MXPA00001520A - Controlled germination using inducible phytate gene - Google Patents

Abstract

The present invention provides a plant containing a phytic acid gene that is expressed in the seed of the plant only when desired. The present invention has two traits that can be induced or switched. The traits are (i) nongerminable seed and, (ii) low or no production of phytic acid.

Description

CONTROLLED GERMINATION USING AN INDUCIBLE FITATO GENE Field of the Invention The present invention provides a plant that contains an inducible gene (phytate gene) that controls the germinability of seeds by regulating the phytic acid content, as well as methods of making and using the plant, the seed thereof. and the progeny of it. Background Mutants with low acid-phytic content in corn are known. The era of recombinant DNA has recently reached financial success in the agricultural industries. It has successfully marketed herbicide-resistant and insect-resistant cotton, as well as soybeans and corn. Before the transformed plants there were some crops that carried herbicide resistance due to mutations in the crops. For example, IT® maize produced by Garst Seed Company is a mutant plant resistant to the herbicide (Imazethapyr) HERBICIDE PURSUIT® (American cyanamide). Mutant plants have also been used to produce value-added traits in various crops. For example, in corn, the amylose extender, waxy and other mutants are used to produce special starch traits, and, in sweet corn, the sugary mutation is used.

A maize mutant with low phytic acid content, Ipal-R, has been produced by Raboy (U.S. Patent No. 5,689,054 (Ipal-1, described therein) .The commercial introduction of this Ipal mutant is expected that is present in 1999. Several companies are currently testing this new mutant with low phytic acid content, several different mutants in corn and in different species that carry this low phytic acid trait can be produced by the use of mutagenic agents and mutagenic methods of maize cultivation Transposon labeling is a well-known method of gene identification and sequencing involving mutant plants with a transposable element.As an advantage, this method provides a means to identify and sequence the genes associated with The mutation, once a gene has been cloned, can be transferred between species. Vegetation to another plant species through the use of gene identification, sequencing and transformation are well known in the industry. Annu. Rev. Plant Physiol. Plant Molecular Biology 1992, 43: 49-82 by Virginia Walbot, entitled "Strategies for Mutagenesis and Gene Cloning Using Transposon Tagging and T-DNA Insertional Mutagenesis". ("Strategies for mutagenesis and cloning of genes using transposon tagging mutagenesis by insertion of T-DNA"). A specific example of using transposon labeling using the mui element for genetic isolation, and the cloning of the amylose extender gene in corn was reported in Plant Cell, Vol. 1555-1566, (November 1993) by Philip Stinard et al., Entitled "Genetic Isolation, Cloning and Analysis of a Mutator-Induced, Dominant Antimorph of the Maize Amylose Extenderl Locus". ("Genetic isolation, cloning and analysis of a dominant antimorph, induced by mutator of place 1 of corn amylose"). This article reports on mutant plants subjected to transposon labeling and the identification of the mutated gene. The gene was sequenced by methods known in the art. The sequenced portion was used as a probe to identify the mutant gene, then the gene was cloned. Gene cloning has been done and is a well-established technique. The DNA recombination techniques used in this invention are known to those skilled in the art and are described in Maniatis et al., MOLECULAR CLONING: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Changeable or inducible transformation constructions are known and can be made by means known in the art. Frequently, after a gene is cloned, it is placed inside a construction that is used for transformation into vegetables. The gene, or allele, or truncated, substituted or deleted variants thereof, can be placed in the sense or antisense position in a transformation vector. One skilled in the art will appreciate that down-regulation of the original genes through the antisense does not require the insertion of the entire gene in the antisense direction but rather only a bit of the sequence that provides up-regulation. That is, fragments of the gene, which contain 30-100, preferably 45-65, nucleic acid bases, can be useful for down-regulating the expression of the gene. Transformation vectors for plant transformation can be purchased from Clontech and other commercial sources. The vectors are preferably selected to provide material suitable for the plant species and the transformation method that is being employed. For example, if the plant species is amenable to transformation by Agrobacterium then the selected vector should include portions of T-DNA in the vector. Promoters, introns, forward sequences, and selectable markers should be selected to give the desired levels of expression of the amino acids encoded by the gene in the desired plant. Moreover, vectors can be optimized by optimizing the specific codon usage for a given host cell, which is being transformed. Recently, inducible promoters have been used to activate genes operably linked to the promoters.

