# Patent

## Title
Method of increasing yield in a plant and genes useful therefor

## Abstract

A method of changing the activity of hexokinases in a plant sink comprising introducing into the plant a gene encoding a fungal hexokinase. In the method, the gene is expressed in the plant seed tissue to produce the protein, thereby changing characteristics of hexokinase activity in the seed of the plant.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 USC §119(e) of U.S. provisional application Serial No. 60/468,518 filed May 7, 2003, the entirety of which is hereby incorporated by reference hereto.


FIELD OF THE INVENTION
The present invention relates generally to the field of plant molecular biology, and more specifically to plant cells or plants transformed to contain and express a DNA molecule encoding a hexokinase that confers changes in carbon containing constituents in the seed of a crop plant.
BACKGROUND OF THE INVENTION
Corn is used in a broad range of applications; from ethanol and animal feed production to production of products directly for human consumption, for example breakfast cereal. On average, 20% of corn produced in the U.S. is used for domestic food and industrial purposes. Material output from processed corn includes starch for direct use or chemical modification, starch used as a degradative feedstock for the manufacture of ancillary products, and coproducts/byproducts such as gluten feed, gluten meal and corn oil. As the list of products containing corn-derived ingredients grows, so does the percentage of the corn that is utilized by the corn processing industry.
A central component in the direct, or indirect, use of corn for many products is starch. The central importance of starch to plant development and to food, feed, and industrial markets has motivated researchers across many years to look for mechanisms that control starch biosynthesis. Mutants of maize that affect seed starch deposition have been instrumental in characterizing the biochemistry of starch synthesis. Considerable research effort continues to explore the metabolic systems involved in synthesizing starch, but in addition molecular techniques are being used to analyze and study genes that encode enzymes known to be critical in starch biosynthesis. In discovering which regions of the genes encode metabolism-controlling aspects of the enzymes, scientists are beginning to manipulate starch metabolism through genetic engineering.
The use of seed starch mutants in various crop plants and the production of transgenic plants that over- or under-express specific proteins has indicated that many proteins/enzymes are capable of affecting starch biosynthesis in storage organs. This can occur directly, by impacting the proteins that: (1) produce the substrate(s) for starch synthesis, (2) initiate the glucose polymerization process and elongate the structure into macromolecules, or (3) alter the structure of the polymers once the elongation process has begun. In addition to a direct impact on starch metabolism, starch production can also be negatively impacted by dysfunction or deficiency of proteins that are catalysts in sugar metabolism or act as transporters of intermediary compounds. Proteins involved in assimilate transport, such as the brittle-1 protein of maize endosperm amyloplast membranes, sucrose carrier proteins or others homologous to the hexose transporter of the chloroplast can also affect starch synthesis by restricting the availability of substrates for normal starch and/or sugar metabolism.
Sucrose is considered to be the primary metabolite utilized in the synthesis of starch, although seed grown in vitro with the reducing sugars, glucose or fructose, also produce starch. In simple terms, the sugars are converted into the sugar nucleotides, ADP-glucose and UDP-glucose, either directly or via phosphorylated carbohydrate intermediates. The sugar nucleotides are substrates for the synthase enzymes that polymerize the glucosyl portion of the molecules into long chains of glucose. The polymers remain essentially linear (amylose) or become branched (amylopectin) and combine in a specific fashion to become granules of starch. The proportion of sugar and other carbohydrates, protein, and oils in seeds and fruits is controlled, at least in part, by the conversion of hexose sugars to hexose-6-phosphate by hexose kinase. The hexose kinases can be divided into three general categories according to their hexose substrate specificity. Hexokinase (HK) can phosphorylate glucose and fructose, while glucokinase (GK) and fructokinase (FK) are relatively specific for the respective hexose isomer. Most genomes contain multiple hexose kinases and multiple isozymes of the enzymes. Many plant tissues express hexose kinases.
The hexose sugars used in the production of starch in the seed are moved in the phloem primarily as sucrose. Sucrose is the primary energy source moved from photosynthetic portions of the plant to areas where the energy is utilized or storage of the energy occurs. Sucrose is translocated from the phloem to the seed via specialized cells. Once to the seed, the sucrose (a disaccharide) is often broken down into monosaccharides. Hexokinases add a high energy phosphate to the monosaccharides making hexose sugars available for use in catabolic and anabolic pathways. It has recently been shown that hexokinase expression in an entire plant (driven by the constitutive cauliflower mosaic virus 35S promoter, CaMV35S) leads to growth repression and decreased true leaf development (Xiao, et al., Plant Molecular Biology 44:451, 2000). It has also been shown that overexpression of hexokinase driven by the CaMV 35S promoter inhibits growth, reduces photosynthesis, and induces rapid senescence in tomatoes (Dai, et al., Plant Cell 11:1253, 1999). When these published data are taken together they show that expression of hexokinases under a constitutive promoter is not advantageous for obtaining plants with increased yield and suggest that an overexpression of hexokinase in seed would lead to seed senescence. The present invention, however, shows that the targeted expression of a fungal hexokinase to seeds does not lead to seed senescence, but rather leads to the augmentation of specific aspects of yield, for example, measures of starch per seed.
SUMMARY OF THE INVENTION
The present invention provides a plant expressing a fungal hexokinase gene preferentially in the seeds of the plant thereby influencing carbon sequestration within the seed. In a preferred embodiment, a polynucleotide encoding a fungal hexokinase is regulated by an operably linked promoter directing enhanced expression of the hexokinase coding sequence in seeds as compared to other plant tissues and provides seeds having increased starch content and decreased sugar content.
More specifically, the invention provides a recombinant DNA molecule that comprises in the 5′ to 3′ direction; a promoter providing enhanced expression in a plant seed, or parts thereof, operably linked to a DNA polynucleotide that encodes a fungal hexokinase, operably linked to a 3′ transcription termination DNA polynucleotide. In a preferred embodiment the DNA polynucleotide encoding the fungal hexokinase is capable of hybridizing to SEQ ID NO: 1, under stringent conditions, or encodes a protein substantially identical to yeast hexokinase A (SEQ ID NO: 2). Moreover, to obtain the desired yield benefits, the promoter that provides enhanced expression in a plant seed is selected from the group consisting of an endosperm enhanced promoter, an embryo enhanced promoter, or an aleurone enhanced promoter.
The invention also provides transgenic plants containing the recombinant DNA molecule herein described. In another embodiment, transgenic plants of the present invention expressing a fungal hexokinase may also contain a second recombinant DNA molecule that expresses a transcription factor, polypeptide, enzyme or the like involved in carbohydrate and sugar metabolism, photosynthesis, or respiration and photorespiration in a manner working cooperatively with the fungal hexokinase expression in the seed to increase starch content, increase or decrease oil, or change the amount of other carbon containing compounds in the seed, and/or decrease sugar content, or increase/decrease other carbon containing compounds. Seed or hybrid seed derived from or directly obtained from a transgenic event containing the hexokinase transgene of the present invention is also an aspect of the present invention and such seed may advantageously be coated with a pesticide for cooperative effect.
A method of producing a plant with increased starch content in its seeds is also provided. This method comprises the steps of inserting into the genome of a plant cell a recombinant DNA molecule comprising;

    
    
        a) a seed-enhanced promoter that functions in plants, operably linked to;
        b) a DNA polynucleotide that encodes a fungal hexokinase, operably linked to;
        c) a transcription termination polynucleotide that functions in plants;
        wherein said promoter is heterologous with respect to the fungal hexokinase polynucleotide;
        obtaining a transformed plant cell containing the recombinant DNA molecule;
        regenerating a transformed plant therefrom; and
        identifying and selecting a transformed plant having an increased starch content in its seed. Similarly, methods for producing transgenic plants having decreased hexose sugar in its seeds or increased oil content in its seeds by introduction of the recombinant DNA molecule of this invention are also possible. Specific embodiments of the above methods could include the methods above wherein said fungal hexokinase is selected from SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30.
    
    


Also provided is a plant according to the invention wherein the number of copies of the recombinant DNA molecule according to the invention is modulated in the seed of said plant through breeding.
A fungal hexokinase that is identical to, hybridizes to, or is 70%, 75%, 80% 85%, 90%, 95%, 98%, or greater identity to at least one polynucleotide selected from the group consisting of SEQ ID NOs: 22, 24, 26, 28, or 30 is also provided herein.
Also provided herein are commodities, food and/or feed ingredients resulting from the milling and processing of seeds or plant tissues of the present invention containing the hexokinase described herein, e.g. oil, meal, flour or other end- or by-product. Food and feed products containing or made with at least one of these commodities, food or feed ingredient are also encompassed herein.



BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING
FIG. 1 shows a plasmid map for pMON 17718.
FIG. 2 shows a plasmid map for pMON45007.
FIG. 3 shows a plasmid map for pMON36176.
FIG. 4 is a graph showing sugar levels for each genotype expressed in units of an internal standard ratio/milligram fresh weight. The metabolites are relative to an internal standard, which therefore produces a ratio. The mean is shown with standard deviation. P-values were calculated to determine statistically significant differences. Differences significant at 90% or greater are shown. Negative control is wildtype elite corn germplasm type B kernels, 20 days after planting. Error bars show standard deviation. A.) Hexose levels B.) Sucrose levels.
FIG. 5 shows a graph of in vivo starch levels for each genotype. The mean is shown with standard deviation. P-values were calculated to determine statistically significant differences. Differences significant at 90% or greater are shown. Negative control is wildtype elite corn germplasm type B kernels, 20 days after planting.
FIG. 6 shows a graph of starch weight. Starch weight was measured by weighing the starch pellet and relating back to fresh weight to determine the percent of fresh weight that is starch. Three events were measured, both positive and negative, as determined by Western analysis. Error bars show standard deviation.
FIG. 7 shows a metabolite analysis of seed, wherein the seed contains one to three copies of the hexokinase transgene in the endosperm. Because the endosperm is triploid, two paternal and one maternal genome, one to three copies of the transgene can be selected by selecting the number of copies and the parents of origin.
FIG. 8 shows Sucrose and starch results for selfed material. Sucrose is expressed in nmol sucrose/mg fresh weight. Starch is expressed in nmol glucose equivalents/mg fresh weight. A: Sucrose in 20 DAP kernels. B: Starch in 20 DAP kernels. C: Sucrose in 45 DAP kernels. D: Starch in 45 DAP kernels. Error bars represent standard error of the mean.
FIG. 9 shows sucrose and starch results for crossed material. Sucrose is expressed in nmol sucrose/mg fresh weight. Starch is expressed in nmol glucose equivalents/mg fresh weight. A: Sucrose in 20 DAP kernels. B: Starch in 20 DAP kernels. C: Sucrose in 45 DAP kernels. D: Starch in 45 DAP kernels. Error bars are standard error of the means. *p<0.05, **p<0.01.
FIG. 10 shows kernel phenotypes of 45 DAP crossed material. A: Oil Content measured by NMR. B: Protein Content measured by NIT.
FIG. 11 shows A) Kernel density measured by Micromeritics AccuPyc Pycnometer, and B) Seed weights.
FIG. 12. Starch in mature kernels. A. Total starch of HC33×LH172. B. Total Starch of LH244×LH172. A and B, Each bar represents measurements from 15 ears. The average with the standard error are graphed. C. Overall total starch in kernels of each tester with the events averaged. P value less than 0.1 are noted with an asterisk.
FIG. 13. Hexokinase Fermentation Results after 48 and 54 hours. The average with the standard error is graphed. P value less than 0.1 are noted. A. Fermentation at 48 hours. B. Fermentation at 54 hours.
FIG. 14. Protein Results by NIR and by Leico. The average with the standard error is graphed. P value less than 0.1 are noted. A. Protein by NIR B. Leico analysis of 4 events in LH244×LH172.
FIG. 15. Oil Results by NIT and by Bulk NMR. The average with the standard error is graphed. P value less than 0.1 are noted. A. Oil by NIT B. Bulk NMR results of 4 events in LH244×LH172.



