WO2010025213A1 - Methods for increasing grain value and compositions thereof - Google Patents

Abstract

The present invention is in the field of plant breeding and commercial production of grain. More specifically, the invention includes methods for obtaining corn plants that produce grain with increased value compared to commodity grain. The invention further provides grain of increased value produced by these methods, as well as methods for handling and treating plants to ensure preferential pollination by pollen containing desirable genes for this purpose.

Description

METHODS FOR INCREASING GRAIN VALUE AND COMPOSITIONS THEREOF

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/092,889 filed on August 29, 2008. The entirety of the application is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention is in the field of plant breeding and grain production. More specifically, the invention includes methods for breeding corn plants containing at least one genetic factor conferring increased economic value to seeds. The invention further provides grain of increased value produced by these methods, and methods for handling and treating plants to ensure preferential pollination by pollen containing desirable genes for this purpose.

BACKGROUND OF THE INVENTION

[0003] Commodity grains are produced for a variety of uses including foodstuffs and food additives for human consumption such as cereal grains and legumes, oils, proteins, starches, sugars and the like; feedgrains and feed additives for animal consumption, such as grain, meal, energy, grain fractionates and the like; or as industrial feedstocks, such as oils, proteins, starches, sugars, pigments, polymers, and other natural products. The economic value of commodity grains is primarily dependent on the amounts and types of starches, oils and proteins. Additional value beyond that offered by commodity grains can be sought by modifying the proportions of various components to increase the yield of those components having greater value in a given market; or by modifying the characteristics of a component to increase nutritional content, functionality, versatility, or efficiency of processing, or to generate novel utility for that component; or by various combinations of such approaches for various components. [0004] Generally, methods for producing seeds having "high value" traits, i.e., traits that confer enhanced economic benefit compared to commodity grains, are complicated and entail special difficulties, conditions or circumstances. For example, in many cases, the genes expressing the desired trait may exhibit xenia, which is the preferential expression of the pollen parent gene in endosperm, seed or fruit. High value traits are also typically associated with reduced plant vigor, grain yield, or seed viability. In other examples, high value traits such as reduced phytate, increased sugar content, and altered starch composition (e.g., ratio of amylose to amylopectin) are conferred by one or more recessive mutations, requiring homozygosity for expression. Further, some high value traits exhibit preferential expression in particular tissues or cell types in the seed, such as endosperm-preferred or embryo-preferred expression.

SUMMARY OF THE INVENTION

[0005] The present disclosure relates to methods and compositions for breeding plants to increase grain value. In various embodiments, the present disclosure provides for methods of obtaining high value grain.

[0006] In one embodiment, a method of the invention for producing high value grain comprises planting at least one plant comprising at least one genetic factor conferring at least one high value trait and growing the at least one plant in a manner to obtain preferential inheritance of the at least one high value trait in the progeny of the plant. The method further comprises harvesting grain from the progeny plant.

[0007] In another embodiment, a method of the invention comprises interplanting a first plant comprising at least one genetic factor conferring at least one high value seed trait with at least one second plant. The method further comprises cultivating the first plant and the at least one second plant to obtain preferential inheritance of the at least one high value seed trait, and harvesting the grain from the plants.

[0008] Further areas of applicability of the present teachings will become apparent from the description provided herein. It should be understood that the description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the present teachings. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0010] FIG. 1 is a listing of exemplary nucleic acids conferring one or more high value grain traits.

[0011] FIG. 2 shows oil, protein and starch content of grain obtained from blends of high oil corn pollinators with elite hybrid grain parents.

[0012] FIG. 3 shows tabulated results of starch fermentation and ethanol production over 54 hr from grain obtained from blends of amylose extender corn pollinators with elite hybrid grain parents.

[0013] FIG. 4 shows tabulated results of starch fermentation and ethanol production over 54 hr from grain obtained from blends of WaxyA corn pollinators with elite hybrid grain parents.

[0014] FIG. 5 shows tabulated results of starch fermentation and ethanol production over 54 hr from grain obtained from blends of High Oil corn pollinators with elite hybrid grain parents.

[0015] FIG. 6 is a graph showing the free lysine content of seed produced by different hybrids comprising a previously reported embryo-expressed transgene that increases lysine biosynthesis.

[0016] FIG. 7 shows the predicted phosphate and phytate content of grain obtained from blends of homozygous lpal/lpal corn pollinators with heterozygous Lpal/lpal elite hybrid grain parents.

[0017] FIG. 8 shows the predicted phosphate and phytate content of grain obtained from a hybrid that is heterozygous for lpal linked to CP4, when the hybrid is treated with glyphosate to eliminate non-CP4 pollen.

[0018] FIG. 9 is a diagrammatic representation of the conventional strategy for obtaining shsh susu kernels on the ears of sweet corn. DETAILED DESCRIPTION

[0019] Unless otherwise noted, the definitions and description below define the methods of the present invention and are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Alberts et al., Molecular Biology of The Cell, 5th Edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer- Verlag: New York, 1991; King et al, A Dictionary of Genetics, 6th ed, Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007. The nomenclature for DNA bases as set forth at 37 CFR § 1.822 is used. [0020] As used herein, an "allele" refers to an alternative sequence at a particular locus; the length of an allele can be as small as 1 nucleotide base, but is typically larger. Allelic sequence can be denoted as nucleic acid sequence or as amino acid sequence that is encoded by the nucleic acid sequence. Alternatively, an allele can be one form of a gene, and may exhibit simple dominant or recessive behavior, or more complex genetic relationships such as incomplete dominance, codominance, conditional dominance, epistasis, or one or more combinations thereof with respect to one or more other allele(s).

[0021] A "locus" is a position on a genomic sequence that is usually found by a point of reference; e.g., a short DNA sequence that is a gene, or part of a gene or intergenic region. The loci of this invention comprise one or more polymorphisms in a population; i.e., alternative alleles present in some individuals.

[0022] As used herein, "marker" means a detectable characteristic that can be used to discriminate between organisms. Examples of such characteristics may include genetic markers, protein composition, protein levels, oil composition, oil levels, carbohydrate composition, carbohydrate levels, fatty acid composition, fatty acid levels, amino acid composition, amino acid levels, biopolymers, pharmaceuticals, starch composition, starch levels, fermentable starch, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics, and agronomic characteristics. As used herein, "genetic marker" means polymorphic nucleic acid sequence or nucleic acid feature.

[0023] As used herein, "genotype" means the genetic component of the phenotype and it can be indirectly characterized using markers or directly characterized by nucleic acid sequencing. Suitable markers include a phenotypic character, a metabolic profile, a genetic marker, or some other type of marker. A genotype may constitute an allele ("haploid genotype") or pair of alleles ("diploid genotype") for at least one genetic marker locus depending on the context. In some embodiments, a genotype may represent a single locus and in others it may represent two or more loci that may be linked and/or unlinked, up to a complete genome-wide set of loci. In another embodiment, the genotype can reflect the sequence of a portion of a chromosome, an entire chromosome, a portion of the genome, and the entire genome. [0024] As used herein, the term "haploid genotype" refers to at least one single allele corresponding to at least one locus.

[0025] As used herein, the term "diploid genotype" refers to at least one pair of alleles corresponding to at least one locus.

[0026] As used herein, the terms "sporophyte" and "sporophytic" refer to the diploid portion of the wild type plant, and/or any diploid cells, tissues and/or organs; and/or the corresponding portion, structures, organs tissues and/or cells of mutants, derivatives and the like. In the wild type plant, certain cells of the sporophyte undergo meiosis to produce the gametophytes. [0027] As used herein, the terms "gametophyte" and "gametophytic" refer to any and/or all wild type pollen grains, egg sacs and/or their progenitor forms, and/or their component cells, that are normally found in the haploid state, i.e., cells and forms following the first or reductive division of meiosis and prior to gamete fusion during fertilization; and the corresponding cells and forms of mutants, derivatives and the like. Because the plants under consideration have highly developed sporophytic forms and much reduced gametic forms, these plants, their parents and their progeny are typically considered to be diploid, referring to their predominant sporophytic forms. Exceptional cases are known in which a plant (sporophyte) may be haploid or partially haploid, or a gamete or gametophyte may be diploid or partially diploid, for example, as the result of one or more mutations, or different forms of reproduction (e.g., certain forms of apomixis).

[0028] As used herein, the term "male gametophyte" refers to the predominantly haploid tissues of the male reproductive structure. This encompasses the microsporocyte starting at the end of first meiosis (meiosis I) through second meiosis (meiosis II), microspores released as a product of meiosis II, the developing microspore through first mitosis, the resulting immature pollen grain comprising a vegetative cell and a generative cell, pollen grains undergoing maturation, which may include a second mitosis to produce two sperm cells from the generative cell (in some species this occurs after germination of the pollen grain), the mature pollen grain, which undergoes germination to produce a pollen tube, and the germinated pollen grain. [0029] As used herein, the term "female gametophyte" refers to the predominantly haploid tissues of the female reproductive structure. This encompasses the megasporocyte starting at the end of meiosis I through meiosis II, megaspores released as a product of meiosis II (of which one per meiosis normally remains viable), and the developing egg sac through multiple mitoses to produce a mature egg sac. A variety of egg sac developmental programs exist; in corn, for example, this entails four mitoses to produce an egg cell, two synergids, a central cell comprising two haploid nuclei, and antipodal cells, which typically proliferate through additional mitoses. Variations in this developmental plan occur in certain mutations of corn, some of which have been used for the production of haploid embryos comprising a genomic complement from either the female parent or the male parent. Note that the sporocytes are considered to be haploid at the end of the reductive division of meiosis I, even though they possess two copies of a haploid genome during meiosis II. Similarly, even though the corn central cell contains two nuclei of the same haploid complement, we consider the entire egg sac to be included when we refer to "predominantly haploid tissues" of the reproductive structures, even if the ploidy of the egg sac or its constituent cells may be considered other than strictly haploid. [0030] As used herein, the term "apomixis" means asexual reproduction through floral structures, with or without fertilization of a central cell, to produce progeny having an essentially identical genotype to the sporophyte of the female floral parent.

[0031] As used herein, "doubled haploid" refers to a diploid plant, embryo, plant tissue or plant cell obtained from at least one cell comprising a haploid genome, via doubling of the haploid genome by spontaneous or induced means. The doubling process typically follows sexual reproduction with a haploid inducer line, in which only one genome is inherited and stably maintained in the zygote. In different systems, either the maternal or paternal haploid genome may be stably inherited, then doubled, to give the genetic equivalent of an inbred diploid progeny.

[0032] As used herein, "phenotype" means the detectable characteristics of a cell or organism which can be influenced by gene expression. [0033] As used herein, the term "homozygous" means having the same allele of a gene at the corresponding locus on each chromosome of the pair in the diploid state. [0034] As used herein, the term "heterozygous" means having different alleles of a gene at the corresponding locus on each chromosome of the pair in the diploid state.

[0035] As used herein, the term "hemizygous" means having an allele of a gene at a given locus on one chromosome in the diploid state, for which there is no corresponding locus on the other chromosome of the pair.

[0036] As used herein, "linkage" refers to the relative frequency at which types of gametes are produced in a cross. For example, if locus A has alleles "A" or "a" and locus B has alleles "B" or "b," a cross between parent I of genotype AaBb and parent J of genotype AaBb will produce four possible gametes in each parent, in which the alleles will segregate to give four gametic classes corresponding to the haploid genotypes AB, Ab, aB and ab. The null expectation is that there will be independent equal segregation into each of the four possible haploid genotypes, i.e., with no linkage, ¹A of the gametes will be of each haploid genotype. Segregation of gametes into haploid genotypes differing in frequency from ¹A may be attributed at least in part to linkage. Complete linkage is expected to result in cosegregation of linked markers. In the above example, if locus A and locus B are completely (or tightly) linked, and if allele A is on the same chromosome as allele b in each parent, so that allele a is on the same chromosome as allele B in each parent, then only two gametes are predicted, namely Ab and aB, each with a predicted frequency of Vi. Incomplete linkage will result in the appearance of recombinant gametes AB and ab as minority classes, depending on the genetic distance between locus A and locus B, typically indicated by the percentage of recombinants observed, and corresponding to the genetic distance in centimorgans (cM).

[0037] As used herein, the term "skewed allelic ratio" refers to any inheritance of different allelic compositions in a ratio that differs from expected for Mendelian inheritance of strictly controlled crosses, after taking into account the mechanism of reproduction, any linkage effects, allelic stability, etc. An uncontrolled pollination, or a partially controlled pollination, in which allelic ratios in progeny are not fixed or cannot be calculated in a straightforward way would be likely to result in a skewed allelic ratio in the progeny, dependent on factors such as: the proportions of different pollinator classes present; overlap of pollen shed with silking of the seed parent which may entail delayed planting of the male or female population, or seed coatings, treatments, encrustments having the effect of altering heat units to germination or emergence, or physical parameters or planting systems that alter time to germination or emergence; pollen viability, function and compatibility with silks; and environmental conditions. For controlled crosses, in normal sexual reproduction, non-Mendelian progeny ratios would be indicative of skewed allelic ratios. Using the example from the definition for "linkage" above, if gametes Ab and aB are expected in equal frequencies due to linkage, the predicted progeny classes for Mendelian inheritance of completely linked markers would be AAbb, AaBb, and aaBB in a 1:2:1 genotypic (and phenotypic) ratio. If the progeny obtained were uniformly AAbb, it would imply gamete inheritance of 100% Ab, an extreme form of skewing. The absence of aB gamete inheritance does not necessarily indicate that the aB gamete is absent per se, only that it is not inherited. For other forms of reproduction, demonstration of a difference between the typical ratio of progeny classes obtained in control experiments and the ratio observed in the system under consideration may indicate a skewed allelic ratio. The allelic ratio, determined from progeny allelic frequencies, is not necessarily a function of the number of viable gametes of each class present, since some classes of gametes may not be inherited as efficiently as others in a given system. [0038] As used herein, the term "preferential inheritance" indicates a higher frequency of inheritance of an allele or haploid genotype than predicted. The higher frequency of inheritance may occur generally, or may occur only under specific conditions. Preferential inheritance of gametes can result in skewed allelic ratios.