The inducible promoter activates or deactivates the gene in response to an activator. For purposes of the present invention a promoter can be either activated and the gene or linked nucleic acid sequence is operably expressed (to include transcription alone or transcription and translation), or the promoter can be deactivated and thus the gene or the linked nucleic acid sequence is operably not expressed (to include only transcription or transcription and translation). A promoter can also be used which, when contacted with the activator, changes the level of expression. These promoters are well known in the art and are referred to as "convertible" or "inducible." Inducible promoters activate the genes in response to an activator such as heat, light, moisture, chemicals and the like. U.S. Patent No. 5,608,143 discloses the use of nucleic acid promoters that are highly responsive to various substituted benzenesulfonamides as activators. Inducible promoters are indicated for use in a recombinant DNA construct, which will allow the expression of a gene to be controlled by an external control chemical substance which acts as an activator. U.S. Patent No. 5,432,068 describes externally inducible promoters that are used in the control of male fertility. International Publication WO 90/08826 describes a sequence of gene promoters (GSTII (glutathione-S-transferase isoform II)) which responds to a protector of vegetable herbicide (N, N, diallyl-2, 2-dichloroacetamide) which it was used as a gene change to allow external control of gene expression. There are other chemically external inducible promoters known in the art. The methods for transforming monocotyledons and dicotyledons are known. The constructions that are adapted for the vegetable species that are going to be transformed are easily bought or made by those who have experience in the technique. These constructions are then transformed into cells or tissues or pollen of the plants. Transformation methods include but are not limited to microparticle bombardment, filiform growths, electroporation, Agrobacterium, and the like. The efficiency of these techniques varies and a person with ordinary skill in the art can select the method according to the type of tissue and the plant species to be transformed. The selective cultivation of desirable traits is known, such as those that have been produced in the commercially available Top Cross® line of corn with high oil content. As soon as a transformant is regenerated (if necessary) it can be used in culture methods with other plants of its kind. A person with ordinary experience will appreciate that cultivation practices depend on the selected species. For example, soybeans develop as crops while corn is produced as a hybrid. A method to produce maize that is patented by Dupont does not use hybrid seed but a sterile male hybrid seed in combination with a male pollinator. The farmer does not plant hybrid seed, but sterile male hybrid seed and a male pollinator (inbred maize seed) that is carrying a high oil content trait. The hybrid canopy is being developed. Attempts have been made to move most crops that are not currently in hybrid production towards hybrid seed instead of crops to gain hybrid vigor but more importantly reduce the risk of germplasm associated with the production of a vegetable that can reproduce itself. same from the seed (segregated from hybrid seed, not the inbred seed). There remains a need for a grain that has added nutritional value due to the reduction of phytic acid produced in the vegetable. There is also a need for a method of making lethal seed to avoid the development of volunteer vegetables. There remains a need for an inducible lethal trait in the seed of several plant species. SUMMARY OF THE INVENTION It is an object of the present invention to provide seeds, specifically corn seed, with a very low content of phytate. Another object of the present invention provides the grain, preferably grain other than corn, which has nutritional value added due to the reduction of the phytic acid produced in the vegetable. It is still another object of the invention to provide a non-germinable seed containing at least one genetic construct that is under the control of and operably linked to a non-wild inducible promoter or to another genetic element where the at least one genetic construct, when induced , produces a germinable seed. The seed of the present invention contains a non-germinable trait or phenotype as well as a genetic element that is under the control of an inducible promoter such that when the promoter is induced, the non-germinable trait is corrected or exceeded and the seed will germinate. It is another object of the invention to provide a germinable seed which contains at least one genetic construct which is under the control of and operably linked to a non-wild inducible promoter or to another genetic element where the at least one genetic construct, when induced , produces a non-germinable seed. The seed of the present invention contains a germinable trait or phenotype as well as a genetic element that is under the control of an inducible promoter so that when the promoter is induced, the germinable trait is destroyed or exceeded and the seed will not germinate. A further object of the present invention is to provide nucleic acid molecules (sequences) which encode proteins involved in the synthesis of phytate as well as the encoded phytate and antibodies thereto. It is an object of the present invention to provide vectors (for expression and cloning), and methods for using same, to make the seed of the present invention preferably containing nucleic acid sequences encoding phytate, or alleles, or truncated variants, replaced, or inserted or fragments thereof. A further object of the present invention includes providing methods for producing inducible non-germinable seed and inducible germinable seeds. Still another object of the present invention is a method to prevent the development of voluntary plants of fallen seeds which include sowing and cultivating the mature germinating seeds of the present invention and applying an activator to the plants produced therefrom, for produce seeds, daughters or embryos, which will not germinate. Still other objects and advantages will become apparent from the consideration of the present description and the accompanying drawings. Brief description of the drawings Figure 1 shows a binary representation of a construction according to the present invention developed for use in dicotyledons and monocotyledons capable of transformation by Agrobacterium, the construct contains the right border of the T-DNA, the promoter NOS, the NPTII marker gene and the NOS terminator, the inducible promoter, in this case the promoter sequence of GSTII, and the phytate gene in the sense orientation and the NOS terminator and the left border of the T-DNA. Figure 2 shows a binary representation of a construction according to the present invention developed for use in dicotyledons and monocotyledons capable of transformation by Agrobacterium. The construct contains the right border of T-DNA, the We promoter, the NPTII marker gene and the NOS terminator, the promoter inducible in this case the promoter region of the gene encoding the 5-2 cDNA clone described in the US Patent. U.S. Patent No. 5,608,143 and the sequence shown in Figure 5 therein (this patent incorporated by reference herein), filed in the American Type Culture Collection (ATCC), Manassas VA, accession number 67804, and the phytate gene in the antisense orientation and the NOS terminator and the left border of T-DNA. Figure 3 shows a construction with the promoter. In this case the promoter region of the gene encoding the 5-2 cDNA clone described in U.S. Patent No. 5,608,143 and the sequence shown in Figure 5 therein deposited in the American Type Culture Collection (ATCC) ), Manassas, VA, accession number 67804, the phytate gene of the present invention and the NOS terminator. The phytate gene is in the sense orientation. Figure 4 shows a construction with the GSTII promoter sequence, the phytate gene of the present invention and the NOS terminator. The phytate gene is in antisense orientation. Figure 5 shows the prior art sequence of the nucleotide sequence of the 5- 2 gene promoter from the gene designated 52,411 in U.S. Patent No. 5,608,143. Figure 6 shows an example of data obtained from 3 inbred lines analyzed to determine the phytate content, plotted as a frequency distribution curve. Figure 7 shows data taken from a low phytate mutant that secretes low phytate content (high in phosphorus). Figure 8 shows the nucleic acid and amino acid sequences of the myo-inositol 1-phosphate synthase, accession number AF056326. Figure 9 shows a linear map of the published sequence of Zea Myo-inositol 1-phosphate synthase from base 86 to base 1620. Detailed description Ethyl Methane Sulfonate (EMS) has been used to induce mutant phytic acid genes in the corn to produce seed with low phytate content. For example, a maize mutant with low phytic acid content, Ipal, has been produced by Raboy (U.S. Patent No. 5,689,054). In some cases the seed may have sufficiently low phytate content to prevent germination of the seed. In other cases it can be found that the seed with low phytate content germinates normally. From these mutated plants the gene of the present invention is isolated and cloned in a repeatable manner. The cloned gene encodes an enzyme that affects the production of phytic acid in the seed. The gene of the present invention includes allelic, truncated, deleted and substituted variants of the cloned gene as well as useful fragments, such as those that can be used to down-regulate the gene in an antisense manner, as described herein. The present invention provides a transformation vector containing the new gene operably linked to an inducible promoter so that the lethal effects of the gene can be managed, exploited or controlled. This vector is useful in non-hybrid crops such as soybeans, wheat, barley, corn, rapeseed and sunflower because the vector can be used to make seed with a very low phytic acid content and also the seed it can only germinate under highly controlled conditions after induction. This vector of the present invention, when present in a vegetable, has the following several advantages: (i) it is useful for farmers because it eliminates volunteer plants in the next season that leaves the seed unable to germinate, (ii) it is useful to a seed company because it maintains the safety of the germplasm by making the seed incapable of being reproduced for breeding purposes; (iii) it prevents the farmer from saving seed by returning to the seed unable to be reproduced for future years; and , (iv) is useful in the food and mill industry due to the fact that it adds nutritional value to the food or ground products having a low phytic acid content. This invention is particularly useful in the grains of flour and in the grains for food. These include, but are not limited to, corn, wheat, soybeans, sunflower, oats, rye and the like. The present invention therefore provides methods of seed safety which includes providing low-phytate, non-germinating daughter seeds or embryos that have been altered to include a gene encoding a functional phytate that is operably linked to, and under the control of, an inducible promoter. Target gene and isolation The genes of the present invention can be isolated, for example, by crossing with a mutant maize plant that has a genetic mutation that lowers phytic acid levels in the seed. From this gene can be identified the original type or wild gene, and the mutant gene. Both the wild type gene with phytic acid and the gene mutated with phytic acid can be used in the present invention. In one embodiment, the present invention provides methods for making and using seeds, as well as seeds and vegetables therefrom, wherein the phytic acid content of the seed is severely eliminated or reduced by inserting an antisense construct of the seed. the genes or fragments of them which encode, in the sense direction, the proteins involved in the synthesis of phytic acid which, even when they are operably linked to a weak promoter, are expected to at least severely reduce phytic acid levels of the seed. In another embodiment, the present invention provides methods for making and using seeds, as well as plants thereof, wherein the phytic acid content of a low phytic acid mutant containing the seed is restored by inserting a construct sense of the wild-type gene or fragments thereof, i.e., the wild-type gene which has been mutated to produce the genotype of low phytic acid content, which even when operably linked to a weak promoter, is expected or known to be increases phytic acid levels of the seed. A method for isolating the nucleic acid sequence encoding the phytate of the present invention is through the use of transposon tagged mutants which allow cloning of the gene, by means known in the art. The transposable element labels the gene, in other words the sequences that flank the transposon insertion site are part of the gene of interest. According to the present invention, which is exemplified herein as corn, a line of mutant inbreeding with low phytic acid content is crossed with a line containing the mutator 1 gene and a mutant gene with low phytic acid content. identifies by the insertion of the mui element in the gene of interest. The mui used in the exemplified embodiments of the present invention is a part of the mui-9 family that shares the terminal inverted repeat structure with approximately 20 base pairs. Multiple copies of the transposon are contained in the mutator lines, which are aggressive mutagens in corn. Most