m⁻²s⁻¹, 28° C., CO2 levels 450 ppm. Ten plants were measured in each event. There were 2 readings for each plant.
A hand-held chlorophyll meter SPAD-502 (Minolta—Japan) was used to measure the total chlorophyll level on live transgenic plants and the wild type counterparts a. Three trifoliates from each plant were analyzed, and each trifoliate were analyzed three times. Then 9 data points were averaged to obtain the chlorophyll level. The number of analyzed plants of each genotype ranged from 5 to 8.

	TABLE 12

	Witchita, KS	Carrollton, IL

		Mean			Mean
pep		SPAD	%	p-	SPAD	%	p-
SEQ ID	construct	vaule	change	value	vaule	change	value

88	pMON68399	ZM_M31143	64.8	2	0.5215	58.87	4	0.0507
		ZM_M31143	64.8	2	0.5828
		ZM_M31146	64	1	0.7624	54	−1.14	0.0337
		ZM_M31146	64	1	0.8319
		ZM_M31147	67.3	6	0.0858	59.84	6	0.0665
		ZM_M31147	67.3	6	0.105
		ZM_M31152	66.6	5	0.1564	58.9	1	0.7965
		ZM_M31152	66.6	5	0.1862
		ZM_M31524	60.4	−5	0.2009	57.44	2	0.5839
		ZM_M31524	60.4	−5	0.1734
		ZM_M32356	61.9	−2	0.5386	59.36	−2	0.4308
		ZM_M32356	61.9	−3	0.4836
		ZM_M34171	62.7	−1	0.7919	60.18	0	0.9203
		ZM_M34171	62.7	−1	0.7255
		ZM_M38646	64.5	2	0.6164	59.89	3	0.3042
		ZM_M38646	64.5	1	0.6819
		ZM_M38660	67.3	6	0.0836	62.35	7	0.004

TABLE 13

PEP			n-	n-	ETR-	%		CER-	%
SEQ ID	Construct	event	trt	ctr	ctr	Change	Pvalue	ctr	Change	Pvalue

105	PMON75524	ZM_M47998	20	40	141.3	3	0.001	45.7	7	0.000
	PMON75524	ZM_M48003	20	40	141.3	8	0.000	45.7	6	0.000
	PMON75524	ZM_M48004	20	40	141.3	−4	0.000	45.7	−8	0.000
	PMON75524	ZM_M48005	20	40	141.3	2	0.008	45.7	4	0.012
	PMON75524	ZM_M48007	20	40	141.3	4	0.000	45.7	−3	0.052
	PMON75524	ZM_M48010	20	40	141.3	6	0.000	45.7	8	0.000
125	PMON81853	ZM_M70887	18	64	136.3	−3	0.298	43.7	−5	0.097
	PMON81853	ZM_M70888	22	64	136.3	15	0.000	43.7	15	0.000
	PMON81853	ZM_M70889	22	64	136.3	−23	0.000	43.7	−18	0.000
	PMON81853	ZM_M70900	22	64	136.3	−14	0.000	43.7	−14	0.000
	PMON81853	ZM_M71630	16	64	136.3	9	0.005	43.7	5	0.119
102	PMON80542	ZM_M57107	20	101	154.1	0	0.863	40.5	5	0.084
	PMON80542	ZM_M57119	20	101	154.1	3	0.002	40.5	5	0.099
	PMON80542	ZM_M57120	20	101	154.1	−6	0.000	40.5	−4	0.112
	PMON80542	ZM_M57121	20	101	154.1	−5	0.000	40.5	−8	0.003
	PMON80542	ZM_M57122	20	101	154.1	10	0.000	40.5	19	0.000
	PMON80542	ZM_M57124	20	101	154.1	1	0.514	40.5	3	0.204
	PMON80542	ZM_M57131	20	101	154.1	6	0.000	40.5	7	0.017
	PMON80542	ZM_M57132	20	101	154.1	9	0.000	40.5	11	0.000
	PMON80542	ZM_M57146	20	101	154.1	9	0.000	40.5	13	0.000

PEP		n-	n-	ETR-	%		CER-	%
SEQ ID	Construct	trt	ctr	ctr	Change	Pvalue	ctr	Change	Pvalue

105	PMON75524	10	42	153.7	−0	0.978	45.8	−2	0.067
	PMON75524	10	42	153.7	1	0.414	45.8	4	0.001
	PMON75524	11	42	153.7	7	0.000	45.8	9	0.000
	PMON75524	12	42	153.7	3	0.004	45.8	5	0.000
	PMON75524	11	42	153.7	1	0.498	45.8	−2	0.072
	PMON75524	10	42	153.7	7	0.000	45.8	9	0.000
125	PMON81853	19	51	151.5	8	0.001	34.8	9	0.012
	PMON81853	10	51	151.5	11	0.000	34.8	22	0.000
	PMON81853	16	51	151.5	10	0.000	34.8	13	0.000
	PMON81853	21	51	151.5	1	0.666	34.8	−0	0.944
	PMON81853	10	51	151.5	12	0.000	34.8	22	0.000
102	PMON80542	9	40	131.7	16	0.000	28.9	18	0.000
	PMON80542	10	40	131.7	−1	0.691	28.9	−3	0.304
	PMON80542	10	40	131.7	18	0.000	28.9	15	0.000
	PMON80542	12	40	131.7	−9	0.000	28.9	−12	0.000
	PMON80542	9	40	131.7	−3	0.126	28.9	−5	0.080
	PMON80542	11	40	131.7	20	0.000	28.9	27	0.000
	PMON80542	10	40	131.7	−3	0.098	28.9	−3	0.276
	PMON80542	11	40	131.7	−4	0.025	28.9	−3	0.191
	PMON80542	10	40	131.7	8	0.000	28.9	5	0.062

When selecting for yield improvement a useful statistical measurement approach comprises three components, i.e. modeling spatial autocorrelation of the test field separately for each location, adjusting traits of recombinant DNA events for spatial dependence for each location, and conducting an across location analysis. The first step in modeling spatial autocorrelation is estimating the covariance parameters of the semivariogram. A spherical covariance model is assumed to model the spatial autocorrelation. Because of the size and nature of the trial, it is likely that the spatial autocorrelation may change. Therefore, anisotropy is also assumed along with spherical covariance structure. The following set of equations describes the statistical form of the anisotropic spherical covariance model.
$C (h; θ) = vI (h = 0) + σ^{2} (1 - \frac{3}{2} h + \frac{1}{2} h^{3}) I (h < 1),$
where I(•) is the indicator function, h=√{square root over ({dot over (x)}²+{dot over (y)}²)}, and
{dot over (x)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]ω_x
{dot over (y)}=[sin(ρπ/180)(x ₁ −x ₂)−cos(ρπ/180)(y ₁ −y ₂)]ω_y
where s₁=(x₁,y₁) are the spatial coordinates of one location and s₂=(x₂,y₂) are the spatial coordinates of the second location. There are 5 covariance parameters, θ=(ν,σ²,ρ,ω_n,ω_j), where ν is the nugget effect, σ²is the partial sill, ρ is a rotation in degrees clockwise from north, ω_nis a scaling parameter for the minor axis and ω_jis a scaling parameter for the major axis of an anisotropical ellipse of equal covariance. The five covariance parameters that defines the spatial trend will then be estimated by using data from heavily replicated pollinator plots via restricted maximum likelihood approach. In a multi-location field trial, spatial trend are modeled separately for each location.
After obtaining the variance parameters of the model, a variance-covariance structure is generated for the data set to be analyzed. This variance-covariance structure contains spatial information required to adjust yield data for spatial dependence. In this case, a nested model that best represents the treatment and experimental design of the study is used along with the variance-covariance structure to adjust the yield data. During this process the nursery or the seed batch effects can also be modeled and estimated to adjust the yields for any yield parity caused by seed batch differences. After spatially adjusted data from different locations are generated, all adjusted data is combined and analyzed assuming locations as replications. In this analysis, intra and inter-location variances are combined to estimate the standard error of yield from transgenic plants and control plants. Relative mean comparisons are used to indicate statistically significant yield improvements.

TABLE 14

PEP SEQ		Transgenic		Mean	Control	Percent	P-
ID NO	construct id	event	control	Transgenic	mean	difference	value

105	pMON75524	ZM_M47998	Negative	173.3	176.1	−1.6	0.392
			segregant
		ZM_M48003	Negative	167.2	176.1	−5.1	0.007
			segregant
		ZM_M48004	Negative	176.2	176.1	0.0	0.990
			segregant
		ZM_M48005	Negative	186.0	176.1	5.6	0.003
			segregant
		ZM_M48007	Negative	177.9	176.1	1.0	0.631
			segregant
		ZM_M48010	Negative	176.8	176.1	0.4	0.841
			segregant
88	pMON68399	ZM_M31146	Negative	179.1	179.9	−0.4	0.778
			segregant
		ZM_M31147	Negative	181.7	179.9	1.0	0.497
			segregant
		ZM_M31524	Negative	179.3	179.9	−0.3	0.829
			segregant
		ZM_M32356	Negative	181.3	179.9	0.8	0.601
			segregant
		ZM_M38646	Negative	180.3	179.9	0.2	0.880
			segregant
		ZM_M38681	Negative	180.2	179.9	0.2	0.894
			segregant
		ZM_M39295	Negative	176.6	179.9	−1.8	0.259
			segregant
		ZM_M39297	Negative	175.6	179.9	−2.3	0.125
			segregant
		ZM_M39298	Negative	184.6	179.9	2.7	0.082
			segregant
		ZM_M39302	Negative	182.0	179.9	1.2	0.440
			segregant
105	pMON75524	ZM_M47998	Negative	173.3	176.1	−1.6	0.392
			segregant
		ZM_M48003	Negative	167.2	176.1	−5.1	0.007
			segregant
		ZM_M48004	Negative	176.2	176.1	0.0	0.990
			segregant
		ZM_M48005	Negative	186.0	176.1	5.6	0.003
			segregant
		ZM_M48007	Negative	177.9	176.1	1.0	0.631
			segregant
		ZM_M48010	Negative	176.8	176.1	0.4	0.841
			segregant
88	pMON68399	ZM_M31146	Negative	179.1	179.9	−0.4	0.778
			segregant
		ZM_M31147	Negative	181.7	179.9	1.0	0.497
			segregant
		ZM_M31524	Negative	179.3	179.9	−0.3	0.829
			segregant
		ZM_M32356	Negative	181.3	179.9	0.8	0.601
			segregant
		ZM_M38646	Negative	180.3	179.9	0.2	0.880
			segregant
		ZM_M38681	Negative	180.2	179.9	0.2	0.894
			segregant
		ZM_M39295	Negative	176.6	179.9	−1.8	0.259
			segregant
		ZM_M39297	Negative	175.6	179.9	−2.3	0.125
			segregant
		ZM_M39298	Negative	184.6	179.9	2.7	0.082
			segregant
		ZM_M39302	Negative	182.0	179.9	1.2	0.440
			segregant