[0039] As used herein, the term "progeny" refers to any plant, plant embryo or seed produced through flowering or in vitro from floral organs. This includes plants produced through sexual reproduction, as well as through apomixis, whether or not pollination occurs, or through aberrant or defective sexual reproduction. It also includes plants produced through anther culture or from flowers or inflorescences cultured or generated in vitro. For example, certain mutations may result in the survival of seed possessing haploid embryos (progeny) of either maternal or paternal origin, which may be recovered and caused to double in genome content to the diploid state. [0040] As used herein, the term "elite" means resulting from breeding and selection for superior agronomic performance, and can refer to any plant or collection of plants having undergone such breeding and selection, whether inbred, hybrid, variety, line or population of plants. An elite plant is any plant from an elite line. [0041] As used herein, the term "partially isogenic" means up to 50% isogenic. [0042] As used herein, the term "substantially isogenic" means greater than 50% isogenic. [0043] As used herein, the term "inbred" means a line that has been bred for genetic homogeneity.

[0044] As used herein, the term "hybrid" means a progeny of mating between at least two genetically dissimilar parents. Without limitation, examples of mating schemes include single crosses, modified single cross, double modified single cross, three-way cross, modified three- way cross, and double cross wherein at least one parent in a modified cross is the progeny of a cross between sister lines.

[0045] As used herein, the term "corn" means Zea mays or maize and includes all plant varieties that can be bred with corn, including wild maize species. In general, the compositions and methods of the present invention can be used with corn plants from the genus Zea. More specifically, corn plants from the species Zea mays and the subspecies Zea mays L. ssp. Mays can be bred using these compositions and methods. In an additional aspect, the corn plant is from the group Zea mays L. subsp. mays Indentata, otherwise known as dent corn. In another aspect, the corn plant is from the group Zea mays L. subsp. mays Indurata, otherwise known as flint corn. In another aspect, the corn plant is from the group Zea mays L. subsp. mays Saccharata, otherwise known as sweet corn. In another aspect, the corn plant is from the group Zea mays L. subsp. mays Amylacea, otherwise known as flour corn. In a further aspect, the corn plant is from the group Zea mays L. subsp. mays Everta, otherwise known as pop corn. Zea or corn plants that can be used with the compositions and methods described herein include hybrids, inbreds, partial inbreds, or members of defined or undefined populations. [0046] As used herein, the term "xenia" refers to the expression in endosperm of a trait encoded by the pollen grain that fertilized that kernel. Some endosperm traits may not exhibit xenia, that is, their expression may be dictated by the genotype of the female parent only. [0047] As used herein, the term "fertilization" encompasses pollen tube interaction with the pistil; pollen tube sensing and/or responding to synergid signaling; pollen tube interaction with synergid; sperm cell transfer into the egg sac; sperm cell movement in the egg sac; sperm cell interaction with synergid, egg cell, and/or central cell; and fertilization of egg cell and/or central cell by sperm cell(s). [0048] As used herein, the term "male-sterile" means failure of the male gametophyte to produce viable and functional pollen grains. Partial sterility refers to reduced functionality and/or numbers of functional pollen grains.

[0049] As used herein, the term "female-sterile" means failure of the female gametophyte to produce viable and functional egg sacs. Partial sterility refers to reduced functionality and/or numbers of functional egg sacs.

[0050] As used herein, a "nucleic acid sequence" comprises a contiguous region of nucleotides of DNA or RNA.

[0051] As used herein, an "endogenous nucleic acid sequence" is a nucleic acid sequence that is native to a species.

[0052] As used herein, an "exogenous nucleic acid sequence" is a nucleic acid sequence that is non-native to a species.

[0053] As used herein, the term "transgene" means a nucleic acid molecule in form of DNA, such as cDNA or genomic DNA, or RNA, such as mRNA or microRNA, which may be single or double stranded, and which has been introduced into an organism. By this definition, a transgene may be, but is not necessarily, integrated as DNA into a chromosome or stably maintained in a cell or host organism.

[0054] As used herein, the term "genetic factor" can refer to a nucleic acid of interest, genetic marker, a gene, a portion of a gene, a DNA-derived sequence, a haplotype, a RNA-derived sequence, a promoter, a 5' untranslated region of a gene, a 3' untranslated region of a gene, microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, ds mRNA, a transcriptional profile, a methylation pattern, and the presence, absence, or variation in copy number of any of the preceding.

[0055] As used herein, the term "nucleic acid of interest" can refer to any nucleic acid in any form known to the art that can appropriately impact at least one trait of interest, when present in an appropriate plant, plant cell, or plant vector. According to the known art, a nucleic acid of interest may comprise endogenous or exogenous nucleic acid sequence. In other aspects, a nucleic acid of interest may comprise single-stranded or double stranded DNA or RNA. A nucleic acid of interest may occur in the nucleus in the hemizygous, heterozygous or homozygous state, as part of a chromosome, artificial chromosome or B-chromosome, in single or multiple copies per locus. A nucleic acid of interest may occur as part of organellar DNA, or an existing extrachromosomal DNA or RNA element, or it may replicate autonomously. A nucleic acid of interest may be transiently maintained and lost or modified after some period of time or through a number of cell divisions or plant generations, by failure to replicate or failure to be partitioned to daughter cells properly or completely, or by recombination, excision, splicing or the like. A nucleic acid of interest may comprise one or more viral sequence(s), replicate as a virus, comprise a helper virus capable of complementing a defective virus, or comprise a defective virus. A nucleic acid of interest may move or be translocated from one plant cell to another plant cell. A nucleic acid of interest may encode or be associated with one or more proteins that function in its integration, replication, expression and/or cell-to-cell transport. A nucleic acid of interest may be copied, transcribed and/or reverse transcribed into a form that integrates into another nucleic acid, replicates autonomously or virally, is transmitted from cell to cell, or is transiently maintained. The manner, copy number, form and permanence with which it is maintained may directly or indirectly impact how it affects the trait of interest, as well as the expected frequency with which it is inherited.

[0056] A nucleic acid of interest may comprise or encode a regulatory nucleic acid sequence, thereby affecting a trait of interest. A nucleic acid of interest may comprise or encode RNA capable of catalytic function, binding to a target protein or nucleic acid, silencing other genes, splicing or self-splicing activity, guide RNA activity, or the like, thereby affecting a trait of interest. A nucleic acid of interest may encode a protein of interest, for example a protein having enzymatic, regulatory, structural, transport, osmotic, pH, electrochemical, redox or permeability function, or the like, or altering the function of other proteins, or being capable of binding nucleic acids, other proteins, or other molecules, thereby affecting a trait of interest. [0057] The functional component(s), genes, and gene products of the nucleic acid of interest may interact with one or more endogenous functional elements, genes or gene products, pathways, or networks, linked or unlinked to the nucleic acid of interest, to produce the effect on the trait of interest. The nucleic acid of interest may encode at least one recessive gene affecting the trait of interest that may be preferentially made homozygous by the method described herein. [0058] The nucleic acid of interest may encode a gene being expressed in a manner that may be constitutive, tissue-preferred, tissue-specific, developmentally regulated, induced by one or more factors or conditions, silenced, genotype-dependent or one or more combinations thereof. [0059] As used herein, the term "commodity seed" means any seed produced as a commodity, such as a grain or an oilseed, that is not valued at or sold for a premium over commodity prices. As such, the seed has properties that generally conform to at least one commercial grade or category as established or generally recognized for that commodity. The seed may be produced directly for commercial use; or to be marketed for sale, for example via a commodities market; or to fulfill a contractual agreement; or according to any other arrangement or series of arrangements.

[0060] As used herein, the term "high value seed" means any seed produced directly for commercial use; or to be marketed for sale, for example via a commodities market; or to fulfill a contractual agreement; or according to any other arrangement; and which is valued at or sold for a premium over commodity prices, generally because of the presence of at least one high value trait not typically associated with commodity seed. The present invention provides seed comprising at least one high value trait.

[0061] As used herein, the term "comprising" means "including but not limited to". [0062] According to the present invention, Applicants have discovered methods and compositions for producing progeny plants comprising at least one high value trait by inter- planting at least two elite populations of plants. In a preferred embodiment, the parental plants reproduce sexually to give diploid progeny containing haploid genomic contributions from each parent. It is anticipated that one or both parents can be either fully fertile or partially sterile. Thus the parent contributing the male gamete to the zygote can be fully fertile, partially male-sterile, partially or fully female- sterile, or any combination of partially or fully female- sterile and partially male-sterile. Similarly, the parent contributing the female gamete to the zygote can be fully fertile, partially or fully male-sterile, partially female-sterile, or any combination of partially or fully male-sterile and partially female-sterile. Moreover, fertility and sterility can be constitutive, inducible or repressible. The preferentially inherited genotype can be inherited from either the maternal parent (egg cell parent) or paternal parent (pollen parent. The preferentially inherited genotype can thus be maternal or paternal in origin, which can affect expression in the zygote and/or developing embryo, depending on whether the gene of interest exhibits any maternal or paternal influence on expression. The origin of the preferentially inherited genotype will also determine its dosage in the triploid endosperm (two doses for a maternally inherited genotype; one dose for a paternally inherited genotype) and its expression characteristics, depending on whether the gene of interest exhibits any maternal or paternal influence on expression. For example, xenia refers to preferential expression of a maternally inherited gene in the developing seed.

[0063] In a preferred embodiment, the parental plants reproduce sexually to give diploid progeny containing haploid genomic contributions from each parent. It is anticipated that one or both parents can be either fully fertile or partially sterile. Thus the parent contributing the male gamete to the zygote can be fully fertile, partially male- sterile, partially or fully female- sterile, or any combination of partially or fully female- sterile and partially male-sterile. Similarly, the parent contributing the female gamete to the zygote can be fully fertile, partially or fully male- sterile, partially female-sterile, or any combination of partially or fully male-sterile and partially female- sterile. Moreover, fertility and sterility can be constitutive, inducible, repressible or reversible. The preferentially inherited genotype can be inherited from either the maternal parent (egg cell parent) or paternal parent (pollen parent). The preferentially inherited genotype can thus be maternal or paternal in origin, which can affect expression in the zygote and/or developing embryo, depending on whether the gene of interest exhibits any maternal or paternal influence on expression. The origin of the preferentially inherited genotype will also determine its dosage in the triploid endosperm (typically two doses for a maternally inherited genotype; one dose for a paternally inherited genotype) and its expression characteristics, depending on whether the gene of interest exhibits any maternal or paternal influence on expression. For example, xenia effects, which refer to preferential expression of a paternally inherited gene in the developing seed, will typically dictate that the high value gene be transmitted through pollen. [0064] In another embodiment, the parental plants reproduce sexually, but the progeny do not receive or do not stably maintain a haploid genomic contribution from one of the parents. This can occur, for example in the progeny of certain mutants of corn. The progeny kernels can thus be haploid and contain a genetic contribution from either the maternal plant or the paternal plant, depending on the particular system. In one aspect, the haploid progeny further undergo haploid doubling, either spontaneously or by any of a number of treatments known to the art. In this instance, the resulting progeny are the equivalent of an inbred diploid line, and are referred to as "doubled haploid" plants. It is therefore possible to obtain a preferentially inherited genotype of interest in the homozygous state in a single generation. It is anticipated that one or both parents can be either fully fertile or partially, inducibly, repressibly or reversibly sterile, by means described herein.

[0065] In yet another embodiment, the parental plants reproduce apomictically, i.e., asexually via flowering parts. Apomixis typically results in the production of progeny embryos genetically identical to the maternal plant. In pseudogamous apomixis, the paternal plant contributes genetic material (normally a haploid genome) to the central cell of the egg sac, which gives rise to the endosperm. Normally the endosperm is triploid, inheriting two haploid genomes from the maternal plant and one from the paternal plant. Apomictically produced plants can vary in their endosperm ploidy, depending on the system. It is anticipated that the preferentially inherited genotype including the gene(s) of interest can be contributed to the endosperm by the paternal plant and contribute to the phenotype of the seed, depending on whether the trait exhibits xenia or not. It is anticipated that one or both parents can be either fully fertile or partially, inducibly, repressibly or reversibly sterile, by means described herein.

[0066] The present invention provides methods of increasing the economic value of grain through skewing of the frequencies or ratios of alleles inherited by the grain in favor of those alleles that are associated with at least one high value trait, without substantially compromising agronomics. The invention encompasses the production of grain comprising at least one high value seed trait obtained by inter-planting at least two elite plant populations. It includes methods of obtaining grain by planting at least a first elite plant or elite plant population comprising at least one genetic factor that confers or contributes to at least one high value seed trait, and at least a second elite plant or elite plant population; growing the at least two elite plants or elite plant populations under conditions that result in preferential inheritance of the at least one high value seed trait; and harvesting the grain. The plants of the invention are elite hybrids, inbreds, varieties or populations. The increased economic value of the grain can be realized by using the grain directly, such as for feed, food, a foodstock, or as an industrial feedstock or the source of an industrial feedstock; or it may be realized by marketing or selling the grain in any fashion. In one embodiment, the method further comprises the steps: sorting the grain based on phenotype for the at least one high value trait; and using or selling the high value and commodity seed separately.

[0067] In another embodiment of the invention, the at least one genetic factor encoding at least one high value seed trait is linked to at least one genetic factor conferring preferential inheritance of at least one haploid genotype. For example, the genetic factor conferring preferential inheritance can confer preferential viability or transmission of a gamete in the presence of a chemical agent; it can encode a gametic incompatibility function that enables transmission of the gamete to progeny in certain pollinations; it can confer fertility or partial fertility to a male- sterile or female- sterile plant; or combinations of the aforementioned allelic skewing genetic factors can be linked to at least one gene of interest. In another embodiment, allelic skewing genetic factors are linked singly or in combination to separate high value genes. Moreover, plants of this embodiment can be self pollinated to achieve allelic skewing, or used in blends as described herein, for example, as the high value trait pollinator of a conventional seed parent. [0068] Types of chemical agents that can be used in this approach include gametocides, antibiotics, phytotoxic agents, nutrients, micronutrients, metabolites, metabolic intermediates, biochemical pathway starting compounds or products, vitamins, cofactors, pesticides, safeners, herbicides or formulations thereof; or other chemicals, compounds, elements, or salts thereof. Herbicides or formulations thereof that can be used in this approach include imidazolinones, sulfonylureas, glufosinate, phosphinothricin, bialaphos, auxins, synthetic auxins, or glyphosate. [0069] In another preferred embodiment of the invention, the conditions giving rise to preferential inheritance of at least one haploid genotype comprise crossing between lines that exhibit self-incompatibility. In a particularly preferred embodiment of the invention, the conditions giving rise to preferential inheritance of at least one haploid genotype comprise crossing between lines that exhibit gametophytic self-incompatibility. Gametophytic self- incompatibility functions applicable to this approach include, for example, the corn Gametophytic 1-S (GaI-S) gene (U.S. Patent No. 6,875,905, U.S. Patent Application US20020104115A1 and U.S. Patent Application US20050198714A1, each of which is incorporated herein by reference in their entirety) or functionally equivalent homolog. In this system, pollen comprising the GaI-S genetic factor is preferentially able to pollinate recipient GaI-S kernels (homozygous or heterozygous) compared to gal pollen, which lacks the GaI-S genetic factor and fails to pollinate GaI-S kernels. Dent corn is homozygous gal/gal, and therefore cannot pollinate GaI-S kernels.