The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing that form a part of this application.
DETAILED DESCRIPTION OF THE INVENTION
It has been discovered that the preferential expression of a polynucleotide encoding a fungal hexokinase (SEQ ID NO:1 and 2) in the seeds and/or seed tissue of a plant unexpectedly increases the yield of the plant as measured by starch content in the seeds. Therefore, in a first aspect the present invention provides nucleic acid molecules encoding a fungal protein having the biological activity of a hexokinase. The nucleic acid of the invention may be in the form of RNA or DNA, including cDNA, synthetic DNA or genomic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded can be either the coding strand or non-coding strand. An RNA analog may be for example, mRNA or a combination of ribo- and deoxy-ribonucleotides. Illustrative examples of a polynucleotide of the present invention are provided. As used herein, “fungus or fungal” refers to any of numerous eukaryotic organisms of the kingdom Fungi, which lack chlorophyll and vascular tissue and range in form from a single cell to a body mass of branched filamentous hyphae that often produce specialized fruiting bodies. The kingdom includes, but is not limited to, the yeasts, molds, smuts, and mushrooms.
A polynucleotide of the invention typically is at least fifteen (15) nucleotides (or base pairs, bp) in length. In some embodiments, a polynucleotide is about 20 to 100 nucleotides in length, or about 100 to 500 nucleotides in length. In other embodiments, a polynucleotide is greater than about 1000 nucleotides in length and encodes a polypeptide having the amino acid sequence of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.
A polynucleotide of the present invention may encode analogs or derivatives of a polypeptide having the deduced amino acid sequence of SEQ ID NO:2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. Such fragments, analogs or derivatives include, for example, naturally occurring allelic variants, non-naturally occurring allelic variants, deletion variants and insertion variants, that do not substantially alter the function of the polypeptide.
The present invention also comprises polynucleotides that hybridize specifically to a fungal hexokinase disclosed herein (SEQ ID NOs: 1, 5, 7, 9, 11, 13, 15 ,17 ,19, 21 ,23, 25, 27, or 29). Such a polynucleotide typically is at least 15 nucleotides in length, and more preferably at least 50 nucleotides in length. These polynucleotides hybridize specifically to a strand of a nucleic acid molecule of this invention, i.e., they do not or only to a small extent hybridize to nucleic acid sequences encoding other proteins. Hybridization typically involves a method by which the presence of DNA sequences in a target nucleic acid mixture are identified by hybridization to a labeled oligonucleotide or DNA fragment probe. Hybridization conditions are sequence dependent and will be different in different circumstances. As used herein “stringent conditions” are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The “thermal melting point” is the temperature (under defined ionic strength and pH) at which 50% of a target molecule hybridizes to a completely complementary molecule. Appropriate stringent conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringent condition of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringent conditions at room temperature, about 22° C., to high stringent conditions at about 65° C. Both temperature and salt concentration may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. For the purposes of this disclosure, stringent conditions include at least one wash in 2.0×SSC at a temperature of at least about 50° C. for 20 minutes, or equivalent conditions. As is understood in the art, high stringency conditions are used to identify nucleic acids that have a high degree of homology to the probe used in the hybridization study.
A polynucleotide within the scope of this invention has at least about 70% sequence identity, preferably about 80% sequence identity, more preferably about 90% sequence identity and most preferably about 95% sequence identity to SEQ ID NOs: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. Optimal alignment of sequences may be conducted by computer programs utilizing algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference nucleic acid or protein may be a full-length molecule or a portion of a longer molecule. Identity is determined typically by comparison of a first nucleic acid or protein sequence, when optimally aligned (with appropriate nucleotide or amino acid insertions or deletions totaling less than 20 percent of the reference sequence over the window of comparison) with the other nucleic acid (or its complementary strand) or protein over a comparison window of at least 20 nucleotide or amino acid positions; at least 50 nucleotide or amino acid positions, at least 100 nucleotide or amino acid positions; or over the entire length of the first nucleic acid or protein.
A polynucleotide of the invention may be obtained by chemical synthesis, isolation and cloning from fungal genomic DNA or other means known to the art, including the use of PCR technology carried out using oligonucleotides corresponding to SEQ ID NO:2, SEQ ID NO:3 or portions of SEQ ID NOs: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. PCR refers to the technique in which a target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, herein incorporated by reference hereto.
The proteins encoded by the various variants of the nucleic acid molecules of this invention exhibit certain common characteristics. Enzyme activity, molecular weight, immunological reactivity, conformation, etc. may be a part of such characteristics as well as physical properties such as mobility in gel electrophoresis, solubility, stability, pH-optimum, temperature optimum, etc.
A polypeptide of this invention comprises an isolated polypeptide having the amino acid sequence of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 as well as derivatives and analogs thereof. It should also be appreciated that certain amino acid substitutions can be made in protein sequences without affecting the function of the protein. Generally, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties. An “isolated” polypeptide or nucleic acid sequence is substantially separated or purified away from other nucleic acid sequences with which the protein or nucleic acid is normally associated in the cell of the organism in which the protein or nucleic acid naturally occurs, i.e., expression of a protein in a non-host organism or other chromosomal or extrachromosomal DNA. The term embraces proteins or nucleic acids that are biochemically purified so as to substantially remove contaminating proteins or nucleic acids and other cellular components. The term also embraces recombinant proteins or nucleic acids and chemically synthesized proteins or nucleic acids.
The invention further relates to vectors, especially plasmids, and other vectors common in genetic engineering, that contain the above-described polynucleotides. Preferably, the polynucleotides contained in the vectors are operably linked to regulatory elements that provide for the transcription and expression of a polypeptide or protein in eukaryotic cells. Typically, the recombinant DNA molecule containing the polynucleotide of the present invention will include in an operably linked manner, in the 5′ to 3′ direction of transcription, a transcription initiation control region capable of promoting, initiating and/or modulating transcription and translation in a plant cell (a promoter region), the polynucleotide encoding a fungal hexokinase, and a transcription and translation termination region (otherwise referred to as a 3′ untranslated region). As used herein, a first nucleic acid sequence is “operably linked” with a second nucleic acid sequence when the sequences are so arranged that the first nucleic acid sequence affects the function of the second nucleic-acid sequence. Preferably, the two sequences are part of a single contiguous nucleic acid molecule and more preferably are adjacent. A “recombinant” nucleic acid or “recombinant DNA molecule” is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. The terms “recombinant DNA construct”, “recombinant vector”, “expression vector” or “expression cassette” refer to any agent such as a plasmid, cosmid, virus, BAC (bacterial artificial chromosome), autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule in which one or more DNA sequences have been linked in a functionally operative manner.
As used herein, a “promoter” refers to a DNA sequence that binds an RNA polymerase (and often other transcription factors as well) and promotes transcription of a downstream DNA sequence. Said sequence can be an RNA that has function, such as rRNA (ribosomal RNA) or tRNA (transfer RNA). Often, the RNA produced is a hetero-nuclear (hn) RNA that has introns that are spliced out to produce an mRNA (messenger RNA). A “plant promoter” is a native or non-native promoter that is functional in plant cells. Constitutive promoters are functional in most or all tissues of a plant throughout plant development. Tissue-, organ- or cell-specific promoters are expressed only or predominantly in a particular tissue, organ, or cell type, respectively. “Specifically” expressed and “enhanced” expression are used inter-changeably herein. “Enhanced expression” is used herein to refer to any promoter that provides an increased expression in a single tissue or developmental stage, or under a particular environmental condition, but causes expression, even significant expression, in other tissue(s), or developmental stage(s), or environmental condition(s).
Temporally regulated promoters are functional only or predominantly during certain periods of plant development or at certain times of day, as in the case of genes associated with circadian rhythm, for example. Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, cold, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
For the most part, any plant promoter can be used as a 5′ regulatory sequence to modulate expression of a particular gene or genes, such as a plant RNA polymerase II promoter. When fused to heterologous DNA sequences, such promoters typically cause the fused sequence to be transcribed in a manner that is similar to that of the gene sequence with which the promoter is normally associated. Promoter fragments that include regulatory sequences can be added (for example, fused to the 5′ end of, or inserted within, an active promoter having its own partial or complete regulatory sequences. Alternatively, heterologous regulatory sequences can be added to the 5′ upstream region of an inactive, truncated promoter, e.g., a promoter including only the core TATA and, sometimes, the CCAAT sequences.
Promoters are typically comprised of multiple distinct “cis-acting transcriptional regulatory elements,” or simply “cis-elements,” each of which can confer a different aspect of the overall control of gene expression. The promoter sequences of the present invention may contain “cis elements” that modulate gene expression. Cis elements can be part of the promoter, or can be upstream or downstream of said promoter. Cis elements (or groups thereof), acting at a distance from a promoter are often referred to as repressors or enhancers. Enhancers act to upregulate the transcriptional initiation rate of RNA polymerase at a promoter, repressors act to decrease said rate. In some cases, the same elements can be found in a promoter and an enhancer or repressor. Cis elements are generally sites where transcription factors bind to the DNA and modulate the rate at which RNA polymerase binds to the promoter.
Examples of constitutive promoters that are active in plant cells include but are not limited to the nopaline synthase (NOS) promoters; the cauliflower mosaic virus (CaMV) 19S and 35S (sometimes called 35S herein, or a derivative of which is called e35S {as described in U.S. Pat. Nos. 5,359,142, 5,196,525, 5,322,938, 5,164,316, and 5,424,200 each of which being hereby incorporated by reference hereto }); the tobacco mosaic virus promoter; the figwort mosaic virus promoters; and actin promoters, such as the Arabidopsis actin gene promoter.
