Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/10—Seeds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
  • A23G1/42—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
  • A23G1/423—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
  • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
  • A21D2/14—Organic oxygen compounds
  • A21D2/18—Carbohydrates
  • A21D2/186—Starches; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/56—Cocoa products, e.g. chocolate; Substitutes therefor making liquid products, e.g. for making chocolate milk drinks and the products for their preparation, pastes for spreading, milk crumb
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
  • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
  • A23G3/346—Finished or semi-finished products in the form of powders, paste or liquids
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
  • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
  • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
  • A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
  • A23G3/366—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
  • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
  • A23L29/212—Starch; Modified starch; Starch derivatives, e.g. esters or ethers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G2200/00—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents
  • A23G2200/06—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents containing beet sugar or cane sugar if specifically mentioned or containing other carbohydrates, e.g. starches, gums, alcohol sugar, polysaccharides, dextrin or containing high or low amount of carbohydrate

Definitions

• the invention relates to a transgenic or mutated plant having genomic material which alters the normal starch synthesis pathway within the plant. More specifically, the present invention relates to a plant having a genotype which creates new forms of starch in significant quantity. Particularly, the invention relates to grain having an embryo with a genotype heterozygous for two or more wild type genes (for example, Aa Bb) and an endosperm having a genotype heterozygous for such genes (for example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced therefrom.
• Aa Bb wild type genes
• an endosperm having a genotype heterozygous for such genes
• Such grain are produced by pollinating a plant having a genotype homozygous recessive for at least one gene and wild type for another gene (for example, aa BB) with pollen from another plant having a genotype homozygous recessive for at least one other gene and wild type for the other gene (for example, AA/bb).
• starch producing plants Most plants produce and store starch. These plants have a starch synthesis pathway for starch production. The amount of starch produced varies with the type of plant. The most commonly known starch producing plants are the cereal grains. These cereals include rice, maize, sorghum, barley, wheat, rye, and oats. Additionally, the potato family, including the sweet potatoes and certain fruits, like the banana, are known as starch producing.
• Starch is an important end-product of carbon fixation during photosynthesis in leaves and is an important storage product in seeds and fruits.
• the starch produced by the edible portions of three grain crops, wheat, rice and maize provide approximately two-thirds of the world's food calculated as calories.
• Starch from plants is used in various ways. For example, it can be extracted and used for cooking and food processing. Starch can be left in the grain or plant and used for animal and human consumption. Starch can also be used in the distillation process for processing alcohols, for example, starch can be converted into ethanol. Additional starch can convert to high-fructose syrup and other industrial components.
• Starch is defined in the dictionary as a granular solid which is chemically a complex carbohydrate which is used in adhesives, sizes, foods, cosmetics, medicine, etc. More generally, starch is comprised of amylose and amylopectin. Amylose and amylopectin is synthesized in the plastid compartment (the chloroplast in photosynthetic cells or the amyloplast in non-photosynthetic cells). Different plants generate differing proportions of amylopectin and amylose. Furthermore, the different branching patterns of amylopectin and different chain lengths of amylose and amylopectin chains gives rise to different starch properties.
• starches with special properties: (i) using starches extracted from different plant species, (ii) using starches extracted from mutant lines of particular plants, (iii) using natural and mutant starches which had been chemically modified, and (iv) using natural and mutant starches which had been physically modified. In all cases the new starches were valuable because of the special properties provided for by the new starch type.
• mutant genes in plants affect the properties of the starch.
• a variety of starch related mutant genes in maize have been identified and some have been cloned. These mutant genes were named according to the physical appearance (phenotype) of the maize kernel or the properties of the starch.
• These recessive mutant genes include waxy (wx), sugary (su) [which includes but is not limited to sugary- 1 (sul), sugary-2 (su2), sugary-3 (su3), sugary-4 (su4)] dull (du), amylose extender (ae), horny (h), shrunken (sh) which includes, but is not limited to, shrunken- 1 (sh-1), shrunken-2 (sh-2).
• Some of these recessive gene lutants produce an isoform of a known enzyme in the starch synthesis pathway.
• the recessive mutant alleles of these genes result in a complete or nearly complete reduction in the activity of a specific isoform of one enzyme (hereinafter defined as complete reduction of enzyme isoform activity) in the pathway when homozygous in a plant or when expressed in sufficient levels in a transgenic plant.