[0070] In one embodiment of the invention, the at least one genetic factor of interest influences at least one kernel-expressed high value trait. In one aspect of the invention, the at least one kernel-expressed trait exhibits xenia. In a preferred embodiment, the at least one kernel- expressed trait is selected from the traits listed in Figure 1. In another preferred embodiment of the invention, the at least one kernel expressed trait is selected from the group consisting of increased oil content, a preferred oil composition, reduced phytate content, increased phosphorous availability, increased protein content, a preferred protein composition, a preferred amino acid composition, waxy traits, a preferred amylose content, a preferred amylopectin content, increased starch content, increased extractable starch, increased fermentable starch, more rapidly fermentable starch, increased soluble carbohydrate digestibility, increased insoluble carbohydrate digestibility, increased sugar content and altered kernel morphology. [0071] In one embodiment of the invention, the transgenic elements conferring increased total lysine in whole grain are provided in U.S. Patent No. 5,258,300, U.S. Patent No. 5,484,956, U.S. Patent No. 5,773,691, U.S. Patent No. 6,459,019, U.S. Patent No. 6,329,574, U.S. Patent No. 7,297,847 and U.S. Patent Application Pub. No. 20070192896, which are all incorporated herein by reference in their entirety. In a preferred embodiment, the present invention comprises grain in which the increased level of total lysine in whole kernels is conferred by a transgene encoding expression of LY038 in grain, as disclosed in U.S. Patent No. 7,157,281, which is incorporated herein by reference in its entirety. In a preferred embodiment, grain of the present invention is corn in which the level of total lysine in whole kernels is increased by at least about 10%, 20%, 30%, 40%, 50% or 55%. In another preferred embodiment, grain of the present invention is corn in which the level of free lysine in whole kernels is increased by at least about 130% or 40-fold. In another preferred embodiment, grain of the present invention is corn in which the level of total lysine in whole kernels is at least about 2800 ppm, 3000 ppm, 3300 ppm, 3600 ppm, 3800 ppm, or 4000 ppm. In another preferred embodiment, grain of the present invention is corn having a reduced requirement for supplemental lysine of at least about 15%, 30%, 50%, 70% or 85%. In another preferred embodiment, grain of the present invention is corn having no requirement for supplemental lysine.

[0072] In another embodiment of the invention, the transgenic elements conferring increased total tryptophan in whole grain are provided in U.S. Patent No. 7,217,865 and U.S. Patent Application Pub. No. US2003/0213010, Pub. No. US2007/0028321 and Pub. No. US2008/0050506, which are incorporated herein by reference in their entirety. In a preferred embodiment, grain of the present invention is corn in which the level of total tryptophan in whole kernels is increased by at least about 300 ppm. [0073] In another embodiment of the invention, the transgenic elements conferring increased oil content in whole grain are provided in U.S. Patent No. 7,179,956 and U.S. Patent Application Pub. No. US2007/0039069, each of which is incorporated herein by reference in their entirety. In a preferred embodiment, grain of the present invention is corn in which the oil content is increased at least about 2% of kernel dry weight.

[0074] In another embodiment of the invention, the transgenic elements conferring decreased phytic acid content and increased free inorganic phosphate in whole grain are provided in U.S. Patent No. 7,186,817, U.S. Patent No. 7,169,595, U.S. Patent No. 7,317,138, U.S. Patent No. 7,141,717, U.S. Patent No. 7,081,563, U.S. Patent No. 6,303,766, U.S. Patent No. 7,339,091, U.S. Patent No. 5,593,963, U.S. Patent No. 5,770,413 U.S. Patent No. 6,022,846, U.S. Patent No. 6,291,224 and U.S. Patent No. 6,197,561; U.S. Patent Application Pub. No. US2005/0202486, Pub. No. US2002/0102681, Pub. No. US2002/0102682 and Pub. No. US2002/0110884; and International Patent Application Pub. No. WO2006/029296, each of which is incorporated herein by reference in their entirety. In a preferred embodiment, grain of the present invention is corn in which the level of inorganic phosphate is increased by at least about fourfold. In another preferred embodiment, grain of the present invention is corn in which the level of inorganic phosphate is at least about 0.6 mg/g dry weight. In another preferred embodiment, grain of the present invention is corn in which the level of phytic acid is reduced by at least about 30%.

[0075] It is anticipated that a trait of interest can be impacted by more than one genetic factor of interest, and that one or more endogenous genes or certain alleles thereof, linked or unlinked to the genetic factor of interest, may be required to express a trait optimally. In any particular genetic background, a given genetic factor of interest can influence a trait of interest in certain combinations, sometimes with other necessary genes. Thus it is anticipated that the genetic background will be an important consideration for some traits of interest, particularly complex polygenic traits of interest.

[0076] It will be understood by those skilled in the art that various combinations and permutations of the above are anticipated by the current invention, and that the above is not intended to be limiting.

EXAMPLES Example 1. Allelic skewing via plant blends and pollen selection to produce high oil corn.

[0077] High oil corn is obtainable by conventional and transgenic approaches, but yield can be severely impacted. Preserving economically competitive yield while enhancing oil content has proven challenging. In the present example, the use of blends of plants and of pollen selection, as methods for skewing alleles inherited by grain, are shown to be applicable to obtaining grain at competitive yields with sufficient oil content to be economically advantageous. [0078] Seed of two fully fertile commercial hybrids, CV024 x CV023Bt and CVO(BBt x CV175, having an average kernel oil content of 5.0% and 4.5% respectively, on a dry matter basis, were blended with seed of CV176 x BHO C14) (HOIOOl, a hybrid oil donor having an average kernel oil content of 15.0% on a dry matter basis. A total of twelve blends were prepared with various ratios of commercial hybrids to hybrid oil donor. The ratios consisted of the hybrid per se and increasing percentages of hybrid high oil pollinator in 10% increments, up to 50% fertile commercial hybrid with 50% fertile hybrid oil donor in order to shift the ratio of high oil parent pollen to total pollen available at the time of seed parent silking. Increasing the percentage of high oil pollinator is expected to skew allelic ratios represented in the progeny, present as kernels on the ears, and thereby alter the composite kernel phenotype, in this case the average oil content of the grain. This invention further anticipates delayed planting or delayed germination to maximize donor pollen shed during the onset of silking through silk emergence in the grain parent, thus ensuring proper nick. Seed was counted and blended to ensure random placement when planted in the farmer's field using a plateless planter.

[0079] Each blend was planted in 12 row plots 500ft. long (0.35Ac.) and allowed to open pollinate. Whenever possible, a pollinator was chosen that would begin to dehisce slightly ahead of silk appearance and contain enough variability to have pollen available throughout the time that silks were produced by the seed parent hybrid. This further increased the likelihood of pollination by the high oil pollinator, i.e., skewing the ratio of alleles present in the progeny kernels toward those carried by the high oil pollinator. Three samples of grain were harvested from the center two rows and submitted for analysis. The averages across the three samples are presented in Figure 2. [0080] Oil content was intermediate between grain parent and pollinator, and increased linearly by about half a percentage point of kernel dry weight with each 10% increase in oil donor relative to total plant population. These results indicate that economically advantageous oil levels were obtained with male-fertile grain parents both by increasing the high oil pollinator populations and by increasing oil content in the seeds of the high oil pollinator line. Previous embodiments of the art demanded the use of a grain parent which would maintain male sterility. A common method of obtaining male- sterile hybrids known to the art is by use of the C or S cytoplasmic male sterility (cms) systems in the female parent, crossed with a male parent that is non-restoring for fertility. A number of high yielding elite hybrids are not readily converted to male-sterile cms C or S hybrids (i.e., the male parents are not readily converted to non-restorer lines). Such lines are thus not readily adapted to use in systems requiring male sterile hybrids, but they can, by contrast, readily be used in the present invention as illustrated by this example. [0081] These results further have application in the reduction of risk in a male-sterile hybrid high oil grain production system. The different genetic backgrounds of grain parents and pollinators can react differently to stresses throughout the growing season. To reduce this risk, a percentage of isogenic pollinator is included, which will flower and react similarly to the grain parent and still obtain increases in oil concentration, as illustrated in Table 1.

Table 1. Use of 5% fertile grain parent with 5% high oil pollinator in male-sterile hybrid high oil grain production.

Blend Pedigree Use Oil Content

90% CV178SDms x CVlWBt Sterile Grain Parent 5%

5% CV178 x CVlWBt Isogenic Pollinator 5% 5% CV176 x BHO) (HOIOOlRR Oil Donor Pollinator 15%

Harvested Grain: 8%

[0082] The per se oil content predicted for the harvested grain assumes that the male-sterile grain parent does not produce pollen. The pollinator portion of the blend is 50% fertile isogenic pollinator and 50% oil donor pollinator. The presence of the isogenic pollinator helps ensure that pollen will be available in a stressed environment. While it does not guarantee the production of high oil grain in all environments, this approach will produce commercial yields of normal corn and avoid risk of loss.

[0083] The oil content of a high-oil field produced by this method is expected to be 7.5%, the same as the 50:50 blend depicted in Table 1. A higher percentage of the elite commercial hybrid (95%) in the field is expected to translate into higher yield potential compared to current production methods of high oil grain (90%).

[0084] In another embodiment of the invention, the blend comprises a high oil pollinator and isogenic pollinator in a more advantageous ratio for production of high oil grain (Table 2). In this blend, 90% of the pollinator is oil donor and 10% is isogenic pollinator. It is predicted the oil content will be increased to about 9.5% of kernel dry weight, since for every 10% increase in pollinator, oil content increases 0.5% of kernel dry weight.

Table 2. Use of 1% fertile grain parent with 9% high oil pollinator in male-sterile hybrid high oil grain production.

Per se Oil

Blend Pedigree Use Content

90% CV178SDms x CV177Bt Sterile Grain Parent 5.0% actual 1% CV178 x CV177Bt Isogenic Pollinator 5.0% actual 9% CV176 x BHO) (HOIOOlRR Oil Donor Pollinator 15.0% actual

Harvested Grain: 9.0% expected [0085] For comparison purposes, a depiction of Cytoplasmic Sterile Grain Parent production without the use of an isogenic pollinator is provided in Table 3.

Table 3. Male-sterile hybrid high oil grain production without fertile grain parent.

Per se Oil

Blend Pedigree Use Content

90% CV178SDms x CVlWBtI Sterile Grain Parent 5.0% actual 10% CV176 x BHO) (HOIOOlRR Oil Donor Pollinator 15.0% actual

Harvested Grain: 10.0% predicted

[0086] This approach can be applied to any male sterility based high value grain system, not just CMS. Some pollen control systems known to the art can be incorporated into high value grain production to provide greater flexibility. For example, pollen control systems that are dominant constitutively sterile but reversible to fertility, described in U.S. Patent No. 5,962,769 and US Patent Application 20020129399A1, each of which is incorporated herein by reference in its entirety, can be used instead of CMS in the grain parent. This approach avoids the problems associated with conversion of male inbreds into non-CMS -restorer lines, and adds the option of treating the field to confer male fertility in the grain parent. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

[0087] Notably, partial male sterility systems, e.g. semi- sterility, shedders or leaky male sterility systems, and chemical treatments such as with glyphosate as described below, can be used beneficially in the system, since reduced pollen shed by the seed parent, as well as reduced shed of undesirable pollen from the pollinator line, directly contribute to ensuring successful competition by pollen comprising the high value genetic factor from the high value pollinator line. This distinguishes the present invention from the prior art, in which male sterility of non- pollinator elite plants is specified. The plant blends and other allele skewing methods described herein can be applied in conjunction with any of the male sterility, incomplete male sterility, partial male sterility, conditional male sterility, reversible male sterility or inducible male sterility options known to the art, for a wide range of endogenous or transgenic high value traits, to provide maximum flexibility in obtaining the highest value of grain under various environmental and economic situations, while safeguarding yield. [0088] In another embodiment of the invention, at least one locus associated with high oil is linked (e.g., within 15 centimorgans) to a genetic factor that confers preferential transmission through pollen in the presence of a gametocide, antibiotic, phytotoxic agent, chemical, compound, element, salt of a chemical, compound or element, nutrient, micronutrient, metabolite, metabolic intermediate, biochemical pathway starting compound or product, vitamin, cofactor, pesticide, safener, inducer, inhibitor, herbicide or formulation thereof. In a preferred embodiment of the invention, the genetic factor confers resistance to glyphosate in plants, including in pollen grains. This can be accomplished with an existing nucleic acid construct described in U.S. Patent No. 7,314,970 and U.S. Patent No. 6,762,344, each of which is incorporated herein by reference in its entirety. This construct, designated CP4, encodes a 5- enolpyruvoylshikimate-3-phosphate synthase (EPSPS) that is expressed and is functional in the plant, including in pollen, in the presence of the herbicide glyphosate. When glyphosate is applied to the field at the V8 to V 12 stage, which is before pollen shed, developing pollen grains that inherit the CP4 transgene will survive, while developing pollen grains that do not inherit the CP4 transgene will be killed. This will result in pollination of plants in the field preferentially or exclusively by the CP4 transgenic pollen grains. With CP4 linked to the at least one high oil locus, the pollen that preferentially survives will be predominantly high oil, with the minority of CP4 transgenic pollen grains lacking the high oil locus arising by meiotic recombination between CP4 and the linked high oil locus.

[0089] One way to obtain a CP4 transgenic event having linkage to at least one locus associated with high oil is to utilize current transformation technologies and to identify a CP4 event that is both functional and in linkage with a locus of interest. For illustrative purposes, the high oil locus of interest is the HOIOOl GBSS allele located on the short arm of chromosome 9, described in U.S. Patent No. 7,179,956, and an event is identified which inserts a functional CP4 transgene 5 cM from the HOIOOl GBSS gene. This hemizygous line can be self pollinated and its progeny screened to identify lines homozygous for CP4.