The term “tissue-specific promoter” means a regulatory sequence that causes an enhancement of transcription from a downstream gene in specific cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots, leaves or stems, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. One skilled in the art will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to expression in other tissues as well.
In a preferred embodiment of the invention, the fungal hexokinase of this invention is operably linked to a seed-specific or seed-enhanced promoter, or promoter that directs expression in the seed or some specific tissue or region therein.
Promoters derived from genes encoding embryonic storage proteins, which includes the gene encoding the 2S storage protein from Brassica napus (Dasgupta et al,,Gene 133:301-302 (1993)); the 2s seed storage protein gene family from Arabidopsis; the gene encoding oleosin 20 kD from Brassica napus (GenBank No. M63985); the genes encoding oleosin A (GenBank No. U09118) and oleosin B (GenBank No. U09119) from soybean; the gene encoding oleosin from Arabidopsis (GenBank No. Z17657); the gene encoding oleosin 18 kD from maize (GenBank No. J05212, Lee, Plant Mol. Biol. 26:1981-1987 (1994)); and the gene encoding low molecular weight sulphur rich protein from soybean (Choi et al., Mol. Gen. Genet. 246:266-268 (1995)), can also be used. Promoters derived from zein encoding genes (including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD, and gamma genes) (Pedersen et al., Cell 29: 1015-1026 (1982)) can be also used. The zeins are a group of storage proteins found in maize endosperm.
Other examples of possible suitable regulatory sequences include introns, 3′ non-coding regions such as poly A sequences, insulator regions and the like. Molecular biological techniques for identifying, obtaining and using such regulatory elements in combination with the polynucleotides of the present invention are known in the art.
The present invention further relates to transgenic plant cells and transgenic plants having been transformed to contain and express the fungal hexokinase polynucleotide of the invention. “Transformed”, “transfected”, or “transgenic” refers to a cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid, such as a recombinant vector. Preferably, the introduced nucleic acid is integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced nucleic acid is inherited by subsequent progeny. A “transgenic” or “transformed” cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a “transgenic” plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a recombinant construct or vector. The method of transformation is not critical to the current invention and various methods of plant transformation are currently known and available. For example, the introduction of DNA sequences into plants and/or plant cells can be accomplished by Agrobacterium mediated transformation, viral vector mediated transformation, electroporation, and microprojectile bombardment mediated transformation (particle gun or biolistics methods. The DNA sequence may also be transformed directly into the plastid genome by plastid transformation. As used herein, the term “plastid” means the class of plant cell organelles that includes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids. These organelles are self-replicating, and contain what is commonly referred to as the “chloroplast genome,” a circular DNA molecule that ranges in size from about 120 to about 217 kb, depending upon the plant species, and which usually contains an inverted repeat region.
This invention is applicable to dicotyledonous and monocotyledonous species alike and is readily applicable to new and/or improved transformation techniques. For efficient production of transgenic plants from plant cells or plant tissue, it is desirable that the plant tissue used for transformation possess a high capacity for regeneration. Techniques are known in the art to regenerate plants from transformed plant tissue cultures of plant cells.
The transgenic plants produced in accordance with this invention may, as previously stated, be any dicotyledonous or monocotyledonous species and are preferably from useful species such as rice, maize (corn), peas, soybean, alfalfa, cassava, potato, cotton, and cereals (e.g., barley, oats, rye, triticale and wheat). Fertile transgenic plants so produced would produce transgenic seed that can be grown and selfed (or outcrossed and selfed) to obtain seeds homozygous for the fungal hexokinase polynucleotide. Seeds can be analyzed in order to identify those homozygotes having the desired expression level and pattern of the DNA molecule containing the selected seed specific promoter and polynucleotide encoding for a fungal hexokinase. The invention also relates to propagules and propagation material of the plants of the invention. As used herein, “propagule” includes all products of meiosis and mitosis, including but not limited to, seed and parts of the plant able to propagate a new plant. For example, propagule includes a shoot, root, or other plant part that is capable of growing into an entire plant. Propagule also includes grafts where one portion of a plant is grafted to another portion of a different plant (even one of a different species) to create a living organism. Propagule also includes all plants and seeds produced by cloning or by bringing together meiotic products, or allowing meiotic products to come together to form an embryo or fertilized egg (naturally or with human intervention). As used herein yeast regularly refers to Saccharomyces cerevissiae but could also include Schizosacchoramyces pombe and other varieties (from the genus Pichia, for example). Corn refers to Zea Mays and all species and varieties that can be bred with it. Wheat refers to all of Triticum aestivum varieties including but not limited to spring, winter, and all facultative wheat varieties. Wheat includes any other wheat species, including but not limited to durum wheat (Triticum durum), spelt (Triticum spelta), emmer (Triticum dicoccum), and wild wheat (Triticum monococcum). Wheat also includes any species that can be bred with any of the aforementioned wheat species. Soybeans refers to Glycine max and any species or variety that can be bred with it. Rice refers to Oriza sativa and any species or variety that can be bred with it. Barley refers to Hordeum vulgare and any species or variety that can be bred with it. Oats refers to Avena sativa and any species or variety that can be bred with it. Canola is a coined name recently given to seed, oil, and meal produced by genetically modified rapeseed plants, oilseed rape (Brassica napus L.) and turnip rape (B. campestris L), herein canola includes all rapeseed plants and organisms that can be bred with them. Agrobacterium tumefaciens as used herein includes all strains and types of this species. Cotton refers to all plants in the genus Gossypium and all plants that can be bred with them.
A method according to the invention comprises introducing a DNA molecule containing a promoter functional in plant cells operably linked to a nucleic acid sequence encoding a fungal, specifically a yeast, hexokinase and producing a plant (as well as fertile progeny plant of such a plant) from the transformed plant cell. Progeny includes fertile descendants of a particular plant or plant line.
The compositions, plants and methods according to the invention described herein are useful in that the transgenic plants produced hereby have desirable yield characteristics in the sink tissues, e.g. seed, manifested by increased starch content. The transgenic plants of the present invention may also be characterized as having decreased sugar content and/or decreased oil content as compared to a native or non-transformed plant of the same species. Plants and other organisms contain multiple forms of hexokinases, and these generally include glucokinases (EC 2.7.1.2) and fructokinases (EC 2.7.1.4). It is demonstrated herein that the activity of a fungal hexokinase, particularly yeast hexokinase A, is not inhibited by increased concentrations of fructose. The expression of fungal hexokinase in sink tissues, such as seeds, provides a way to maintain and enhance sucrose utilization by alleviating any buildup of hexose that may otherwise feedback inhibit sucrose synthase. This enhancement of sucrose synthase increases the flux of carbon through the pathway leading to increases in starch, sugar, and/or other carbohydrates leading to an increase in yield as measured by increasing seed weight and/or starch production.
Hexokinase is believed to function via the phosphorylation of hexose sugars within the seed. These phopohorylated sugars are then available for any catabolic or anabolic pathway. By introducing other transgenes that cause expression of other enzymes within the seed these sugars can be directed toward specific pathways or functions. One possible mechanism for this is the expression of a sucrose phosphorylase gene (gtfA) such as the gene from Strepococcus mutans. It has previously been shown that the expression of this enzyme in maize endosperm can result in an increase in starch accumulation (U.S. Pat. Nos. 6,235,971, 6,222,098, and 5,716,837 herein incorporated by reference hereto for a description of genes encoding sucrose phosphorylase). The addition of a fungal hexokinase to a plant or seed already expressing gtfA may further enhance the effects of gtfA by alleviating any fructose pool that potentially could inhibit sucrose synthase. Thus, a nucleic acid sequence encoding a fungal hexokinase may be used in conjunction with other useful genes to further enhance the yield of a plant as measured by starch accumulation or carbon assimilation in the seeds. Other useful genes for use in combination with yeast hexokinase includes, but is not limited to, sucrose phosphorylase, ADP- or UDP-glucose pyrophosphorylase, sedoheptulose 1,7-bisphosphatase enzyme, soluble starch synthase, starch branching enzyme, granule bound starch synthase, starch phosphorylases, starch debranching enzymes, isoamylases, disproportionating enzymes, fructose 1,6 biphosphate aldolase, acetyl coA carboxylase, oleate hydroxylase, 3-ketoacetyl coA sythase III, 3-ketoacetyl coA sythase II, 3-ketoacetyl coA sythase I, Malonyl CoA:ACP transcyclase, 3-ketoacetyl-ACP reductase, sucrose synthase, sucrose phosphate synthase, sucrose phosphate phosphorylase, phosphoglucomutases, phosphoglucoisomerases 2,3-trans-enoyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase, β-GDH, and α-GDH, KAS I dependent FAS, KAS II dependent FAS, stearoyl-ACP desaturase, Palmitoyl-ACP thioesterase, oleoyl-ACP thioesterase, oleate elongase, acyl-CoA:glycerol-3-phosphate acyltransferase, acyl-CoA:lysophosphotidate acyltransferase, phosphotidate phosphatase, CDP-choline:diacylglycerol cholinephosphotransferase, oleate desaturase FAD2, linoleate desaturase FAD3, acyl-CoA:sn-1 acyllysophosphatidylcholine acyltransferase, and oleosin.
It may also be desirable to provide a transgenic seed of the present invention whereby the copy number of the transgene in the transgenic event is modulated in a manner to enhance yield. Modifications of the number of copies of a transgene in a seed can be modulated by controlling whether the transgene is present in a heterozygous or homozygous state within the parent plants, or is not present at all (nullizygous). This modification of copy number can be controlled, for example, by using a homozygous male and nullizygous female. This combination would create a seed wherein one copy of the transgene was present in the embryo and two copies would be present in the endosperm. The number of copies of the transgene can be controlled to create plants, wherein the seeds can have different numbers of copies of the transgene in the endosperm. Changing the number of copies of the transgene in the endosperm can lead to changes in the amount of transgene protein produced within the seed, and change aspects of yield. The copy number can vary from one to three copies if there is one transgene with one insertion site per haploid genome. The yield of the transgenic event is then modulated by the change in the amount and ratio of the carbon containing compounds in the seed resulting from the change in copy number of the transgene.