• complete reduction of enzyme isoform activity a specific isoform of one enzyme
• starch Several crop varieties are known which produce different types of starch.
• the type of quality of starch makes it suitable for certain purposes, including particular methods of processing or particular end-uses.
• Naturally-occurring maize mutants produce starches of differing fine structure suitable for use in various food products and other applications.
• known mutants produce altered starch, some of these lines are not suitable for crop breeding and/or for the farmers' purposes. For example, they can give relatively poor yields, and/or are difficult to process and/or can have poor germination.
• a single mutant is a plant that is homozygous for one recessive mutant gene.
• waxy maize, waxy rice, waxy barley, and waxy sorghum have the homozygous mutant waxy (wx) gene.
• wx waxy
• starches from waxy genotypes have very little or no amylose
• amylose extender (ae) results in starch with high amylose.
• a double mutant is a single plant that has homozygous (or full expression) of two recessive mutant genes. For example, the wxfll double mutant is taught in U.S. patent 4,789,738.
• starch 95 Normal starch is defined as starch which is not chemically modified (by people) or which is produced from a plant that has the expected genes (wild type) regulating the starch synthesis pathway.
• double lower-case letters for example aa
• double upper-case letters for example AA
• AA homozygous non-mutant gene
• one upper-case and one lower-case letter, for example Aa shall refer to a non- homozygous set of genes, one mutant, one non-mutant. Different letters in the same size shall mean different genes; "aa/bb” would be a double mutant; “aa/bB” would be a single homozygous mutant gene and a heterozygous mutant gene in the genome of the plant. For purposes of this application, the order of any three letters on one side of the
• AAa/bbB is defined to be equivalent to aAA/bBb, AaA/Bbb, AaA/bBb, aAA/Bbb, and the like.
• maize plants and the embryo are diploid, maize endosperm is triploid. 110
• the endosperm genotype has two gene doses which are inherited from the female plant portion and one gene dose which is inherited from the pollen or male plant portion.
• aaA the endosperm in the kernel of this female plant.
• a non-mutant plant "AA” is crossed to a mutant plant "aa” with the non- 115 mutant as the female, the endosperm on the kernel of the female plant will be "Aaa", because two gene doses come from the female and one from the male plant.
• a first object of the present invention is to provide a method of developing hybrid plants having altered complex carbohydrate content of the grain which does not 150 require the crossing of double mutant inbreds.
• An object of the present invention is to provide plants that produce grain having altered starch properties.
• Another object of the present invention is to provide transgenic plants that produce grain having altered starch properties.
• Yet another object of the present invention is to provide a maize plant that produces both altered starch and larger quantities of starch than the associated mutant 160 plants produce.
• Still another object of the present invention is to provide new plants which contain genes which produce incomplete reduction of the activity of at least two isoforms of the specific enzymes in the starch synthesis pathway of the plant.
• a further object of the present invention is to provide maize plants which have endosperms with the genotype "AAa/BBb or AAa/bbB or aaA BBb or aaA/bbB".
• Yet another object of the present invention is a plant producing the following 170 endosperm genotype "wxwxWX/AeAeae”.
• Still an additional object of the present invention is to provide the altered starch which can be produced by plants having the genotype "AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB". 175
• Yet another object of the present invention is to provide novel uses of the starch obtained from the Maize plants of the present invention.
• the present invention broadly covers a method of producing intermutants
• the method of producing grain with altered starch qualities includes the steps of planting the female acting parent which is capable of flowering.
• the female parent having substantially complete reduction of at least one specific isoform enzyme in the
• starch synthesis pathway This can be by a homozygous recessive mutant or by the partial down regulation of the wild type gene through the use of a cloned gene using techniques generally known as antisense or co-suppression or sense-down regulation. Additionally the female has incomplete reduction of at least one specific isoform enzyme in starch synthesis pathway. This can be by a heterozygous recessive mutant
• a step includes eliminating the first parent's capability to produce pollen.
• the method includes the step of pollinating the female acting parent with the pollen of the male acting parent which is a non-mutant parent. Harvesting the grain produced by said first parent. Additionally, the method can
• 195 include the extraction of starch from the grain.