[0090] Through the use of traditional backcrossing or marker-assisted backcrossing, lines homozygous for both the CP4 gene and HOIOOl GBSS can be isolated. These lines can be used as pollinators of a female line in a production field to produce hybrid seed that is heterozygous HOIOOl GBSS. [0091] The hybrid plants produced from the hybrid seed in a producer's field will have a desired phenotype because they will be heterozygous for HOIOOl GBSS. Glyphosate is applied at about the V8 to V12 growth stage, killing nontransgenic developing pollen grains and allowing only pollen grains containing the CP4 gene to survive. Since the CP4 gene is only 5 cM from the HOIOOl GBSS locus and in coupling phase with the HOIOOl GBSS allele, 95% of all pollen shed will carry the HOIOOl GBSS allele while only 5% will carry the wild type allele. The 5% CP4, non-HOIOOl GBSS pollen grains are due to meiotic crossing over between the CP4 locus and the HOIOOl GBSS locus. The female gametes will segregate 1:1 for HOIOOl GBSS : non- HOIOOl GBSS, with linkage to CP4 being immaterial because no selective pressure is exerted on the female gametes. Therefore the progeny grain are predicted to segregate 47.5% homozygous HOIOOl GBSS, 50% heterozygous and 2.5% homozygous for non-HOIOOl GBSS. By comparison, in the absence of glyphosate-resistant pollen selection, the segregation ratio in the kernels is expected to be 25% : 50% : 25%, or 1:2:1.

[0092] In yet another embodiment of the invention, the above hybrid with CP4 linked to HOIOOl GBSS is used as a pollinator in a blend, in which the grain parent hybrid is optionally heterozygous for HOIOOl GBSS and comprises a gene for glyphosate resistant EPSPS that confers resistance to glyphosate in plants but not in pollen (not necessarily linked to HOIOOl GBSS). Glyphosate resistance selection of pollen produced by the pollinator line is carried out by spraying with glyphosate at about V8 to V12 stage. The female gametes of the grain parent will segregate 1:1 HOIOOl GBSS : non-HOIOOl GBSS and approximately 95% of the pollen will be HOIOOl GBSS. The kernels formed will segregate in the same ratios as given above. The advantage over using just the hybrid that is heterozygous HOIOOl GBSS linked to the CP4 transgene (with glyphosate resistance pollen selection) is that the grain parent can be a high yielding elite line, such as a hybrid, and the pollinator can be a heavy pollen shedding line. In addition, the blend can offer greater flexibility to the farmer. For example, the grain parent can optionally be non-HOIOOl GBSS, and it can comprise transgenes in addition to non-CP4 glyphosate resistant EPSPS, adding further flexibility to the system.

[0093] In yet another embodiment, the blends include a male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a dominant, reversibly male-sterile line, transgenic for CP4; the HOIOOl GBSS CP4 pollinator; and a male- fertile pollinator that is isogenic to the grain hybrid, comprising the CP4 transgene, to ensure high yield under environmental conditions in which the HOIOOl GBSS CP4 pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

[0094] In another embodiment of the invention, at least one corn plant comprises at least one transgene comprising allele HOIOOl GBSS, described in U.S. Patent No. 7,179,956, that confers high oil content in seeds. Allelic skewing can be used to maximize the flexibility of the system in obtaining a high yield of high oil grain. The at least one transgenic line can be hemizygous or homozygous for the high oil transgene(s) and linked or unlinked to a pollen selection marker to provide opportunities for and flexibility in farmer choices. The at least one transgenic line thus obtained can be used to produce at least one high oil grain hybrid or blend. Used as a grain hybrid homozygous for HOIOOl GBSS, the system would require engineering of both parents as transgenic for HOIOOl GBSS, and yield of the hybrid is not certain to be competitive. Thus, although the homozygous transgenic hybrid can be used as a high oil hybrid, using a hybrid in the hemizygous state for the HOIOOl GBSS transgene would require only one HOIOOl GBSS transgenic parent, and the transgenic hybrid would be more likely to have suitable agronomic characteristics and yield. Such hybrids can potentially be used as high oil hybrids per se, or as grain parents in blends with other pollinator lines. Moreover, hemizygous or homozygous transgenic hybrids, inbreds, lines and populations comprising the HOIOOl GBSS transgene can be used as high oil pollinator lines in blends with at least a grain parent.

[0095] Linkage to a pollen-selectable marker such as CP4 further extends options to include the use of selecting pollen grains transgenic for the high oil transgene. This can be useful when homozygous or hemizygous high oil plants are present in blends with at least one plant that is transgenic for glyphosate resistant EPSPS that confers resistance to glyphosate in plants but not in their pollen. In one embodiment of the invention, the HOIOOl GBSS CP4 transgenic pollinator is used in a blend with a glyphosate resistant transgenic (non-CP4) grain parent that produces glyphosate sensitive pollen. In another embodiment, the blend includes a male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a dominant, reversibly male-sterile line, transgenic for CP4; the transgenic HOIOOl GBSS CP4 pollinator; and a male-fertile pollinator that is isogenic to the grain hybrid, comprising the CP4 transgene, to ensure high yield under environmental conditions in which the HOIOOl GBSS CP4 pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field. [0096] In another embodiment, the at least one HOIOOl GBSS CP4 plant or plant population is used as a high oil line or hybrid per se, without any other seed parent or pollinator. In the case where the HOIOOl GBSS CP4 transgenic is hemizygous for the linked transgenes, herbicide resistant pollen selection is useful to remove segregating nontransgenic pollen, ensuring that essentially all kernels are pollinated with HOIOOl GBSS transgenic pollen and therefore express a higher oil phenotype on average than would be obtained in the absence of glyphosate herbicide resistance selection of pollen.

[0097] In yet another embodiment, both the grain parent and the high oil pollinator line comprise the HOIOOl GBSS allele or transgene, linked to CP4, so that all plants shed pollen, that essentially all comprises HOIOOl GBSS allele or transgene, resulting in essentially all kernels being pollinated with pollen conferring the high oil trait. The advantage of this arrangement compared to using just a high oil line as the grain parent undergoing self pollination is that it allows a high yielding grain parent to be combined with a heavy pollen- shedding pollinator. [0098] In the above embodiments involving the HOIOOl GBSS transgene linked to CP4, glyphosate is applied to the field at the V8 to V12 stage, so that developing pollen grains that inherit the CP4 transgene will survive, while developing pollen grains that do not inherit the CP4 transgene will be killed. This will permit the CP4 transgenic pollen grains to preferentially survive and pollinate plants in the field. In the case of the sole pollinator line being CP4 linked to the at least one high oil transgene (HOIOOl GBSS CP4), the pollen that preferentially survives will be transgenic for HOIOOl GBSS CP4. In the maternal gametes segregating 1:1 HOIOOl GBSS CP4 : nontransgenic (nt), the kernels will segregate very close to Vi HOIOOl GBSS CP4/nt and !Z₂ HOIOOl GBSS CP4/HOI001 GBSS CP4. In the absence of glyphosate herbicide resistance selection of pollen, the kernels will segregate ¹A HOIOOl GBSS CP4/HOI001 GBSS CP4, Vi HOIOOl GBSS CP4/nt and ¹A nt/nt. The glyphosate herbicide resistance selection of pollen thus results in a ratio of progeny kernels that is favorable to increased oil content. [0099] In yet another embodiment of the invention, at least one additional transgene contributing to or conferring at least one high value trait discussed in the subsequent examples is present in the transgenic insertion or through a separate insertion that is linked. In yet another embodiment of the invention, transgenic and endogenous high value traits can be stacked in a single plant, either linked or unlinked, by methods known in the art. In yet another embodiment of the invention, different high value traits, different endogenous genes, alleles or transgenes contributing to the same high value traits, or the same genes, alleles or transgenes can be present in both the pollinator and the seed parent.

Example 2: Modifying starch characteristics to improve fermentation kinetics.

[00100] Starch mutants of crop plants, particularly in corn, are well-known for kernel morphology changes accompanied by poor germination and lack of seedling vigor. They often exhibit altered kernel starch grain size and content. Smaller starch grains may be more readily fermented to ethanol than wild type starch grains. Thus, even if they are lower in overall starch content, such mutants may provide as much ethanol during fermentation as wild type corn; and the conversion to ethanol may occur more quickly, providing an economic incentive to devise methods for producing grain with altered starch characteristics at competitive yields. The present example provides methods to produce grain from corn blends and teaches the use of pollen selection as another useful allele- skewing methodology.

[00101] Ethanol production plants use a fermentation process based on a 54 hour time frame. Although starch is the main component converted to sugars and alcohol, High Extractable Starch hybrids (HES) have generally not been the best ethanol, or High Fermentable Starch (HFS) producers, in the 54 hour time frame. It is postulated that hybrids having smaller starch granule size provide greater surface area exposure and therefore can be converted to ethanol at a faster rate, resulting in resource savings for ethanol production facilities. Smaller starch granules are expected to have a greater ratio of surface area to volume than larger starch granules, and to have greater potential for enzymatic breakdown at an earlier stage in the fermentation process. Smaller starch granules are also expected to undergo hydrolysis at lower temperature, thus being more responsive to cold cook applications. [00102] The effect of starch composition on ethanol fermentation kinetics was evaluated by crossing hybrids as grain parents with materials known to have smaller starch granule size (i.e., as blends, in a manner similar to that described in Example 1). Fermentation rates were monitored at two or more time points. Grain from specific blends produced as much as 13% more ethanol in a 24 hour time frame compared to grain from the normal counterparts, and the available starch was completely converted to ethanol between 24 and 48 hrs. Increases in fermentation speed can greatly enhance ethanol plant production efficiency, providing increased market value to grain capable of being fermented more quickly.

[00103] Four hybrids with known fermentability and extractability were sib mated within a row by hand to estimate what would occur in a farmer's field. (Table 4).

Table 4. Grain parent hybrids used in blends with pollinators having altered starch characteristics

Pedigree Extractability Fermentability

CV179 x CV023 Low Low

CV014 x CV006 High Low

CV007 x CV180 High High

CV024 x CV008 Low Med

[00104] Mutants that express differences in starch granule size such as amylose extender (ae) and others such as waxy A (wxA) were used as pollen donors onto each of the hybrids. These were also handled as above. In addition, each hybrid was crossed with each other hybrid to determine if hybrid interactions through a xenia effect could exist for fermentability and handled in the same manner as listed above.

[00105] All processed grain samples were evaluated for proximate analysis and fermentation. Results are presented in Figures 3 and 4. Proximate analyses of oil, protein, starch, moisture, extractable starch and ethanol production were conducted on grain samples before grinding (in bold, Figures 3-4). The comparison hybrids are listed in each table. Crosses with a pollen donor contain "++NAME OF DONOR" in the pedigree. For example, in Figure 3, the hybrids with pollen donor CVl 81 amylose extender contain "++CV181-REC-AE" in the pedigree. [00106] In Figure 3, amylose extender (ae) has been used as a pollen donor to study the effect of starch granule size on ethanol fermentation kinetics. It has been documented that ae materials contain very small starch granule size. Amylose extender functions by eliminating starch branching to create a higher percentage of linear chain starch.

[00107] Homozygous ae hybrids are grown in the marketplace but are generally 35% lower yielding than commercial hybrids. Heterozygous ae hybrids, on the other hand, exhibit less yield reduction and so were consequently used to minimize this adverse effect on yield. Starch granules of amylose (homozygous ae) have lower swelling capacity, possibly making them more amenable to breakdown.

[00108] Proximate analyses of crosses containing ae were quite similar to their normal counterparts in oil and starch components. Hybrids containing ae were predicted to be 1.42% higher in ethanol production as seen in the MC-54%DMB column.

[00109] In the 24 hour time period, grain from all ae crosses was 12.48% higher in fermentation. Available glucose was reduced by 29.73%. These results indicate that grain from crosses of germplasm having smaller starch granule size were more rapidly converted to ethanol. In the 48 hour time period, grain from ae crosses was still slightly higher in fermentation (2.32%) compared to grain from their normal counterparts but available glucose was lower. In the 54 hour time frame, grain from ae hybrids offered slightly more ethanol (0.79%). Proximate analysis predicted an increase of 1.42%. Thus, crosses containing ae fermented faster but they also produced more ethanol.

[00110] Figure 4 shows results for conversions of OH43 to waxy A (wxA). This variation of the waxy mutant is known to have a different pasting curve and is much more resistant to thermal breakdown. Pasting curves of starch derived from this naturally occurring mutant rival that of chemically treated starches. Traditional iodine testing of this mutant yields a reaction somewhat in between waxy and normal.

[00111] Grain from OH43wxA crosses exhibited slightly higher oil content and starch content (Figure 4). In the 24 hour time frame, ethanol production increased 8.38% and available glucose decreased 14.63%. At 48 hours, ethanol production was 1.38% higher and available glucose was 11.88% higher. At 54 hours, ethanol production was quite similar but glucose was much higher (78.45) in wxA crosses. Grain from the wxA crosses have been shown to give more ethanol produced by the 24 hour time point and a tendency to have more glucose present at the 54 hour period than grain from their normal counterparts. [00112] In another embodiment of the invention, hybrids or inbreds comprising at least one endogenous gene or gene mutation that impacts starch fermentability are used to generate hybrids that are transgenic for a herbicide resistance gene that confers resistance to the entire plant including pollen, such as CP4. Many transgenics for CP4 are screened to identify at least one line with CP4 linked (within about 15cMorgans) to the gene or mutation conferring rapidly fermentable starch. The resulting line is then used with glyphosate resistance pollen selection, alone or in blends as described above for transgenic rapidly fermentable starch, CP4 lines. [00113] In another embodiment of the invention, hybrids or inbreds can be generated that are transgenic for at least one gene that impacts starch fermentability. The rapidly fermentable starch hybrid is used as a hybrid in and of itself, typically hemizygous for the transgene in cases where yield is adversely impacted by the transgene in the homozygous state; or as a pollinator in blends as described above. In another embodiment of the invention, the at least one transgene is linked to the CP4 transgene that confers glyphosate resistance to plants including pollen. As a grain hybrid itself, this hybrid is grown and treated at about V8 to V12 with glyphosate. Only transgenic pollen survives, so that the kernels produced by the crop segregate ^xh homozygous transgenic and ^xh hemizygous transgenic. In another embodiment of the invention, the transgenic CP4, rapidly fermentable starch hybrid or inbred is used as a pollinator in blends as described above. All other hybrids or inbreds in the blends carry a glyphosate resistance transgene to ensure that they survive treatment with glyphosate. Glyphosate is applied to the field at about V8 to V12. Pollen grains that inherit the transgene survive, while pollen grains that do not are killed. The transgenic pollen grains that preferentially survive pollinate plants in the field. As in the case of hybrids hemizygous for high oil transgenes, glyphosate resistance pollen selection increases the percentage of progeny kernels having the at least one transgene for the trait of interest, in this case, conferring a rapidly fermentable starch phenotype. [00114] In yet another embodiment of the invention, the hybrid or inbred plants in the blend other than the transgenic rapidly fermentable starch hybrid comprise a glyphosate resistant EPSPS that does not confer resistance in pollen. This ensures that the only pollen surviving glyphosate herbicide resistance selection comprises the CP4 transgene linked to the at least one transgene that impacts starch fermentability, which is produced by the transgenic, rapidly fermentable starch pollinator hybrid. This ensures that every kernel produced in the field contains the transgene conferring rapidly fermentable starch. [00115] In yet another embodiment of the invention, the grain parent and rapidly fermentable starch pollinator line each comprise at least one gene, allele or transgene contributing to the rapidly fermentable starch phenotype. In a preferred embodiment, the at least one gene, allele or transgene in each parent is linked to CP4, such that the only pollen surviving glyphosate herbicide resistance selection comprises the CP4 transgene linked to the at least one gene, allele or transgene that impacts starch fermentability. Glyphosate resistance pollen selection ensures that essentially every kernel produced in the field contains at least one gene allele or transgene conferring rapidly fermentable starch, in addition to the at least one gene, allele or transgene inherited by 50% of the kernels through the female gametes. The advantage of using a blend of rapidly fermentable starch grain parent and rapidly fermentable starch pollinator as opposed to a rapidly fermentable starch grain parent alone, undergoing self pollination, is that one can use a high yielding grain parent with a heavy pollen shedding pollinator.