A seed of the present invention comprising a transgenic event as described herein may also be treated with a pesticide. It is believed that the combination of a transgenic seed exhibiting increased starch content and thereby increased yield as a result of the production of a fungal hexokinase of the invention within the cells of the transgenic seed or plant grown from the seed coupled with treatment of the seed with certain chemical or protein pesticides may provide unexpected synergistic advantages to seeds having such treatment, including unexpectedly superior efficacy for yield potential.
When it is said that some effects are “synergistic”, it is meant to include the synergistic effects of the combination on the pesticidal activity (or efficacy) of the combination of the transgenic event and the pesticide.
Pesticides suitable for use in seed treatments in the invention include pyrethrins and synthetic pyrethroids; oxadizine derivatives; chloronicotinyls; nitroguanidine derivatives; triazoles; organophosphates; pyrrols; pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentation products; and carbamates. Known pesticides within these categories are listed in The Pesticide Manual, 11th Ed., C. D. S. Tomlin, Ed., British Crop Protection Council, Farnham, Surry, UK (1997). Particularly preferred synthetic pyrethroids are tefluthrin, lambda cyhalothrin, bifenthrin, permethrin and cyfluthrin. Even more preferred synthetic pyrethroids are tefluthrin and lambda cyhalothrin, and yet more preferred is tefluthrin. Insecticides that are oxadiazine derivatives are useful in the seed treatments and coatings along with compositions and seeds of the present invention. The oxadizine derivatives that are preferred for use in the present invention are those that are identified in U.S. Pat. No. 5,852,012.
Chloronicotinyl insecticides are also useful in the subject invention. Chloronicotinyls that are preferred for use in the subject composition are described in U.S. Pat. No. 5,952,358, and include acetamiprid ((E)-N-[(6-chloro-3-pyridinyl)methyl]-N′-cyano-N-methyleneimidamide, CAS RN 135410-20-7), imidacloprid (1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2-imidazolidinimime, CAS RN 138261-41-3), and nitenpyram (N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N′-methyl-2-nitro-1,1-ethenediamine, CAS RN 120738-89-8).
Nitroguanidine insecticides are useful in the present invention. Such nitroguanidines can include those described in U.S. Pat. Nos. 5,633,375, 5,034,404 and 5,245,040.
Pyrrols, pyrazoles and phenyl pyrazoles that are useful in the present invention include those that are described in U.S. Pat. No. 5,952,358. Diacylhydrazines that are useful in the present invention include halofenozide (4-chlorobenzoate-2-benzoyl-2-(1,1-dimethylethyl)-hydrazide, CAS RN 112226-61-6), methoxyfenozide (RH-2485; N-tert-butyl-N′-(3-methoxy-o-toluoyl)-3,5-xylohydrazide, CAS RN 161050-58-4), and tebufenozide (3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2,(4-ethylbenzoyl)hydrazide, CAS RN 112410-23-8).
Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate are useful in the present invention. A preferred triazole is triazamate (ethyl [[1-[(dimethylamino)carbonyl]-3-(1,1-dimethylethyl)-1H-1,2,4-triazol-5-yl]thio]acetate, CAS RN 112143-82-5).
Biological/fermentation products, such as avermectin (abamectin, CAS RN 71751-41-2) and spinosad (XDE-105, CAS RN 131929-60-7) are useful in the present invention.
Organophosphate insecticides are also useful as one of the components of seed treatments and coatings in the present invention. Preferred organophophate insecticides include acephate (CAS RN 30560-19-1), chlorpyrifos (CAS RN 2921-88-2), chlorpyrifos-methyl (CAS RN 5598-13-0), diazinon-(CAS RN 333-41-5), fenamiphos (CAS RN 22224-92-6), and malathion (CAS RN 121-75-5). In addition, carbamate insecticides are useful in the present invention. Preferred carbamate insecticides are aldicarb (CAS RN 116-06-3), carbaryl (CAS RN 63-25-2), carbofuran (CAS RN 1563-66-2), oxamyl (CAS RN 23135-22-0) and thiodicarb (CAS,RN 59669-26-0).
When an insecticide is described herein for use in a seed treatment and/or seed coating, it is to be understood that the description is intended to include salt forms of the insecticide as well as any isomeric and/or tautomeric form of the insecticide that exhibits the same insecticidal activity as the form of the insecticide that is described.
The insecticides that are useful in the seed treatments and coatings of the present invention can be of any grade or purity that pass in the trade as such insecticide. Other materials that accompany the insecticides in commercial preparations as impurities can be tolerated in the subject methods and compositions, as long as such other materials do not destabilize the composition or significantly reduce or destroy the activity of any of the activities or components or the transgenic event. One of ordinary skill in the art of the production of insecticides can readily identify those impurities that can be tolerated and those that cannot.
It is preferred that the seed treatments and coatings described herein be used along with transgenic seeds of the present invention. Although it is believed that the seed treatments can be applied to a transgenic seed in any physiological state, it is preferred that the seed be in a sufficiently durable state that it incurs no damage during the treatment process. Typically, the seed would be a seed that had been harvested from the field; removed from the transgenic plant; and separated from any other non-seed plant material. The seed would preferably also be biologically stable to the extent that the treatment would cause no biological damage to the seed. In one embodiment, for example, the treatment can be applied to seed corn that has been harvested, cleaned and dried to a moisture content below about 15% by weight. In an alternative embodiment, the seed can be one that has been dried and then primed with water and/or another material and then re-dried before or during the treatment with the pesticide. Within the limitations just described, it is believed that the treatment can be applied to the seed at any time between harvest of the seed and sowing of the seed. As used herein, the term “unsown seed” is meant to include seed at any period between the harvest of the seed and the sowing of the seed in the ground for the purpose of germination and growth of the plant.
When it is said that unsown seed is “treated” with the pesticide, such treatment is not meant to include those practices in which the pesticide is applied to the soil, rather than to the seed. For example, such treatments as the application of the pesticide in bands, “T”-bands, or in-furrow, at the same time as the seed is sowed are not considered to be included in the present invention.
The pesticide, or combination of pesticides, can be applied “neat”, that is, without any diluting or additional components present. However, the pesticide is typically applied to the seeds in the form of a pesticide formulation. This formulation may contain one or more other desirable components including but not limited to liquid diluents, binders to serve as a matrix for the pesticide, fillers for protecting the seeds during stress conditions, and plasticizers to improve flexibility, adhesion and/or spreadability of the coating. In addition, for oily pesticide formulations containing little or no filler, it may be desirable to add to the formulation drying agents such as calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth or any other adsorbent material. Use of such components in seed treatments is known in the art. See, e.g., U.S. Pat. No. 5,876,739. The skilled artisan can readily select desirable components to use in the pesticide formulation depending on the seed type to be treated and the particular pesticide that is selected. In addition, readily available commercial formulations of known pesticides may be used, as demonstrated in the examples below.
The seeds may also be treated with one or more of the following ingredients: other pesticides, including compounds which act only below the ground; fungicides, such as captan, thiram, metalaxyl, (methoxam=resolved isomer of metalaxyl), fludioxonil, oxadixyl, and isomers of each of those materials, and the like; herbicides, including compounds selected from carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; fertilizers; and biocontrol agents such as naturally-occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichodenna, Glomus, Gliocladium and mycorrhizal fungi. These ingredients may be added as a separate layer on the seed or alternatively may be added as part of the pesticide composition.
Preferably, the amount of the novel composition or other ingredients used in the seed treatment should not inhibit generation of the seed, or cause phytotoxic damage to the seed.
The pesticide formulation that is used to treat the transgenic seed in the present invention can be in the form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., water); wettable powder; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation is preferably about 0.5% to about 99% by weight (w/w), preferably 5-40% or as otherwise formulated by those skilled in the art of applying such a formulation to the seed of a particular crop plant.
As mentioned above, other conventional inactive or inert ingredients can be incorporated into the formulation. Such inert ingredients include but are not limited to: conventional sticking agents, dispersing agents such as methylcellulose (Methocel A15LV or Methocel A15C, for example, serve as combined dispersant/sticking agents for use in seed treatments), polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P), polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPIVA S-630), thickeners (e.g., clay thickeners such as Van Gel B to improve viscosity and reduce settling of particle suspensions), emulsion stabilizers, surfactants, antifreeze compounds (e.g., urea), dyes, colorants, and the like. Further inert ingredients useful in the present invention can be found in McCutcheon's, vol. 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996. Additional inert ingredients useful in the present invention can be found in McCutcheon's, vol. 2, “Functional Materials,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.
The pesticides and pesticide formulations of the present invention can be applied to seeds by any standard seed treatment methodology, including but not limited to mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. Any conventional active or inert material can be used for contacting seeds with pesticides according to the present invention, such as conventional film-coating materials including but not limited to water-based film coating materials such as Sepiret (Seppic, Inc., Fairfield, N.J.) and Opacoat (Berwind Pharm. Services, Westpoint, Pa.).
The subject pesticides can be applied to a seed as a component of a seed coating. Seed coating methods and compositions that are known in the art are useful when they are modified by the addition of one of the embodiments of the combination of pesticides of the present invention. Such coating methods and apparatus for their application are disclosed in, for example, U.S. Pat. Nos. 5,918,413, 5,891,246, 5,554,445, 5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coating compositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356, 5,882,713, 5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103, 5,622,003, 5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587, 4,383,391, 4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.
Useful seed coatings contain one or more binders and at least one of the subject combinations of pesticides. Binders that are useful in the present invention preferably comprise an adhesive polymer that may be natural or synthetic and is without phytotoxic effect on the seed to be coated. The binder may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.