• This invention also encompasses a plant having genomic material which includes genes which give incomplete reduction of the activity of at least two specific isoforms of the enzymes in the starch synthesis pathway of said plant. And the starch it 200 produces which has altered structure when compared with the starch formed by a similar plant as described but which comprises genomic material which does not form isoforms of the enzymes in the starch synthesis pathway of the plant.
• a plant which forms said starch in grain such as cereal grains.
• the present invention is a starch producing plant comprising
• genomic material which includes genes which give incomplete reduction of the activity of at least two specific isoforms enzymes in the starch synthesis pathway of said plant whereby said plant produces substantially more starch than said plant would produce if said genes gave complete reduction of the activity of the same two specific isoforms of the enzymes within the starch synthesis pathway.
• the invention encompasses grain having endosperm genotypes of wxwxWx/AeAeae, or
• aeaeAe/WxWxwx or wxwxWx/DuDududu, or duduDu/WxWxwx, or aeaeAe/DuDududu, or duduDu/AeAeae, or wxwxWx/SuSusu, or susuSu/WxWxwx, or aeaeAe/SuSusu, or susuSu/AeAeae, or duduDu/SuSusu, or susuSu/DuDududududu and the like.
• the starch from a grain having a genotype of wxwxWx/AeAeae.
• the starch 225 from a grain having a genotype of Aeaeae/WxWxwx.
• a and b can be selected from ae, wx, sh, bt, h, su, fl, op and B and A can be selected from Ae, Wx, Sh, Bt, H, Su, Fl, Op.
• the starch obtained in accordance with the present invention produces a strong resilient gel which clears from the mouth uniquely fast.
• the starch of the present invention produces a strong resilient gel which clears from the mouth uniquely fast.
• 235 invention produces a gel with a unique and distinctive texture compared to conventional starches.
• the unique and distinctive texture makes the starch of the present invention suitable as a replacement for conventional gelling gums such as natural gums and gelatin, in whole or in part in food formulations.
• the starch of the present invention has also been found to produce a more resilient gel than common
• cornstarch produced from maize produces a gel which has improved clarity compared to a gel made from a common starch. Such improved clarity is visible to the human eye and lends itself to a more appetizing foodstuff.
• FIG 1 is a graph of enzyme activity for different gene dosages of a single mutant
• FIG 2a is a graph of the DSC scan of waxy, amylose extender and common
• FIG 2b is a graph of the DSC scan of a double mutant (aeaeae/wxwxwx);
• FIG 2c is a graph of the DSC scan of starch from an intermutant
• FIG 2d is a graph of the DSC scan of starch from another intermutant
• FIG 3a is a graph of Brabender data of common starch in various pH.
• FIG 3b is a graph of Brabender data of waxy starch in various pH.
• FIG 3c is a graph of Brabender data of 70% amylose starch in various pH.
• FIG 3d is a graph of Brabender data of double mutant starch in various pH.
• FIG 3e is a graph of Brabender data of a first intermutant starch in various pH.
• FIG 3f is a graph of Brabender data of a second intermutant starch in various pH.
• FIG 4a is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels of branching 280 enzyme I.
• FIG 4b is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels of branching enzyme IL 285
• FIG 4c is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels of bound starch synthase (waxy).
• FIG 4d is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels soluble starch synthase.
• FIG 5 is a plot of the elastic modulus (G') over time comparing a gel made
• FIG 6 is a plot of the elastic modulus (G') plotted against strain for both the present invention and an aewx starch.
• the present invention is an improved crop line which has manipulated expression of at least two starch-synthesizing enzymes which alter the 305 amount and type of starch, and, consequently, alters the grain produced by the plant.
• Specialty maize or mutant plants differ from "normal" maize because of its altered endosperm.
• the changed endosperm gives rise to a high degree of starch branching, or changed sugar content, or different kernel structure.
• 315 course is formed by the sperm and ovule, and the selection of both parents effects the endosperm's makeup.
• the present invention can be formed by two principle methods.
• the invention can be formed within a selected crop species by the use of mutant breeding. And the
• 320 invention can be formed in various plants by the use of transformation of the plants with genes which partially down regulate two or more enzymes in the starch synthesis pathway. More particularly, down regulation of one of the isoforms of the enzyme in the starch synthesis pathway to approximately 1/3 of the normal activity and 2/3 of the normal activity in the other isoform enzyme or down regulation of both isoform
• the present invention encompasses a method of producing grain with altered starch qualities which includes the steps of planting a parent which is capable of flowering, this parent having substantially complete reduction of at least one specific isoform of an enzyme (A) in the starch synthesis pathway and having no reduction of at least one other specific isoform of an enzyme (B) in the starch synthesis pathway.