mu insertion events produce a null genotype because the insertion of the transposable element does not result in altered essential proteins. With targeted transposon labeling, the insertion event is targeted to specific known mutations, such as the gene with low phytic acid content. Transposon events occur in the range of approximately 10"4 to 10" 6 insertions per site per chromosome tested. To identify when the phytic acid gene has a DNA transposon inserted in it, it is required that there is an identification of a mutant phenotype from the insertion event and a restoration of the partial or complete function in the gametes from the events from separation. The labeled mutant can then be crossed with a mutant plant with low phytic acid content carrying the recessive gene with low phytic acid content. If the transposable element has landed or has been inserted into the gene of interest then the grain of the cross may be identified as grain with low phytic acid content or low phytate grain. The gene is recessive and its effects are only present if both parents carry the low phytic acid gene. The allelism is verified in a similar breeding cross manner with similar known low phytic acid mutants. To find the transposable events, several hundred thousand progeny are required if the less active elements are used. The use of mui substantially reduces the required number of progeny that needs to be analyzed to determine the grain trait. Depending on the transposable element used, it may be useful to cross over again the endogamous ones that have the mutant gene destined. Crossing again was used in the present invention of the invention described herein because the mui line was used. The mui line can be identified by having lost mutator activity through the cross-over procedure again by monitoring the reporter gene of the anthocyanin pathway that produces a purple coloration in the seed. Crossing it again with the standard inbred corn simplifies the segregation analysis because the number of copies will be greatly reduced. The allelism is verified with similar known mutants. The muI element is cloned using the transposon element as a probe to clone the mutant alleles or alternatively to probe the original wild-type gene. This method identifies the original and mutant genes with low phytic acid content in the seed, which can then be isolated, purified and sequenced, by means known in the art. The method to find mutable alleles, as an efficient means to clone functional genes, requires a mutant labeled with an insertion element, a clone of the element, tests to verify the recovered clone, and simple genetic tests. The loss of activity of the reporter gene is used to show loss of activity of the mutator. In general, the process involves a process called co-segregation. This involves selecting and harvesting tissue for molecular cloning; prepare DNA from the progeny of crosses of various lines; and making samples of several m / m individuals (homozygous recessive mutant genes) and several M / M individuals (homozygous wild type genes) of each line. Then, Southern stains prepared from DNA taken from the mu-labeled material are prepared after digesting the samples with various 5-methyl cytosine insensitive enzymes that do not digest the muI element that is labeling the mutant; the bands that are present in all the m / m individuals that are not present in the M / M individuals are identified and identified; and a DNA hatchery of the m / m individuals is prepared and three samples of each different line are sampled. The darker band is searched for and selected as well as the only shared diploid copy fragment. This co-segregation process will identify the mu element that is labeling the mutant gene. The results were verified by matching the fragment with the segregation analysis. The fragment is identified from Southern staining by locating the line with the smallest number of segregating bands that has no evidence of a band of comigrant. A large progeny sample of m / m and M / M from this line is analyzed to evaluate the statistical significance between the band and the phenotype. Several restriction enzymes are used for this confirmation. Significant samples of this population test are subjected to digestion by several enzymes, in order to select enzymes that do not digest the bands. This is done as a double digestion with the initial restriction enzyme as well as with enzymes that badly digest the bands, in combination with high resolution agarose gels. Then the m / m hatchery of DNA that was not digested is multiplied and the DNA is selected by size and cloned. Cloned gene The genes that are cloned by the process described above are genes that encode amino acids that produce proteins whose function is to alter phytic acid levels in the plant. These genes are designated as phytate genes (phytic acid genes). Inducible promoter Several compounds have been used in the control of gene expression in plants. The inducer of the expression of the gene must be safe and have no harmful effects on the desired agronomic traits of the plant. Natural products are known, including hormones, and other chemicals, which affect the expression of the gene in vegetables. Plant growth regulators that include AAA, ethylene, abscisic acid, auxin, salicylic acid, and other plant hormones affect gene expression. These natural chemicals seem to induce the expression of the gene. Hormones, whether natural or synthetic, which affect gene expression in plants, can be used with the present invention. However, care must be taken to avoid unwanted activation of plant growth by the coactivation of the plant's internal systems or the activation of lethal genes present in the plant. Clearly, the use of hormones as induction substances must be linked to a hormone that will not activate the metabolic system that is harmful to the final product. The environmental inducers to regulate external genes include light, heat, low temperature and different levels of gas in the air. These regulators may be used, individually or in combination, according to the present invention. These regulators are slightly less practicable since there is no currently known method to control many of these environmental inductors. It is not possible to have a cold growth season or a nebulous growth season in which these inducers may not be strongly activated or may be activated in a final manner. So although these types of inductors will work with the present invention, they must be selected with care. Other plant genes have been induced by oligosaccharides that are present in wounds and pathogenic infection, for example, the glucan induction of phenylalanine-ammonia-lyase and chalcone synthase in soy, or the induction of an inducible inhibition gene similar to potato wound. This induction requires more effort since the vegetable has to be injured to induce the foreign gene. The inductor of choice, which is safe or has little or no effect on the vegetable to which it is applied, is the most useful inducer in many cases. The preferred external control of the inducer is a substance that induces the expression of the desired gene in any tissue at any time in the life cycle of the plant. This requires that a promoter be activated or deactivated after the application of the activating material such as a chemical. This regulation is carried out controlling this response in several plant species with little effect on the growth of the plant. The ideal activator is a chemical that is applicable with standard field equipment, in combination with something else, such as a herbicide, which is usually applied to the field to avoid repeated steps on the field. Alternatively, the material could be applied aerially using crop-type sprinkling equipment. At least one embodiment of the present invention utilizes a promoter that is known to allow (drive) the production of enzymes that protect the plant when activated by an activator, generally referred to as chemical insurers, commonly used in conjunction with the herbicides. It has been determined that vegetables are more protected from herbicidal activity when they are insured. An example of an insurer is one that acts to conjugate the herbicide with glutathione. This is because an increase in glutathione-S-transferase (GST) activity increases the glutathione-S-transferase mRNA in the insured plants. In this way, this insurance treatment increases the product of the gene but does not negatively affect the plant. It has been shown that maize can be ensured by a wide variety of substances, including, but not limited to, naphthalic anhydride, N, N-diallyl-2, 2-dichloroacetamide or cymiminyl when sulfonylurea herbicides are employed. Chlorosulphoron and metasulfuron methyl metabolism rates increase within hours of insurer application. In the present invention, it is the glutathione-S-transferase enzyme promoter that is used as a changeable promoter linked to the phytate gene of the present invention. The change is achieved by using a chemical that induces the change. The inducer of the gene useful in the present description can be any of the inducing or activated substances. The inducing substances are clearly useful in any of US Pat. Nos. 5,608,143 (incorporated by reference) and / or International Publication WO 90/08826, (hereby incorporated by reference) entitled, "Gene Switch" (" Change of Gen "). The method of using an inducing substance to change a gene is known in the art and can be implemented in accordance with the teachings of the present invention. These methods are also described in International Publication WO93 / 09237. The preferred promoters of the present invention are those that respond to insurers used in herbicidal formulations. In nature these promoters and their associated genes are induced by an insurer in the herbicide to protect the plant from the damage that the herbicide can inflict on the plant in the absence of the insurer. In the present invention, these promoters and heterologous genes are preferably activated and deactivated by the insurer. The insurer is preferably applied in a herbicide and a marker gene can make the plant resistant to this herbicide if it is not yet resistant. Alternatively, the chemical, such as the insurer, can be applied separately to the vegetable. This can be useful if the time of primary application of the herbicides is significantly earlier than the development of the seed. The present invention therefore provides, methods for making and using seed, as well as seeds and plants thereof, wherein the phytic acid content of the seed is severely eliminated or reduced by inserting an antisense construct of genes or fragments thereof which, in the sense direction, they encode proteins or protein fragments involved in the synthesis of phytic acid which, although operably linked to a weak promoter, is expected to at least severely reduce phytic acid levels of the seed; the construction being operably linked to a promoter or to another genetic element which is induced by a chemical or an environmental factor, preferably an insurer, so that the application of the chemical or environmental factor to the vegetables produced from the seed produces seeds with a severely reduced amount of phytic acid compared to the original seed. Preferably, the seed produced from the vegetable that has received the factor contains less than about 5 weight percent of the control phytate content, more preferably less than about 2 weight percent of the control phytate content, more preferably less about 1 weight percent phytate control content of phytic acid than the seed produced by the vegetable that received the factor. A person with ordinary experience will appreciate that the common means of measuring phytic acid requires the destruction of the seed so that these comparison values are the most common average values of a seed sample produced as described herein. Moreover, an aspect of the present invention is to provide a non-germinable seed after induction of the parent plant, independently of the decrease in the amount of phytic acid compared to the parent seed. In another embodiment, the present invention provides methods for making and using seeds, as well as plants thereof, wherein the phytic acid content of a mutant with low phytic acid content containing the seed is restored by inserting a construct sense of an original gene or fragments thereof, that is, the original gene that has been mutated to produce the genotype with low phytic acid content of the mutant with low phytic acid content, which, even when operably linked to a Weak promoter is expected or known to increase phytic acid levels of the seed; the construction is operably linked to a promoter or to another genetic element which is induced by a chemical or an environmental factor, preferably an insurer, so that the application of the chemical or environmental factor to the vegetables produced from the seed they produce daughter seeds with an increased amount of phytic acid compared to the original seed. Preferably, the seed produced from the vegetable that has received the factor contains more than about 5 weight percent of control phytate content, more preferably more than about 8 weight percent, more preferably more than about 10 weight percent of phytic acid that the seed that produced the vegetable that has received the factor. A person of ordinary skill in the art will appreciate that the plants and seeds with reduced or low phytic acid of the present invention can be produced by transformation with phytase-encoding nucleic acid molecules, preferably operably linked to an inducible promoter of the present invention, either together with the phytate genes and variants described herein, or alone.