TABLE 15

		Mean	Mean	Percent	P-
Construct	Event	Transgenic	Control	change	value

PEP SEQ ID
127	PMON78911	ZM_M45101	167.9	176.1	−4.7	0.015
127	PMON78911	ZM_M59413	175.4	176.1	−0.4	0.832
127	PMON78911	ZM_M59778	161.2	176.1	−8.5	0.000
127	PMON78911	ZM_M59783	191.0	176.1	8.4	0.000
127	PMON78911	ZM_M59784	182.6	176.1	3.7	0.053
127	PMON78911	ZM_M62810	180.2	176.1	2.3	0.212
130	PMON77895	ZM_M61016	171.5	176.1	−2.6	0.163
139	PMON77895	ZM_M61017	173.4	176.1	−1.6	0.397
130	PMON77895	ZM_M61033	184.1	176.1	4.5	0.015
131	PMON79152	ZM_M64367	162.9	176.1	−7.5	0.000
131	PMON79152	ZM_M65978	184.5	176.1	4.7	0.012
131	PMON79152	ZM_M65982	175.0	176.1	−0.6	0.733
131	PMON79152	ZM_M65986	139.7	176.1	−20.7	0.000
131	PMON79152	ZM_M65992	171.8	176.1	−2.5	0.182
132	PMON80921	ZM_M63833	184.2	176.1	4.6	0.015
133	PMON75505	ZM_M49384	183.6	176.1	4.2	0.023
134	PMON80925	ZM_M60505	183.4	176.1	4.1	0.039
134	PMON80925	ZM_M82005	179.8	176.1	2.1	0.268
134	PMON80925	ZM_M62007	178.5	176.1	1.3	0.489
134	PMON80925	ZM_M63594	180.1	176.1	2.3	0.229
106	PMON79163	ZM_M45011	177.0	176.1	0.5	0.792
106	PMON79163	ZM_M48217	179.8	176.1	2.1	0.289
106	PMON79163	ZM_M81816	183.5	176.1	4.2	0.033
106	PMON79163	ZM_M61822	168.1	176.1	−4.6	0.023
136	PMON79164	ZM_M44045	172.1	176.1	−2.3	0.217
136	PMON79164	ZM_M59749	180.6	176.1	2.5	0.175
136	PMON79164	ZM_M59750	181.8	176.1	3.2	0.087
136	PMON79164	ZM_M61349	169.5	176.1	−3.8	0.042
136	PMON79164	ZM_M81889	175.0	176.1	−0.6	0.738
136	PMON79164	ZM_M61890	145.4	176.1	−17.4	0.000
136	PMON79164	ZM_M82983	175.7	176.1	−0.3	0.881
136	PMON79164	ZM_M83003	185.0	176.1	5.0	0.007
107	PMON75533	ZM_M47453	183.4	176.1	4.1	0.027
107	PMON75533	ZM_M47460	178.4	176.1	1.3	0.491
107	PMON75533	ZM_M49275	183.9	176.1	4.4	0.018
107	PMON75533	ZM_M49278	177.0	176.1	0.5	0.790
137	PMON79853	ZM_M49833	174.6	176.1	−0.9	0.633
137	PMON79853	ZM_M65281	183.4	176.1	4.1	0.030
138	PMON81228	ZM_M59931	169.3	176.1	−3.9	0.055
138	PMON81228	ZM_M80825	185.8	176.1	5.5	0.003
148	PMON82223	ZM_M70571	185.8	176.1	5.5	0.007
161	PMON79665	ZM_M51224	171.9	176.1	−2.4	0.198
161	PMON79665	ZM_M53787	172.2	176.1	−2.2	0.233
161	PMON79665	ZM_M55078	184.2	176.1	4.6	0.019
139	PMON79430	ZM_M50221	181.1	176.1	2.8	0.137
139	PMON79430	ZM_M50222	178.6	176.1	1.4	0.477
139	PMON79430	ZM_M50223	180.8	176.1	2.7	0.153
139	PMON79430	ZM_M50727	177.7	176.1	0.9	0.637
139	PMON79430	ZM_M50729	179.0	176.1	1.6	0.377
139	PMON79430	ZM_M51479	171.7	176.1	−2.5	0.198
139	PMON79430	ZM_M51481	185.4	176.1	5.2	0.008
139	PMON79430	ZM_M51490	178.5	176.1	1.3	0.492
140	PMON79731	ZM_M52239	187.5	176.1	6.5	0.001
140	PMON79731	ZM_M52245	172.2	176.1	−2.2	0.230
140	PMON79731	ZM_M52252	174.6	176.1	−0.9	0.638
140	PMON79731	ZM_M52255	172.4	176.1	−2.1	0.248
140	PMON79731	ZM_M52375	173.3	176.1	−1.6	0.396
140	PMON79731	ZM_M52802	173.6	176.1	−1.5	0.447
140	PMON79731	ZM_M52812	166.6	176.1	−5.4	0.004
141	PMON78229	ZM_M55961	176.0	176.1	−0.1	0.963
141	PMON78229	ZM_M55962	182.3	176.1	3.5	0.065
141	PMON78229	ZM_M55964	175.1	176.1	−0.6	0.743
141	PMON78229	ZM_M56184	187.2	176.1	6.3	0.001
141	PMON78229	ZM_M56185	181.8	176.1	3.2	0.083
141	PMON78229	ZM_M59082	176.1	176.1	0.0	0.984
SEQ ID NO
116	PMON79697	ZM_M53938	171.6	176.1	−2.6	0.171
116	PMON79697	ZM_M53939	180.2	176.1	2.3	0.236
116	PMON79697	ZM_M54371	175.0	176.1	−0.6	0.733
116	PMON79697	ZM_M54372	185.1	176.1	5.1	0.009
116	PMON79697	ZM_M54374	181.2	176.1	2.8	0.127
144	PMON78240	ZM_M53464	184.1	176.1	4.5	0.015
144	PMON78240	ZM_M53465	175.2	176.1	−0.5	0.785
144	PMON78240	ZM_M53470	174.4	176.1	−1.0	0.611
144	PMON78240	ZM_M53471	166.7	176.1	−5.4	0.005
144	PMON78240	ZM_M53478	173.6	176.1	−1.4	0.456
144	PMON78240	ZM_M53673	175.8	176.1	−0.2	0.917
144	PMON78240	ZM_M53674	172.5	176.1	−2.1	0.269
144	PMON78240	ZM_M53684	179.4	176.1	1.8	0.342
122	PMON80500	ZM_M56549	173.4	176.1	−1.6	0.408
122	PMON80500	ZM_M56560	173.4	176.1	−1.6	0.394
122	PMON80500	ZM_M56565	175.4	176.1	−0.4	0.811
122	PMON80500	ZM_M56567	177.9	176.1	1.0	0.599
122	PMON80500	ZM_M56568	185.9	176.1	5.6	0.003
122	PMON80500	ZM_M58003	169.4	176.1	−3.8	0.047
145	PMON80283	ZM_M58140	174.6	176.1	−0.9	0.641
145	PMON80283	ZM_M58141	179.7	176.1	2.0	0.294
145	PMON80283	ZM_M58143	183.8	176.1	4.4	0.024
146	PMON80866	ZM_M58256	177.6	176.1	0.8	0.651
146	PMON80866	ZM_M59441	183.3	176.1	4.1	0.028
146	PMON80866	ZM_M60646	174.8	176.1	−0.7	0.692
147	PMON80292	ZM_M57487	180.8	176.1	2.6	0.159
147	PMON80292	ZM_M58571	184.2	176.1	4.6	0.021
147	PMON80292	ZM_M58578	177.5	176.1	0.8	0.717
142	PMON79696	ZM_M53849	177.6	179.1	−1.2	0.431
142	PMON79696	ZM_M53849	190.3	179.1	5.8	0.0003
142	PMON79696	ZM_M53849	178.5	179.1	−0.7	0.0635
150	PMON81857	ZM_M67504	178.8	176.1	1.5	0.415
150	PMON81857	ZM_M70000	182.7	176.1	3.7	0.047
150	PMON81857	ZM_M71064	172.1	176.1	−2.3	0.229
150	PMON81857	ZM_M71065	184.6	176.1	4.8	0.011
150	PMON81857	ZM_M72550	174.3	176.1	−1.0	0.589
149	PMON83553	ZM_M71131	150.7	176.1	−14.5	0.000
149	PMON83553	ZM_M71140	187.4	176.1	6.4	0.001
149	PMON83553	ZM_M71156	150.3	176.1	−14.7	0.000
149	PMON83553	ZM_M71161	172.7	176.1	−1.9	0.298
150	PMON81857	ZM_M67504	178.8	176.1	1.5	0.415
150	PMON81857	ZM_M70000	182.7	176.1	3.7	0.047
150	PMON81857	ZM_M71064	172.1	176.1	−2.3	0.229
150	PMON81857	ZM_M71065	184.6	176.1	4.8	0.011
150	PMON81857	ZM_M72550	174.3	176.1	−1.0	0.589
151	PMON82212	ZM_M67581	171.1	176.1	−2.8	0.126
151	PMON82212	ZM_M67583	186.1	176.1	5.6	0.002
151	PMON82212	ZM_M69111	173.2	176.1	−1.7	0.368
PEP SEQ ID NO
108	PMON79709	ZM_M51983	184.3	176.1	4.7	0.037
108	PMON79709	ZM_M51985	180.1	176.1	2.3	0.231
108	PMON79709	ZM_M52052	185.6	176.1	5.3	0.013
108	PMON79709	ZM_M52710	175.5	176.1	−0.4	0.862
108	PMON79709	ZM_M52720	175.2	176.1	−0.6	0.765
129	PMON73787	ZM_M55089	162.6	176.1	−7.7	0.000
128	PMON73787	ZM_M61950	186.4	176.1	5.8	0.002
128	PMON73787	ZM_M61953	164.7	176.1	−6.5	0.001
129	PMON73787	ZM_M61958	165.9	176.1	−5.8	0.003
129	PMON73787	ZM_M61965	134.3	176.1	−23.8	0.000
129	PMON73787	ZM_M61966	172.6	176.1	−2.0	0.280
135	PMON78942	ZM_M66312	176.2	176.1	0.0	0.997
135	PMON78942	ZM_M66316	173.1	176.1	−1.7	0.362
135	PMON78942	ZM_M66318	164.1	176.1	−5.9	0.000
135	PMON78942	ZM_M66331	183.3	176.1	4.1	0.029

C. Selection for Enhanced Water Use Efficiency (WUE)

Described in this example is a high-throughput method for greenhouse selection of transgenic corn plants to wild type corn plants (tested as inbreds or hybrids) for water use efficiency. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height are measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIH). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above may be adjusted for +/−˜one day for each step given the situation.
To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)2)*100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)*100]. Fully watered corn plants of this age run around 98% RWC.
The transgenic plants provided by this invention were selected through the selection process according to the standard procedure described above and the performance of these transgenic plants are shown in Table 16 below.

TABLE 16

PEP SEQ		N	Perc,	Pvalue,	Perc,	Pvalue,	Perc,	Pvalue,	Perc,	Pvalue,
ID NO	Construct	Event	SAH	SAH	RGR	RGR	SDM	SDM	RWC	RWC

88	PMON68399	18	−2.9129	0	4.6104	0	−1.2282	0.0534	2.0799	0
87	PMON72494	2	−2.6854	0	3.3347	0.0034	−3.178	0.0258	2.8177	0.0001
	PMON72494	2	−1.4189	0	4.5389	0	1.0503	0.2808	1.8075	0.0272
	PMON72494	6	−2.8912	0	5.0217	0	−3.0056	0.0032	3.0684	0
	PMON72494	1	−3.2736	0	1.4026	0.2741	0.0968	0.9545	−2.3654	0.0194
97	PMON76342	1	−3.6096	0.0003	8.9657	0	−2.9332	0.2317	2.1037	0.1252
	PMON76342	2	−0.9997	0.0384	4.9006	0	−1.7424	0.1472	−0.8155	0.2552
117	PMON78237	4	−2.0513	0	2.1335	0.0011	3.2477	0.0002	0.5998	0.2456
104	PMON78936	2	0.2781	0.3727	1.3631	0.0165	2.1849	0.023	1.4237	0.0744
	PMON78936	4	−2.3342	0	6.1784	0	−2.5964	0.0336	2.5358	0.0003
103	PMON78949	4	−1.6398	0	4.5323	0	2.2077	0.0112	0.9068	0.08
109	PMON79422	4	−2.0016	0	2.8698	0	−1.3511	0.0488	1.8883	0.0009
116	PMON79697	2	−1.0829	0.1252	2.9806	0.0225	−0.0495	0.9771	0.0115	0.9907
	PMON79697	3	−1.5704	0	2.1663	0	−0.4949	0.5582	1.7787	0.0073
120	PMON80452	1	−1.7626	0.0032	2.1476	0.2778	2.1702	0.3832	−1.914	0.1164
	PMON80452	8	−0.2756	0.0645	−1.0206	0.0002	0.4707	0.3101	−0.072	0.8521
	PMON80452	11	−0.7077	0.0258	2.1403	0.0003	1.4477	0.0623	−0.0405	0.9267
115	PMON80489	6	−0.895	0.0001	3.7262	0	−1.5941	0.0442	1.4212	0.0038
102	PMON80542	8	−2.5925	0	1.1234	0.0254	2.1829	0.0013	3.2415	0
	PMON80542	1	−5.5931	0	2.5902	0.0486	−2.1444	0.2158	9.5238	0

Transgenic plants transformed with pMON67754 comprising the recombinant DNA as set forth in SEQ ID NO: 3 were tested in field with moderate drought conditions in Satanta, Ill. and Dixon Calif. SPAD readings on leaves under a moderate drought stress showed a significant increase in chlorophyll level in the transgenic plants as compared to the control plants. Two events showed a significant increase in SPAD reading for chlorophyll level, indicating an improvement in drought tolerance. In replicated field trials, 2 events (ZM_M16396 and ZM_M16401) out of 6 tested, showed significantly (p<0.1) improved leaf SPAD readings in two
different locations, indicating an improvement in drought tolerance.

D. Selection for Growth Under Cold Stress

(1) Cold Germination Assay—

Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.
Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C for 24 days (no light) in a Convrion growth chamber (Conviron Model PGV36. Controlled Environments. Winnipeg. Canada). Two hundred and fifty milliliters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.
The germination index is calculated as per:
Germination index=(Σ([T+1−n _i ]*[P _i −P _i-1]))T
Where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.