[00116] In yet another embodiment, the blends include a male-sterile grain parent, such as a CMS line in a non-restorer background, or a dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent; and a rapidly fermentable starch pollinator. This arrangement is to ensure high yield under environmental conditions in which the transgenic, rapidly fermentable starch pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field. [00117] In yet another embodiment, the blends include a glyphosate resistant male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a CP4-transgenic dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent, comprising the CP4 transgene; and a rapidly fermentable starch pollinator that is transgenic for CP4, linked either to an endogenous rapidly fermentable starch allele or a rapidly fermentable starch transgene, to ensure high yield under environmental conditions in which the transgenic, rapidly fermentable starch pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that allows for glyphosate herbicide resistance selection of pollen and mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

[00118] The present example demonstrates that hybrid crossing strategies can be used to shorten the time required to produce ethanol. It has been demonstrated that the 54 hour time frame commonly used by ethanol producers can be reduced to 30 to 36 hours with essentially the same output, potentially greatly improving the efficiency of a functioning ethanol plant. A further aspect of this embodiment of the invention is that the output from the plant, either from the fractionization application or the direct output after fermentation, is further used as the base material for the production of bio-based fuels such as bio-butanol. It is envisioned that this material will be more amenable to the production of bio-based fuels such as bio-butanol than currently used cellulosic fractions and whole plant breakdown.

Example 3: Production of high oil corn with improved starch fermentation kinetics.

[00119] High oil corn is known to have smaller starch grains compared to wild type. This provides an opportunity to obtain two high value grain traits, i.e., high oil and rapidly fermentable starch, from the same grain. The various blends and pollen selection methods are taught as for the previous examples.

[00120] HOI002, which is very high in oil content (close to 20%), also possesses exceptionally small starch granule size. Also, HOI002 is unusual since it creates oil bodies in endosperm that contribute to overall oil content. HOI002 was derived from MGC915E, which has an opaque phenotype. When used as a pollinator, HOI002 and many of its relatives contribute a noticeable effect on grain composition that may indicate large expression effects on both endosperm and embryo. It is of note that HOIOOl is closely related to HOI002.

[00121] Figure 5 shows grain ethanol fermentation results using HOI002 as a pollen donor in fertile blends. It was chosen for the extreme effect it was expected to have. When used as a pollen donor, HOI002 demonstrates large xenia effects and greatly reduces starch accumulation and test weight, while increasing oil expression in the embryo. The comparison hybrids are listed first in Figure 5 and contain ":@#". Crosses containing HOI002 as a pollen donor contain "++HOI002" in the pedigree. [00122] In proximate analyses, crosses containing HOI002 gave kernels 10.25% lower in starch and 23.44% lower in extractable starch, but 8.78% higher in oil content compared to their normal counterparts. Grain from HOI002 crosses was predicted by proximates to be 8.26% lower in ethanol production as seen in the MC-54%DMB column.

[00123] In the 24 hour time period, grain from all HOI002 crosses gave much more fermentation (12.36% average across the 4 hybrids versus 10.91% average of the standard hybrids = 13.30% increase averaged over all hybrids). This was verified by the glucose levels which were reduced by 41.44%, indicating that conversion to ethanol occurred at a faster rate. This result is remarkable considering available starch was much less. These results indicate that the smaller starch granules inherited from HOI002 were more rapidly converted to ethanol than wild type starch granules. Coupled with the results from Example 2, they also indicate that modifications of starch granule size occurred through a number of different approaches. [00124] In the 48 hour time period, grain from HOI002 crosses gave 1.64% lower fermentation compared to their normal counterparts. As judged by low glucose levels present at 48hrs, conversion of starch to ethanol in the HOI002-derived samples was essentially complete by this time (but after 24 hrs). The standard hybrids still contained some glucose that could be further used to produce more ethanol. While grain from the HOI002 crosses produced slightly less ethanol overall than the standards, it was accomplished much more quickly, as much as 24 hours faster. Existing ethanol plants consume large amounts of energy, so the ability to increase the amount of energy produced per kilowatt hour/man hour expended to run the facility, enabled by grain produced as described above, should greatly enhance profit potential. [00125] In the 54 hour time period, grain from HOI002 crosses were unchanged from their 48 hour totals. Overall, actual ethanol production was 4.34% less than the standards (proximates predicted -8.26%).

[00126] The oil content of grain from the HOI002 crosses was greatly enhanced (oil was increased by 116% versus the standards) which can provide excellent value if fractionization technology is applied. Generally, Distillers Dried Grains (DDG' s) are placed into fractions that have direct application to swine and cattle feeding operations. Value is generated for this high oil fraction in the poultry industry or, as an alternative, without fractionization. One embodiment of the present invention provides high oil grain produced by blends and comprises starch that is rapidly fermented to ethanol. In another aspect of this invention, the fractionized portion is entered into Bio-butanol production with the DDG' s. The bio-butanol is then blended with gasoline or diesel fuel to capture the available carbon from the grain. [00127] Although grain from HOI002 crosses were much lower in starch (-10.25%) and extractable starch (-23.44%), the crosses generated only 4.34% less ethanol and did so in much less time than their normal counterparts, demonstrating value capture opportunities in ethanol production.

[00128] In another embodiment of the invention, at least one transgene conferring both high oil and small starch grain size is introduced into corn and tested for confirmation of rapidly fermentable starch phenotype. Alternatively, at least one transgene conferring high oil content and at least one transgene affecting starch fermentability are introduced into corn, either together on a single transgenic insertion, or independently into sites that are linked, by methods known to the art. In another embodiment of the invention, at least one high oil transgene is introduced into at least one high fermentable starch genetic background. In yet another embodiment of the invention, at least one transgene affecting starch fermentability is introduced into at least one high oil genetic background. In each of the above cases, the resulting lines are used as pollinators, seed parents or both in blends to enhance oil content in commercially produced grain and improve starch fermentation kinetics. Typically, if any of the high oil and rapidly fermentable starch genetic factors confer adverse phenotypes in the homozygous state, they are used in the heterozygous or hemizygous state in grain parent hybrids. Homozygous, heterozygous or hemizygous conditions of the high value trait genetic factors can be used in high value trait pollinator lines depending on the needs of the system.

[00129] In another embodiment of the system, the blends include a male- sterile grain parent, such as a CMS line in a non-restorer background, or a dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent; and a high oil, rapidly fermentable starch pollinator. This arrangement is to ensure high yield under environmental conditions in which the transgenic, rapidly fermentable starch pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male- sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field. [00130] In still another embodiment of the invention, the at least one transgene of the above embodiments is linked to the CP4 transgene. The resulting line can be used as a high oil, rapidly fermentable starch line per se if the agronomic traits and yield are competitive, as may be expected for a hybrid in the hemizygous or heterozygous condition for the high value seed trait genetic factors. The field is sprayed with glyphosate at V8 to V12 to select for transgenic pollen, giving essentially 100% of pollen conferring the linked high value trait and increasing the percentage of kernels produced in the field that have genes encoding both high oil and high fermentable starch, in some embodiments to essentially 100%. This in turn increases the value of the grain obtained. In another embodiment, blends are prepared in which the other hybrids or inbreds in the blend comprise a transgene encoding a glyphosate resistant EPSPS, to ensure they will survive treatment with glyphosate. Pollen selection via differential glyphosate resistance increases the percentage of kernels produced in the field that have genes encoding high oil and high fermentable starch, increasing the value of the grain obtained. Yield is maintained at competitive levels while enhancing the value of the grain, in this case, through increased oil content and more rapidly fermented starch. In yet another embodiment, plants in the blend other than the high oil, rapidly fermentable starch plants, comprise a gene encoding a glyphosate resistant EPSPS that confers glyphosate resistance to the plant but not in pollen. Glyphosate resistance pollen selection in this instance gives essentially 100% of pollen conferring the linked high value trait. In yet another embodiment of the invention, the grain parent and high oil, rapidly fermentable starch pollinator line each comprise at least one gene, allele or transgene contributing to the high oil, rapidly fermentable starch phenotype. In a preferred embodiment, the at least one gene, allele or transgene in each parent is linked to CP4, such that the only pollen surviving glyphosate herbicide resistance selection comprises the CP4 transgene linked to the at least one gene, allele or transgene that impacts starch fermentability. Glyphosate resistance pollen selection ensures that essentially every kernel produced in the field contains at least one genetic factor contributing to the high oil, rapidly fermentable starch phenotype in addition to the at least one genetic factor inherited by 50% of the kernels through the female gametes. The advantage of using a blend of rapidly fermentable starch grain parent and rapidly fermentable starch pollinator as opposed to a rapidly fermentable starch grain parent alone, undergoing self pollination, is that one can use a high yielding grain parent with a heavy pollen shedding pollinator. [00131] In yet another embodiment of the system, the blends include a glyphosate resistant male- sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a dominant, reversibly male-sterile line, comprising the CP4 transgene; a male-fertile pollinator that is isogenic to the grain parent also comprising the CP4 transgene; and a high oil, rapidly fermentable starch pollinator, with the transgenic high value trait(s) linked to CP4. The male-fertile pollinator need not necessarily be isogenic and could also utilize a means to delay either germination or pollination, such as a polymer coating. This arrangement allows for glyphosate resistance pollen selection while ensuring high yield under environmental conditions in which the transgenic, rapidly fermentable starch pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

Example 4. Production of grain with improved traits using RNAi.

[00132] Plants expressing increased levels of essential amino acids, such as lysine, tryptophan or methionine, have been observed to suffer deleterious phenotypes related to stand establishment and seedling phenotype; and in the case of hybrid crops such as corn, high essential amino acid expression in an inbred may not translate to high essential amino acid content in the hybrid. In addition, production of hybrids homozygous for a transgene typically requires that the transgene be maintained in both male and female inbreds, whereas production of hybrids hemizygous for the transgene require only that it be maintained in a single parent of choice. The present invention provides methods for obtaining skewed allelic ratios, i.e., pollen selection, to increase the average dosage of one or more genetic factors impacting amino acid content in seeds to improve the commercial production of these traits in corn.

[00133] LysRNAi encodes an endosperm-expressed transgene (cordapA) that reduces zein expression, increasing free lysine content (U.S. Patent Application 11/077089). CV137 x CV182, which represents the wild type, expresses approximately 50 ppm free lysine. CV137LysRNAi x CV182LysRNAi (homozygous) expresses approximately 2250 ppm free lysine. The hemizygous hybrid is predicted to expresses approximately 1150 ppm free lysine, assuming an additive effect of LysRNAi.

[00134] The self -pollinated hemizygous hybrid contains a combination of different dosages of LysRNAi in the kernels of the ear. LysRNAi gene dosages for the population of kernels arising on the hybrid ear are shown in Table 5.

Table 5. LysRNAi endosperm dosage and expected lysine content of grain from a self pollinated hemizygous hybrid

Free Lysine

Pedigree Endosperm Dosage (ppm)

CV137 x CV182 - - / - 50

¹A: - - I - 50 1A: - - /LysRNAi 783 1A: LysRNAiLysRNAi/ - 1950

CV137LysRNAi x CV182

¹A: LysRNAiLysRNAi/ 2250 LysRNAi

Average: 1258

CV137LysRNAi x CV182LysRNAi LysRNAiLysRNAi/LysRNAi 2250

[00135] In one embodiment of the invention, the LysRNAi transgene is cloned and combined into a vector stack containing CP4 (the gene encoding a glyphosate -resistant EPSPS that confers glyphosate herbicide resistance in plants, including pollen). For demonstration purposes, the combined vector is incorporated into a male inbred such as CV 177 to create CV 177 (CP4 LysRNAi). A hybrid is produced by crossing this inbred to another corn inbred and this hemizygous cross is planted in a farmer's field. At about V8 to V12, the field is sprayed with glyphosate to select transgenic pollen. Spraying with glyphosate at this growth stage reduces the number of genetic classes of kernels produced from 4 to 2 since the pollen donor cannot contribute the wild type (nontransgenic) allele. Since the additive relationship is present, we are able to predict the classes, as indicated in Table 6. Table 6. Endosperm dosage of LysRNAi linked to CP4, and expected lysine content of grain, from a self pollinated hemizygous hybrid sprayed with glyphosate.

[00136] In another embodiment of the invention, an RNAi transgene designed to suppress or increase the expression of a trait of interest is cloned and combined into a vector stack containing CP4. For demonstration purposes, the combined vector is incorporated into a male inbred such as CV177 to create CV177 (CP4 + transgene). A hybrid is produced by crossing this inbred to another corn inbred and this hemizygous cross is planted in a farmer's field. At about V8 to V12, the field is sprayed with glyphosate to select transgenic pollen. Spraying with glyphosate at this growth stage reduces the number of genetic classes of kernels produced from 4 to 2 since the pollen donor cannot contribute the wild type (nontransgenic) allele.