• 385 other parent has no reduction of one isoform of an enzyme (A) and substantially complete reduction of at least one other specific isoform of an enzyme (B) in the starch synthesis pathway. It is then necessary to eliminate said first parents capability to produce pollen and allow pollination to proceed from said second mutant parent, and finally harvesting the grain produced by said first parent. Additionally, the method can include the
• a slurry is prepared which comprises water and an effective amount of starch of the present invention
• the sol contains the starch of the present invention in the amount of about 1 to about 20% by weight total sol.
• the slurry is cooked at a temperature of about 90° C and above to provide thickening characteristics prior to adding to the foodstuff. Cooking time is about 10 minutes.
• the sol in accordance with the present invention need not be cooked if the starch has already been subjected to a process which makes it cold water swellable. Cooking generally comprises raising the temperature of an aqueous slurry of the starch of the present
• a sol or a thickener composition of the starch of the present invention is added to a foodstuff in a conventional manner in order to provide the benefits of the starch of the 410 present invention to the foodstuff.
• a sol made in accordance with the present invention is combined with a foodstuff and the composition is cooked to the necessary degree to provide a thickened foodstuff.
• Conventional mixing is employed to prepare the thickened foodstuff.
• Cooking of the sol and foodstuff composition is also carried out in a conventional manner.
• starch of the present invention is mixed with the foodstuff or a slurry comprising the starch of the present invention and water is mixed with a foodstuff 420 and the resulting mixture is cooked to the desired degree to obtain a thickened foodstuff.
• the starch itself or a slurry containing the starch itself is mixed with a foodstuff, the resulting mixture must be cooked in order to provide a thickened foodstuff.
• the mixing as well as the cooking is accomplished in a conventional manner. Cooking is carried out at a temperature of about 90° C and above. Cooking time is about 10 minutes but may 425 vary depending on the amount of foodstuff present and the amount of shear that the mix is subject to during cooking.
• Such a thickener composition can provide considerable economic advantage to the user.
• Those familiar with the art have long used a variety of gelling gums for their clean
• starch of the present invention can replace all or a portion of these conventional gelling gums.
• a weight ratio of about 1:1, starch of the present inventio gelling gum can be employed. Larger or smaller amounts
• Such gelling gums include gelatin, pectin, carrageenin, gum arabic, tragacanth, guar, locust bean, zanthan, agar, algin and carboxymethyl cellulose.
• the starch of the present invention can be used in any food formulation, 445 where there is a need to provide gel characteristics and a clean break from the mouth.
• the starch of the present invention can be used in a food formulation which had heretofore employed a common starch, thereby providing the food with improved properties, i.e. clean break when compared to the same food formulation using a common starch. 450
• the clean break of a gel made with the starch of the present invention is useful in a variety of food applications.
• the clean break of the starch gel has value in a variety of bakery applications, for example cream or fruit fillings for pies such as lemon, banana cream or Bavarian cream; and in low or reduced fat high solids fruit centers for cookies, 455 for example, in fig bars.
• the starch of the present invention also creates an improved texture in mousses, egg custards, flans and aspics.
• starch as used in the specification and claims means not only the substantially pure starch granules as extracted from a starch bearing plant but also grain 460 products of the starch granule such as flour, grit, hominy and meal.
• 465 but may not be limited to, the preparation of foodstuff, paper, plastics, adhesives, paints, production of ethanol and co syrup products.
• the Brookfield Viscometer measures shear-strength (in centipoise, cP) and stability of starch pastes.
• Pasting temperature denotes the temperature of paste formation.
• Peak Viscosity denotes the temperature needed to provide a useable paste. Viscosity at 95C denotes the ease of cooking of the starch. Viscosity at 50C denotes the setback in paste viscosity during cooling of a hot paste. 540 Viscosity after 1 hour at 50C denotes the stability of the cooked paste.
• Percentages of oil starch and protein in starch give a measure of how well purified the starch is and indicates millability.