	TABLE 17

	Germination index

PEP			Percent		Mean of
SEQ ID	Construct ID	Event ID	change	Mean	controls	P-value

85	PMON69456	ZM_M15392	−27	23.4	32.07	0.0718
	PMON69456	ZM_M15392	12	47.88	42.93	9.00E−04
	PMON69456	ZM_M15392	13	48	42.44	0.0756
	PMON69456	ZM_M17042	−9	29.2	32.07	0.4
	PMON69456	ZM_M17042	17	49.5	42.44	0.0248
	PMON69456	ZM_M17042	16	49.89	42.93	0
	PMON69456	ZM_M17042	−6	28.14	30.07	0.6526
	PMON69456	ZM_M17044	−38	19.25	30.88	0.019
	PMON69456	ZM_M17044	9	46.17	42.44	0.2317
	PMON69456	ZM_M17044	7	46.88	43.86	0.0297
	PMON69456	ZM_M17044	14	34.14	30.07	0.3445
107	PMON75533	ZM_M47453	3	46.88	45.38	0.3782
	PMON75533	ZM_M47453	25	49.75	39.69	0.002
	PMON75533	ZM_M47460	23	48.83	39.69	0.0047
	PMON75533	ZM_M47460	3	46.88	45.38	0.3782
	PMON75533	ZM_M49275	14	45.08	39.69	0.0914
	PMON75533	ZM_M49275	11	50.46	45.38	0.0031
	PMON75533	ZM_M49278	15	45.83	39.69	0.055
	PMON75533	ZM_M49278	14	51.75	45.38	2.00E−04
119	PMON78235	ZM_M53641	16	48.25	41.72	4.00E−04
	PMON78235	ZM_M53641	23	45	36.5	0.0508
	PMON78235	ZM_M53641	1	48.42	48.08	0.9116
	PMON78235	ZM_M53641	5	42.17	40.24	0.5629
	PMON78235	ZM_M53994	26	46	36.5	0.0294
	PMON78235	ZM_M53994	15	47.92	41.72	7.00E−04
	PMON78235	ZM_M53994	1	48.67	48.08	0.8459
	PMON78235	ZM_M53994	−4	38.58	40.24	0.6196
	PMON78235	ZM_M53997	16	48.21	41.72	4.00E−04
	PMON78235	ZM_M53997	15	42	36.5	0.2036
104	PMON78936	ZM_M45248	25	48.25	38.69	0.0221
	PMON78936	ZM_M45248	14	48.29	42.21	0.0013
	PMON78936	ZM_M45274	15	48.33	42.21	0.0012
	PMON78936	ZM_M45274	24	48.08	38.69	0.0245
	PMON78936	ZM_M45275	5	40.5	38.69	0.6613
	PMON78936	ZM_M46485	11	42.92	38.69	0.3066
	PMON78936	ZM_M46516	−1	38.33	38.69	0.9301
	PMON78936	ZM_M46516	−4	40.38	42.21	0.3274
	PMON78936	ZM_M47276	11	43.08	38.69	0.288
110	PMON79425	ZM_M50823	4	42.79	41.31	0.3848
	PMON79425	ZM_M50823	18	42.83	36.25	0.0378
	PMON79425	ZM_M50856	4	42.88	41.31	0.3589
	PMON79425	ZM_M50856	13	40.83	36.25	0.1462
	PMON79425	ZM_M51300	7	44.25	41.31	0.087
	PMON79425	ZM_M51300	−3	35.16	36.25	0.7282
	PMON79425	ZM_M51302	23	44.54	36.25	0.0093
	PMON79425	ZM_M51302	17	48.17	41.31	1.00E−04
	PMON79425	ZM_M51313	12	46.33	41.31	0.004
	PMON79425	ZM_M51313	23	44.7	36.25	0.008
	PMON79425	ZM_M51608	24	45.08	36.25	0.0057
	PMON79425	ZM_M51608	11	45.88	41.31	0.0086
	PMON79425	ZM_M51623	21	43.7	36.25	0.0189
	PMON79425	ZM_M51623	14	47.21	41.31	8.00E−04
	PMON79425	ZM_M52067	−5	39.13	41.31	0.2033
	PMON79425	ZM_M52067	8	39.08	36.25	0.368
116	PMON79697	ZM_M53938	7	47.04	43.93	0.0587
	PMON79697	ZM_M53938	5	42	40.17	0.6198
	PMON79697	ZM_M53939	18	47.25	40.17	0.0575
	PMON79697	ZM_M53939	11	48.58	43.93	0.0049
	PMON79697	ZM_M54371	11	48.88	43.93	0.0028
	PMON79697	ZM_M54371	15	46.25	40.17	0.1019
	PMON79697	ZM_M54372	1	40.75	40.17	0.8745
	PMON79697	ZM_M54374	12	49.21	43.93	0.0022
	PMON79697	ZM_M54374	18	47.25	40.17	0.0575
111	PMON79718	ZM_M50838	6	45.25	42.78	0.331
	PMON79718	ZM_M51591	−3	42.67	43.93	0.4409
	PMON79718	ZM_M51591	−18	35.08	42.78	0.0031
	PMON79718	ZM_M51592	−3	41.42	42.78	0.5919
	PMON79718	ZM_M51594	6	46.46	43.93	0.1241
	PMON79718	ZM_M51594	13	48.15	42.78	0.0545
	PMON79718	ZM_M51598	11	48.96	43.93	0.0024
	PMON79718	ZM_M51598	11	47.58	42.78	0.0606
	PMON79718	ZM_M51615	6	46.46	43.93	0.1241
	PMON79718	ZM_M51615	11	47.33	42.78	0.075
	PMON79718	ZM_M51618	2	43.5	42.78	0.7759
	PMON79718	ZM_M52797	−6	40.17	42.78	0.3047
	PMON79718	ZM_M52937	16	49.67	42.78	0.0077
	PMON79718	ZM_M52937	12	49.04	43.93	0.0021
96	PMON80270	ZM_M55967	10.19	50.63	45.94	6.00E−04
	PMON80270	ZM_M55968	7.38	49.33	45.94	0.0129
	PMON80270	ZM_M55969	3.27	47.44	45.94	0.2678
	PMON80270	ZM_M55970	10.56	50.79	45.94	4.00E−04
	PMON80270	ZM_M55971	7.38	49.33	45.94	0.0129
	PMON80270	ZM_M55972	2.66	47.17	45.94	0.3663
	PMON80270	ZM_M56524	3.81	47.7	45.94	0.1952
	PMON80270	ZM_M56526	−7.6	42.46	45.94	0.0105
	PMON80270	ZM_M56527	−19.87	36.82	45.94	0
120	PMON80452	ZM_M53452	13	41.83	37.08	0.1902
	PMON80452	ZM_M53452	19	49.63	41.56	0
	PMON80452	ZM_M53452	7	51.42	48.08	0.2683
	PMON80452	ZM_M53452	0	40.25	40.24	0.9971
	PMON80452	ZM_M53455	−3	36	37.08	0.7642
	PMON80452	ZM_M53455	17	48.67	41.56	0
	PMON80452	ZM_M53455	−9	43.67	48.08	0.1434
	PMON80452	ZM_M53455	−1	39.92	40.24	0.9231
	PMON80452	ZM_M53456	18	49.17	41.56	0
	PMON80452	ZM_M53456	18	43.83	37.08	0.0639
	PMON80452	ZM_M53469	14	47.54	41.56	1.00E−04
	PMON80452	ZM_M53469	18	43.75	37.08	0.0672
	PMON80452	ZM_M53694	14	42.42	37.08	0.1418
	PMON80452	ZM_M53694	13	46.92	41.56	4.00E−04
	PMON80452	ZM_M53695	21	50.08	41.56	0
	PMON80452	ZM_M53695	22	45.25	37.08	0.0256
	PMON80452	ZM_M53696	21	50.42	41.56	0
	PMON80452	ZM_M53696	31	48.5	37.08	0.002
	PMON80452	ZM_M54104	13	41.75	37.08	0.198
	PMON80452	ZM_M54104	13	47.17	41.56	2.00E−04
	PMON80452	ZM_M54106	8	39.92	37.08	0.4332
	PMON80452	ZM_M54106	12	46.38	41.56	0.0015
118	PMON80461	ZM_M52932	17	48.67	41.56	0
	PMON80461	ZM_M52932	32	48.17	36.5	0.0079
	PMON80461	ZM_M52932	−8	43.25	46.86	0.1944
	PMON80461	ZM_M52932	9	43.92	40.24	0.271
	PMON80461	ZM_M53218	16	42.42	36.5	0.1717
	PMON80461	ZM_M53218	7	44.58	41.56	0.0448
	PMON80461	ZM_M53218	−6	44.08	46.86	0.3172
	PMON80461	ZM_M53218	4	41.92	40.24	0.6145
	PMON80461	ZM_M53235	22	50.71	41.56	0
	PMON80461	ZM_M53235	24	45.25	36.5	0.0445
	PMON80461	ZM_M53503	13	46.79	41.56	6.00E−04
	PMON80461	ZM_M53503	28	46.83	36.5	0.0181
	PMON80461	ZM_M53504	12	41	36.5	0.2975
	PMON80461	ZM_M53504	14	47.5	41.56	1.00E−04
	PMON80461	ZM_M53848	24	51.57	41.56	0
	PMON80461	ZM_M53848	15	41.92	36.5	0.2104
	PMON80461	ZM_M54282	22	50.75	41.56	0
	PMON80461	ZM_M54282	29	47	36.5	0.0164
	PMON80461	ZM_M54284	21	44.33	36.5	0.0714
	PMON80461	ZM_M54284	22	50.71	41.56	0
	PMON80461	ZM_M55266	7	50.22	46.86	0.2268
	PMON80461	ZM_M55957	10	51.53	46.86	0.0945
	PMON80461	ZM_M56233	9	51.18	46.86	0.1217
	PMON80461	ZM_M56728	2	47.92	46.86	0.7033
122	PMON80500	ZM_M56549	−0.52	45.71	45.94	0.8613
	PMON80500	ZM_M56560	8.29	49.75	45.94	0.0053
	PMON80500	ZM_M56565	2.2	46.96	45.94	0.4535
	PMON80500	ZM_M56567	9.19	50.17	45.94	0.002
	PMON80500	ZM_M56568	10.82	50.92	45.94	3.00E−04
	PMON80500	ZM_M58003	4.2	47.88	45.94	0.1542

(2) Cold Shock Assay—

The experimental set-up for the cold shock assay was the same as described in the above cold germination assay except seeds were grown in potted media for the cold shock assay.
The desired numbers of 2.5″ square plastic pots were placed on flats (n=32, 4×8). Pots were filled with Metro Mix 200 soil-less media containing 19:6:12 fertilizer (6 lbs/cubic yard) (Metro Mix, Pots and Flat are obtained from Hummert International, Earth City, Mo.). After planting seeds, pots were placed in a growth chamber set at 23° C., relative humidity of 65% with 12 hour day and night photoperiod (300 uE/m2-min). Planted seeds were watered for 20 minute every other day by sub-irrigation and flats were rotated every third day in a growth chamber for growing corn seedlings.
On the 10^thday after planting the transgenic positive and wild-type negative (WT) plants were positioned in flats in an alternating pattern. Chlorophyll fluorescence of plants was measured on the 10^thday during the dark period of growth by using a PAM-2000 portable fluorometer as per the manufacturer's instructions (Walz, Germany). After chlorophyll measurements, leaf samples from each event were collected for confirming the expression of genes of the present invention. For expression analysis six V1 leaf tips from each selection were randomly harvested. The flats were moved to a growth chamber set at 5° C. All other conditions such as humidity, day/night cycle and light intensity were held constant in the growth chamber. The flats were sub-irrigated every day after transfer to the cold temperature. On the 4^thday chlorophyll fluorescence was measured. Plants were transferred to normal growth conditions after six days of cold shock treatment and allowed to recover for the next three days. During this recovery period the length of the V3 leaf was measured on the 1^stand 3^rddays. After two days of recovery V2 leaf damage was determined visually by estimating percent of green V2 leaf.
Statistical differences in V3 leaf growth, V2 leaf necrosis and fluorescence during pre-shock and cold shock can be used for estimation of cold shock damage on corn plants.

(3) Early Seedling Growth Assay—

Three sets of seeds were used for the experiment. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention were expressed in the seed. The second seed set was nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third seed set consisted of two cold tolerant and two cold sensitive commercial check lines of corn. All seeds were treated with a fungicide “Captan”, (3a,4.7,a-tetrahydro-2-[(trichloromethly)thio]-1H-isoindole-1,3(2H)-dione, Drex Chemical Co. Memphis, Tenn.). Captan|(0.43 mL) was applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.
Seeds were grown in germination paper for the early seedling growth assay. Three 12″×18″ pieces of germination paper (Anchor Paper #SD7606) were used for each entry in the test (three repetitions per transgenic event). The papers were wetted in a solution of 0.5% KNO₃and 0.1% Thyram.
For each paper fifteen seeds were placed on the line evenly spaced down the length of the paper. The fifteen seeds were positioned on the paper such that the radical would grow downward, for example longer distance to the paper's edge. The wet paper was rolled up starting from one of the short ends. The paper was rolled evenly and tight enough to hold the seeds in place. The roll was secured into place with two large paper clips, one at the top and one at the bottom. The rolls were incubated in a growth chamber at 23° C. for three days in a randomized complete block design within an appropriate container. The chamber was set for 65% humidity with no light cycle. For the cold stress treatment the rolls were then incubated in a growth chamber at 12° C. for twelve days. The chamber was set for 65% humidity with no light cycle.
After the cold treatment the germination papers were unrolled and the seeds that did not germinate were discarded. The lengths of the radicle and coleoptile for each seed were measured through an automated imaging program that automatically collects and processes the images. The imaging program automatically measures the shoot length, root length, and whole seedling length of every individual seedling and then calculates the average of each roll.
After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold selection is conducted in the same manner of the primary selection only increasing the number of repetitions to five. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.