[00137] In another embodiment, the hybrid hemizygous for the LysRNAi transgene is used as a pollinator in a blend, in a manner analogous to that described for high oil corn in Example 1. In yet another embodiment, the hybrid hemizygous for the LysRNAi transgene linked to the CP4 transgene is used as a pollinator in a blend in conjunction with glyphosate resistance selection, in a manner analogous to that described for high oil corn in Example 1. All other hybrids or inbreds in the blend comprise a transgene encoding a glyphosate resistant EPSPS, to ensure they will survive treatment with glyphosate. At about V8 to V12, the field is sprayed with glyphosate to select transgenic pollen. Spraying with glyphosate at about V8 to V12 reduces the number of genetic classes of kernels produced since the nontransgenic pollen from the pollen donor does not survive. This results in most kernels having an endosperm genotype of - - /(CP4LysRNAi). For an event and genotype having activity similar to that described above, this corresponds to a maximum lysine content of about 783 ppm predicted in ears of the grain parent, if essentially all pollination were by the pollinator parent (1517 ppm predicted in ears of the pollinator line itself). Yield is maintained at competitive levels while enhancing the value of the grain, in this case, through increased lysine content. In yet another embodiment, hybrids or inbreds in the blend other than the high lysine hybrid comprise a gene encoding a glyphosate resistant EPSPS that confers glyphosate resistance to the plant but not in pollen. This further ensures that only pollen comprising the CP4 transgene linked to the LysRNAi transgene survives, resulting in kernels of the grain parent having an endosperm genotype of exclusively - - - - /LysRNAiCP4. In yet another embodiment, the blends include a pollinator that is isogenic to the grain parent, but comprising the CP4 transgene, as well as a high lysine pollinator, to ensure high yield under environmental conditions in which the high lysine pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments.

[00138] Another lysine construct is an embryo-expressed transgene encoding a feedback- insensitive enzyme of the lysine biosynthesis pathway, dihydrodipicolinic acid synthase (DHDPS) from Corynebacterium, for which an event designated LY038 has been reported (U.S. Patent 7,157,281). Commodity corn has from about 0.26% to about 0.27% (-2575 - 2700 ppm) total lysine, mostly from germ protein; however, high-oil lines generally have larger embryos, resulting in as much as about 3000 ppm total lysine. Figure 6 shows free lysine levels for a number of inbred conversions of LY038 in various hybrid combinations. On average, homozygous LY038 hybrids had around 60% more free lysine than hemizygous LY038 hybrids. Note that some very high lysine hybrids were in fact homozygous LY038 in this test, but most were hemizygous. Typical values for free lysine content in whole kernels are given in Table 6 for hybrids related to CHOOl that were either homozygous for LY038, hemizygous with the gene transmitted via the female parent or hemizygous with the gene transmitted via the male parent. Free lysine values for wild type (nontransgenic for LY038) CHOOl were typically in the 50 ppm range. Table 7. Inheritance of Increased Lysine Content in Corn Hybrids (α = 0.05).

Lysine Lysine 95% LCL

Hybrid Source (mean ppm) (ppm)

CH002 hemi Female 1631 1523

CH003 hemi Female 1576 1470

CHOOl hemi Female 948 842

CHOOl hemi Male 836 730

CHOOl homo Both 1425 1328

[00139] For CHOOl, assuming the hemizygous class is the average of the two reported values, the same kind of analysis as was used for LysRNAi gives the results shown in Table 8.

Table 8. LY038 embryo dosage and expected lysine content of grain from a self pollinated hemizygous hybrid

Pedigree Embryo Dosage Free Lysine (ppm)

CHOOl (normal) +/+ 50

VA: +/LY038 892 (737 predicted)

CHOOl Hemizygous VA: LY038/+ 892 (737 predicted)

Average: 815 (737 predicted)

CHOOl Homozygous LY038/LY038 1425

[00140] For demonstration purposes, the combined vector is incorporated into a male inbred such as CV177 to create CV177(CP4LY038). A hybrid is produced by crossing this inbred to the female inbred of CV009 and this hemizygous cross is planted in a farmers' field. At about V8 to V12, the field is sprayed with glyphosate to select transgenic pollen. Spraying with glyphosate at this growth stage reduces the number of genetic classes of kernels produced from 4 to 2 since the pollen donor cannot contribute the wild type (nontransgenic) allele. For purposes of demonstration, analysis in the same way would give results as shown in Table 9. Table 9. Embryo dosage and expected lysine content of grain from a self pollinated hemizygous hybrid containing the CP4 LY038 vector stack and sprayed with glyphosate between V8 and V12.

Pedigree Embr o Dosa e Free L sine

[00141] In another embodiment, a hybrid or inbred comprising the LY038 event is used as a pollinator in a blend, in a manner analogous to that described for high oil corn in Example 1. In yet another embodiment, the hybrid hemizygous for the LY038 transgene linked to the CP4 transgene is used as a pollinator in a blend in conjunction with glyphosate resistance selection, in a manner analogous to that described for high oil corn in Example 1. All other hybrids or inbreds in the blend comprise a transgene encoding a glyphosate resistant EPSPS to ensure they will survive treatment with glyphosate. At about V8 to V12, the field is sprayed with glyphosate to select transgenic pollen which reduces the number of genetic classes of kernels produced since the nontransgenic pollen from the pollen donor does not survive. This results in most kernels having an embryo genotype of -/(CP4LY038). For an event and genotype with activity similar to that described above, this corresponds to a maximum lysine content of about 836 ppm predicted in ears of the grain parent provided that essentially all pollination was by the pollinator parent (1131 ppm predicted in ears of the pollinator line itself). Yield is maintained at competitive levels while enhancing the value of the grain, in this case, through increased lysine content. In yet another embodiment, hybrids or inbreds in the blend other than the high lysine grain parent comprise a gene encoding a glyphosate resistant EPSPS that confers glyphosate resistance to the plant but not in pollen. This further ensures that only pollen comprising the CP4 transgene linked to the LY038 transgene survives, resulting in kernels of the grain parent having an embryo genotype of exclusively - - /LY038 CP4. In yet another embodiment, the blends include a pollinator that is isogenic to the grain parent, but comprising the CP4 transgene, as well as a high lysine pollinator, to ensure high yield under environmental conditions in which the high lysine pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. [00142] The results predicted for grain from hybrids hemizygous for a transgene impacting lysine content that is linked to CP4 and treated with glyphosate to skew the pollen population to transgenic classes only are thus similar for endosperm-expressed LysRNAi and embryo- expressed LY038. In another embodiment of the invention, these two transgenic constructs are combined to generate increases in free lysine due to transgene expression in both embryo and endosperm. In yet another embodiment, LysRNAi and LY038 are linked to CP4 to enable glyphosate selection of transgenic pollen grains. A vector is constructed which combines LysRNAi and LY038 with CP4. Increases in lysine content due to LysRNAi expression in endosperm and LY038 expression in embryo are expected to be greater than increases due to either transgene alone. Such results have been obtained for LysRNAi and LY038 unlinked to CP4, as reported in U.S. Patent Application Serial No. 11/077,089 reproduced in part in Table 10.

Table 10. Kernel lysine content (ppm) for LysRNAi, LY038 and LysRNAi/LY038 transgenics (reproduced from U.S. Patent Application 11/077089).

Construct LysRNAi/LY038 LysRNAi LY038 Nontransgenic

2908^a 64^a 1838^C 43

Whole kernel free lysine 2498^b 67^b

Average: 2703 66

23159^a 302^a 16630^c 238

Embryo free lysine 21581^b 43 l^b

Average: 22550 367

119^a 28^a 58^C 10

Endosperm free lysine 105^b 20^b

Average: 112 24

6160^a 2930^a 4290^c 2575

Whole kernel total lysine 5475^b 3320^b

Average: 5817 3125

^a LysRNAi event PQ15 LysRNAi event PQ71 ^c LY038 event M27908 AU transgenes were hemizygous.

[00143] To compare the effects of the transgenes independently and in combination, the two constructs were introduced into plants independently and then crossed together. Thus they likely segregate independently, resulting in more genetic classes of kernels than expected if the transgenes were linked. This would change the levels of lysine expected for many of the individual kernels, but would likely not impact the range of lysine levels in individual kernels (both linked and unlinked genes are expected to give nontransgenic as well as homozygous transgenic kernels, which are expected to be the two extremes) and may not significantly affect the average lysine level. It is possible to produce plants with any two transgenic constructs in linkage using method known in the art for direct insertion and/or recombination, i.e., Cre-lox or site specific meganucleases or by screening multiple events for events located within a certain proximity on a chromosome to ensure linkage, followed by trait integration methods known in the art to obtain a plant with the two or more constructs in genetic linkage. [00144] Although the two transgenes are differentially expressed with respect to embryo and endosperm, lysine levels in both parts of the seed are impacted by each transgene alone, as well as by the two transgenes in combination. Moreover, these two genes act synergistically, giving lysine levels in combination that are greater than the sum of the lysine levels obtained individually. These complications notwithstanding, glyphosate resistance selection for pollen transgenic for linked LysRNAi, LY038 and CP4 transgenes will increase the expected average level of lysine in progeny kernels by eliminating nontransgenic pollen. The four genetic classes of kernels expected in the absence of glyphosate resistance selection are reduced to two genetic classes in the presence of glyphosate resistance selection, as shown in Table 11.

Table 11. Predicted genetic classes of kernels from self -pollinated lines hemizygous for linked trans genes.

Progeny (Embryo)

Fraction Genotype Endosperm Genotype

VA + +/ LY038 LysRNAi + + + +/ LY038 LysRNAi

VA LY038 LysRNAi/+ + LY038 LysRNAi LY038 LysRNAi/+ +

LY038 LysRNAi/LY038 LY038 LysRNAi LY038

VA LysRNAi LysRNAi/LY038 LysRNAi

[00145] In another embodiment, the hybrid hemizygous for the linked LysRNAi LY038 transgenes is used as a pollinator in blends, in a manner analogous to that described for high oil corn in Example 1. In yet another embodiment, LysRNAi, LY038 and CP4 are linked, and at least one hybrid or inbred hemizygous for the linked LysRNAi LY038 CP4 transgenes is used as a pollinator in blends in conjunction with glyphosate resistance selection, in a manner analogous to that described for high oil corn in Example 1. All other hybrids or inbreds in the blend comprise a transgene encoding a glyphosate resistant EPSPS, to ensure they will survive treatment with glyphosate. At about V8 to V12, the field is sprayed with glyphosate to select transgenic pollen. Spraying with glyphosate at about V8 to V12 reduces the number of genetic classes of kernels produced since the nontransgenic pollen from the pollen donor does not survive. This results in most kernels having genotype of /LysRNAi LY038 CP4

(endosperm: /LysRNAi LY038 CP4). Yield is maintained at competitive levels while enhancing the value of the grain, in this case, through increased lysine content. In yet another embodiment, hybrids or inbreds in the blend other than the high lysine hybrid comprise a gene encoding a glyphosate resistant EPSPS that confers glyphosate resistance to the plant but not in pollen. This further ensures that only pollen comprising the CP4 transgene linked to the LysRNAi and LY038 transgenes survives, resulting in kernels of the grain hybrid parent having an endosperm genotype of exclusively /LysRNAi LY038 CP4.

[00146] In yet another embodiment of the invention, the grain parent and high lysine pollinator line each comprise at least one gene, allele or transgene contributing to the high lysine phenotype. In a preferred embodiment the at least one high lysine genetic factor in each parent is linked to CP4 such that the only pollen surviving glyphosate herbicide resistance selection comprises the CP4 transgene linked to the at least one high lysine genetic factor. Glyphosate resistance pollen selection ensures that essentially every kernel produced in the field contains at least one high lysine genetic factor, in addition to the at least one high lysine genetic factor inherited by 50% of the kernels through the female gametes. The advantage of using a blend of high lysine grain parent and high lysine pollinator as opposed to a high lysine grain parent alone, undergoing self pollination, is that one can use a high yielding grain parent with a heavy pollen shedding pollinator.

[00147] In yet another embodiment, the blends include a male-sterile grain parent, such as a CMS line in a non-restorer background, or a dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent; and a high lysine pollinator. This arrangement is to ensure high yield under environmental conditions in which the transgenic, high lysine pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

[00148] In yet another embodiment, the blends include a glyphosate resistant male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a CP4-transgenic dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent, comprising the CP4 transgene; and a high lysine pollinator that is transgenic for CP4 linked to the high lysine transgene to ensure high yield under environmental conditions in which the transgenic, high lysine pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that allows for glyphosate herbicide resistance selection of pollen and mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field. [00149] These methods are also useful for improving commercial production of grain having increased tryptophan content. Expression of transgenic anthranilate synthase (Trp), which catalyzes the first committed step from aromatic amino acids to tryptophan synthesis in higher plants, fungi and bacteria, can result in higher levels of free tryptophan compared to nontransgenic plants through a variety of mechanisms (U.S. Patent No. 7,217,865). [00150] Data from U.S. Patent Application No. 11/836,690 indicate that nontransgenic corn kernels typically contain less than 25 ppm of free tryptophan (usually 8-10 ppm), whereas free tryptophan levels in corn kernels homozygous for transgenic Trp ranged from no better than nontransgenic to as high as -1350 ppm average for the top event and -960 ppm average for the top construct (14 events), with average expression over fifteen test constructs of -300 ppm (3 to 17 events/construct). Expressed in the embryo, one can predict for an "average" construct that gave -302 ppm (five events) a hemizygous expression level of 156 ppm, assuming linear response to gene dosage. That is, grain that is 100% hemizygous would be expected to have [(302 - 10)/2] + 10 = 156 ppm tryptophan by gene dosage. Alternatively, for a hemizygous average transgenic Trp line showing Mendelian segregation, self pollination would give 25% homozygous transgenic (Trp/Trp) : 50% hemizygous (Trp/-) : 25% nontransgenic (-/-), which gives (25%)(302 ppm) + (50%)(~156 ppm) + (25%)(10 ppm) = 75.5 ppm + 78 ppm + 2.5 ppm = 156 ppm. Of the fifteen constructs tested, four averaged at least enough free tryptophan (>= 589 ppm in seeds homozygous for the construct) to reach a target level of about 300 ppm in the hemizygous state, making the strategy highly feasible. Using the highest expressing Trp line (1350 ppm in the homozygous condition), if one obtains as little as 45% hemizygous transgenic and 55% nontransgenic kernels from planting a blend, it would meet the 300 ppm product concept.