The expression of any of these phytases (phytic acid degradation enzymes) during grain development can be used to degrade the phytic acid that is being synthesized in the developing seed. The sequence encoding the phytase is known, such as the one that has been published by the NCBI, and wherever it may be, under any of the access numbers: P34752, gi ¡464382 | sp | P34752 | PHYA_ASPNG [464382]; P34753, gi | 46438l | sp | P347531 PHYA_ASPAW [464381]; 2599490 (AF029053) phybl3 precursor [Bacillus subtilis] gi | 2599490

[2599490]; 1IHP gi 12981783 | pdb | 1IHP | [2981783]; 3150040 (U85968) gi | 3150040 [3150040]; 2943981 (U92439) phytase [Enterobacter cloacae] gi | 2943981 [2943981]; P34754 gi | 464385 | sp | P34754 | PHYB_ASPNG [464385]; P34755 gi | 464384 | sp | P34755 | PHYB_ASPAW [464384]; P07102 gi | l30735 | sp | P07102 | PPA_ECOLI [130735]; 2108356 (U59802) phytase [Talaromyces thermophilus] gi | 2108356 [2108356]; 2108354 (U59804) phytase [Aspergillus fumigatus] gi | 2108354 [2108354]; 2108352 (U59803) phytase [Emericella nidul 2108352

[2108352]; 2300890 (A46793) gi | 2300890 e306401 [2300890]; 2300889 (A46793) gi | 2300889 e306184 [2300889]; 2300887 (A46791) gi | 2300887 e306400 [2300887]; 2300885 (A46789) gi | 2300885 e306183 [2300885]; 2300883 (A46787) gi | 2300883 e306182 [2300883]; 2300881 (A46785) gi | 2300881 e306399 [2300881]; 2300879 (A46783) gi | 2300879 e306181 [2300879]; 2148991 (U60412) phytase [Aspergillus terreus] gi | 2148991 [2148991]; JN0715 3-phytase (EC 3.1.3.8) precursor B - Aspergillus ficuum gi] 542378 | pir | | JN0715 [542378]; JN0890 acid phosphatase (EC 3.1.3.2) precursor - Aspergillus awamori gi | 542377 | pir | | JN0890 [542377]; JN0889 3-phytase (EC 3.1.3.8) precursor A - Aspergillus awamori gi | 542376 | pir | | JN0889 [542376]; JN0656 3-phytase (EC 3.1.3.8) precursor A Aspergillus niger gi | 484414 | pir | | JN0656 [484414]; JN0482 3-phytase (EC 3.1.3.8) A - Aspergillus ficuum gi | 419906 | pir | | JN0482 [419906]; PQ0641 3-phytase (EC 3.1.3.8) - Aspergillus ficuum (fragments) gi | 542374 | pir | | PQ0641 [542374]; S33278 phytase P2 - Escherichia coli (fragment) gi | 421153 | pir | | S33278 [421153]; B36733 acid phosphatase (EC 3.1.3.2) precursor - Escherichia coli gi | 96267 | pir | B36733 [96267]; S18408 alkaline phosphatase (EC 3.1.3.1) - rat gi | 111353 | pir | | S18408 [111353]; 1943870 (U59806) phytase [Thielavia heterothallica] gi | l943870 [1943870]; 1943868 (U59805) phytase [Aspergillus terreus] gi | l943868 [1943868]; 1938256 (U75531) phytase [Zea mays] gi | l938256

[1938256]; all sequences and the text of the U.S. Patent Number: 5593963, including, gi | l831488 | pat | us | 5593963 | 20 [1831488]; 408990 phytase. { EC 3. 1.3.26} [Aspergillus ficuum, Peptide, 441 aa] [Aspergillus ficuum] gi | 40899? | Bbs | 130910 [408990]; 235916 gi | 235916 | bbs | 58159 [235916]; 235915 gi | 235915 | bbs | 58160

[235915]; 2393 gi | 2393 [2393]; all sequences and text of the Patent of the United States of North America Number: 5436156, including gi | 912286 | pat | uS¡ 5436156 | 32 [912286]; 583196 (A19452) gi¡583196 [583196]; 583194 (A19451) gi | 583194

[583194]; 166521 (M94550) gi | l66521 [166521]; 166519 (L02421) phytase [Aspergillus niger] gi | l66519 [166519]; 166482 (L02420) acid phosphatase [Aspergillus niger] gi | l66482 [166482]; 304997 (L20567) phyB [Aspergillus niger] gi | 304097 [304097]. The methods and constructions useful in the present invention can also be found in the Patents of the States United States of America numbers 5,770,413 and 5,593,963. In another embodiment, the construct and the promoter or other genetic elements are operably linked to a marker gene as is known in the art.