• Starch particle size gives an indication of starch yield and recoverability through
• Figure 1 is a graph of enzyme activities for individual gene-dosages (e.g., MMM,
• ADPG-PP 565 glucose pyrophosphorylase
• SSS soluble starch synthase
• BE branching enzyme
• BSS bound starch synthase
• Figure 2 is a graph of the DSC scan of starches extracted from grain taken from waxy, amylose extender and common (wild type) com.
• DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta H, Peak ⁇ Temperature, Onset Temperature and Endset
• FIG. 2b is a graph of the DSC scan of starches extracted from grain taken from the double mutant (aeaeae/wxwxwx) com.
• DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta 585 H, Peak II Temperature, Onset Temperature and Endset Temperature).
• the profile of the double mutants is different from the data provided in Figure 2a on common starch and the single mutants, waxy and high amylose.
• Figure 2c is a graph of the DSC scan of starches extracted from grain taken from intermutant (aeaeAe/WxWxwx) com.
• Such DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta H, Peak II Temperature, Onset Temperature and Endset Temperature). It is particularly noteworthy that the profile of the intermutant starch is different from the starch of the
• Figure 2d is a graph of the DSC scan of starches extracted from grain taken from intermutant (wxwxWx/AeAeae) com.
• Such DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta H, 600 Peak ⁇ Temperature, Onset Temperature and Endset Temperature). It is particularly noteworthy that the profile of the intermutant starch is different from the starch of the double mutant and appears to be similar to that of waxy starch.
• Figure 3a is a graph of Brabender data taken from common starch in either neutral 605 or acid conditions. Common com starch shows substantial breakdown in viscosity using acid conditions.
• Figure 3b is a graph of Brabender data taken from waxy starch in either neutral or acid conditions.
• the waxy mutation most particularly affects viscosity of the starch 610 in neutral conditions.
• Figure 3c is a graph of Brabender data taken from amylose extender (70% amylose) starch in either neutral or acid conditions. High amylose starches increase in viscosity in either acid or neutral conditions. 615
• Figure 3d is a graph of Brabender data taken from double mutant (aeaeae/wxwxwx) starch in neutral conditions. Double mutant starches maintain viscosity despite being homozygous for the waxy mutation.
• Figure 3e is a graph of Brabender data taken from intermutant (aeaeAe/WxWxwx) starch in neutral conditions. It is particularly noteworthy from these data that the new intermutant starches provide an increasing strength of viscosity similar to that seen with high amylose mutants, despite containing no increase in apparent amylose content.
• Figure 3f is a graph of Brabender data taken from intermutant (wxwxWx/AeAeae) starch in neutral conditions. It is particularly noteworthy from these data that the new intermutant starches provide an increasing strength of viscosity similar to that seen with high amylose mutants, despite containing no increase in apparent amylose
• This example illustrates the production of maize grain 635 possessing starch of the present invention.
• Maize plants of various backgrounds can be converted to mutant genotypes using either traditional breeding and/or backcrossing techniques or else using mutagenesis such as chemical treatments of pollen.
• waxy inbreds and 640 hybrids can also be purchased from a number of suppliers and foundation seed companies. Any maize line with good agronomic traits and relatively high yield can be employed.
• normal inbred lines are converted to mutant inbred lines using chemical mutagenesis followed by
• 650 valuable inbred line may be used for this process.
• the lines were confirmed to carry the mutation of interest by an allelism test in which the line may be crossed with a known mutant line, a process well known to those skilled in the art. Furthermore, the kernels from the line will have the
• the male and female hybrids can be made up of the same or different genetic
• the 670 done by a variety of methods including, but not limited to, hand pollination, hand and mechanical detasseling, introgressing genetic or cytoplasmic male sterility into the female plants, introducing male sterility through genetic transformation and use of chemical detasseling agents.
• the 675 grain of this cross contains the present invention with a genotype in the endosperm of aaA/BBb, with the starch from this genotype called intermutant starch. It is well known to those skilled in the art that the genetic background can be optimized for best starch qualities.
• Starch may be extracted from grain by a number of different methods. The most commonly used method involves a "wet
• the basic principle involves steeping and starch separation.
• the key step in this process involves softening the grain in a steep tank a process which has been optimized to permit optimal separation of the com grain components. This method
• the grain was steeped for 30-40 hrs at 48-52°C in tanks usually holding 50-90 metric tons of grain.