[00151] Accordingly, in various embodiments of the invention, a transgenic Trp line is used as a pollinator, a grain parent or both in a blend analogous to what has been described in the previous examples. Typically a hemizygous Trp hybrid is preferred for use as an elite grain parent if there are adverse effects on agronomic characteristics or yield associated with the homozygous state of the transgene. In other embodiments a line comprising Trp linked to CP4 is used in blends as a pollinator, grain parent or both. All non-Trp plants in the blend comprise a transgene encoding a glyphosate resistant EPSPS to ensure they will survive treatment with glyphosate. The field is sprayed with glyphosate at V8 to V12, resulting in survival of only CP4-transgenic pollen and skewing the frequencies of the genetic classes of kernels produced towards Trp positive (hemizygous or both hemizygous and homozygous, depending on whether the Trp line is used solely as the pollinator, or as the seed parent).

[00152] In some embodiments, all non-Trp plants in the blends are transgenic for a non-CP4 glyphosate-resistant EPSPS encoding gene that confers glyphosate resistance in plants but not in the pollen, resulting in essentially 100% of the grain comprising the Trp and linked CP4 transgenes in the hemizygous state when the Trp line is the pollinator only, under glyphosate resistance pollen selection. For an event and genotype having activity of at least about 589 ppm in the homozygous state, this corresponds to a maximum tryptophan content of about 300 ppm predicted in ears of the grain parent (444 ppm predicted in ears of the pollinator line itself). Yield is maintained at competitive levels while enhancing the value of the grain, in this case, through increased tryptophan content. When the seed parent comprises Trp in the hemizygous condition in this embodiment, the grain segregates 50% homozygous : 50% hemizygous for Trp. The advantage of using a Trp pollinator line with a hemizygous Trp seed parent under glyphosate resistance pollen selection is that a high yielding seed parent and a heavy pollen shedding pollen parent can be used.

[00153] In yet another embodiment the blends include a male-sterile grain parent, such as a CMS line in a non-restorer background, or a dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent; and a high tryptophan pollinator. This arrangement is to ensure high yield under environmental conditions in which the transgenic, high tryptophan pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field. [00154] In yet another embodiment, the blends include a glyphosate resistant male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a CP4-transgenic dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent, comprising the CP4 transgene; and a high tryptophan pollinator that is transgenic for CP4, linked to the high tryptophan transgene to ensure high yield under environmental conditions in which the transgenic, high tryptophan pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that allows for glyphosate herbicide resistance selection of pollen and mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

Example 5. Production of grain with low levels of phytate.

[00155] In traditional corn hybrids, phosphorus is tightly bound in the form of phytic acid and is accumulated in the aleurone layer (<20%) and the germ (>80%) of a corn kernel. Phytic acid is the major form of phosphorus in grains (as much as 80% of total), and is maintained in a form not readily available to non-ruminants. Phytic acid in animal waste contributes to odor and to surface and ground water pollution. For these reasons, it is desirable to increase available phosphorus while decreasing phytic acid content of grain used in animal diets. [00156] To address this concern, EMS treatments were used to create low phytic acid mutants, including lpal-1, lpa2-2, Ipa3-1 in corn, here referred to generally as lpa. These are described in U.S. Patent No. 6,111,168 and U.S. Patent No. 5,689,054, both of which are incorporated herein by reference in their entirety. The mutants were recessive and were originally employed on both sides of a commercial pedigree to obtain uniformly low phytic acid grain. Grain produced with homozygous lpa/lpa hybrids was high in available phosphorus (HAP), which was quite promising. However, when lpa was in a homozygous state, lower stand establishment and lower yield occurred, making commercial production impractical by this approach. [00157] One approach to improving the commercial viability of the lpa mutants, and an embodiment of the current invention, is with blends, in a way that is analogous to that described in the previous examples. In this case, the grain parent hybrid is heterozygous for lpa and the pollinator line is homozygous for lpa. The pollinator is somewhat over-planted compared to other blends to ensure sufficient pollen shed. The pollinator and grain hybrids are chosen to ensure maximum pollination by the pollinator line and minimum pollination by the grain hybrid. Under these conditions, the grain will predominantly segregate 1/2 Lpa/lpa to 1/2 lpa/lpa in the embryos. The corresponding endosperm genotypes will be LpaLpa/lpa and lpalpa/lpa, respectively.

[00158] Based on public data and assuming Mendelian segregation and dominant gene action, grain produced by this approach can be calculated to have an average 37% decrease in phytic acid and a 634% increase in available inorganic phosphorus in the kernel compared to wild type

(Figure 7). The stand establishment issue with the original mutants is circumvented by using only one dose in the hybrid form. Hybrid vigor hides any problem that may be associated with the heterozygous condition at the Lpa locus.

[00159] Another embodiment of the invention employs linkage (e.g., within 15 centimorgans) between glyphosate-resistant EPSPS (CP4) as a selectable marker and any of the recessive mutations that confer reduced levels of phytate in corn kernels. One way to identify such an event is to utilize current transformation technologies and to perform CP4 insertions until an event is identified that is both functional and in close proximity to the locus of interest. For illustrative purposes, we present the case in which the locus of interest is the lpal gene located on the short arm of chromosome 1, and an event is identified which inserted a functional CP4 gene 5 cM from the lpal gene. This hemizygous line is self pollinated and its progeny are screened to identify lines homozygous for the CP4 gene.

[00160] Through the use of traditional backcrossing or marker-assisted backcrossing, lines homozygous for both the CP4 gene and the mutant lpal gene are isolated. Such lines are used as pollinators of a female line in a production field to produce hybrid seed that is heterozygous

Lpal/lpal.

[00161] The hybrid seed, when grown in a producer's field, has a normal phenotype because of the presence of a wild type Lpal allele. Glyphosate is applied at about the V8 to V12 growth stage, killing nontransgenic developing pollen grains and allowing only pollen grains containing the CP4 gene to survive. Since the CP4 gene is only 5 cM from the lpal locus and in coupling phase with the lpal allele, 95% of all pollen shed will carry the lpal allele while only 5% will carry the Lpal allele. The 5% CP4/Lpal pollen grains are due to meiotic crossing over between the CP4 locus and the Lpal locus. Phytate in the grain is reduced by about 33% and inorganic phosphate levels increase about 690% in the grain as shown in Figure 8.

[00162] In yet another embodiment of the invention, a line with CP4 linked to lpal is used as a pollinator in a blend, in which the grain parent elite hybrid is heterozygous for lpal and comprises a gene for glyphosate resistant EPSPS that confers resistance to glyphosate in plants but not in pollen. Glyphosate resistance selection of pollen produced by the pollinator line is carried out by spraying with glyphosate at about V8 to V12 stage. The female gametes of the grain parent will segregate 1:1 Lpal:lpal and approximately 95% of the pollen will be lpal. The kernels formed will segregate at the same ratios as those given in Figure 8. The advantage over using the lpal CP4 hybrid by itself (with glyphosate resistance pollen selection) is that a high yielding seed parent and a heavy pollen shedding pollen parent can be used. Notably, the heterozygous Lpa/lpa seed parent may comprise the CP4 transgene, so long as it is linked to the lpa allele, and the same outcome will be obtained, but with potentially a higher amount of pollen available in the field.

[00163] Since both the seed parent and the pollinator line must be at least heterozygous Lpa/lpa, if CP4 is linked to the lpa allele in both parents, the use of a male sterility system in addition to glyphosate resistance pollen selection offers no advantage in this instance. If the seed parent comprises CP4 unlinked to the lpa allele, or if glyphosate resistance pollen selection is not used, then a male sterility system such as a CMS line in a non-restorer background or a dominant, reversibly male sterile line, would be helpful to reduce the background of undesired pollen. In this situation, an isogenic, but wild type Lpa/Lpa, male fertile, CP4-transgenic pollinator line would be useful insurance in obtaining pollination if the heterozygous Lpa/lpa condition has any adverse affects on agronomic characteristics or yield.

[00164] Studies have indicated that DDG' s contain lower amounts of phytic acid compared to grain before fermentation. It has also been found that phosphorus is in a more available form (60% available vs. 20% in whole grain), reducing the need for monocalcium phosphate in the diet by 5 lb./ton of feed if 20% DDGS is included in the diet. Fermentation of grain in which the phytic acid profile is already significantly improved before distillation would make DDG' s much preferred over raw grain as a food source, especially in areas where environmental concerns of phosphorus and water quality are issues.

Example 6. Production of grain with multiple high value traits.

[00165] Transgene combinations conferring different high value traits linked to CP4 are also anticipated by the present invention. In one aspect, a single construct was previously generated that contains CP4 as a glyphosate- selectable marker linked to transgenes conferring enhanced economic value through high-level expression of lysine and tryptophan. In another aspect, two or more constructs are generated and are obtained in linkage using methods known in the art such as Cre-lox. The transgenic line is crossed with a high oil inbred to produce grain that has high levels of both lysine and tryptophan, two essential amino acids normally at insufficient levels in corn, as well as high levels of oil and high fermentable starch as sources of additional value. Pollen selection for the CP4 gene occurs when spraying with glyphosate at about V8 to V12, making a new production system for high value corn possible:

{CV176 with CP4 Lys/Lys, Trp/Trp} x HOI002 as a pollinator -Blend with a glyphosate Ready sterile grain parent -Spray with glyphosate at about V8 to select pollen -All pollen will be High Oil + Lys + Trp -Grain produced will be high value corn

[00166] Without pollen selection, the grain produced is a mix of higher and lower lysine and tryptophan kernels. In this manner, the pollen selection greatly increases the effect of the two genes in concert with one another.

[00167] In another embodiment of the invention, the transgenes are inserted into HOI002 to achieve high value corn. In addition, this allows the use of the hybrid as a pollinator for a grain hybrid in fertile blends, in conjunction with glyphosate resistance selection of pollen, to ensure competitive yields and agronomic characteristics for commercial production of high value grain. [00168] In yet another embodiment, the blends include a glyphosate resistant male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a CP4-transgenic dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent, but comprising the CP4 transgene; and a transgenic CP4-linked high lysine, high tryptophan pollinator. This type of blend is designed to ensure high yield under environmental conditions in which the high lysine, high tryptophan pollinator fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field. [00169] In yet another embodiment of the invention, the combination of the low phytic acid trait (embryo and endosperm) with amylose extender (endosperm) provides value capture for the Renessen fractionization process. One way of accomplishing this is by a conventional mutation + linked transgenic vector stack. An RNAi expressed specifically in embryos against the Lpal gene, which encodes an ABC transporter involved in phytic acid uptake and/or transport, has been reported to reduce phytate in corn seeds by 32% to 87% and to increase free inorganic phosphate (Shi, J. et al., Nature Biotechnology 25: 930-937 (2007)). Some of the transgenics had significantly reduced kernel weight and experienced germination delays of 1-2 days and modest reductions in germination rates (70%-90% compared to 90% for controls). Assuming an appropriate event is obtained that confers relatively high efficacy without compromising yield and agronomic characteristics in an elite background, the Lpal-RNAi transgene is usable in allelic skewing systems to obtain grain with rapidly fermentable starch, low phytate and high available phosphate. A transgenic construct comprising a CP4 transgene linked to Lpal-RNAi is introduced in large numbers into corn, to identify an event that maps within about 15 cMorgans of the AeI locus on chromosome 5. The transgenic line is introduced into an ael background, and a recombinant is obtained linking the transgenic insert to the mutant allele. The mutant ael allele and linked CP4 Lpal-RNAi transgenic insertion are introgressed together into at least one elite germplasm and the resulting at least one line is used in blends as a pollinator parent, with at least a seed parent that is resistant to glyphosate. The latter is preferably transgenic for a glyphosate-resistant EPSPS gene that confers resistance to the plant but not in pollen. The plants are treated with glyphosate at about V8 to V12 to select for glyphosate resistance in pollen. If CP4 and Lpal-RNAi are 15 cMorgans apart, the resulting grain segregates 85% CP4 Lpal-RNAi ael / - - Ael and 15% CP4 Lpal-RNAi Ael / - - Ael. The fractionated grain has lower phytic acid, higher available phosphate and ferments starch at a faster rate, benefiting both ethanol production and increasing value of the resulting DDG' s. Example 7. Development of sweet corn with increased sucrose content.

[00170] The introduction of homozygous recessive mutations into sweet corn that increase the sugar content of immature kernels has met with enthusiastic consumer acceptance. This preference has driven efforts to further enhance sugar content by combining two such homozygous recessive mutations in a single hybrid. One of the difficulties encountered is that the combination of two such homozygous recessive mutations leads to much worse germination and plant vigor. The current art relies on a strategy that delivers hybrid seed with one homozygous recessive mutation and one heterozygous mutation, which gives acceptable hybrid seed germination and plant vigor, and results in 3: 1 segregation of the heterozygous mutation in the kernels, i.e. 25% of the kernels on the hybrid ear will be homozygous recessive for both mutations. However, the trade-off for acceptable seed germination and plant vigor is that the sweetness of the resulting ears is not as great as if both mutation are homozygous in the hybrid. The present example provides an improved strategy over the state of the art, resulting in a higher percentage of double homozygous recessive kernels on the hybrid ear and thus enhanced sweetness without compromising germination and plant vigor.

[00171] Two alleles of the gametophytic incompatibility gene Gal play a role in incompatibility between dent corn and popcorn. These are the dominant allele GaI-S and recessive allele gal. Pollen containing either allele can pollinate gal/gal dent corn silks, but only GaI-S pollen can pollinate Gal-S/gal or Gal-S/Gal-S silks, the latter being the genotype of most popcorns. For dent corns, this provides a means to skew pollen ratios toward those containing a nucleic acid of interest, such as a gene of interest (GOI), by linkage of the genetic factor of interest with GaI-S. In heterozygous or hemizygous crosses, pollen containing Gal cannot pollinate the heterozygous (or hemizygous) silks, eliminating one class of pollen from the cross. For a simple single locus cross, this results in only two gametic combinations instead of four, resulting in the elimination of at least one progeny class in the F2 grain from Fl hybrids. In one aspect, a conventional approach is described wherein a GaI-S donor and GOI donor are crossed followed by selection for GaI-S and GOI in addition to agronomic traits of interest using methods known in the art. Application of target individuals comprising Gal-S:GOI are detailed with respect to pollen selection utility. In another preferred aspect, a transgenic approach is used to closely link GaI-S and a GOI, maximizing coinheritance. [00172] In this example, GaI-S is used to enhance the flavor of sweetcorn. Currently, it is common practice for sweetcorn breeders to use both the sugary (su) gene and the shrunken (sh) gene. However, when both genes are in the homozygous state (shsh susu), the resulting kernels are extremely affected with regards to vigor and germination. Therefore, a common practice in sweetcorn breeding is to make the Fl using one parent that is fixed for both traits (shsh/susu) and the other parent fixed for just one trait (shsh/SUSU). The resulting Fl plant then does not suffer the severe deleterious effects, but the ear still gets some benefit of enhanced flavor, since the different alleles at the heterozygous locus segregate in the F2 kernels, giving homozygosity for the mutant allele in 25% of the kernels, as shown in Figure 9.