In one embodiment of the present invention the plants and vegetables described in the embodiments of the invention are any among corn, soybeans, rice, oats, sunflower, wheat, barley, forage grasses, and the like. Constructions The present invention includes a construct having a promoter associated with a phytic acid gene, the promoter is under external control so that when activated it makes the seed so low in phytic acid that the seed is unable to germinate (ie it can not form a maturing plant). This construction can be transformed into several plants that are transformable. The placement of the gene within the construction determines whether the gene is going to be up-regulated, the orientation in the direction, if it is going to be down-regulated, the antisense orientation. Figure 1 shows a binary representation of a construction according to the present invention. This construction is developed primarily for use with processing by Agrobacterium. This is mainly developed for use in dicotyledons and monocotyledons capable of transformation by Agrobacterium. The construct contains the right border of T-DNA, the promoter Nos the NPTII marker gene and the NOS terminator, the promoter inducible in this case the GSTII promoter sequence and the phytate gene in sense orientation and the NOS terminator and the left border of T-DNA This construction is ideal for soybeans and similar crops. The marker gene may be selected to be a gene resistant to the herbicide such as the glyphosate-resistant gene. Figure 2 shows a binary representation of a construction according to the present invention developed for use in dicotyledons and monocotyledons capable of transformation by Agrobacterium. The construct contains the right border of T-DNA, the Nos promoter, the NPTII marker gene and the NOS terminator, the inducible promoter, in this case the promoter region of the gene, encodes the 5-2 cDNA clone described in the US Pat. United States of America number 5,608,143, the sequence shown in Figure 5 and the phytate gene in the antisense orientation and the NOS terminator and the left border of T-DNA. This insurer is applied to activate the promoter and thus expresses the product encoded by the gene through the application of the herbicide that contains the insurer or only by the application of the insurer. Figure 3 shows a construction with the promoter in this case the promoter region of the gene encoding the 5-2 cDNA clone described in U.S. Patent No. 5,608,143, the sequence shown in Figure 5 therein, this patent incorporated by reference in. present, deposited with the American Type Culture Collection (ATCC), Manassas, VA, accession number 67804. The phytate gene of the present invention and the NOS terminator. The phytate gene is in the sense orientation. Thus when the activator is applied the promoter changes and the gene will express the amino acids it encodes. Figure 4 shows a construction with the promoter in this case the GSTII promoter sequence and the phytate gene of the present invention and the NOS terminator. The phytate gene is in antisense orientation. These constructions in their different forms can be transformed into corn, soybeans, rice, oats, sunflower, wheat, barley, forage grasses, and the like. The present invention includes constructs that have been optimized for expression in each of these plants, such as, for example, with respect to the use of codon, promoters, etc. and other processes that increase expression as well as optimized to produce an active enzyme. or optimal antisense or cosuppression effects. Additionally, the constructions of the present invention can be transformed into plants that are already transformed or that are already mutants. For example, this gene construct can be used to make the mutants with low phytic acid content externally regulated by the application of the activator. The promoter in the construct can act to induce the gene to express or the activator can stop the expression of the gene by inducing the promoter to deactivate. In the same way, the orientation of the gene in the construction can be in the direction sense or antisense, up-regulating the sense orientation of the gene expression and down-regulating the expression of the antisense orientation gene. In this way the same effect can be produced by changing the promoters from inducible to non-inducible or changing the orientation of the gene. Transformation Representative diagrams according to the present invention are presented in the first four figures. The first two figures show a construction that is adapted for transformation by Agrobacterium. The second two figures show a plasmid for use with other transformation techniques such as microparticle bombardment. These transformation techniques are well known to those skilled in the art. Additionally, these constructions are excellent for use with the filiform growth transformation system shown in U.S. Patent No. 5,302,523, and U.S. Patent No. 5,464,765 incorporated herein by reference. EXAMPLE 1 There are four preferred ways to produce the seed material with low phytic acid content of the present invention. The preferred method provides the farmer with a seed product that is both low in phytic acid content and non-germinable so that the seed is not capable of producing a mature plant. The preferred seed of the present invention ends up as seed saved by the farmer in the case of non-hybrid crops. Currently, most rapeseed and wheat and soybeans are not sold as hybrids although both wheat and rapeseed have undergone research to develop hybrid capabilities. If the plant is not normally marketed as hybrid seed but as seed such as wheat and soybeans then the material according to the present invention is easily produced. Transformative reproduction with inbred material If the promoter is induced to activate the gene, and the gene is in antisense orientation then the application of the activator will produce seed with extremely low levels of phytic acid. This seed will not germinate. The activator is preferably applied when the grain is full or slightly before this. The activator preferably in the form of an insurer which may be in a herbicide if the promoter is the GSTII or an insulator-induced promoter. In this way, during the increase of the seed, the seed is not sprayed with the activator. This allows the production of seeds with normal levels of phytic acid. However, when the seed is sold to the farmer for the production of grain with low phytic content then the activator must be applied to the plant to produce a seed with low content or without phytic acid. Due to the low content of phytate in the seed, the activator will also make the seed unable to germinate. In this way, soybeans or other seeds of plant species will not grow in the next season as voluntary plants and the seeds will not be saved for use in the following year for sowing purposes. In this embodiment, the present invention provides a mature seed that contains a sufficient amount of phytic acid to germinate; the mature seed contains a genetic construct containing an inducible promoter operably linked to nucleic acid molecules which, in the direction of sense, encode phytic acid or fragments thereof or allelic variations thereof, the nucleic acid molecules being placed in the construction in the antisense direction to the promoter, so that when the promoter is activated by an activator in a plant grown from the mature seed, a developing seed of the product or embryo is produced which will not germinate. The present invention provides the plant produced from mature seed and methods for reducing the growth of volunteer plants and reducing the amount of germinable stored seed which includes applying an activator to the plant.