The amount of pesticide that is used for the treatment of the seed will vary depending upon the type of seed and the type of active ingredients, but the treatment will comprise contacting the seeds with an amount of the combination of pesticides that is pesticidally effective. When insects are the target pest, that amount will be an amount of the insecticide that is insecticidally effective and when fungi are the target pest, the amount will be an amount of the fungicide that is fungicidally effective. As used herein, an insecticidally effective amount means that amount of insecticide that will kill insect pests in the larvae or pupal state of growth, or will consistently reduce or retard the amount of damage produced by insect pests and a fungicidally effective amount means that amount that will kill, reduce, retard or inhibit fungal damage produced the target fungi.
In general, the amount of pesticide that is applied to the seed in the treatment will range from about 10 gm to about 2000 gm of the active ingredient of the pesticide per 100 kg of the weight of the seed. Preferably, the amount of pesticide will be within the range of about 50 gm to about 1000 gm active per 100 kg of seed, more preferably within the range of about 100 gm to about 600 gm active per 100 kg of seed, and even more preferably within the range of about 200 gm to about 500 gm of active per 100 kg of seed weight. Alternatively, it has been found to be preferred that the amount of the pesticide be over about 60 gm of the active ingredient of the pesticide per 100 kg of the seed, and more preferably over about 80 gm per 100 kg of seed.
The pesticides that are used in the treatment must not inhibit germination of the seed and should be efficacious in protecting the seed and/or the plant during that time in the target insect's life cycle in which it causes injury to the seed or plant. In general, the coating will be efficacious for approximately 0 to 120 days after sowing.
The pesticides of the subject invention can be applied to the seed in the form of a coating. The use of a coating is particularly effective in accommodating high pesticidal loads, as can be required to treat typically refractory pests, such as corn rootworm, while at the same time preventing unacceptable phytotoxicity due to the increased pesticidal load.
The coatings formed with the pesticide are preferably of the type that are capable of effecting a slow rate of release of the pesticide by diffusion or movement through the matrix to the surrounding medium.
In addition to the coating layer, the seed may be treated with one or more of the following ingredients: other pesticides including fungicides and herbicides; herbicidal safeners; fertilizers and/or biocontrol agents. These ingredients may be added as a separate layer or alternatively may be added in the pesticidal coating layer.
The pesticide formulation may be applied to the seeds using conventional coating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The seeds may be presized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.
The pesticide-treated seeds may also be enveloped with a film overcoating to protect the pesticide coating. Such overcoatings are known in the art and may be applied using conventional fluidized bed and drum film coating techniques.
In another embodiment of the present invention, a pesticide can be introduced onto or into a seed by use of solid matrix priming. For example, a quantity of the pesticide can be mixed with a solid matrix material and then the seed can be placed into contact with the solid matrix material for a period to allow the pesticide to be introduced to the seed. The seed can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus seed can be stored or planted directly. Solid matrix materials which are useful in the present invention include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the pesticide for a time and releasing that pesticide into or onto the seed. It is useful to make sure that the pesticide and the solid matrix material are compatible with each other. For example, the solid matrix material should be chosen so that it can release the pesticide at a reasonable rate, for example over a period of minutes, hours, or days.
The present invention further embodies imbibition as another method of treating seed with the pesticide. For example, plant seed can be combined for a period of time with a solution comprising from about 1% by weight to about 75% by weight of the pesticide in a solvent such as water. Preferably the concentration of the solution is from about 5% by weight to about 50% by weight, more preferably from about 10% by weight to about 25% by weight. During the period that the seed is combined with the solution, the seed takes up (imbibes) a portion of the pesticide. Optionally, the mixture of plant seed and solution can be agitated, for example by shaking, rolling, tumbling, or other means. After imbibition, the seed can be separated from the solution and optionally dried, for example by patting or air drying.
In yet another embodiment, a powdered pesticide can be mixed directly with seed. Optionally, a sticking agent can be used to adhere the powder to the seed surface. For example, a quantity of seed can be mixed with a sticking agent and optionally agitated to encourage uniform coating of the seed with the sticking agent. The seed coated with the sticking agent can then be mixed with the powdered pesticide. The mixture can be agitated, for example by tumbling, to encourage contact of the sticking agent with the powdered pesticide, thereby causing the powdered pesticide to stick to the seed.
The present invention also provides a transgenic corn seed that has been treated with a pesticide by the method described above.
The treated seeds of the present invention can be used for the propagation of corn plants in the same manner as conventional treated corn seed. The treated seeds can be stored, handled, sowed and tilled in the same manner as any other pesticide treated seed. Appropriate safety measures should be taken to limit contact of the treated seed with humans, food or feed materials, water and birds and wild or domestic animals.
Agronomically and commercially important products and/or compositions of matter including but not limited to animal feed, commodities, and seed products and by-products that are intended for use as food for human consumption or for use in compositions that are intended for human consumption including but not limited to flour, meal, syrup, oil, starch, foods containing seeds or seed parts and seed by-products, and the like are intended to be within the scope of the present invention if these products and compositions of matter contain detectable amounts of the nucleotide sequences set forth herein or are derived from or obtained directly from a seed containing a transgenic event of the present invention. Such products and/or compositions are also referred to herein as biological samples. The biological samples can be derived from the transgenic event of the present invention, either from the plant, the plant tissue, or the seed produced by the plant.
All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.
The following examples are included to demonstrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings and examples is to be interpreted as illustrative and not in a limiting sense.
EXAMPLES
Example 1
PCR of Hexokinase From Yeast and Cloning to Create pMON45006
The complete coding sequence of yeast hexokinase (yHxkA) (SEQ ID NO:1) was PCR amplified from yeast genomic DNA based upon publicly available sequence data (Genbank gi:171736, using primers ATAGGATCCATGGTTCATTTAGGT [SEQ ID NO:2] and AAACTCGAGTTAAGCGCCAATGAT [SEQ ID NO:3]). The 5′ and 3′ ends of the nucleotide sequence were modified to include a BamHI and XhoI restriction sites, respectively. This PCR product was isolated and cloned into the commercially available pET30a+vector (Moffatt, B. A. and Studier, F. W. (1986) J. Mol. Biol. 189, 113-130; Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S., Dunn, J. J., and Studier, F. W. (1987) Gene 56, 125-135; Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Meth. Enzymol. 185, 60-89) from Invitrogen (Carlsbad, Calif.), using restriction enzymes. The pET30a+ vector and related vectors are available from Novagen, an affiliate of Merck KgAa, Darmstadt, Germany and details about said vector are available at the Novagen website. This vector provides a means to overexpress ykxkA in E.coli. Crude preparations of the recombinant protein from E. coli containing this vector have been shown to be insensitive to substrate inhibition by glucose and fructose up to 50 mM.
Example 2
Creation of Plasmid pMON17718 and 45007
pMON17718 (SEQ ID NO:33): This plasmid was used as the plant transformation vector for the creation of transgenic corn plants discussed in later examples. A portion of this sequence is shown in SEQ ID NO:34. The plasmid consists of a gene of interest expression cassette consisting of a glutelin-1 promoter (gt1), wild-type (wt) wheat cab (chlorophyll a/b binding protein) leader, rice actin 1 (ractin1) intron, yeast hexokinase A coding region (HXK) (SEQ ID NO:1) and an NOS 3′ terminator. The plasmid also contains the selectable marker (NPTII). The vector was constructed by combining the Not 1 fragment containing gt 1 promoter, wt cab leader, rice actin1 intron/HXK, and NOS 3′ terminator from pMON 45007 (SEQ ID NO: 32; FIG. 2) with the NotI fragment of pMON 36176 (FIG. 3) containing the selectable marker (NPTII). The plasmid also contains the right and left border required for agrobacterium transformation. This vetor was used to transform corn plants via an agrobacterium mediated transformation process.
PMON45007 (SEQ ID NO:32) consists of a gene of interest expression cassette consisting of a glutelin-1 promoter (gt1), wild-type (wt) wheat cab (chlorophyll a/b binding protein) leader, rice actin 1 (ractin1) intron, yeast hexokinase A coding region (HXK) (SEQ ID NO:1) and an NOS 3′ terminator. The plasmid also contains the selectable marker (NPTII). The vector was constructed by combining the Not I fragment containing gt 1 promoter, wt cab leader, rice actinl intron/HXK, and NOS 3′ terminator from pMON 45007 (SEQ ID NO:32) with the Notl fragment of pMON 36176 (FIG. 3) containing the selectable marker (NPTII). An MluI fragment from this plasmid was used for gun based transformation of corn.
All plasmids discussed above can be propagated in E. coli and transformed into E. coli using methods known to those skilled in the art. All plasmids can be isolated from E. coli via published protocols. For methods required to propagate bacteria, isolate DNA from bacteria, create and run agarose gels, isolate DNA from said gels, set up ligations, do restriction digests, transform bacteria, and for other required techniques please see Maniatis et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and myriad other texts and references that discuss molecular biology techniques.
Example 3
Selection of Events in Corn. (pMON45007)
pMON45007 was transformed into corn plants using a particle gun mediated transformation.
The expression of yeast hexokinase A (HXK) in elite corn was confirmed by Western blot analysis using a rabbit antibody raised against HXK (Garvey, et al., Methods in Immunology, 3rd edition, W.A. Benjamin, Inc., Reading Mass., 1977, Part I). Homozygous material was identified from R2 seed by selfing plants identified as positive or negative at the R1 generation. Once homozygous material was obtained, experiments were initiated to determine the efficacy of HXK in the seed of a plant.
Example 4
Carbon-14 Labeling Experiment
Efficacy Studies (Elite Corn Germplasm Type B Genotype)
In order to determine whether the expression of hexokinase and GtfA (glucosyltransferase) in the seed of a plant had an effect on the seed, the following experiments was undertaken.
Crosses between gtfA containing and HXK containing plants were made to evaluate the efficacy of both genes together in a greenhouse experiment. It has been postulated that increased carbon in the seed due to the presence of a fungal hexokinase could be utilized by gtfA to increase aspects of yield. An independent event expressing the gtfA gene was used as the female parent and events expressing the HXK gene were used as the male parent in the following manner:












1 event gtfA(female)

2 events HXK(male)







pos
X
pos


pos
X
neg


neg
X
pos


neg
X
neg









Both genes are targeted for expression in the endosperm. Endosperm tissue is triploid, receiving 2 polar nuclei from maternal and 1 from paternal gametes. Therefore, resulting positive HXK material will have 1 dose of HXK and the resulting positive gtfA material will have 2 doses of gtfA. This material was harvested on ice at 20 days post pollination (20DAP). Multiple ears from each cross were harvested to obtain a sample set that was representative of all crosses.

To evaluate HXK homozygous selfed material, HXK homozygous positive and negative material was selfed. The resulting HXK positive material contained 3 doses of HXK. This allowed us to evaluate metabolic differences between 1 and 3 doses of HXK.

    Metabolite Profiling: Endosperm tissue was dissected, sectioned, and the fresh weight was recorded. The tissue was ground using 3×3 mm steel beads and shaking for 2 minutes in a 4° C. paint shaker. The metabolites were extracted using 80% ethanol with 0.1% formic acid. After centrifugation for 5 minutes at 8000 rpm, the soluble fraction (supernatant) was used to analyze the changes in sugars that are involved in the starch biosynthetic pathway. These metabolites were measured using an in-house method developed using LC/MS/MS (see below). The insoluble fraction (pellet) was analyzed for in vivo starch content by enzymatic digestion followed by glucose determination using a kit from Boehringer Mannheim (Ingelheim, Germany).
    Endosperm Feeding: Endosperm tissue was sliced and dissected away from the other components of the seed. The carbon incorporation into starch was measured in vitro by using a sugar feeding assay which has been described previously (Felker, et al., Plant Physiology 94:996, 1990). Endosperm slices were fed 200 mM 14C-sucrose for 2 and 4 hours. The radioactivity incorporated in sucrose, fructose, glucose, and starch was determined. The amount of label in the sugars was determined by extracting the sugars in 80% EtOH and separating using a Supercosil 5 um, 4.6×250 mm LC-NH2 column on an HPLC, in line with a radiometric detector. The mobile phase was 75% Acetonitrile:25% water with a flow rate of 1 ml/min. The amount of label was quantified by constructing a standard curve using sucrose. The results are reported in micromole Glucose equivalents/micrograms fresh weight, therefore using the sucrose standard curve the result was multiplied by 2 to get to glucose equivalents. The amount of labeled carbon incorporation into starch was determined as well as the percentage of starch weight/fresh weight.
    Activity Measurements: To determine the activity difference between material that contains 1 dose of HXK and 3 doses of HXK, activity assays were done. The method is the NADP coupled reaction shown below:






Results

    Metabolite Profiling: In order to evaluate the effect of HXK on the starch biosynthesis pathway, it was important to look at any alterations in metabolites found in kernels expressing HXK. The data showed that there were no significant differences in any of the sugars when comparing gtfA+/HXK+ to gtfA−/HXK+ at this 20 DAP timepoint. The data for hexoses and sucrose is shown in FIG. 4.