TABLE 18

Root length	Shoot length	Seedlling length

Mean

of

PEP

Percent

con-

P-

Percent

con-

P-

Percent

con-

P-

SEQ ID

Construct ID

Event ID

change

Mean

trols

value

change

Mean

trols

value

change

Mean

trols

value

88	PMON68399	ZM_M31143	−4	9.5	9.85	0.5479	−1	7.94	8.04	0.7666	−3	17.45	17.9	0.6024
	PMON68399	ZM_M31143	7	11.15	10.41	0.1158	8	9.42	8.69	0.0536	8	20.57	19.1	0.0607
	PMON68399	ZM_M31146	11	11.52	10.41	0.0186	0	8.67	8.69	0.9668	6	20.2	19.1	0.1593
	PMON68399	ZM_M31146	14	9.9	8.66	0.011	6	7.37	6.99	0.2969	10	17.27	15.65	0.0396
	PMON68399	ZM_M31147	13	11.75	10.41	0.0051	12	9.69	8.69	0.0088	12	21.43	19.1	0.0034
	PMON68399	ZM_M31147	14	11.25	9.85	0.0185	4	8.33	8.04	0.3961	9	19.58	17.9	0.0513
	PMON68399	ZM_M31152	−20	8.4	10.45	2.00E−04	−7	7.44	7.96	0.3265	−14	15.84	18.41	0.0087
	PMON68399	ZM_M31152	1	10.48	10.41	0.8793	6	9.17	8.69	0.1965	3	19.66	19.1	0.4697
	PMON68399	ZM_M31524	15	12.01	10.41	9.00E−04	10	9.54	8.69	0.0242	13	21.55	19.1	0.0021
	PMON68399	ZM_M31524	12	11.08	9.85	0.0385	8	8.69	8.04	0.0569	11	19.77	17.9	0.0306
	PMON68399	ZM_M32356	12	10.99	9.85	0.0533	−1	7.99	8.04	0.8731	6	18.98	17.9	0.2052
	PMON68399	ZM_M32356	12	11.7	10.41	0.0068	7	9.32	8.69	0.096	10	21.01	19.1	0.0153
	PMON68399	ZM_M34171	−24	8.6	11.39	4.00E−04	−13	7.35	8.48	0.0331	−20	15.95	19.87	0.0016
	PMON68399	ZM_M34171	13	11.72	10.41	0.006	6	9.23	8.69	0.1486	10	20.95	19.1	0.0187
	PMON68399	ZM_M38646	10	12.63	11.52	0.032	3	10.38	10.05	0.4864	7	23.01	21.57	0.106
	PMON68399	ZM_M38660	10	12.68	11.52	0.0249	3	10.37	10.05	0.4953	7	23.06	21.57	0.0947
	PMON68399	ZM_M38681	6	12.2	11.52	0.1829	3	10.31	10.05	0.5738	4	22.52	21.57	0.2835
	PMON68399	ZM_M38697	7	12.35	11.52	0.1053	0	10.03	10.05	0.9751	4	22.38	21.57	0.3563
	PMON68399	ZM_M39295	11	12.84	11.52	0.0115	11	11.12	10.05	0.0264	11	23.97	21.57	0.0084
	PMON68399	ZM_M39297	20	13.84	11.52	0	7	10.79	10.05	0.1203	14	24.63	21.57	0.001
	PMON68399	ZM_M39298	7	12.29	11.52	0.1342	−1	9.91	10.05	0.7669	3	22.19	21.57	0.4785
	PMON68399	ZM_M39299	6	12.17	11.52	0.2051	1	10.13	10.05	0.8674	3	22.29	21.57	0.4118
	PMON68399	ZM_M39302	−44	6.44	11.52	0	−31	6.98	10.05	0	−38	13.42	21.57	0
87	PMON72494	ZM_M26428	22	17.55	14.42	0	4	12.4	11.87	0.21	14	29.95	26.29	1.00E−04
	PMON72494	ZM_M26428	46	15.57	10.67	0	12	11.3	10.11	0.0033	29	26.86	20.78	0
	PMON72494	ZM_M26428	23	14.1	11.43	0	13	9	7.98	0.0704	19	23.1	19.4	8.00E−04
	PMON72494	ZM_M26428	−6	10.7	11.43	0.2402	9	8.71	7.98	0.1938	0	19.41	19.4	0.9925
	PMON72494	ZM_M26428	3	11.02	10.67	0.5208	9	11.07	10.11	0.0163	6	22.09	20.78	0.1209
	PMON72494	ZM_M49327	8	12.13	11.23	0.2163	5	10.44	9.93	0.271	7	22.57	21.16	0.2103
	PMON72494	ZM_M49327	17	11.22	9.61	0.0189	4	8.28	7.93	0.5332	11	19.51	17.53	0.0853
	PMON72494	ZM_M49327	22	14.04	11.54	4.00E−04	21	9.73	8.06	0.0039	21	23.77	19.59	1.00E−04
	PMON72494	ZM_M49328	4	11.7	11.23	0.5112	11	11.03	9.93	0.0196	7	22.74	21.16	0.1618
	PMON72494	ZM_M49328	28	12.31	9.61	1.00E−04	17	9.27	7.93	0.0206	23	21.58	17.53	6.00E−04
	PMON72494	ZM_M49328	27	14.61	11.54	0	37	11.07	8.06	0	31	25.68	19.59	0
	PMON72494	ZM_M60546	−2	12.67	12.95	0.7032	6	9.48	8.95	0.4795	1	22.15	21.91	0.8478
89	PMON73765	ZM_M35084	10	10.56	9.61	0.1621	−2	7.8	7.93	0.8286	5	18.36	17.53	0.4667
	PMON73765	ZM_M35084	30	14.51	11.2	1.00E−04	27	9.25	7.27	0.0015	29	23.76	18.46	0
	PMON73765	ZM_M54013	42	13.61	9.61	0	13	8.96	7.93	0.0717	29	22.57	17.53	0
	PMON73765	ZM_M54013	32	14.78	11.2	0	49	10.82	7.27	0	39	25.6	18.46	0
	PMON73765	ZM_M54016	33	12.82	9.61	0	7	8.51	7.93	0.3051	22	21.33	17.53	0.0013
	PMON73765	ZM_M54016	34	14.98	11.2	0	39	10.09	7.27	0	36	25.07	18.46	0
91	PMON73816	ZM_M37183	21	12.1	9.96	0.0378	14	10.65	9.35	0.0587	18	22.75	19.31	0.0348
	PMON73816	ZM_M37183	33	11.5	8.66	0	21	9.82	8.09	0	27	21.32	16.75	0
	PMON73816	ZM_M37188	18	11.78	9.96	0.076	21	11.3	9.35	0.0051	20	23.08	19.31	0.021
	PMON73816	ZM_M37188	24	10.71	8.66	0	16	9.41	8.09	3.00E−04	20	20.11	16.75	0
	PMON73816	ZM_M37197	30	12.93	9.96	0.0044	6	9.88	9.35	0.4306	18	22.82	19.31	0.0313
	PMON73816	ZM_M37197	30	11.26	8.66	0	13	9.11	8.09	0.0047	22	20.37	16.75	0
90	PMON73829	ZM_M37805	29	9.46	7.32	1.00E−04	13	6.58	5.8	0.0171	22	16.04	13.12	1.00E−04
	PMON73829	ZM_M37805	18	11.78	9.96	0.076	15	10.74	9.35	0.0436	17	22.52	19.31	0.0484
	PMON73829	ZM_M37815	30	12.92	9.96	0.0046	13	10.57	9.35	0.0756	22	23.49	19.31	0.0109
	PMON73829	ZM_M37815	11	8.14	7.32	0.1117	13	6.54	5.8	0.0225	12	14.68	13.12	0.0241
	PMON73829	ZM_M38768	13	11.26	9.96	0.201	−1	9.25	9.35	0.8842	6	20.51	19.31	0.4543
	PMON73829	ZM_M38768	−2	7.2	7.32	0.8084	2	5.93	5.8	0.6854	0	13.13	13.12	0.9914
	PMON73829	ZM_M38797	−39	4.49	7.32	0	−19	4.68	5.8	8.00E−04	−30	9.16	13.12	0
	PMON73829	ZM_M38797	−11	8.83	9.96	0.2685	0	9.36	9.35	0.9827	−6	18.2	19.31	0.4895
	PMON73829	ZM_M38798	−62	3.75	9.96	0	−35	6.07	9.35	0	−49	9.82	19.31	0
	PMON73829	ZM_M38798	−50	3.67	7.32	0	−41	3.41	5.8	0	−46	7.08	13.12	0
	PMON73829	ZM_M39692	3	7.54	7.32	0.6671	−3	5.62	5.8	0.5857	0	13.16	13.12	0.9475
	PMON73829	ZM_M39692	17	11.69	9.96	0.0919	3	9.59	9.35	0.7181	10	21.28	19.31	0.2211
92	PMON75305	ZM_M35696	26	14.78	11.77	0	18	11.74	9.97	3.00E−04	22	26.52	21.74	0
	PMON75305	ZM_M35696	33	11.51	8.66	0	15	9.33	8.09	7.00E−04	24	20.84	16.75	0
	PMON75305	ZM_M36703	27	14.94	11.77	0	13	11.25	9.97	0.007	20	26.19	21.74	0
	PMON75305	ZM_M36703	40	12.15	8.66	0	22	9.84	8.09	0	31	21.99	16.75	0
	PMON75305	ZM_M36711	26	14.88	11.77	0	9	10.91	9.97	0.0455	19	25.78	21.74	2.00E−04
	PMON75305	ZM_M36711	35	11.68	8.66	0	16	9.38	8.09	4.00E−04	26	21.06	16.75	0
93	PMON75306	ZM_M35601	29	11.19	8.66	0	33	10.76	8.09	0	31	21.94	16.75	0
	PMON75306	ZM_M35601	11	13.05	11.77	0.0507	12	11.2	9.97	0.0097	11	24.24	21.74	0.0159
	PMON75306	ZM_M35604	24	14.64	11.77	0	16	11.57	9.97	9.00E−04	21	26.21	21.74	0
	PMON75306	ZM_M35604	42	12.29	8.66	0	35	10.92	8.09	0	39	23.21	16.75	0
	PMON75306	ZM_M35605	47	12.72	8.66	0	30	10.49	8.09	0	39	23.2	16.75	0
	PMON75306	ZM_M35605	18	13.92	11.77	0.0013	22	12.12	9.97	0	20	26.04	21.74	1.00E−04
94	PMON75309	ZM_M35865	21	10.45	8.66	0	3	8.3	8.09	0.5545	12	18.75	16.75	0.0017
	PMON75309	ZM_M35865	22	11.75	9.66	0.0038	17	10.68	9.1	0.0064	20	22.43	18.76	0.0031
	PMON75309	ZM_M35878	23	10.6	8.66	0	26	10.17	8.09	0	24	20.78	16.75	0
	PMON75309	ZM_M35878	18	11.38	9.66	0.0163	13	10.3	9.1	0.0362	16	21.68	18.76	0.017
	PMON75309	ZM_M36160	19	11.51	9.66	0.0099	19	10.79	9.1	0.0037	19	22.31	18.76	0.0041
	PMON75309	ZM_M36160	32	11.41	8.66	0	19	9.6	8.09	0	25	21.01	16.75	0
95	PMON75312	ZM_M35649	22	14.37	11.77	1.00E−04	12	11.18	9.97	0.0107	18	25.55	21.74	3.00E−04
	PMON75312	ZM_M35649	28	11.06	8.66	0	13	9.15	8.09	0.0034	21	20.21	16.75	0
	PMON75312	ZM_M37099	9	9.46	8.66	0.0458	13	9.11	8.09	0.0049	11	18.57	16.75	0.0042
	PMON75312	ZM_M37099	23	14.42	11.77	1.00E−04	10	10.97	9.97	0.0343	17	25.39	21.74	6.00E−04
	PMON75312	ZM_M37100	37	11.9	8.66	0	22	9.83	8.09	0	30	21.73	16.75	0
	PMON75312	ZM_M37100	9	12.85	11.77	0.0979	5	10.45	9.97	0.3064	7	23.29	21.74	0.1298
101	PMON75515	ZM_M43539	26	12.88	10.19	0	13	10.12	8.98	0.0097	20	23	19.17	0
	PMON75515	ZM_M43546	−3	9.87	10.19	0.5762	−5	8.55	8.98	0.3141	−4	18.43	19.17	0.3786
	PMON75515	ZM_M50136	16	10.41	8.98	0.0441	14	7.42	6.51	0.2064	15	17.84	15.48	0.085
	PMON75515	ZM_M50136	24	13.2	10.68	0.0015	25	9.27	7.42	0.0053	24	22.47	18.1	4.00E−04
	PMON75515	ZM_M50142	25	11.25	8.98	0.0018	17	7.61	6.51	0.1294	22	18.87	15.48	0.0145
	PMON75515	ZM_M50142	31	13.94	10.68	1.00E−04	35	10	7.42	1.00E−04	32	23.94	18.1	0
105	PMON75524	ZM_M47998	17	11.23	9.61	0.0452	35	9.69	7.17	0.0012	25	20.91	16.79	0.0043
	PMON75524	ZM_M47998	15	13.3	11.54	0.0101	38	11.15	8.06	0	25	24.45	19.59	0
	PMON75524	ZM_M48003	4	9.99	9.61	0.6366	9	7.78	7.17	0.4187	6	17.77	16.79	0.4837
	PMON75524	ZM_M48003	28	14.77	11.54	0	15	9.22	8.06	0.0414	22	24	19.59	1.00E−04
	PMON75524	ZM_M48004	19	11.44	9.61	0.0245	29	9.24	7.17	0.007	23	20.68	16.79	0.0069
	PMON75524	ZM_M48004	5	12.11	11.54	0.3919	1	8.17	8.06	0.8374	4	20.28	19.59	0.5062
	PMON75524	ZM_M48005	18	11.37	9.61	0.0303	19	8.57	7.17	0.0654	19	19.93	16.79	0.0276
	PMON75524	ZM_M48005	33	15.38	11.54	0	29	10.4	8.06	1.00E−04	32	25.78	19.59	0
	PMON75524	ZM_M48007	20	11.51	9.61	0.0195	7	7.66	7.17	0.5152	14	19.17	16.79	0.0927
	PMON75524	ZM_M48007	28	14.78	11.54	0	46	11.78	8.06	0	36	26.55	19.59	0
	PMON75524	ZM_M48010	22	11.77	9.61	0.0083	12	8.05	7.17	0.2443	18	19.81	16.79	0.0339
	PMON75524	ZM_M48010	18	13.62	11.54	0.0026	25	10.08	8.06	6.00E−04	21	23.7	19.59	2.00E−04
107	PMON75533	ZM_M47453	55	14.93	9.61	0	54	11.03	7.17	0	55	25.96	16.79	0
	PMON75533	ZM_M47453	39	14.99	10.8	0	44	10.24	7.12	0	41	25.23	17.92	0
	PMON75533	ZM_M47460	15	11.03	9.61	0.0782	5	7.53	7.17	0.63	11	18.56	16.79	0.208
	PMON75533	ZM_M47460	36	14.65	10.8	0	21	8.6	7.12	0.0037	30	23.25	17.92	0
	PMON75533	ZM_M49275	23	11.82	9.61	0.0069	20	8.58	7.17	0.0636	22	20.4	16.79	0.0119
	PMON75533	ZM_M49275	30	14.09	10.8	0	21	8.65	7.12	0.0028	27	22.74	17.92	0
	PMON75533	ZM_M49278	14	10.96	9.61	0.093	7	7.68	7.17	0.4982	11	18.64	16.79	0.1885
	PMON75533	ZM_M49278	18	12.79	10.8	0.0014	13	8.01	7.12	0.0757	16	20.8	17.92	0.0023
114	PMON75980	ZM_M53387	17	13.08	11.23	0.0122	11	10.99	9.93	0.0247	14	24.08	21.16	0.0109
	PMON75980	ZM_M53389	13	12.69	11.23	0.0463	9	10.85	9.93	0.0503	11	23.54	21.16	0.0363
	PMON75980	ZM_M53390	5	11.8	11.23	0.4269	4	10.33	9.93	0.3908	5	22.13	21.16	0.3859
	PMON75980	ZM_M53392	20	13.42	11.23	0.0033	13	11.19	9.93	0.0079	16	24.62	21.16	0.0028
	PMON75980	ZM_M53396	14	12.75	11.23	0.0383	4	10.38	9.93	0.338	9	23.12	21.16	0.0831
	PMON75980	ZM_M53397	6	11.92	11.23	0.3398	−4	9.59	9.93	0.455	2	21.51	21.16	0.7576
	PMON75980	ZM_M53398	4	11.66	11.23	0.5533	3	10.27	9.93	0.4659	4	21.93	21.16	0.4944
113	PMON78232	ZM_M55911	−3	12.1	12.44	0.652	12	9.85	8.82	0.1004	3	21.94	21.27	0.5616
	PMON78232	ZM_M55911	−5	13.18	13.83	0.3591	2	9.43	9.27	0.8057	−2	22.61	23.09	0.6774
	PMON78232	ZM_M56069	14	14.13	12.44	0.031	7	9.44	8.82	0.3213	11	23.56	21.27	0.0511
	PMON78232	ZM_M56069	11	15.39	13.83	0.0296	12	10.38	9.27	0.0932	12	25.77	23.09	0.0237
	PMON78232	ZM_M56206	−14	10.75	12.44	0.0307	−9	8	8.82	0.1837	−12	18.75	21.27	0.0333
	PMON78232	ZM_M56206	1	14.03	13.83	0.7776	5	9.73	9.27	0.4808	3	23.76	23.09	0.5663
	PMON78232	ZM_M56428	12	13.9	12.44	0.0606	11	9.83	8.82	0.1065	12	23.73	21.27	0.0367
	PMON78232	ZM_M56428	13	15.55	13.83	0.0164	18	10.91	9.27	0.0143	15	26.46	23.09	0.0048
106	PMON79163	ZM_M45011	16	11.88	10.25	0.0215	7	8.54	8	0.4508	12	20.42	18.26	0.0941
	PMON79163	ZM_M45011	20	12.98	10.8	0.0017	23	8.74	7.12	0.0046	21	21.71	17.92	4.00E−04
	PMON79163	ZM_M48217	16	11.89	10.25	0.0213	18	9.42	8	0.0487	17	21.3	18.26	0.0197
	PMON79163	ZM_M48217	28	13.81	10.8	0	20	8.51	7.12	0.0062	24	22.32	17.92	0
98	PMON79174	ZM_M47171	13	11.58	10.25	0.0602	20	9.61	8	0.0259	16	21.18	18.26	0.0247
	PMON79174	ZM_M47171	28	13.84	10.8	0	24	8.82	7.12	0.001	26	22.65	17.92	0
	PMON79174	ZM_M47941	18	12.09	10.25	0.0101	6	8.48	8	0.4971	13	20.57	18.26	0.0734
	PMON79174	ZM_M47941	25	13.53	10.8	0	16	8.24	7.12	0.026	21	21.77	17.92	1.00E−04
99	PMON79413	ZM_M48525	44	13.83	9.61	0	30	9.34	7.17	0.0049	38	23.17	16.79	0
	PMON79413	ZM_M48525	26	13.66	10.8	0	32	9.41	7.12	0	29	23.07	17.92	0
	PMON79413	ZM_M50333	25	12.05	9.61	0.0031	25	8.95	7.17	0.0197	25	21	16.79	0.0036
	PMON79413	ZM_M50333	27	13.75	10.8	0	34	9.55	7.12	0	30	23.3	17.92	0
	PMON79413	ZM_M53171	18	11.34	9.61	0.0331	27	9.13	7.17	0.0107	22	20.46	16.79	0.0106
	PMON79413	ZM_M53171	21	13.04	10.8	3.00E−04	37	9.78	7.12	0	27	22.82	17.92	0
112	PMON79447	ZM_M53825	16	12.45	10.71	0.0079	17	9.12	7.83	0.0281	16	21.57	18.53	0.0077
	PMON79447	ZM_M53825	30	14.57	11.2	1.00E−04	34	9.75	7.27	1.00E−04	32	24.32	18.46	0
	PMON79447	ZM_M53826	11	11.87	10.71	0.0705	0	7.84	7.83	0.9839	6	19.71	18.53	0.2903
	PMON79447	ZM_M53826	34	15	11.2	0	42	10.31	7.27	0	37	25.32	18.46	0
	PMON79447	ZM_M53835	6	11.31	10.71	0.342	−5	7.42	7.83	0.4779	1	18.73	18.53	0.8568
	PMON79447	ZM_M53835	32	14.83	11.2	0	47	10.66	7.27	0	38	25.49	18.46	0