If the promoter is induced to deactivate the gene, the gene is in sense orientation, then the plant, without the construct containing the promoter and the gene, would have to contain a mutant form of the gene that would produce very low levels of phytic acid in the seed so that the seed, without the construction that contains the promoter and the gene, does not germinate. The application of the activator to the plant produced from the mature seed containing the construction of this modality will produce non-germinating developing seed or embryo that contains extremely low levels of phytic acid. The activator is preferably in the form of an assurer of a herbicide if the promoter is or contains a fragment of the promoter shown in Figure 5. The activator is preferably a substituted benzenesulfonamide. In this way, during the increase of the seed, that is, the growth of the progeny of the mature seed of this embodiment of the invention to increase the number of mature seeds of this modality, the plant produced from the mature seed of this mode is not sprayed with the activator. However, when the mature seed of this modality is growing to provide seed or grain to farmers or end users, for the production of products with low phytic content, then the activator must be applied to the plants produced from the mature seed of this embodiment of the invention for producing the non-germinable developing seed, with low phytic content (embryo seed or girl). This will result in the production of a seed with very low phytic acid content which will also make the seed non-germinable. In this way, soybeans or other seeds of plant species will not grow in the next season as volunteer plants and can not be saved for use in the following year for planting purposes. In this embodiment, the present invention provides a mature seed that contains a sufficient amount of phytic acid to germinate wherein at least a portion of the sufficient amount of phytic acid is produced from a genetic construct containing an inducible promoter operably linked to nucleic acid molecules that encode phytic acid, so that when the promoter is activated by an activator in a plant that grows from the mature seed, a developing seed or embryo is produced by the plant that will not germinate. The mature seed of this modality, without the construction, does not produce enough phytic acid to germinate. The present invention provides the plant produced from mature seed and methods for reducing the growth of volunteer plants and reducing the amount of germinable stored seed that includes applying an activator to the plant. If the promoter is induced to activate the gene, then the gene is in sense orientation, then the plant would have to contain a mutant gene that is producing extremely low levels of phytic acid in the seed. This seed would be unable to germinate. The application of the activator in this mode will restore the germinability since there will be increased levels of phytic acid. The activator is preferably in the form of an assurer of a herbicide if the promoter is or contains a fragment of the promoter shown in Figure 5. The activator is preferably a substituted benzenesulfonamide. During the increase of the seed, that is, the growth of the progeny of the mature seed of this embodiment of the invention to increase the number of mature seed of this modality, the plant produced by the mature seed of this modality is sprayed with activator . However, when the seed of this modality is growing to produce seed or grain to the farmer or another user as seed or grain with low phytic acid content then the activator should not be applied. This will result in seed production with very low phytic acid content that also makes the seed non-germinable. In this way, soybeans or other seeds of plant species will not grow the next season as volunteer plants and can not be saved for use in the following year for planting purposes. In this modality, the present invention provides a mature seed containing a sufficient amount of phytic acid to germinate wherein at least a portion of the sufficient amount of phytic acid is produced from a genetic construct containing an inducible promoter operably linked to acid molecules. nucleic acid encoding phytic acid, so that when the promoter is activated by an activator in a plant grown from mature seed, a developing seed or embryo is produced by the plant which will germinate. The mature seed of this modality, without construction, does not produce enough phytic acid to germinate. The present invention provides the plant produced from mature seed and methods for reducing the growth of volunteer plants and reducing the amount of germinable stored seed that includes applying an activator to the plant. If the promoter is induced to deactivate the gene, and the gene is in the antisense orientation, then the plant, without the promoter and operably linked to the gene, would have to contain a normal or original gene that was capable of producing normal levels of phytic acid in the seed This seed, with the promoter and the linked gene operably, will not germinate. The application of the activator will restore the production of phytic acid and therefore the seed with the promoter and the operably linked gene of this mode will normally germinate. The activator is in the form of an insurer in a herbicide if the promoter is or contains the fragments shown in Figure 5. The activator is preferably a substituted benzenesulfonamide. Thus during the increase of the seed as described above, the plant produced from the mature seed of this mode is sprayed with the activator. However, when the seed is sold to the farmer or to another end user, as described above, as low in phytic, then the activator should not be applied. This will result in the production of a seed with low phytic acid content and thus low phytate in the seed and the absence of the activator will also make the seed non-germinable. In this way, the soy bean or other seed of vegetable species will not grow the next season as a voluntary plant and can not be saved for use in the following year for planting purposes. In this embodiment, the present invention provides a mature seed that contains a sufficient amount of phytic acid to germinate; the mature seed containing a genetic construct containing an inducible promoter operably linked to nucleic acid molecules which, in the direction of sense encodes phytic acid O- fragments thereof or allelic variations thereof, the nucleic acid molecules being placed in the construction in the direction antisense to the promoter, so that when the promoter is activated by an activator in a plant grown from mature seed, a developing seed or embryo is produced which will germinate. The mature seed of the present modality, without the construction, is able to make at least phytic acid sufficient to germinate. The antisense arrangement of the nucleic acid molecules in the construct reduces the amount of phytic acid to the extent that the seed in development or embryos of a plant produced from the mature seed will not germinate unless activator is applied to the plant. . The present invention provides the plant, produced from mature seed and methods for reducing the growth of volunteer plants and reducing the amount of germinable stored seed that includes applying an activator to the plant. Other combinations of gene orientation and inducibility of promoter and other activators are known. The insurer or a pesticide activator is preferred since it decreases the time and expense of covering the field more than once. In other words it provides the dual utility of acting as a herbicide or pesticide and inducer of the genes. Transformative reproduction with hybrid material The number of combinations of gene orientation and of mutant and wild-type gene useful in the present invention increases when the plants are hybrid plants. A hybrid transformative reproduction method employs hybrids formed with two inbred low-phytic acid mutants, as described above, at least one of the mutants bears a genetic construct that contains a transforming gene, or an allelic variation, substituted, deleted or truncated thereof which was able to encode amino acid sequences necessary to produce phytic acid, preferably the construct contains the gene in the sense direction encoding the wild-type phytic acid. During production, the lethal mutant carrying the transformed wild-type gene is exposed to the dew that induces the gene during the period of seed production. This induces the promoter and the sense gene is activated and the gene produces the phytic acid necessary to maintain the germination capacity of the seed. This acts as the protector of the line and also as the maintainer of the line. Preferably, the seed producer applies the activator as opposed to the end user, or farmer. The end user or farmer needs only to plant the mature seed. When the inducible promoter is activated by an insurer, as in the preferred embodiments, an experienced person will appreciate that a number of plant herbicides can carry the activator that induces the gene and care must be taken to limit or discourage the use of herbicides that include the insurer during the production of end-user or farmer of the hybrid seed. Alternatively, herbicides not containing the activator of the present invention may be formulated to be applied separately from the activator / insurer. In this case, it is preferable that the herbicide and activator be applied separately. Another method of the present invention uses a wild-type plant transformed with a wild-type gene, which, when placed in a sense direction, encodes normal levels of phytic acid. In this embodiment of the present invention, however, the wild type gene is in the antisense direction and operably linked to an inducible promoter, as described herein. The gene is placed in the antisense position to trim the expression of the wild-type gene in the plant when the plant is exposed to the activating chemical. This antisense gene is induced in the field by the application of a chemical inducer or an environmental factor before the production of the grain or during the development of the developing seed or embryo. Alternatively, a wild mutant plant having lethal phytic acid levels can be used and modified to include the wild type gene in the sense orientation operably linked to an inducible promoter, as described herein, inserted so that when the The promoter is induced in the plant expresses upwards this gene and is capable of producing daughter seed or embryos that will germinate. The inducible promoter is deactivated by the chemical. The chemical activator is applied, such as by spray, on the plant by the end user or farmer before the outbreak of senescence of the plant. In this way, the mature seed contains the phytic acid genes needed to produce necessary phytate levels during germination, but the gene is deactivated and the chemical is not applied and the wild-type gene is not expressed. The present invention therefore provides an operably changing gene linked to nucleic acid molecules that encode the amino acid sequences necessary to produce phytic acid. The direction of the gene within the construction and the specific promoter change can be selected according to the present invention by a person with ordinary experience depending on the genetic and phenotypic background of the tissue to be transformed as well as the desired product (seed daughter or embryo) that is going to be produced. EXAMPLE 2 The method for producing low phytate mutants useful for transposon labeling is a known method called mutagenesis. The process is presented in the work of Neuffer: Maize Genetic Newsletter 45: 146. The mutations were induced in the inbred line by treating the pollen with ethyl methane sulfonate in paraffin oil according to the procedure described by Neuffer (1974). This treatment was carried out on several inbreds of several cereal plant genotypes. This example will focus on the development of low phytate corn mutants through this process. This process of mutagenesis has been used to make several cereal mutants. The general steps of the process of the present invention include treating inbred pollen (in this case corn) with ethyl methane sulfonate hereinafter "EMS". Inbred pollen is placed in ethyl methane sulfonate in oil for 45 minutes. A brush is used and the pollen is brushed on the stigmas of a receiving corn cob. This forms the seed Mutant- 1 (Mi). This seed is grown and self-pollinated to produce Mutant-2 (M2) grains. The resulting M2 grains are tested to determine the phenotype of low phytate content. A preferred maize mutant of the present invention that can be used as a basis for transposon labeling and phytic acid gene isolation is the maize inbred line (Zea Mays) EX 1965PY deposited in the American Type Culture Collection (Manassas, VA) under conditions of the Budapest treaty, on July 7, 1998, designation number 203034. Alternatively, Zea maize seeds from Ipal (Ipal-1) and Ipa2 (Ipa2-1) mutant homozygotes described in US Patent No. 5,689,054, which were deposited on August 15, 1996, under the terms of the Budapest Treaty in the American Type Culture Collection and assigned access numbers ATCC 97678 and ATCC 97679, respectively . Preferred phytic acid genes of the present invention are those obtainable from these deposited materials, preferably by transposon labeling methods known in the art. Other preferred nucleic acid sequences of the present invention include SEQ ID NO: 1, a sequence encoding the protein shown in SEQ ID NO: 2, and FIGS. 8 and 9, and sequences that have been published by the National Center for Biological Information under the access numbers g3108052 and g3108053. The nucleic acid sequences encoding the amino acid sequences of SEQ ID NO: 2 and those shown in Figures 8 and 9, as well as the allelic, substituted, deleted or truncated variations thereof are also included that are capable of encoding the amino acid sequences necessary to produce phytic acid. EXAMPLE 3 The HVPE method is a common test for mutants with low phytate content. (Faboy, Maydica 35: 383 (1990)). The method is based on differential migration of phosphorus compounds. After fractionating the compounds electrophoretically, a chromatogram allows a semiquantitative assessment of phytic acid relative to other compounds. An alternative method involves the analysis of higher levels of inorganic phosphorus in the grain. For example, samples of the grain can be ground (to pass a 2-millimeter screen in a Wiley mill) followed by the addition of either 50 milligrams of gram germ or one gram of endosperm in 15 milliliters of 0.4 M HCl in 0.7 Na2S04. Phytic acid precipitates as an iron salt. The phosphorus in the ferric phytate is precipitated and the total phosphorus is determined. Phytic acid P (milligram) is converted to phytic acid by a conversion factor of 3.5. The principles of phytate measurement are known. In the method used in the present study, a solution of 5-sulfosalicylic acid and FeCl3 (Wade's reagent) forms a pink chromophore. Phytic acid binds with iron in this solution decreasing the level of pink. The measurement of this color loss can be used as an indication of phytic acid levels. Since the control does not contain phytate, all the readings of the samples that do contain phytate will be negative numbers. If there is too much phytate, however, the iron-phytate complex can precipitate as a milky white substance. In this case the pink color will not be present but the milky white matter absorbs the light and will result in falsely high readings. In this way some visual observation may be necessary. This may need to use a minor aliquot (less than 25 microliters) of the corn extract if the corn variety has high levels of phytate. A rapid analysis procedure, as described below, can be used to keep an account of presumptive seeds with low phytate content. In this procedure, a single-knife shaver is used to cut the tip cap of the grain just behind the black layer. The cut should transect the scutellum at a point at or near the tip of the radicle. Usually, eight representative grains of each ear were selected. The grains are then placed, with the surface cut up, in a microplate having the surface covered with cellophane tape (with the sticky side facing up). The dyeing procedure is completed after the dissection of at least 100 families. The dyeing is done with the use of a repeating pipette to place a drop of 10 microliters of Wade reagent (as described below) on the cut surface of each grain. After a few minutes the color disappears as the phytic acid of the scutellum binds to the iron in Wade's reagent. Observations were made for families that segregate by the slower disappearance of the color pink in relation to the others (perhaps 5 percent of the total). These slower families were reanalyzed by the quantitative procedure described here. The phytate was quantified as follows. Individual grains (from 7 to 10 of each family) were crushed in steel plate of a Carver manual pump press (better results were obtained when the wells of the plate were lined with glycine paper and crushed with a pressure of approximately 250 kilograms) and placed in 1.5 milliliter microcentrifuge tubes. 1 milliliter of 0.65 N hydrochloric acid was added and allowed to stand overnight. The combination was mixed by inversion the next day and allowed to settle for 5 minutes. An aliquot of 15 microliters of the supernatant was added to a microcentrifuge tube with 100 microliters of a regulator A (as described herein) and mixed. Mutants with low phytate content turned a very blue color due to the high levels of phosphorus in the seeds. The mutants were generally re-tested the next day. The 0.65 N hydrochloric acid extraction solution was made by adding 216 milliliters of 12.1 N hydrochloric acid to 3784 milliliters of water. Reagent A was made new every day and included two parts (by volume) of deionized water, one part (one volume) of ascorbic acid solution, one part (by volume) of ammonium molybdate solution and one part (by volume) of H2SO4 solution. The ammonium molybdate solution was made by adding 25 grams of (NH4) 6M07024x 4H20 to make one liter with water. The H2S04 solution was made by adding 167 milliliters of 36 N of sulfuric acid to 833 milliliters of water. The ascorbic acid solution was made by adding 100 grams of ascorbic acid L to make one liter with water. The ascorbic acid solution was stable with refrigeration for about seven weeks but only about two hours without refligation. Phytic acid standards were prepared by means known in the art. The corn kernel (M2) without phosphate was visually analyzed as follows. Grains were selected, phenotypes were scored and placed in a multi-well crusher plate. The grains were crushed in the multi-well crusher plate using a hydraulic press. The crushed grains were transferred to 1.5 milliliter Eppendorf microcentrifuge tubes. 0.5 milliliter of reagent A was added. After leaving two hours of reaction time, 0.5 milliliter of reagent-B was added. The tubes were covered and mixed by inverting them. The reactions were scored visually to determine the blue tone after one hour, using a light box when necessary, and the bluest samples were selected as having the highest phosphate content. Frequently, the bluest samples of each family (cob) were selected and compared for the final selection. Reagent A of this test was prepared from 50 milliliters of dimethyl sulfoxide and 50 milliliters of reagent B. Reagent B was made fresh each day and prepared from 60 milliliters of distilled water; 30 milliliters of 10 percent ascorbic acid solution (10 grams of ascorbic acid with water to a total volume of 100 milliliters, the ascorbic acid solution was refrigerated and left stable for one week); 30 milliliters of 3.5 percent solution of ammonium molybdate (2.5 grams (NH4) 6 Mo7 024 * 4H20 water was added for a total volume of 100 milliliters); 30 milliliters of 6N sulfuric acid solution (170 milliliters of water plus 25 milliliters of concentrated sulfuric acid adjusted to a total volume of 200 milliliters with water). The phytate (Red Test) was measured quantitatively as follows. Approximately 12 mature seeds were crushed in a steel crushing plate of a Carver manual pump press. The best results were obtained when the wells of the plate were lined with waxed paper. The grains were crushed with a pressure of 2500 to 5000 kilograms and transferred to Eppendorf tubes. One milliliter of 0.65 N of hydrochloric acid was added to them, allowing it to settle overnight and mixing the next day by inverting the tube. To test, 200 milliliters of Wade A reagent (described below) was combined with 10 milliliters of the corn juice / hydrochloric acid extract obtained above in individual wells of a microtiter plate. Any change in color was noted and the samples that followed red were noted as having low phytate content since the phytate binds to iron and turns the solution white. The quantification can be completed with a spectrophotometer with measurement at 490 nm. Wade reagent A used herein was prepared by adding 25.4 grams of 5-sulfosalicylic acid and 350 milligrams of FeCl3.6H20 (ground with mortar and pestle if necessary) to 1.5 liters of deionized water. NaOH was used to adjust the pH to 3.05 and the volume was adjusted with deionized water to two liters. This reagent was stable in refrigeration for about a month. 0.65 N HCl was prepared by adding 216 milliliters of HCl (12.1N) to 3784 milliliters of deionized water. These tests allowed the selection of the desired maize plants containing the desired alleles. Other details of these methods and modalities in accordance with the present invention are found in the joint pending TPC Application number (unassigned), filed July 7, 1998 entitled "Animal Feed with Low Phytic Acid, Oil Burdened and Protein Laden Grain. "(" Animal feed with grains with low phytic acid content, loaded with oil and loaded with protein "), which is based on the Provisional Patent Applications of the United States of America numbers 60 / 051,854 and 60 / 051,855, filed On July 7, 1997, the entire contents of which are incorporated herein by reference. Other test methods for phosphorus are known and can be used to select plants. Then seeds containing the desired levels of phytic acid are increased. This process was used in the present invention and several inbred lines carrying the mutation with low phytic acid content were selected. These included several inbred lines with low phytate content with good combination ability which crossed each other to form a hybrid that produced the grain of the present invention. Thus, the developed endogams of the present invention were produced from rigid stem, from Lancaster and other versatile heterotic patterns so that the endogenous ones when crossed with each other with the appropriate heterotic pattern make excellent hybrid material. It was also discovered that several developed mutations of the present invention but with low phytic acid content were not the same mutant as Ipal-R and Ipa2-R mutations described in U.S. Patent No. 5,689,054 as Ipal-1 and Ipa2-1, respectively. Additionally, the seed was analyzed to determine its germinability in standard seed germination tests. It was found that some mutants with low phytate content were unable to germinate while others would germinate normally. Only the seed with good germination characteristics was maintained. Figure 6 shows an example of some data obtained from three of these endogenous lines analyzed to determine the phytate content. It was plotted as a frequency distribution curve and it is clearly evident that there were samples with very low phytate content (less than 0.5 units (percent by weight)) while the volume of the samples had a higher phytate content. The line known as line U095 was selected as initial material for its higher levels than the average protein and oil. This line produces seeds that are loaded with oil and protein and that will germinate normally. U095py retains these characteristics with the low phytate levels described above. When they cross with certain other inbred lines, the resulting hybrid produces grain that is loaded with oil and protein and contains low levels of phytic acid. The following table represents the phytic acid content (milligram / gram of seed) for mutant corn according to the present invention compared to the wild-type seed. - - The following provides an example of an inbred line according to the present invention.