There is a significant difference in the hexose and sucrose levels when 1 dose of HXK is present compared to gtfA alone or the negative control. The results indicate that the change seen in hexoses and sucrose is due to the presence of HXK at this 20 DAP timepoint
The graph in FIG. 5 shows the in vivo starch measurement of the combination material.
The in vivo starch data indicates that the presence of 1 dose of HXK can increase the starch content relative to the negative control and that the level of increase is equivalent to that seen when gtfA is present. However, no synergistic gain is seen at 20 days after pollination when gtfA is combined with HXK. The remaining metabolites measured in the starch biosynthetic pathway are shown in FIG. 6.
The results of analysis of the homozygous HXK material containing 3 doses of HXK, is remarkably different from the results seen when 1 dose of HXK is present. There were no significant changes in the hexoses or sucrose levels across 3 events comparing positive to its' negative isoline. There was a trend towards a decrease in in vivo starch in 2 of the 3 events. Under the conditions of this experiment (20 DAP), there was an average 37% decrease in % starch weight/fresh weight across 3 events as shown in FIG. 6.
The remaining metabolites measured are shown in FIG. 7 with the fold decrease compared to the negative. It appears that the level of expression of HXK is important based on the different results seen depending on the dosage of HXK. When 1 dose of HXK is present, it appears that levels of several carbon metabolites are increased and that the presence of the transgene is having the desired effect on hexose, sucrose, and starch. The level of HXK with 1 dose is sufficient to enhance the flux through the pathway without triggering any regulation that may decrease starch biosynthesis. The level of HXK with 3 doses appears to be reducing the production of starch and causing a transient backup of other metabolites in the pathway, under the conditions of this experiment.
In material containing 1 dose of HXK, sucrose and fructose are decreased as described in a previous figure. The levels of the hexose phosphates and APDG are elevated along with starch levels. The flux through the pathway is enhanced sufficiently enough to enhance starch synthesis but perhaps not enough to trigger any regulatory mechanisms. The levels of hexose phosphates and ADPG are elevated to a higher magnitude compared to 1 dose of hexokinase, however starch is decreased. The levels of triose phosphates are also much higher when 3 doses of HXK is present compared to 1 dose. It appears that when 3 doses of HXK is present, the starch biosynthesis pathway shuts down under the conditions of this experiment at 20 DAP. It may be due to regulatory mechanisms within the glycolytic or other pathways.
The metabolite analysis provides insight to what is occurring within the starch biosynthesis pathway in the endosperm. This reflects only what is occurring at 20 DAP and does not reflect what would happen at maturity. Since a positive effect on starch was seen when one dose of HXK is present, we want to determine what the effect is at seed maturity. Since we see an increase in starch at this timepoint, we will investigate if this translates into an increase in seed weight at maturity, due to the increase in starch. Also, to better understand the effect of the transgene on the pathway, it is important to measure the metabolites and starch levels at several stages of development. This would also be important for the material that is expressing 3 doses of HXK. We may be able to learn what stages of development are critical for increased starch in order to translate into increased seed weight. We can also learn which metabolites are altered and to what magnitude throughout development, when the transgene is present.
To further evaluate if HXK can enhance the effects seen with gtfA, similar types of experiments will be performed. As stated earlier, an increase in fructose was seen when gtfA was present compared to the negative control. However, the increase seen at 20 days after pollination was minimal compared to the increase seen later in development. The efficacy studies done thus far on material expressing both HXK and gtfA have been done at 20 DAP. At this timepoint we may not see a positive effect with both genes, because the fructose levels are not considerably increased. At later timepoints, when the fructose levels are higher, we may see a positive effect because HXK could act to alleviate any buildup of fructose and enhance the flow of carbon through the pathway. Again, if HXK could alleviate the buildup of fructose, then sucrose synthase would remain active and more sucrose would be hydrolyzed. Therefore, we will evaluate sugar and starch levels of material containing both HXK and gtfA at later stages of development.
In the future, we will also evaluate this approach in elite germplasms, LH 172, LH244 and possibly others. We will generate homozygous HXK material to perform similar efficacy studies to evaluate HXK alone and in combination with gtfA.
Example 6
Selection of Events in Elite Corn. (pMON 17718)
Expression of HXK in corn was confirmed in RO plants by Western analysis using a rabbit antibody raised against yeast HXK. The R1/F1 material was advanced and Taqman® (Applied Biosystems, Foster City, Calif.) analysis was done on leaf tissue to determine copy number and to identify putative homozygous material. The R2 seed was screened by Western analysis to confirm Taqman results. The R2/F2 seed was advanced and again sampled for Taqman® to identify additional homozygous events.
Example 9
An efficacy experiment was conducted in a field to investigate the effect of YHXKA on kernel metabolism in the LH172 genotype. The gene was evaluated in selfed material and also crossed onto LH244 (since the endosperm tissue is triploid, receiving 2 polar nuclei from maternal and 1 from paternal gametes, selfed YHXKA material will have 3 doses of YHXKA in the endosperm and the crossed material will have only 1 dose of YHXKA in the endosperm).
Planting & Sampling
Positive and negative selections of 3 homozygous events (YAD 016, 055, 081) were planted (2 rows per selection). All material was hand pollinated (selfed and crossed onto LH244). Kernel samples were harvested onto dry ice at 20 days after planting (DAP) and at 45 DAP. Multiple ears per source were harvested to obtain a sample set that was representative of all crosses. YHXKA expression was checked by Western analysis to confirm expression level and to unequivocally determine the transgenic status of the seed.
Sugar & Starch Extraction
20 DAP kernels were dissected to remove the pericarp and embryo, leaving only the endosperm. The kernels were cut in half, weighed, and placed in a 96 deep well plate (termed the extraction plate). 45 DAP kernels were ground into a powder using a Cemotec 1090 Sample Mill. The powder was weighed and transferred into a 96 deep well plate (termed the extraction plate). Two steel bars and 400 μl of 80% ethanol were added to each well in the extraction plate. The extraction plates were sealed with a capmat and placed in a Harbil 5G-HD paint shaker for 2 cycles of 3 minutes. The extraction plates were incubated in a 55° C. oven for 10 minutes, inverting by hand to mix 1-2 times during incubation. The plates were centrifuged for 5 minutes at 3000 rpm. 200 μl of the supernatant was removed into a new 96 deep well plate (the assay plate) which was used to measure sucrose. 250 μl of 80% ethanol was added to the extraction plates and mixed, incubated, and centrifuged as described above. 250 μl of the supernatant was removed and added to the assay plate for sucrose analysis. An additional 150 μl of 80% ethanol was added and the extraction plateand mixing, incubation, and centrifugation was repeated. 150 μl of the supernatant was removed and added to the assay plate for sucrose analysis. The assay plate for sucrose analysis was placed in the Speedvac overnight to remove the ethanol. The plates were stored at −80° C. until ready to assay. 200 μl of water was added when ready to conduct the assay.
The pellet left from the ethanol extractions was washed with 200 μl of 100% ethanol, placed in a Harbil 5G-HD paint shaker for 3 minutes, and centrifuged for 5 minutes at 3000 rpm. The supernatant was discarded. This ethanol-washing procedure was repeated 4 times. The resulting pellet was placed in the SpeedVac for 1.5 hours and then placed in a 65° C. oven for 2 days to dry the pellet completely. The pellet was then used for starch measurements.
Assays
Sucrose was assayed using the procedure from Bergmeyer, H. U. & Bernt, E. (1974) in Methods of Enzymatic Analysis (2d ed., vol.3, pp. 1176-1179, Verlag Chemie, Weinheim/Academic Press Inc., New York and London).
The dried pellet was digested as follows to determine the starch content. 250 μl of 0.2 N potassium hydroxide was added to the dried pellet, inverted to mix, and placed at 80° C. for 2 hours. 200 μl of 0.25 M sodium acetate buffer (pH 4.65) was added, inverted to mix, and lo centrifuged for 5 minutes at 3000 rpm. 5 units of amyloglucosidase (in 100 mM acetate buffer, pH 4.65) was added to each well and mixed thoroughly. The plates were incubated at 55° C. overnight. The plates were centrifuged for 10 minutes at 3000 rpm. The extract was used to assay for glucose using the protocol from Bergmeyer, H. U. & Bernt, E. (1974) in Methods of Enzymatic Analysis.
Additional Kernel Analysis
Kernels from 45 DAP elite corn hybrids were dried to a moisture content of 8.5-11% in a 37° C. oven. The material was then used to measure kernel weight, protein, oil, and density.
Iodine Staining
The kernels were cut in half and stained using Kent Lugol's solution (potassium iodide and iodine) diluted 1:10. (can be ordered from Sigma-Aldrich Co., St. Louis, Mo. catalog # L6146).
Results
In kernels of the selfed ears at 20 DAP, sucrose levels were significantly reduced, and starch levels were increased in transgene-positive plants for all three events (FIG. 8). However, at maturity (45 DAP), sucrose levels were higher in two events and starch levels were reduced in two events. Starch levels were significantly increased in the YAD8 1 transgene positives at 45 DAP compared to the isogenic negative control (YAD8 1 is a specific insertion site created by a single insertion site, and the progeny of that plant, often this is referred to as an event).
The effect of the transgene on sucrose levels of kernels resulting from the cross were similar to those of the selfed ears (FIG. 9). In kernels from the crossed ears, sucrose levels were reduced in 2 of 3 events at 20 DAP, but sucrose levels were increased in all three events at maturity. However, the effect of the transgene on starch accumulation was more positive in kernels from the crossed ears than in kernels from the selfed ears. Starch levels were higher in transgene positive plants of 2 events at 20 DAP and in 2 events at 45 DAP.
Analysis of kernel protein and oil of mature kernels from the crossed ears was conducted by NIT and NMR, respectively. Interestingly, kernels from events that had increased starch at 45 DAP (FIG. 9) also had reduced oil content (FIG. 10A). Transgene positive kernels of these same events had increased protein (FIG. 10B).
To determine whether the difference in starch accumulation corresponded with differences in starch distribution, mature kernels were stained with iodine. In all three events, we observed that the iodine-resistant “hard” endosperm was reduced in kernels that contain the YHXKA transgene compared to transgene-negative controls (FIG. 12).
Density was measured for mature kernels of crossed ears, and was found to be reduced for all three events (FIG. 11). The decrease ranged from 4.3% to 4.9%. Seed weight was reduced for Event YAD16, but was unchanged for the other two events (FIG. 11). Note small sample size for seed weight data. The corn kernel densities were measured using Micromeritics AccuPyc 1130 Pycnometer. The AccuPyc works by measuring the amount of displaced gas. The pressures observed upon filling the sample chamber and then discharging it into a second empty chamber allow computation of the sample solid phase volume. Gas molecules rapidly fill the tiniest pores of the sample; only the truly solid phase of the sample displaces the gas.
Discussion
The results indicate that endosperm-targeted expression of the YHXKA transgene can result in an increase in starch in corn kernels at maturity (FIGS. 8 and 9). An increase in starch was observed in mature kernels of two events from crossed ears and in one event from selfed ears. The fact that starch was more consistently increased in kernels from the crossed ears could be due to an effect of dosage of the transgene (although other mechanisms are possible), since kernels of the selfed ears would have 3 doses of the transgene, while kernels of the crosses would have only one dose. This mechanism could also explain the differences in events in the selfed ears, if YAD81 has lower enzyme activity than the other events. Alternatively, the difference in results from selfed and crossed ears could be due to differences in the genetic backgrounds (although other mechanisms are possible). The selfs would be homozygous for LH172 alleles at all loci, while the crosses were heterozygous for all LH172 and LH244 at all alleles. The difference in kernel quality due to the different genetic background of the selfs and crosses was readily observed by kernel size.
The increase in kernel starch was accompanied by a small increase in kernel protein, and a decrease in kernel oil (FIG. 10). These composition changes resulted in an approximately 5% decrease in density of the kernel.
Fermentation Assay
The Fermentation method uses High Performance Liquid Chromatography (HPLC) with refractive index detection (RID) to separate and quantify ethanol. The fermentation requires a two step process. The first step is the liquefaction of the starch to soluble dextrins by adding water and alpha amylase to the 25.0 g flour sample and placing in an 85° C. water bath. The second step is the simultaneous hydrolysis of the dextrins and the fermentation of glucose using additional enzymes and yeast. The relative standard deviation for this method is  ̃2%.
Protein Assays
First pass protein analysis was conducted by NIT. To confirm the results, a sub-set of the events in one tester (Events 2, 21, 32, 82 in the LH244×LH172) were analyzed using Elemental Analysis from Leico which uses combustion GC analysis.
Oil Assays
First pass oil analysis was conducted by NIT. To confirm the results, a sub-set of the events from one tester (Events 2, 21, 32, 82 in the LH244×LH172) were analyzed by bulk NMR.
Results
Sucrose & Starch
There were no overall differences in sucrose in mature kernels when comparing positive to negative in either tester (data not shown). The measurement of total starch did reveal differences (FIG. 12). In the HC33 tester, one event (86) had a significant increase in total starch (FIG. 12A). Another event (13) had a trend towards an increase in starch, but the difference was not quite statistically significant. In the LH244 tester, 4 events (2, 13, 81, and 86) had a significant increase in total starch (FIG. 12B). One event (21) had a significant decrease in starch. When the starch values from all the events were averaged across each tester, there was a 9.8% increase in LH244 tester when comparing transgene negative and positive seeds (FIG. 12C). There was no statistical difference in the HC33 tester (FIG. 12C).
Fermentation
Fermentation results are shown in FIG. 13 for 48 and 54 hours. Results are given in % ethanol yield on a moisture corrected basis. Standard calibration curve(s) were used. Three events (2, 13, 86) had a statistically significant increase (2-3%) in ethanol at both 48 and 54 hours in the HC33×LH172 background. One event (86) had an increase in ethanol at both timepoints in the LH244×LH172 background.
Protein Results
Protein results from NIT and Leico methods are shown in FIG. 14. All events tested by NIT showed an increase in protein ( ̃15%) when comparing the positive to its negative isoline. Four events were chosen for confirmation of protein content using the Leico method. 3 events out of the 4 tested by Leico showed an increase in protein (10-13%).
Oil Results
Oil results from NIT and bulk NMR are shown in FIG. 15. All events tested by NIT showed an increase in oil ( ̃10%) when comparing positive to its negative isoline. Four events were chosen for confirmation by NMR. 2 events out of the 4 showed an increase in oil by bulk NMR (10-14%). These two methods produced different values for the percent oil. These differences are due to the different calibrations used for the two methods.
These data indicate that endosperm-targeted expression of YHXKA results in composition changes in mature kernels. Kernels from hybrid material were used in which 1⁄4 of the kernels would be negative for the transgene and the other 3⁄4 of the kernels would have different doses of HXK. One advantage to using this material is that it gives a clear picture of how the transgene behaves in true field conditions in the hybrid setting. The experiment showed that total starch was increased in mature kernels, specifically in the LH172×LH244 hybrid. Several events also had an increase in ethanol produced by fermentation of kernel extracts. These increases may be due to an increase in the amount of extractable starch, so that more starch was available for the assay.

## Claims

1. A corn plant containing in its genome a recombinant DNA molecule comprising, in the 5′ to 3′ direction, a promoter providing expression in a plant seed operably linked to DNA encoding a fungal hexokinase.

2. A seed from a corn plant of claim 1 wherein said seed has in its genome a recombinant DNA molecule comprising, in the 5′ to 3′ direction, a promoter providing expression in a plant seed operably linked to DNA encoding a fungal hexokinase.