4. Cold Field Efficacy Trial

This example sets forth a cold field efficacy trial to identify gene constructs that confer enhanced cold vigor at germination and early seedling growth under early spring planting field conditions in conventional-till and simulated no-till environments. Seeds are planted into the ground around two weeks before local farmers are beginning to plant corn so that a significant cold stress is exerted onto the crop, named as cold treatment. Seeds also are planted under local optimal planting conditions such that the crop has little or no exposure to cold condition, named as normal treatment. The cold field efficacy trials are carried out in five locations, including Glyndon Minn., Mason Mich., Monmouth Ill., Dayton Iowa, Mystic Conn. At each location, seeds are planted under both cold and normal conditions with 3 repetitions per treatment, 20 kernels per row and single row per plot. Seeds are planted 1.5 to 2 inch deep into soil to avoid muddy conditions. Two temperature monitors are set up at each location to monitor both air and soil temperature daily.
Seed emergence is defined as the point when the growing shoot breaks the soil surface. The number of emerged seedling in each plot is counted everyday from the day the earliest plot begins to emerge until no significant changes in emergence occur. In addition, for each planting date, the latest date when emergence is 0 in all plots is also recorded. Seedling vigor is also rated at V3-V4 stage before the average of corn plant height reaches 10 inches, with 1=excellent early growth, 5=Average growth and 9=poor growth. Days to 50% emergence, maximum percent emergence and seedling vigor are calculated using SAS software for the data within each location or across all locations.
The following table lists the data that were collected and analyzed based on the procedure illustrated above. The analyzed data across all locations only include those from Glyndon Minn., Mason Mich. and Mystic Conn.

	TABLE 19

	Days to 50% Emergence

Across Black

Dirt Trts

Warm Trts

Mason_Trt 2

Glyndon_Trt 2

Mystic_Trt 2

PEP SEQ ID			P		P		P		P		P
construct	Event1	Delta	value	Delta	value	Delta	value	Delta	value	Delta	value

88	ZM_M31146	1.46	0.106	0.04	0.979	0.51	0.755	2.17	0.079	0.99	0.551
pMON68399	ZM_M31147	1.29	0.153	0.34	0.81	0.59	0.721	1.47	0.234	1.64	0.322
	ZM_M31524	−0.41	0.649	0.23	0.873	−2.09	0.205	−0.13	0.919	0.69	0.676
	ZM_M32356	−0.21	0.814	0.33	0.815	−1.18	0.472	−1.59	0.197	3.52	0.034
	Construct	0.53	0.302	0.24	0.772	−0.54	0.563	0.48	0.495	1.71	0.071
90	ZM_M37805	0.95	0.293	−0.04	0.977	−0.4	0.808	2.28	0.065	−0.35	0.831
pMON73829	ZM_M37815	−1.24	0.169	0.06	0.965	−0.84	0.611	−1.4	0.258	−1.35	0.417
	ZM_M38768	2.79	0.002	0.7	0.621	0.64	0.696	2.11	0.087	6.3	0
	Construct	0.83	0.145	0.24	0.788	−0.2	0.849	1	0.2	1.53	0.144
92	ZM_M35696	1.75	0.053	0.14	0.922	−1.93	0.24	4.17	0.001	0.61	0.715
pMON75305	ZM_M36703	−0.47	0.603	0.4	0.777	−2.34	0.155	−0.83	0.502	2.12	0.202
	ZM_M36711	−0.92	0.31	0.32	0.823	−1.23	0.454	−1.5	0.223	0.57	0.731
	Construct	0.12	0.832	0.29	0.749	−1.84	0.078	0.61	0.432	1.1	0.295
93	ZM_M35601	−0.53	0.56	−0.36	0.803	−0.25	0.877	−0.22	0.861	−1.42	0.392
pMON75306	ZM_M35604	−0.92	0.309	0.45	0.752	0.1	0.951	−1.89	0.125	−0	1
	ZM_M35605	1.46	0.105	−0.08	0.958	−0.74	0.654	2.89	0.019	0.82	0.623
	Construct	0.01	0.992	0.01	0.994	−0.3	0.776	0.26	0.738	−0.2	0.847
94	ZM_M35865	−0.31	0.735	−0.27	0.849	−2.91	0.078	0.25	0.84	1.18	0.475
pMON75309	ZM_M35878	−0.1	0.916	0.33	0.817	0.3	0.858	−0.48	0.698	0.28	0.867
	ZM_M36160	−0.58	0.519	−0.46	0.748	−1.84	0.264	−0.65	0.597	0.81	0.625
	Construct	−0.33	0.566	−0.13	0.882	−1.48	0.155	−0.29	0.707	0.76	0.47
107	ZM_M49275	−3.72	0.001	2.39	0.343	−5.47	0.004	−5.14	0	X	X
pMON75533	ZM_M49278	−2.37	0.042	2.08	0.409	−7.87	0	−1.9	0.185	X	X
	Construct	−3.04	0.001	2.24	0.241	−6.67	0	−3.52	0.003	X	X
119	ZM_M53641	1.25	0.166	0.04	0.978	2.88	0.081	−0.31	0.804	2.74	0.099
pMON78235	ZM_M53994	−0.56	0.536	−0.13	0.926	−1.04	0.526	0.06	0.962	−1.31	0.429
	ZM_M53997	−0.8	0.376	0.11	0.937	0.38	0.816	−1.82	0.139	0.07	0.968
	Construct	−0.04	0.95	0.01	0.994	0.74	0.478	−0.69	0.376	0.5	0.635
104	ZM_M45248	−1.52	0.211	2.65	0.294	−4.77	0.013	−2.01	0.188	X	X
pMON78936	ZM_M45274	−3.87	0.001	2.58	0.307	−5	0.009	−5.59	0	X	X
	Construct	−2.69	0.004	2.61	0.171	−4.89	0.002	−3.8	0.002	X	X
110	ZM_M50823	−2	0.057	−0.17	0.921	−5.85	0.002	−2.51	0.08	2.87	0.136
pMON79425	ZM_M50856	0.01	0.993	−0.6	0.714	−5.19	0.007	−0.31	0.839	6.23	0.001
	ZM_M51300	−1.91	0.068	−0.02	0.989	−4.43	0.021	−2.42	0.091	1.61	0.402
	ZM_M51302	−3.5	0.001	−0.21	0.899	−6.08	0.002	−5.38	0	2.85	0.139
	ZM_M51313	−4.06	0	−0.12	0.94	−4.16	0.03	−5.38	0	−1.31	0.496
	ZM_M51608	−2.84	0.007	−0.27	0.87	−3.74	0.051	−4.88	0.001	2.15	0.265
	ZM_M51623	−2.09	0.047	−0.15	0.926	−5.14	0.007	−3.15	0.028	3.09	0.11
	Construct	−2.34	0.001	−0.22	0.838	−4.94	0	−3.43	0	2.5	0.048
116	ZM_M53939	−2.66	0.022	2.55	0.313	−3.3	0.085	−4.63	0.001	X	X
pMON79697	ZM_M54371	−1.02	0.378	2.71	0.282	−3.56	0.063	−2.04	0.154	X	X
	ZM_M54374	−2.79	0.016	2.67	0.29	−4.36	0.023	−4.3	0.003	X	X
	Construct	−2.16	0.01	2.64	0.11	−3.74	0.009	−3.66	0.001	X	X
111	ZM_M51598	−2.19	0.071	2.23	0.376	−4.51	0.019	−3.25	0.033	X	X
pMON79718	ZM_M52937	−1.8	0.138	3.07	0.224	−5.32	0.006	−2.14	0.162	X	X
	Construct	−2	0.037	2.65	0.165	−4.92	0.002	−2.69	0.028	X	X
120	ZM_M53455	0.14	0.873	0.29	0.838	3.04	0.065	−1.71	0.166	0.95	0.565
pMON80452	ZM_M53456	−0.56	0.532	−0.51	0.719	0.97	0.555	−1.18	0.337	−0.86	0.602
	ZM_M53694	0.88	0.332	0.25	0.859	2.06	0.211	1.04	0.401	−0.62	0.706
	ZM_M53695	1.47	0.104	0	0.998	3.07	0.062	0.22	0.857	2.37	0.154
	ZM_M53696	0.95	0.295	−0.2	0.888	0.46	0.78	0.74	0.55	1.85	0.265
	Construct	0.57	0.23	−0.03	0.965	1.92	0.028	−0.18	0.783	0.74	0.402
118	ZM_M53218	−1.55	0.087	−0.02	0.988	−3.54	0.032	−2.09	0.09	1.55	0.351
pMON80461	ZM_M53235	−1.42	0.117	0.34	0.808	−0.5	0.761	−1.86	0.131	−1.44	0.386
	ZM_M53848	−0.36	0.69	−0.02	0.988	−1.11	0.5	−0.6	0.624	0.88	0.595
	ZM_M54282	−0.98	0.279	0.16	0.909	−3.97	0.016	0.32	0.796	−0.58	0.727
	ZM_M54284	−1.06	0.24	0.05	0.972	−0.35	0.832	−1.21	0.328	−1.49	0.37
	Construct	−1.07	0.025	0.1	0.891	−1.89	0.03	−1.09	0.095	−0.21	0.806

Maximum Percent Emergence

Across Black

Dirt Trts

Warm Trts

Mason_Trt 2

Glyndon_Trt 2

Mystic_Trt 2

PEP SEQ ID			P		P		P		P		P
construct	Event1	Delta	value	Delta	value	Delta	value	Delta	value	Delta	value