• the steep water contains 0.2% sulphur dioxide (SO 2 gas is bubbled-in) and so is mildly acidic (pH 4.0). The sulphur dioxide helps break-up
• Various intermutant grains can have the starch purified and prepared in this manner is suitable for a variety of food, feed and industrial uses. It may be used directly as unmodified com starch. It may be modified by chemical or physical treatments
• the starch 720 that preserve granule structure and granules may be washed to remove residual reactants. Bleaching is sometimes used to create super-white starches.
• the starch can be gelatinized using a high temperature treatment and sold directly as gelatinized starch. Such starch may be chemically modified
• the polymer itself may be hydrolysed partially or completely to produce maltodextrins or glucose.
• Such products can be further modified by fermentation to produce ethanol for the gasoline industry, or the glucose can be converted to high-fructose co syrup for the sweetener
• sh2 shrunken-2
• bt2 brittle-2
• dull 735 du
• sugary su
• waxy wx
• amylose extender ae
• Shrunken-2 encodes one subunit of ADP glucose pyrophosphorylase
• Brittle-2 encodes one subunit of ADP glucose pyrophosphorylase
• Waxy encodes granule bound starch synthase
• 745 Amylose Extender encodes an isoform of branching enzyme
• Dull alters expression of an isoforms of soluble starch synthase and branching enzyme
• Plant transformation vectors for use in the method of the invention may be 785 constructed using standard techniques. Since these enzymes are localized in the amyloplast compartment of the cell, the gene construct requires the presence of an amyloplast transit peptide to ensure its correct localization in the amyloplast.
• the transformation construct may carry the gene either in the partial sense orientation or in the antisense orientation. Expression of said gene in the plant results in a reduction 790 in expression of the enzyme by effects well known in the art as "sense cosuppression or antisense”. When only a reduction in expression is needed the transit peptide is not required. However, when enzyme overexpression is required then a correct plastid targeting sequence is needed in the construct.
• Key enzymes required for this invention include branching enzyme and soluble and bound starch synthase.
• Branching enzyme [1,4- ⁇ -D-glucan: 1,4- ⁇ -D-glucan 6- ⁇ -D-(l,4- ⁇ -D-glucano ) transferase] converts amylose to amylopectin, (a segment of a 1,4- ⁇ -D-glucan chain is transferred to a primary hydroxyl group in a similar glucan chain) sometimes called Q-enzyme.
• Soluble starch synthase [ADPglucose: 1 ,4- ⁇ -D-glucan 4- ⁇ -D- glucosyltransferase] extends the chain-length of amylopectin and perhaps also
• Bound starch synthase [ADPglucose: 1 ,4- ⁇ -D-glucan 4- ⁇ -D- glucosyltransferase] extends the chain length of amylose and perhaps also amylopectin.
• chloroplast transit peptides have similar sequences (Heijne et al describe a database of chloroplast transit peptides in 1991, Plant Mol Biol Reporter, 9(2): 104- 126).
• Other potential transit peptides are those of ADPG pyrophosphorylase (1991, Plant Mol Biol Reporter, 9:104-126), small subunit RUBISCO, acetolactate synthase, glyceraldehyde-3P-dehydrogenase and nitrite 830 reductase.
• ADPG pyrophosphorylase (1991, Plant Mol Biol Reporter, 9:104-126
• small subunit RUBISCO small subunit RUBISCO
• acetolactate synthase glyceraldehyde-3P-dehydrogenase
• nitrite 830 reductase the consensus sequence of the transit peptide of small subunit RUBISCO from many genotypes has the sequence:
• the co small subunit transit peptide of RUBISCO has the sequence: 835 MAPTVMMASSATATRTNPAQAS AVAPFQGLKSTASLPVARRSSRSLGN
• VASNGGRIRC The transit peptide of leaf starch synthase from com has the sequence:
• the selection is a particle bombardment.
• constructs of the various maize mutant genes are available from depositories in the U.S. and Europe. Attached are a few examples of some of these constructs as shown in Figures 4a - d.
• Figure 4c shows a promoter, which is CaMv
• Figure 4d is similar but shows the soluble starch syntheses first isoform gene in the construct.
• Figure 4a again has the same construct but shows the branching enzyme 855 first isoform.