Protein and oil content were measured by near infrared analysis on a Dickey-John Reflectance Near Infrared Spectrometer. The grain of the present invention can also be used as a substitute source for the corn grain or flour used to make corn tortilla, corn meal, and corn flakes by replacing the grain of the present invention in the recipe and baking or processing usually. The grain of the present invention can also be used as a substitute for the wet corn milling industry by replacing the grain of the present invention in order to increase the milling efficiency and recoverable starch content. The animal feed made as a by-product of the milling process is also substantially reduced in phytate content.

EXAMPLE 4 Methods for selecting low phytate have been described above. Figure 7 shows data taken from a mutant with low phytate content that secretes low phytate content (high phosphorus content). Clear evidence of Mendialian segregation is apparent. EXAMPLE 5 Crossing mutants with low phytate content with a mutant-containing population (labeled with transposon) it is possible to identify a rare case of a gene labeled with mutator. First, homozygous plants with low phytate content cross with a population labeled with transposon. Biochemical assays are then used to analyze the resulting seed to determine the low phytate content (phosphate content in this case). Using this method it is possible to identify a phytate gene tagged with transposon that specifies the gene for the mutation with low phytate content. The results of the biochemical analyzes are shown in Figure 7. Here the data for the distribution of seed frequencies of tested transposon labeled material are displayed to determine their phosphorus content. A rare event was found in this example. Germination tests revealed that some mutants with low phytate content had acceptable germination while others would not germinate. Similar results can be obtained by experts with ordinary experience using the methods described herein.

Of these non-germinating materials of preferred low phytate content, it is possible, using known methods, to clone, sequence and manipulate the phytic acid mutants useful in the present invention. The entire contents of the references referenced herein are incorporated by reference in their entirety.

Claims (32)

1. NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property 1. A transgenic plant containing a genetic construct comprising a heterologous nucleic acid sequence for a selected gene product that regulates the production of these phytic acid plants.
2. 2. A transgenic plant according to claim 1, characterized in that the nucleic acid sequence is operably linked to a promoter that is inducible by an activator.
3. 3. A transgenic plant according to claim 1, characterized in that the plant also contains a mutant allele which results in the plant forming low levels of phytic acid.
4. 4. A transgenic plant according to claim 1, characterized in that the plant also contains a wild-type phytate gene which results in the plant forming phytic acid levels sufficient for the plant, without said construction, to produce seed daughter which will germinate.
5. 5. A transgenic plant according to claim 3, characterized in that the promoter is active until the activator is applied to the plant and the nucleic acid sequence is operably linked in the sense direction.
6. 6. A transgenic plant according to claim 3, characterized in that the promoter is not active until an activator is applied to the plant and the nucleic acid sequence is operably linked in the sense direction.
7. 7. A transgenic plant according to claim 4, characterized in that the promoter is not active until an activator is applied to the plant and the nucleic acid sequence is operably linked in the antisense direction.
8. 8. A transgenic plant according to claim 4, characterized in that the promoter is active until an activator is applied to the plant and the nucleic acid sequence is operably linked in the antisense direction.
9. 9. A transgenic plant according to claim 3, characterized in that the nucleic acid sequence encodes a mutant phytate gene product obtainable from a maize mutant plant selected from the group consisting of U095py, Ipal-1 ( ATCC Access number 97678), Ipa2-1 (ATCC Access number 97679) and allelic variants, truncated, substitution and deletion thereof.
10. 10. A transgenic plant according to claim 9, characterized in that the product of the gene is obtainable by mu transposon labeling.
11. 11. A transgenic plant according to claim 4, characterized in that the wild type phytate gene is obtained by mu transposon labeling of plants containing phytic acid.
12. 12. A transgenic plant according to claim 4, characterized in that the wild-type phytate gene is a nucleic acid sequence that encodes SEQ ID NO: 2.
13. 13. A transgenic plant according to the claim claimed in the claim. 4 characterized in that the wild-type phytate gene is SEQ ID N0: 1.
14. 14. A transgenic plant according to claim 1, characterized in that the plant is selected from the group consisting of corn, soybean, rice, oats, sunflower, wheat, barley, rye, and forage grasses.
15. 15. A transgenic plant according to claim 2, characterized in that the activator is a compound selected from the group consisting of: 2-chloro-N- (methylaminocarbonyl) benzenesulfonamide, 1- (n-butyl) -3-methylsulfonylourea , methyl-2- [(aminocarbonyl) aminosulfonyl] benzoate, N-isopropylcarbamoylbenzenesulfonamide, N- (aminocarbonyl) -2-chlorobenzenesulfamide and N'- [2 - (n-butylaminocarbonyl)] -6-chloro-N, N-dimethyl-l, 2-benzenedisulfonamide.
16. 16. A recombinant genetic construct capable of transforming a plant, comprising a nucleic acid sequence encoding the gene product regulating the production of phytic acid operably linked 3 'to a nucleic acid promoter sequence.
17. 17. A recombinant genetic construct according to claim 16, characterized in that the nucleic acid promoter sequence is as shown in Figure 5.
18. 18. A recombinant genetic construct according to claim 16, characterized in that the nucleic acid promoter sequence is inducible by the application of N, N-diallyl-2,2-dichloroacetamide.
19. 19. A recombinant genetic construct according to claim 16, characterized in that the nucleic acid promoter sequence is inducible by a compound selected from the group consisting of N, N-diallyl-2,2-dichloroacetamide, benzyl-2- chloro-4- (trifluoromethyl) -5-thiazole carboxylate, naphthalene-1,8-dicarboxylic anhydride, 2-dichloromethyl-2-methyl-1,3-dioxolane.
20. 20. A method for generating seeds comprising the following steps: seeding a mature seed containing an externally inducible recombinant DNA vector which can be activated by an activator, said recombinant DNA vector containing at least one nucleic acid sequence encoding a gene product selected that results in a lethally low production of phytic acid; applying the activator to the plant generated by said mature seed whereby the nucleic acid sequence is induced to produce said selected gene product; harvest the daughter seed produced from the plant after the activator has been applied.
21. 21. A method for generating seeds comprising the following steps: seeding a mature seed containing an externally inducible recombinant DNA vector that can be inactivated by an activator, said recombinant DNA vector containing at least one nucleic acid sequence encoding a gene product selected which results in a lethally low production of phytic acid; apply to the plant generated by the mature seed, the activator by which the nucleic acid sequence is induced to stop the coding of said selected gene product; harvest the daughter seed produced from the plant after the activator has been applied.
22. 22. The daughter seed produced by the transgenic plant in accordance with that claimed in claim 5.
23. 23. The daughter seed produced by the transgenic plant in accordance with that claimed in claim 6.
24. 24. The daughter seed produced by the transgenic plant in accordance with what is claimed in claim 7.
25. 25. The daughter seed produced by the transgenic plant in accordance with that claimed in claim 8.
26. 26. A plant in accordance with claim 1 in claim 1 which is dicotyledone.
27. 27. A plant in accordance with claim 1 in claim 1 which is monocotyledone.
28. 28. A plant in accordance with claim 1 of claim 1 which is of the Gramineae family.
29. 29. A hybrid plant of which at least one parent is a plant in accordance with claim 1. -
30. 30. Seed daughter produced by a plant in accordance with claim 1 claim
31. 31. Non-germinating grain produced from a plant in accordance with the claim in claim 1.
32. 32. An isolated, purified, nucleic acid sequence encoding phytate produced according to a method comprising: crossing a homozygous mutant plant with low phytate content with a population of plants containing a mutant; identify the daughter seed tagged with mutator that contains the low phytate content; and cloning and isolating the nucleic acid sequence of the daughter seed.