[00173] GaI-S is linked to the sugary gene (Su), but not tightly; GaI-S maps at 4S- 13 and SuI maps at 4S-47 as listed in Neuffer, M. G., Coe, E.H. and Wessler, S.R., Eds., Mutants of Maize, Cold Spring Harbor Laboratory Press: Woodbury, New York, 1997. Therefore the two loci are 34 cMorgans apart, giving 34% recombinant gametes and 66% nonrecombinant gametes. These will be divided into two classes of each; at 33% for each nonrecombinant class and 17% for each recombinant class, as summarized in Table 12.

Table 12. Gamete classes and predicted frequencies for self-pollination of GaI-S su / gal Su individuals

I 33% Gal-S:su (frequency = 0.33)

II 33% ga:SU (frequency = 0.33)

III 17% Gal-S:SU (frequency = 0.17)

IV 17% ga:su (frequency = 0.17)

[00174] Gamete classes II and IV will not be inherited through pollen, since only GaI-S pollen can pollinate Gal-S/ga silks. Therefore su/su (sugary) progeny will arise from class I pollen fertilizing class I or class IV egg sacs. The frequency of these progeny will be

[(0.33 x 0.33) + (0.33 x 0.17)] x 2 = 0.33

All other progeny will be SU/SU or SU/su (non-sugary) and their frequency is given by

1- 0.33 = 0.67

[00175] The ratio of sugary to non-sugary progeny kernels is thus predicted to be 33%:67% (1:2) instead of the 50%:50% ratio (1:1) predicted in the absence of recombination; this is still a higher proportion of sugary kernels than is obtainable by the standard method, described above, which gives 25% sugary to 75% non- sugary kernels (1:3).

[00176] In another embodiment of the invention, reduced recombination is obtained from a source with tighter linkage between GaI-S and su. This can be achieved by one of various methods known to the art, for example, introduction of a transgenic SU allele closer to gal in a transformable germplasm. This transgenic event, in which su is closely linked to the GaI-S allele, is then moved into an sh- containing background. The final step is to make the resulting source homozygous for GaI-S, su and sh. With the reduced rates of recombination expected for this scenario, the system could obtain much closer to a 1:1 ratio for sugary to non-sugary kernels. [00177] The above example deals with using the Gal system to skew pollen for the purpose of enhancing the sugar content of sweet corn, but from the preceding examples, it is evident to one skilled in the art that a Gal-based allelic skewing system can be used in a manner analogous to the use of the CP4 glyphosate resistance pollen selection system to obtain field corn with grain having enhanced value.

Example 8. Production of grain with pollen-conferred, non-pollen-transmissible high value traits.

[00178] Apomixis, the asexual reproduction of plants through floral structures, may be harnessed for use in production of seeds. The methods described in previous examples can be employed in several possible versions of apomictic seed or grain production, providing economic opportunities in the production of high value grain. The present example shows how the pollen skewing methods described herein could be applied using apomixis.

[00179] Apomictic plants are capable of producing seeds asexually through floral structures. Both facultative and obligate apomicts are known to the art. Embryo sacs or embryos may arise by various mechanisms, from different cell types, both sporophytic and unreduced gametophytic of strictly maternal origin, and at various stages of floral development. The resulting embryos are genetically identical to the maternal parent. Most apomictic species tolerate imbalances in parental gene dosage in their endosperm and still set seed. By comparison, many obligate sexually reproducing species, including crop plants such as corn, have a strict requirement for a specific ratio of parental genomes in the endosperm, typically two maternal genomes to one paternal genome, for proper seed development. Atypical ratios of parental genomes in the endosperm can lead to low seed weight, aberrant seed morphology, and poor germination or in viability. Some apomicts produce viable seed despite having a requirement for a specific parental gene ratio, and one way around this problem is through fertilization-induced endosperm development (pseudogamy). Genes controlling important steps of apomixis are beginning to be identified in various species. Genetic engineering methods may be required to develop apomictic crop plants such as corn.

[00180] In one embodiment, at least one plant comprises a transgene that confers or programs the plant to undergo pseudogamous apomixis. The apomixis system can optionally be inducible or repressible to alter functionality of the system. The at least one plant further comprises at least one gene conferring at least one high value endosperm-expressed trait. Such traits can include: high fermentable starch, altered seed protein type or content, elevated lysine and high oil content in the endosperm. The at least one high value endosperm-expressed trait can be conferred by at least one endogenous gene, which is optionally linked to a herbicide resistance gene that confers herbicide resistance to the entire plant including in pollen. This can be accomplished through screening of many transgenic events to obtain an insertion that is linked to a gene that confers the high value trait or contributes a major effect to the high value trait. The at least one high value endosperm-expressed trait can alternatively or additionally be conferred by at least one transgene, which is preferably linked to a herbicide resistance gene that confers herbicide resistance to the entire plant including in pollen. The herbicide resistance gene can be a glyphosate resistance transgene that confers resistance to the entire plant including pollen, such as CP4. For herbicide resistance gene linked systems, the at least one plant is sprayed at V8 to V12, killing nontransgenic pollen, while herbicide resistant pollen survives. Self pollination in a way that facilitates pseudogamous apomixis results in skewing of the allelic ratio in favor of the high value trait in the endosperm but not the embryo. This gives expression of the high value endosperm-expressed trait, but the progeny remain genetically the same regardless of the zygosity of any genes. The constitutive pseudogamous apomictic system allows progeny seed to be set aside for planting in the future, while the inducible or repressible pseudogamous apomixis system can be managed in much the same way as hybrid seed systems. In this embodiment, a skewed allelic ratio does not occur in the embryo, nor in absence of pollen selection. [00181] In another embodiment, at least one plant comprises a transgene that confers or programs the plant to undergo pseudogamous apomixis. At least one pollinator plant conferring at least one high value endosperm-expressed trait is used in a blend with the at least one pseudogamous apomictic plant, in a manner that confers preferential pollination by the pollinator line. The endosperm of the developing kernels undergoes allelic skewing favoring the high value endosperm-expressed trait from the pollen-derived genome, such as the aforementioned traits.

[00182] In another embodiment, at least one first plant comprises at least one transgene that confers or programs pseudogamous apomictic reproduction in developing kernels of at least one second plant upon pollination by the at least one first plant. The at least one first plant further comprises at least one gene conferring at least one high value endosperm expressed trait, such as the aforementioned traits. In this embodiment, the high value pollinator serves to cause pseudogamous apomixis in the kernels it pollinates, thereby preventing the at least one transgene conferring or programming pseudogamous apomixis from being transmitted heritably via pollen to the next generation, i.e., the embryo.

[00183] Pseudogamous apomixis is envisioned to involve blocking sexual reproduction or development of sexually derived embryos, and initiating and maintaining apomictic reproduction; or converting or adapting one or more sexual reproductive functions to apomictic reproduction; or a combination in part or entirely of these functions. Proper regulation of reproductive functions to produce a pseudogamous apomictic plant may involve more than one transgene. In another embodiment, at least one transgene involved in conferring or programming pseudogamous apomixis is present in a grain parent and at least one additional transgene involved in conferring or programming pseudogamous apomixis is present in a companion pollinator line. The latter further comprises at least one high value endosperm-expressed trait, such as the aforementioned traits.

[00184] In additional embodiments, the pollinator line of each of the above embodiments involving blends comprises at least one high value endosperm-expressed trait conferred by at least one endogenous gene. In further embodiments, the pollinator line of each of the above embodiments involving blends comprises at least one high value endosperm-expressed trait conferred by at least one transgene. [00185] In further embodiments, the pollinator lines of the above embodiments involving blends comprise a herbicide resistance transgene conferring resistance to the entire plant including pollen and linked to at least one gene associated with or conferring the high value endosperm- expressed trait in the pollinator line. In these embodiments, the grain parents of the above embodiments involving blends comprise a transgene which encodes herbicide resistance to the entire plant except the pollen. The herbicide can be glyphosate, and the herbicide resistance transgenes can be a glyphosate-resistant EPSPS that confers resistance to plants including their pollen, such as CP4, in the pollinator line; and a glyphosate-resistant EPSPS that confers resistance to plants except their pollen in the seed parent. The at least one grain parent plant and at least one pollinator plant are interplanted and at V8 to V 12 the plants are sprayed with glyphosate. CP4 transgenic pollen survives and pollinates the plants, resulting in grain from the grain parent being preferentially pollinated by pollen conferring the high value trait in the endosperm only.

[00186] In yet another embodiment, the blends include a glyphosate resistant male-sterile grain parent, such as a CMS line in a non-restorer background transgenic for glyphosate resistant EPSPS, or a CP4-transgenic dominant, reversibly male-sterile line; a male-fertile pollinator that is isogenic to the grain parent, but comprising the CP4 transgene; and a pollinator comprising a high value, endosperm-expressed trait linked to CP4 to ensure high yield under environmental conditions in which the pollinator comprising the high value, endosperm-expressed trait fails to shed pollen while receptive silks are present on ears of the grain parent. The result is a system that mitigates the risk of crop failure under high stress environments. The advantage of including the isogenic pollinator and a reversibly male-sterile grain parent in a blend is that it provides more than one way of ensuring pollen in the field.

[00187] It will be understood by one skilled in the art that various modifications and complications of the pseudogamous apomictic system are possible, particularly in the regulation of the system, which may involve, for example, inducible or repressible expression, non-cell- autonomous gene interactions, and the like, without changing the underlying basis of the methods herein described.

Claims

WHAT IS CLAIMED IS:

1. A method for producing high value grain, the method comprising: interplanting a first plant and at least one second plant, the first plant comprising at least one genetic factor conferring at least one high value trait; growing the first plant and the at least one second plant to obtain preferential inheritance of at least one high value trait in the progeny of the first and at least one second plant; and harvesting grain from the progeny.

2. The method of claim 1 wherein preferential inheritance is obtained by linking the at least one genetic factor conferring at least one high value trait to at least one second genetic factor conferring preferential inheritance of at least one haploid genotype.

3. The method of claim 2 wherein preferential inheritance of the haploid genotype comprises preferential inheritance of at least one genotype of the male gametophyte.

4. The method of claim 3 wherein preferential inheritance is due to at least one selective agent selected from the group consisting of a gametocide, antibiotic, phytotoxic agent, chemical, compound, element, nutrient, micronutrient, metabolite, metabolic intermediate, biochemical pathway starting compound, biochemical pathway product, vitamin, cofactor, pesticide, safener, herbicide, salts thereof, combinations thereof, and formulations thereof.

5. The method of claim 4 wherein the selective agent comprises a herbicide.

6. The method of claim 5 wherein the herbicide comprises glyphosate.

7. The method of claim 2 wherein preferential inheritance of the haploid genotype comprises preferential inheritance of at least one genotype of the female gametophyte.

8. The method of claim 7 wherein preferential inheritance is due to the presence of at least one selective agent selected from the group consisting of a gametocide, antibiotic, phytotoxic agent, chemical, compound, element, nutrient, micronutrient, metabolite, metabolic intermediate, biochemical pathway starting compound, biochemical pathway product, vitamin, cofactor, pesticide, safener, herbicide, a salt of any of the aforementioned, and formulation of any of the aforementioned.

9. The method of claim 1 wherein the step of generating progeny comprises sexually crossing at least one first plant with at least one second plant.

10. The method of claim 9 wherein at least one of the first plant or second plant is at least partially male-sterile.

11. The method of claim 2 wherein at least one of the genetic factors is linked to at least one element selected from the group consisting of an exogenous genetic factor, an endogenous genetic factor and a modified endogenous genetic factor.

12. The method of claim 2 wherein the genetic factor conferring at least one high value trait encodes at least one recessive trait.

13. The method of claim 2 wherein the second genetic factor is a gametophytic incompatibility genetic element.

14. The method of claim 2 wherein the second genetic element is a GaI-S genetic element conferring preferred pollination of at least a second plant that comprises a GaI-S genetic element.

15. The method of claim 1 wherein preferential inheritance is obtained by delaying planting, germination or emergence of one plant relative to another plant.

16. The method of claim 1 wherein preferential inheritance is obtained by the second plant shedding pollen later than most of its silks have emerged.

17. The method of claim 16 wherein the method comprises impacting the timing of pollen shed relative to silking with at least one element selected from the group consisting of an endogenous genetic element, an exogenous genetic element and a chemical compound.

18. The method of claim 1 wherein at least one plant is selected from the group consisting of a hybrid plant or an inbred plant.

19. The method of claim 1 wherein the plant is interplanted with an elite inbred, hybrid or cultivar to generate progeny.

20. The method of claim 1 wherein the at least one first plant is partially isogenic to the at least one second plant.

21. A method of claim 1 wherein the at least one first plant is substantially isogenic to the at least one second plant.

22. Grain produced by the method of claim 1 wherein the high value trait is selected from the group consisting of increased oil content, a preferred oil composition, reduced phytate content, increased phosphorous availability, increased protein content, a preferred protein composition, a preferred amino acid composition, waxy traits, a preferred amylose content, a preferred amylopectin content, increased starch content, increased extractable starch, increased fermentable starch, increased soluble carbohydrate digestibility, increased insoluble carbohydrate digestibility, increased sugar content and altered kernel morphology.

23. Grain produced by the method of claim 1, wherein the grain is corn and the at least one high value trait is selected from the group consisting of increased lysine, increased tryptophan, increased amount of oil, increased efficiency of conversion of starch to ethanol, increased conversion of starch to ethanol, and increased amount of inorganic phosphate as compared to commodity corn.

24. A method for producing high value grain, the method comprising planting at least one plant comprising at least one genetic factor conferring at least one high value trait; growing the at least one plant in a manner to obtain preferential inheritance of the at least one high value trait in the progeny of the plant; and harvesting grain from the progeny.