88	ZM_M31146	−2.7	0.428	1.42	0.601	0.19	0.97	−7.53	0.125	4.07	0.503
pMON68399	ZM_M31147	−6.31	0.064	−5.8	0.033	−6.48	0.184	−9.75	0.047	0.74	0.903
	ZM_M31524	−2.7	0.428	−1.91	0.481	5.19	0.288	−4.2	0.393	−7.59	0.212
	ZM_M32356	3.55	0.297	−1.91	0.481	8.52	0.081	5.8	0.237	−5.93	0.33
	Construct	−2.04	0.293	−2.05	0.185	1.85	0.505	−3.92	0.161	−2.18	0.53
90	ZM_M37805	−4.18	0.22	5.83	0.032	1.67	0.733	−9.01	0.067	−0.37	0.951
pMON73829	ZM_M37815	4.71	0.167	−3.62	0.183	8.33	0.088	2.1	0.669	6.3	0.301
	ZM_M38768	−6.27	0.066	−2.51	0.356	−1.67	0.733	−5.68	0.247	−12.04	0.048
	Construct	−1.91	0.374	−0.1	0.954	2.78	0.368	−4.2	0.177	−2.04	0.596
92	ZM_M35696	−5.02	0.141	2.49	0.359	10	0.041	−12.35	0.012	−5.37	0.377
pMON75305	ZM_M36703	0.4	0.906	−1.95	0.473	6.67	0.172	0.99	0.841	−7.04	0.248
	ZM_M36711	3.6	0.291	1.38	0.611	1.67	0.733	6.54	0.183	−0.37	0.951
	Construct	−0.34	0.875	0.64	0.709	6.11	0.048	−1.6	0.605	−4.26	0.268
93	ZM_M35601	−2.52	0.46	3.6	0.185	1.67	0.733	−5.68	0.247	−0.37	0.951
pMON75306	ZM_M35604	3.04	0.372	−3.06	0.26	5	0.305	5.43	0.269	−3.7	0.543
	ZM_M35605	−3.49	0.306	−1.4	0.607	8.33	0.088	−10.12	0.039	−2.04	0.738
	Construct	−0.99	0.647	−0.28	0.869	5	0.105	−3.46	0.266	−2.04	0.596
94	ZM_M35865	−2.1	0.538	−3.06	0.315	1.67	0.733	−2.35	0.633	−5.37	0.377
pMON75309	ZM_M35878	−0.99	0.772	1.38	0.611	−0	1	−0.12	0.98	−3.7	0.543
	ZM_M36160	0.82	0.81	1.38	0.611	8.33	0.088	0.99	0.841	−7.04	0.248
	Construct	−0.76	0.726	−0.1	0.955	3.33	0.28	−0.49	0.874	−5.37	0.163
107	ZM_M49275	10.25	0.019	5.28	0.274	17.5	0.002	15	0.009	X	X
pMON75533	ZM_M49278	4.88	0.265	−1.39	0.773	19.17	0.001	6.11	0.284	X	X
	Construct	7.56	0.03	1.94	0.594	18.33	0	10.56	0.024	X	X
119	ZM_M53641	−1.27	0.71	1.38	0.611	5	0.305	0.99	0.841	−12.04	0.048
pMON78235	ZM_M53994	1.65	0.628	−1.4	0.607	3.33	0.494	0.99	0.841	1.3	0.831
	ZM_M53997	5.26	0.122	3.05	0.262	1.67	0.733	9.88	0.044	−0.37	0.951
	Construct	1.86	0.382	1.01	0.557	3.33	0.28	3.95	0.203	−3.7	0.336
104	ZM_M45248	−3.09	0.481	1.94	0.687	10.83	0.056	−1.67	0.77	X	X
pMON78936	ZM_M45274	9.88	0.024	6.94	0.15	14.17	0.013	16.11	0.005	X	X
	Construct	3.39	0.331	4.44	0.223	12.5	0.007	7.22	0.121	X	X
110	ZM_M50823	4.65	0.24	−0.83	0.792	10.83	0.056	7.22	0.206	−6.67	0.346
pMON79425	ZM_M50856	−6.88	0.082	−0.83	0.792	5.83	0.304	−8.33	0.144	−16.67	0.019
	ZM_M51300	3.54	0.371	−0.83	0.792	4.17	0.462	8.33	0.144	−6.67	0.346
	ZM_M51302	12.85	0.001	0.83	0.792	14.17	0.013	19.44	0.001	−1.67	0.814
	ZM_M51313	9.51	0.016	−0.83	0.792	15.83	0.005	12.78	0.025	−3.33	0.637
	ZM_M51608	5.49	0.166	0.83	0.792	10.83	0.056	7.22	0.206	−3.33	0.637
	ZM_M51623	1.6	0.687	3.06	0.333	17.5	0.002	2.78	0.626	−16.67	0.019
	Construct	4.39	0.09	0.2	0.923	11.31	0.002	7.06	0.059	−7.86	0.09
116	ZM_M53939	6.36	0.147	1.94	0.687	12.5	0.028	11.67	0.041	X	X
pMON79697	ZM_M54371	0.06	0.989	3.61	0.454	2.5	0.659	7.22	0.206	X	X
	ZM_M54374	10.06	0.022	−1.39	0.773	12.5	0.028	17.22	0.003	X	X
	Construct	5.49	0.081	1.39	0.66	9.17	0.03	12.04	0.005	X	X
111	ZM_M51598	4.13	0.345	−1.39	0.773	19.17	0.001	5	0.381	X	X
pMON79718	ZM_M52937	−1.42	0.745	6.94	0.15	15.83	0.005	−1.67	0.77	X	X
	Construct	1.36	0.698	2.78	0.446	17.5	0	1.67	0.72	X	X
120	ZM_M53455	1.65	0.628	−1.95	0.473	−3.33	0.494	7.65	0.119	−5.37	0.377
pMON80452	ZM_M53456	3.04	0.372	0.27	0.921	−5	0.305	8.77	0.074	−0.37	0.951
	ZM_M53694	−0.15	0.964	0.83	0.761	−1.67	0.733	−0.12	0.98	1.3	0.831
	ZM_M53695	−3.9	0.252	1.38	0.611	1.67	0.733	−3.46	0.481	−10.37	0.089
	ZM_M53696	0.96	0.779	2.49	0.359	6.67	0.172	2.1	0.669	−7.04	0.248
	Construct	0.32	0.86	0.6	0.675	−0.33	0.897	2.99	0.25	−4.37	0.175
118	ZM_M53218	3.46	0.31	−0.84	0.757	8.33	0.088	8.77	0.074	−12.04	0.048
pMON80461	ZM_M53235	3.6	0.291	0.83	0.761	−3.33	0.494	9.88	0.044	−2.04	0.738
	ZM_M53848	4.98	0.143	3.05	0.262	6.67	0.172	7.65	0.119	−2.04	0.738
	ZM_M54282	−0.57	0.867	−3.62	0.183	6.67	0.172	−3.46	0.481	−2.04	0.738
	ZM_M54284	4.98	0.143	−1.19	0.679	10	0.041	0.99	0.841	7.96	0.191
	Construct	3.29	0.068	−0.35	0.807	5.67	0.028	4.77	0.067	−2.04	0.527

E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels

This example sets forth a high-throughput selection for identifying plant seeds with improvement in seed composition using the Infratec 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.
Infratec Model 1221, 1225, or 1227 with transport module by Foss North America is used with cuvette, item #1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Software. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.

TABLE 20

Typical sample(s):	Whole grain corn and soybean seeds
Analytical time to run method:	Less than 0.75 min per sample
Total elapsed time per run:	1.5 minute per sample
Typical and minimum	Corn typical: 50 cc; minimum 30 cc
sample size:	Soybean typical: 50 cc; minimum 5 cc
Typical analytical range:	Determined in part by the specific
	calibration.
	Corn - moisture 5-15%, oil 5-20%,
	protein 5-30%, starch 50-75%, and
	density 1.0-1.3%.
	Soybean - moisture 5-15%, oil 15-25%,
	and protein 35-50%.

	TABLE 21

	Kernel Protein Content of Transgenic plant seeds in Midwest Hybrid Trials in 2003, 2004, and 2005.

Hybrid 2003

Hybrid 2004

Hybrid 2005

Mean

PEP

Trans-

Mean

%

Pval-

Trans-

Mean

%

Pva-

Trans-

Mean

%

Pval-

SEQ ID

Construct

Event

genic

Control^a

Change

ue

genic

Control^a

Change

lue

genic

Control^b

Change

ue

84	PMON69462	ZM_M17475	9.2	8.7	6.9	0.00	8.8	8.1	8.8	0.00	9.5	9.0	6.4	0.00
	PMON69462	ZM_M17512	9.4	8.7	8.0	0.00	8.9	8.1	10.3	0.00	9.6	9.0	6.8	0.00
	PMON69462	ZM_M19779	8.6	8.7	−1.1	0.37	8.0	8.1	−1.8	0.20	—	—	—	—
	PMON69462	ZM_M19792	8.9	8.7	2.3	0.17	8.1	8.1	−0.1	0.92	—	—	—	—
	PMON69462	ZM_M19775	8.5	8.7	−2.3	0.17	8.0	8.1	−1.4	0.32	—	—	—	—
	PMON69462	ZM_M19755	—	—	—	—	8.1	8.1	0.3	0.83	8.7	9.0	−2.5	0.09
	PMON69462	ZM_M19263	—	—	—	—	7.9	8.1	−2.1	0.12	—	—	—	—
	PMON69462	ZM_M19752	—	—	—	—	8.1	8.1	0.0	0.97	—	—	—	—
126	PMON83769	ZM_M75771	—	—	—	—	—	—	—	—	9.6	9.1	5.9	0.00
	PMON83769	ZM_M73623	—	—	—	—	—	—	—	—	9.1	9.1	0.2	0.92
	PMON83769	ZM_M73624	—	—	—	—	—	—	—	—	9.8	9.1	7.7	0.00
	PMON83769	ZM_M74392	—	—	—	—	—	—	—	—	9.6	9.1	5.0	0.00
	PMON83769	ZM_M74394	—	—	—	—	—	—	—	—	9.9	9.1	8.8	0.00
	PMON83769	ZM_M74395	—	—	—	—	—	—	—	—	9.5	9.1	4.5	0.01
	PMON83769	ZM_M75255	—	—	—	—	—	—	—	—	9.8	9.1	8.0	0.00
	PMON83769	ZM_M75260	—	—	—	—	—	—	—	—	9.5	9.1	4.1	0.01
124	PMON80868	ZM_M59335	—	—	—	—	—	—	—	—	9.2	9.0	2.1	0.24
	PMON80868	ZM_M59391	—	—	—	—	—	—	—	—	9.3	9.0	3.0	0.10
	PMON80868	ZM_M59764	—	—	—	—	—	—	—	—	9.0	9.0	0.0	0.98

Kernel protein reported on a 100% dry matter basis
^aControl for 2003 and 2004 was recurrent parent
^bControl for 2005 trial was pollinator for pMON69462 and recurrent parent for pMON83769 and pMON80868

	TABLE 22

	Kernel Protein Content of Transgenic plant seeds in Hawaii Inbred Trials^a

PEP				Mean	Mean	%
SEQ ID	Construct	Event	Year	Transgenic	Control^b	Change	Pvalue

84	PMON69462	ZM_M17475	2002	14.2	10.7	32.7	0.02
	PMON69462	ZM_M17512	2002	12.6	11.8	6.8	0.10
	PMON69462	ZM_M19779	2002	11.4	10.7	6.5	0.10
	PMON69462	ZM_M19792	2002	12.5	11.6	7.8	0.10
	PMON69462	ZM_M19775	2002	12.9	11.9	8.4	0.10
	PMON69462	ZM_M19755	2003	12.0	11.3	6.4	0.44
	PMON69462	ZM_M19263	2003	10.8	11.0	−2.2	0.77
	PMON69462	ZM_M19752	2003	11.1	11.9	−7.0	0.23
	PMON69462	ZM_M19270	2002	13.0	10.5	23.8	0.02
	PMON69462	ZM_M19781	2002	12.4	10.3	20.4	0.02
	PMON69462	ZM_M19257	2003	12.7	11.4	11.4	0.30
126	PMON83769	ZM_M73624	2004	13.4	9.4	42.9	0.00
	PMON83769	ZM_M74380	2004	11.9	11.7	1.4	0.88
	PMON83769	ZM_M74392	2004	10.7	12.0	−10.5	0.21
	PMON83769	ZM_M74394	2004	11.8	10.7	10.5	0.05
	PMON83769	ZM_M74395	2004	13.6	11.8	14.8	0.00
	PMON83769	ZM_M75255	2004	12.5	11.0	13.2	0.27
	PMON83769	ZM_M75771	2004	12.3	12.5	−2.2	0.83
124	PMON80868	ZM_M59335	2004	13.3	12.1	10.4	0.07
	PMON80868	ZM_M59764	2004	12.8	11.5	10.8	0.27
	PMON80868	ZM_M59765	2004	13.7	11.8	15.5	0.00

^aKernel protein reported on a 100% dry matter basis
^bControl was negative isoline for each event

Example 6

This example illustrates the preparation of transgenic plant cells containing recombinant DNA (SEQ ID NO:82) expressing a maize phytochrome A protein (PHYA). A full-length cDNA encoding a corn PHYA protein was cloned from corn. The cDNA clone contained 3396 bp of nucleotides encoding a 1131 amino acid PHYA protein with molecular weight at 125.2 kD. Based on the cDNA sequences, primers were designed to clone a genomic DNA, from a maize inbred LH172 genomic library. Recombinant DNA comprising a rice actin promoter operably linked to the genomic DNA encoding the corn PHYA protein followed by a Hsp17 terminator was inserted into transformation vector of pMON74916 as set forth in SEQ ID NO: 10030. Corn plant cells were transformed with recombinant DNA expressing PHA using pMON74916 and used to regenerate a population of transgenic plants. Transgenic plants were regenerated from about 100 events of transformed plant cells; plants from 90 of the events with various expression levels were selected for pollination to produce R1 and F1 seeds; and plants from 31 events were selected for screening for an enhanced trait.

Seed Germination and Seedling Development

Five events were selected to be analyzed phenotypic effect on seed germination and seedling development in the dark condition along with other transgenic material. 12 inbred seeds of each wild-type and transgenic maize events were germinated in a wetted and rolled germination paper in a complete dark growth chamber for 10 days. The length of mesocotyl, coleoptiles and root were measured for every seedling. The transgenic maize seedlings were identified showing great elongation growth of both mesocotyl and expanded coleoptiles imparted from recombinant DNA expressing PHYA protein as compared to non-transgenic controls.
Density Study
Transgenic plants were grown in fields at three densities: high density at 42,000 plants per acre; medium density at 35,000 plants per acre; and low density at 28,000 plants per acre. Plants from three plant cell events expressing PHYA were selected for studying physiological and yield responses to different densities. The physiological data from the density trial Y1130 is summarized in the Table 23 shown below. Event ZM_S83483 under high planting density showed significant decrease in plant height, ear height, and internode length and had a significant increase in chlorophyll content.

	TABLE 23

	Low Density YI130 JV 2004	High Density YI130 JV 2004

Event			Stem	Internode			Internode		Photo
ID	Plant Height	Ear Height	Diameter	Length	Plant Height	Ear Height	Length	SPAD	rate

ZM_S83483	not	Significant	not	Significant	not	Significant	increase not	Significant	NA
	significant	Decrease	significant	Decrease	significant	Decrease	significant	Increase
	P = 0.727	P = 0.085	P = 0.9436	P = 0.0370	P = 0.5866	P = 0.0185	P = 0.2412	P = 0.0762
ZM_S83897	not	decrease not	not	Significant	Significant	not	not	Significant	Significant
	significant	significant	significant	Increase	Decrease	significant	significant	Decrease	Decrease
	P = 0.8778	P = 0.1937	P = 0.2517	P = 0.0421	P = 0.0306	P = 0.6542	P = 0.5206	P = 0.0153
ZM_S83907	Highly	increase not	Significant	Highly	Significant	Significant	not	increase not	not
	Significant	significant	Increase	Significant	Increase	Increase	significant	significant	significant
	Increase	P = 0.2426	P = 0.0633	Increase	P = 0.0016	P = 0.015	P = 0.89	P = 0.3208
	P = 0.0021			P = 0.001

Kernel Trait Analysis

As shown in Table 24, events ZM_S83444, ZM_S83446, ZM_S83473, ZM_S83480, ZM_S83483, and ZM_S83907 show significant increases in single kernel weight. Event ZM_S83452 shows significant increases in single kernel weight and total kernel weight. The screening data show that plant cells with stably-integrated, non-natural, recombinant DNA expressing a phytochrome A protein can be regenerated into plants exhibiting increased yield as compared to control plants.

TABLE 24

event	Trait	Mean_TRAN	Mean_CON	TRAN-CON	% change	Pvalue	Result

ZM_S83416	Total kernel weight, g	151.3	140.21	11.09	8	0.1452	Non Signifincant
	Total kernel number	876	830.22	45.78	6	0.3118	Non Signifincant
	Singel kernel weight, g	0.17	0.17	0.01	6	0.3551	Non Signifincant
ZM_S83444	Total kernel weight, g	147.14	144.65	2.49	2	0.753	Non Signifincant
	Total kernel number	664.38	930.47	−266.1	−29	0	Highly Significant
	Singel kernel weight, g	0.25	0.16	0.09	56	0	Highly Significant
ZM_S83446	Total kernel weight, g	152.12	158.27	−6.15	−4	0.3931	Non Signifincant
	Total kernel number	718.88	918.94	−200.07	−22	0	Highly Significant
	Singel kernel weight, g	0.2	0.17	0.03	18	0.0008	Highly Significant
ZM_S83452	Total kernel weight, g	166.94	140.21	26.72	19	0.0014	Highly Significant
	Total kernel number	888.89	830.22	58.67	7	0.2123	Non Signifincant
	Singel kernel weight, g	0.19	0.17	0.02	12	0.0045	Highly Significant
ZM_S83473	Total kernel weight, g	145.87	146.47	−0.6	−0	0.9451	Non Signifincant
	Total kernel number	784.71	885.21	−100.5	−11	0.0099	Highly Significant
	Singel kernel weight, g	0.18	0.16	0.02	13	0.0618	Signifincant at 10%
ZM_S83480	Total kernel weight, g	157.23	149.44	7.79	5	0.3769	Non Signifincant
	Total kernel number	856.67	924.28	−67.61	−7	0.0982	Signifincant at 10%
	Singel kernel weight, g	0.18	0.16	0.02	13	0.0018	Highly Significant
ZM_S83483	Total kernel weight, g	164.86	158.27	6.6	4	0.3599	Non Signifincant
	Total kernel number	820.4	918.94	−98.54	−11	0.0165	Significant
	Singel kernel weight, g	0.19	0.17	0.02	12	0.0317	Significant
ZM_S83897	Total kernel weight, g	132.62	149.44	−16.83	−11	0.0617	Signifincant at 10%
	Total kernel number	743.5	924.28	−180.78	−20	0.0001	Highly Significant
	Singel kernel weight, g	0.18	0.16	0.02	13	0.0125	Significant
ZM_S83907	Total kernel weight, g	146.23	146.47	−0.24	−0	0.9807	Non Signifincant
	Total kernel number	733.44	833.41	−99.97	−12	0.0703	Signifincant at 10%
	Singel kernel weight, g	0.19	0.17	0.02	12	0.0792	Signifincant at 10%
ZM_S83416	Total kernel weight, g	157.3	146.47	10.83	7	0.2666	Non Signifincant
	Total kernel number	881.8	833.41	48.39	6	0.3558	Non Signifincant
	Singel kernel weight, g	0.18	0.17	0	0	0.6827	Non Signifincant

Example 7

This example illustrates the preparation of transgenic plant cells containing recombinant DNA (SEQ ID NO:77) expressing a soybean MADS box transcription factor protein and identified as G1760.
The DNA encoding the soybean MADS box transcription factor was cloned from a soybean library and inserted into a recombinant DNA construct comprising a CaMV 35S promoter operably linked to the DNA encoding the transcription factor followed by a terminator. The recombinant DNA construct was inserted into a transformation vector plasmid to produce plasmid pMON74470, as set forth in SEQ ID NO: 10029 which was used for Agrobacterium-mediated transformation of soybean plant cells.
Soybean plant cells were transformed with recombinant DNA expressing the MADS box transcription factor using MON74470 and used to regenerate a population of transgenic plants. Transgenic soybean plants were regenerated and selected for screening for an enhanced trait.
Transgenic soybean plants exhibited flowers with highly enlarged sepals and a winding stem. The main stem exhibited reduced lateral branching and increased raceme formation. Flowering time was decreased by about 2 to 4 days as compared to control plants under short day (10 hr) and long day (14 hr) conditions. Transgenic plants also flowered by 5 weeks when placed under non-inductive 20 hr light; wild-type control plants did not flower under such conditions. Floral and pod abscission was greatly reduced in the transgenic plants resulting in an increase in the number of pods per plant. Wild type control plants produced on the order of 100 pods, specific transgenic plants produced at least 125 pods per plant and plants regenerated from plant cells of one transgenic event produced greater than 200) pods per plant. There was also a delay in maturity ranging from one week exhibited by plants from single copy event A29204 to a month exhibited by plants from a multi-copy event A28877. Over 95% of the pods on transgenic plants from event A29204 mature in a time period; but only 50% of the pods on transgenic plants from event A28877 mature in the same time period. Seeds from transgenic plants were smaller than seed from control plants and greater in number than seeds from control plants, e.g. about 1800 more seed per pound. Transgenic plants were also shown to be have enhanced water use efficiency.
In testing soybeans for drought tolerance, 4.5″ pots were prepared with Metromix 200 and the pots were adjusted to the same weight. Pots were saturated with water. R2 or R3 homozygous seeds were placed in the soil in the pots, 15 pots per event, 3 to 6 events per construct. Plants were grown with a light intensity of 600 μEM⁻²S⁻¹; Temperature: 28° C.; Relative humidity (RH): 60%. A gene check with gene check strip (Trait RUR Lateral Flow 50 tests, from Strategic Diagnostics, Inc.) for the presence of the CP4 gene was done on selected plants. Unwanted negative plants were discarded. When plants reached the V1 stage. Pots were saturated with water by thorough irrigation. A picture was taken of the plant in the water saturated pot. Excess water was drained and further water was withheld until the pot water content of 50% and 10% of the water capacity for well watered controls and drought treated plants, respectively (monitor the water content by measuring soil moisture or pot weight every 3-5 days). At approximately 10% of the saturated water weight, the plants began to show the onset of the wilting phenotype. Limited-watering was continued every 1-2 days to maintain pot water content at 50 or 10%. The drought injury phenotype was determined for next 14 days (see the table of measurements). Photograph of plants and physiological assays were run on each at 14 days after the onset of drought treatment. Theses included, but were not limited to, plant height, leaf relative water content, leaf water potential, chlorophyll content and chlorophyll fluorescence. Pot were saturated with nutrient solution and resume regular watering schedule after 14 days.

TABLE 25

Measurement	Protocol

Agronomic measurements	Emergence, early season vigor, height (cm)
Visual drought score	Score of 1 to 4: 1. Healthy plants, no
	difference from control plants; 2. On sight
	of wilting, leaves become wilt; 3. Wilted
	plants, still green and recoverable; 4.
	Severely wilted, chlorotic and not
	recoverable

Drought assay measurements as described in Table 25 taken on transgenic soybean plants showed that transgenic soybean plants from transgenic plant cells of event GM 29204 exhibited enhanced water use efficiency.

R0 plants regenerated from one transgenic plant cell event (28877) of 41 transgenic plant cells events produced a large number of pods per node and seeds/plant—531 R1 seeds per plant compared to an average of 150 seeds per plant, i.e. increased yield.

Example 6. Consensus Sequence

This example illustrates the identification of consensus amino acid sequence for the proteins and homologs encoded by DNA that is used to prepare the transgenic seed and plants of this invention having enhanced agronomic traits.
ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 136 and its nine homologs, and SEQ ID NO: 151 and its 11 homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. FIGS. 1A-1G show an alignment of the sequences of SEQ ID NO: 136, its homologs and the consensus sequence (SEQ ID NO: 10031) at the end. FIGS. 2A-2G show an alignment of the sequences of SEQ ID NO: 151, its homologs and the consensus sequence (SEQ ID NO: 10032) at the end. The symbols for consensus sequence are (1) uppercase letters for 100% identity in all positions of multiple sequence alignment output; (2) lowercase letters for >=70% identity; symbol; (3) “X” indicated <700/o identity; (4) dashes “−” meaning that gaps were in >=70% sequences.
The consensus amino acid sequence can be used to identify DNA corresponding to the full scope of this invention that is useful in providing transgenic plants, for example corn and soybean plants with enhanced agronomic traits, for example improved nitrogen use efficiency, improved yield, improved water use efficiency and/or improved growth under cold stress, due to the expression in the plants of DNA encoding a protein with amino acid sequence identical to the consensus amino acid sequence.

Example 7. Identification of Amino Acid Domain by Pfam Analysis

The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein families for the proteins of SEQ ID NO:84 through 166 are shown in Table 26. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 91 is characterized by two Pfam domains, i.e. SRF-TF and K-box; and, the protein with amino acids of SEQ ID NO:165 is characterized by six Pfam domains, i.e. GAF, Phytochrome, PAS, a repeated PAS. HisKA, and HATPase.

3	86	Pkinase	79	337	343	4.30E−100
5	88	FA_desaturase	99	319	206.2	6.60E−59
2	85	Ras	10	178	297.9	1.60E−86
1	84	Glyoxalase	27	171	130.1	5.40E−36
8	91	SRF-TF	9	59	121.4	2.30E−33
8	91	K-box	75	176	151.7	1.70E−42
7	90	K-box	4	104	145.6	1.20E−40
83	166	SRF-TF	9	59	99.2	1.10E−26
83	166	K-box	75	172	92.4	1.20E−24
82	165	GAF	219	404	105.6	1.30E−28
82	165	Phytochrome	415	595	407.6	1.60E−119
82	165	PAS	622	738	88.9	1.40E−23
82	165	PAS	753	878	101.1	2.80E−27
82	165	HisKA	898	957	27.6	4.00E−05
82	165	HATPase_c	1012	1124	66.9	5.80E−17
9	92	Homeobox	97	158	68	2.80E−17
10	93	AP2	5	68	127.5	3.30E−35
11	94	GATA	196	231	71.3	2.70E−18
12	95	AT_hook	57	69	7.4	1.1
12	95	DUF296	84	208	183.6	4.30E−52
24	107	Synaptobrevin	128	215	137.6	2.90E−38
31	114	Pyridoxal_deC	28	381	194.6	2.10E−55
36	119	Metallophos	63	258	161	2.80E−45
21	104	Pkinase	12	267	346	5.40E−101
21	104	Pkinase_Tyr	12	265	88.5	1.80E−23
21	104	NAF	310	369	98.6	1.60E−26
26	109	MtN3_slv	9	98	96.7	6.10E−26
26	109	MtN3_slv	132	218	116.8	5.70E−32
27	110	Lactamase_B	94	252	125.1	1.80E−34
33	116	HSP20	53	157	159.9	5.80E−45
28	111	RTC	3	353	275.2	1.10E−79
28	111	RTC_insert	184	300	120.8	3.40E−33
37	120	PDZ	200	284	37.6	3.80E−08
37	120	Peptidase_S41	320	483	244.5	1.90E−70
35	118	E2F_TDP	167	232	131	2.90E−36
41	124	Pkinase	63	341	199.5	7.00E−57
41	124	Pkinase_Tyr	63	341	243	5.60E−70
43	126	zf-C2H2	72	94	25.6	0.00016
43	126	zf-C2H2	149	171	20.5	0.0054
4	87	zf-C2H2	85	107	22.1	0.0018
17	100	PRA1	10	161	181.8	1.50E−51
22	105	AAA	154	352	85	2.10E−22
14	97	CBFD_NFYB_HMF	31	96	134.4	2.80E−37
34	117	Peptidase_C15	11	219	−72.2	3.50E−07
20	103	Pkinase	13	267	345.5	7.80E−101
20	103	Pkinase_Tyr	13	265	75.2	1.80E−19
20	103	NAF	312	371	104.7	2.50E−28
32	115	HSF_DNA-bind	49	225	212.2	1.00E−60
19	102	Pkinase	37	291	353.9	2.30E−103
19	102	RIO1	50	208	−88.1	0.0038
19	102	NAF	375	432	101.8	1.80E−27
40	123	Aldo_ket_red	7	284	448.1	1.00E−131
42	125	FBPase	13	337	691.6	5.30E−205
6	89	SRF-TF	9	59	119.7	7.20E−33
18	101	DNA_photolyase	6	173	163.3	5.70E−46
18	101	FAD_binding_7	205	476	425.8	5.50E−125
30	113	Pkinase	41	327	326.6	3.80E−95
23	106	NIF	95	291	90.6	4.10E−24
15	98	Got1	30	130	237	3.60E−68
16	99	RRM_1	21	89	67.1	5.00E−17
29	112	Di19	13	206	365.4	8.00E−107
25	108	CorA	90	467	408.2	1.00E−119
39	122	SPC25	12	190	252.3	9.00E−73
44	127	Response_reg	18	139	151.1	2.60E−42
44	127	HisKA	320	385	101.5	2.30E−27
44	127	HATPase_c	432	565	138.4	1.70E−38
44	127	Response_reg	740	862	128	2.40E−35
44	127	Hpt	922	1013	63.4	6.60E−16
45	128	Response_reg	18	139	151.1	2.60E−42
45	128	HisKA	320	385	101.5	2.30E−27
45	128	HATPase_c	432	565	138.4	1.70E−38
45	128	Response_reg	740	862	128	2.40E−35
45	128	Hpt	922	1013	63.4	6.60E−16
46	129	NAM	9	135	313.7	2.90E−91
47	130	Aminotran_1_2	183	576	55.7	1.40E−13
48	131	Catalase	18	401	960.1	7.80E−286
49	132	BRO1	10	172	177.8	2.40E−50
69	152	Got1	30	130	211.8	1.40E−60
70	153	Got1	30	130	174.9	1.80E−49
71	154	Cystatin	36	124	87.6	3.40E−23
72	155	Cystatin	36	124	87.6	3.40E−23
73	156	RRM_1	22	87	32.4	1.40E−06
74	157	Pkinase_Tyr	55	304	86.2	9.10E−23
74	157	Pkinase	55	306	362	8.40E−106
75	158	SPX	1	167	88.9	1.30E−23
75	158	zf-C3HC4	238	286	17	0.0024
76	159	Pkinase_Tyr	19	271	70.8	4.00E−18
76	159	Pkinase	19	273	359.7	4.10E−105
76	159	NAF	324	381	105.6	1.30E−28
77	160	SRF-TF	9	59	100.8	3.60E−27
77	160	K-box	73	173	95.3	1.60E−25
50	133	Peptidase_S10	1	227	−42.7	6.00E−11
51	134	Ank	44	76	47.3	4.70E−11
51	134	Ank	77	109	33.5	6.40E−07
51	134	Ank	111	144	15.7	0.14
51	134	Ank	185	217	39.7	9.00E−09
51	134	Ank	228	260	30.7	4.50E−06
52	135	Pkinase_Tyr	51	341	158.7	1.40E−44
52	135	Pkinase	63	341	104.4	3.00E−28
54	137	GATase_2	2	162	11.8	6.10E−12
54	137	Asn_synthase	211	479	334.3	1.80E−97
55	138	HSP20	56	164	168.2	1.90E−47
78	161	Lactamase_B	93	251	129	1.20E−35
56	139	UPF0057	11	62	102.9	8.40E−28
57	140	Oxidored_FMN	6	341	302.1	9.10E−88
58	141	Pkinase	39	325	309.2	6.40E−90
59	142	Pyridoxal_deC	33	381	546	3.40E−161
60	143	Pyridoxal_deC	33	381	546	3.40E−161
61	144	HSP20	57	160	178.8	1.20E−50
38	121	PDZ	200	284	37.6	3.80E−08
38	121	Peptidase_S41	320	483	244.5	1.90E−70
62	145	Cpn60_TCP1	59	562	578.6	5.40E−171
63	146	DSPc	50	188	142.9	7.70E−40
64	147	Isoamylase_N	61	149	94.9	2.10E−25
64	147	Alpha-amylase	209	589	−36.4	1.30E−07
79	162	Pkinase	45	299	360.3	2.80E−105
79	162	NAF	384	441	105.2	1.70E−28
65	148	DUF1685	38	146	184.5	2.40E−52
80	163	GAF	219	404	108.4	1.90E−29
80	163	Phytochrome	415	595	409.1	5.70E−120
80	163	PAS	622	737	96.6	6.50E−26
80	163	PAS	752	877	107.4	3.80E−29
80	163	HisKA	897	956	26.7	7.10E−05
80	163	HATPase_c	1011	1123	64.4	3.30E−16
66	149	Glyco_hydro_1	74	558	1024.9	0
67	150	ArfGap	17	133	174.4	2.50E−49
81	164	AP2	6	69	132	1.50E−36

TABLE 27

	accession	gathering
pfam domain name	number	cutoff	domain description

AAA	PF00004.17	10	ATPase family associated with various
			cellular activities (AAA)
AP2	PF00847.9	0	AP2 domain
Aldo_ket_red	PF00248.10	−97	Aldo/keto reductase family
Alpha-amylase	PF00128.11	−93	Alpha amylase, catalytic domain
Aminotran_1_2	PF00155.9	−57.5	Aminotransferase class I and II
Ank	PF00023.17	21.6	Ankyrin repeat
ArfGap	PF01412.8	−17	Putative GTPase activating protein for Arf
Asn_synthase	PF00733.10	−52.8	Asparagine synthase
BRO1	PF03097.6	25	BRO1-like domain
CBFD_NFYB_HMF	PF00808.12	18.4	Histone-like transcription factor (CBF/NF-
			Y) and archaeal histone
Catalase	PF00199.8	−229	Catalase
CorA	PF01544.8	−61.3	CorA-like Mg2+ transporter protein
Cpn60_TCP1	PF00118.13	−223.4	TCP-1/cpn60 chaperonin family
Cystatin	PF00031.10	17.5	Cystatin domain
DNA_photolyase	PF00875.7	−10	DNA photolyase
DSPc	PF00782.9	−21.8	Dual specificity phosphatase, catalytic
			domain
DUF1685	PF07939.1	25	Protein of unknown function (DUF1685)
DUF296	PF03479.4	−11	Domain of unknown function (DUF296)
Di19	PF05605.2	25	Drought induced 19 protein (Di19)
E2F_TDP	PF02319.9	17	E2F/DP family winged-helix DNA-
			binding domain
FAD_binding_7	PF03441.3	25	FAD binding domain of DNA photolyase
FA_desaturase	PF00487.13	−46	Fatty acid desaturase
FBPase	PF00316.9	−170.3	Fructose-1-6-bisphosphatase
GAF	PF01590.14	23	GAF domain
GATA	PF00320.15	28.5	GATA zinc finger
GATase_2	PF00310.10	−106.2	Glutamine amidotransferases class-II
Glyco_hydro_1	PF00232.8	−301.8	Glycosyl hydrolase family 1
Glyoxalase	PF00903.14	12.1	Glyoxalase/Bleomycin resistance
			protein/Dioxygenase superfamily
Got1	PF04178.2	25	Got1-like family
HATPase_c	PF02518.13	22.4	Histidine kinase-, DNA gyrase B-, and
			HSP90-like ATPase
HSF_DNA-bind	PF00447.7	−70	HSF-type DNA-binding
HSP20	PF00011.9	13	Hsp20/alpha crystallin family
HisKA	PF00512.13	10.2	His Kinase A (phosphoacceptor) domain
Homeobox	PF00046.17	−4.1	Homeobox domain
Hpt	PF01627.11	25	Hpt domain
Isoamylase_N	PF02922.7	−6.5	Isoamylase N-terminal domain
K-box	PF01486.7	0	K-box region
Lactamase_B	PF00753.15	22.3	Metallo-beta-lactamase superfamily
Metallophos	PF00149.16	22	Calcineurin-like phosphoesterase
MtN3_slv	PF03083.5	−0.8	MtN3/saliva family
NAF	PF03822.4	25	NAF domain
NAM	PF02365.5	−19	No apical meristem (NAM) protein
NIF	PF03031.7	−81	NLI interacting factor-like phosphatase
Oxidored_FMN	PF00724.8	−147.7	NADH:flavin oxidoreductase/NADH
			oxidase family
PAS	PF00989.12	20	PAS fold
PDZ	PF00595.11	12.1	PDZ domain (Also known as DHR or
			GLGF)
PRA1	PF03208.8	25	PRA1 family protein
Peptidase_C15	PF01470.7	−100	Pyroglutamyl peptidase
Peptidase_S10	PF00450.11	−198	Serine carboxypeptidase
Peptidase_S41	PF03572.7	−25.8	Peptidase family S41
Phytochrome	PF00360.9	11	Phytochrome region
Pkinase	PF00069.14	−70.8	Protein kinase domain
Pkinase_Tyr	PF07714.4	65	Protein tyrosine kinase
Pyridoxal_deC	PF00282.8	−158.6	Pyridoxal-dependent decarboxylase
			conserved domain
RIO1	PF01163.11	−89.1	RIO1 family
RRM_1	PF00076.10	15.2	RNA recognition motif. (a.k.a. RRM,
			RBD, or RNP domain)
RTC	PF01137.11	−36.9	RNA 3′-terminal phosphate cyclase
RTC_insert	PF05189.3	25	RNA 3′-terminal phosphate cyclase
			(RTC), insert domain
Ras	PF00071.11	18	Ras family
Response_reg	PF00072.11	−14.4	Response regulator receiver domain
SPC25	PF06703.1	25	Microsomal signal peptidase 25 kDa
			subunit (SPC25)
SPX	PF03105.9	−20	SPX domain
SRF-TF	PF00319.8	11	SRF-type transcription factor (DNA-
			binding and dimerisation domain)
Synaptobrevin	PF00957.9	25	Synaptobrevin
UPF0057	PF01679.7	25	Uncharacterized protein family UPF0057
zf-C2H2	PF00096.14	19	Zinc finger, C2H2 type
zf-C3HC4	PF00097.12	16.9	Zinc finger, C3HC4 type (RING finger)

Example 8. Selection of Transgenic Plants with Enhanced Agronomic Trait(s)

This example illustrates the preparation and identification by selection of transgenic seeds and plants derived from transgenic plant cells of this invention where the plants and seed are identified by screening a having an enhanced agronomic trait imparted by expression of a protein selected from the group including the homologous proteins identified in Example 4. SEQ ID NO: 121, 128, 152-160, 162 and 164. Transgenic plant cells of corn, soybean, cotton, canola, wheat and rice are transformed with recombinant DNA for expressing each of the homologs identified in Example 4. Plants are regenerated from the transformed plant cells and used to produce progeny plants and seed that are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Plants are identified exhibiting enhanced traits imparted by expression of the homologous proteins.

Pfam domain name

What is claimed is:

1. A plant cell with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names consisting of AAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 12, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60 TCP1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP, FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro 1, Glyoxalase, Gotl, HATPase c, FISF DNA-bind, HSP20, HisKA, Homeobox, Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 slv, NAF, NAM, NIF, Oxidored FMN, PAS, PDZ, PRA1, Peptidase C15, Peptidase S10, Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1, RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-TF, Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4; wherein the Pfam gathering cuttoff for said protein domain families is stated in Table 28; wherein said plant cell is selected from a population of plant cells with said recombinant DNA by screening plants that are regenerated from plant cells in said population and that express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

2. A plant cell of claim 1 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO:84 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO:166 and homologs thereof listed in Table 2.

3. A plant cell of claim 1 wherein said protein is selected from the group of proteins identified in Table 1.

4. A plant cell of claim 1 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.

5. A plant cell of claim 4 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.

6. A transgenic plant comprising a plurality of the plant cell of claim 1

7. A transgenic plant of claim 6 which is homozygous for said recombinant DNA.

8. A transgenic seed comprising a plurality of the plant cell of claim 1.

9. A transgenic seed of claim 8 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.

10. Non-natural, transgenic corn seed of claim 9 wherein said seed can produce corn plants that are resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi. fungus or both.

11. A transgenic pollen grain comprising a haploid derivative of a plant cell of claim 1.

12. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names consisting of AAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 1 2, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60 TCP1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP, FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro 1, Glyoxalase, Gotl, HATPase c, HSF DNA-bind, HSP20, HisKA, Homeobox, Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 slv, NAF, NAM, NIF, Oxidored FMN, PAS, PDZ, PRA1, Peptidase C15, Peptidase S10, Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1, RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-TF, Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4; wherein the gathering cutoff for said protein domain families is stated in Table 28; and wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil, said method for manufacturing said seed comprising:

(a) screening d population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA,

(b) selecting from said population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants,

(c) verifying that said recombinant DNA is stably integrated in said selected plants,

(d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO:1-83; and

(e) collecting seed from a selected plant.

13. A method of claim 12 wherein plants in said population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells, and wherein said selecting is effected by treating said population with said herbicide.

14. A method of claim 13 wherein said herbicide comprises a glyphosate, dicamba, or glufosinate compound.

15. A method of claim 12 wherein said selecting is effected by identifying plants with said enhanced trait.

16. A method of claim 12 wherein said seed is corn, soybean, cotton, alfalfa, wheat or rice seed.

17. A method of producing hybrid corn seed comprising:

(a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names consisting of AAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 1 2, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60 TCP1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP, FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro 1, Glyoxalase, Gotl, HATPase c, HSF DNA-bind, HSP20, HisKA, Homeobox, Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 slv, NAF, NAM, NIF, Oxidored FMN, PAS, PDZ, PRA1, Peptidase C15, Peptidase S10, Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1, RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-TF, Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4; wherein the gathering cuttoff for said protein domain families is stated in Table 28;

(b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA;

(c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide;

(d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants;

(e) repeating steps (c) and (d) at least once to produce an inbred corn line;

(f) crossing said inbred corn line with a second corn line to produce hybrid seed.

18. The method of selecting a plant comprising cells of claim 1 wherein an immunoreactive antibody is used to detect the presence of said protein in seed or plant tissue.

19. Anti-counterfeit milled seed having, as an indication of origin, a plant cell of claim 1.

20. A method of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of claim 1 which are selected for enhanced water use efficiency.

21. A method of claim 20 comprising providing up to 300 millimeters of ground water during the production of said crop.

22. A method of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of claim 1 which are selected for enhanced nitrogen use efficiency.