• Figure 4b shows the second branching enzyme second isoform.
• other constructs associated with the gene mutants used in maize breeding are also available.
• the purpose of this experiment is to form an inbred that has partial down regulation of the waxy gene. If the inbred selected is already a mutant for ae, then the grain produced by crossing with a non-mutant inbred will be the grain of an intermutant. Depending on the strength of the down regulation, the female inbreds grain will resemble the mm*/mm* or the mm*/m** type of starch and grain. Clearly, the inbred selected is already a mutant for ae, then the grain produced by crossing with a non-mutant inbred will be the grain of an intermutant. Depending on the strength of the down regulation, the female inbreds grain will resemble the mm*/mm* or the mm*/m** type of starch and grain. Clearly, the inbred selected is already a mutant for ae, then the grain produced by crossing with a non-mutant inbred will be the grain of an intermutant. Depending on the strength of the down regulation, the female inbreds grain
• the transformation target tissue is immature zygotic embryos, through embryogenic
• 870 callus can also be employed. Immature zygotic embryos from A 188 plants 12 days after pollinated with the B73 ae inbred can be selected. The medium for the callus was 6 mM L-proline, 2% (w/v) sucrose, 2 mg/1 2,4- dichlorophenoxyacetic acid (2,4- D) and 0.3% (w/v) Gelrite (Caroline Biological Supply) (pH 6.0). Callus is grown and suspension cultures were initiated.
• This plasmid contains a 355- ladh-pat nos 3' selectable gene expression cassette.
• the cell suspensions are sieved and then suspended in 5 ml of suspension medium and placed on filter paper through vacuum. The constmct was coated into particles as is know in the art. The plates were then bombarded. The cells are then transferred to a N-6 medium and after 14 days transformed cells are selected by 1 mg/1 bialaphos. The cells are then suspended in a medium containing .6% (w/v) (Sea-
• the plant is bred and developed to an inbred having the mutant and the down 900 regulated pathway.
• the selected inbred can have the mutant crossed onto a transgenic after transformation to form the desired starch in the grain when the transgenic plant is employed as the female.
• Sample 1 was a commercial product sold by American Maize-Products Company of Hammond, Indiana. The percent amylose and the gelatinization temperature for
• Sample 1 above are mean values determined by random sampling of product.
• AMY V and AMY VII are commercial high amylose co starches sold by 920 American Maize-Products Company of Hammond, Indiana.
• the percent amylose and the gelatinization temperatures in Table 3 above are mean values determined from a random sampling of product.
• the 99% confidence interval for the percent amylose in AMY V and AMY VU was 53.4 to 62.5 and 65.5 to 73.8, respectively.
• the 99% confidence interval for the gelatinization temperature for AMY V and 925 AMY V ⁇ was 72.8 to 84.4 and 83.1 to 90.8, respectively.
• V ⁇ were grown in native maize.
• Starch Sample 4 corresponds to the Present Invention, while Sample 5 corresponds to the average values from Example 1 of U. S. Patent No. 5,009,911. 930 Sample 6 corresponds to a commercial waxy starch sold by American Maize-
• the percent amylose was determined using standard calorimetric iodine procedures wherein the starch is first gelatinized with sodium hydroxide and then reacted with an iodine solution and the resulting sample measured using a spectrophotometer in a 1 cm cell at 600 nm against a blank of 2% iodine solution.
• the DSC gelatinization temperature was measured using a scanning calorimeter manufactured by Mettler Moddle No. 300 using 30% starch solids following the procedure outlined in the owner's manual for that model.
• This example illustrates the gel strength of a sol made from
• Waxy Co Starch 16.0 In order to perform the gel strength test reported in Table 4 above, sols were prepared by mixing water with starch and subjecting the slurry to a rapid heat mode in the Brabender Visco-Amylograph to heat the sample to 50° C. Once 50° C was
• the instrument was set at a controlled rate of heating, 1.5°C/minute, until a temperature of 95° C was reached. The sample was then held at 95° C for 30 minutes. Next, the sample was cooled at 1.5° C to a temperature of 50° C for 30 minutes. Portions of these sols were added separately to 4 ounce jars into which a plumger was placed. The sols were then allowed to stand at ambient conditions for
• This example illustrates the difference between aewx starch, a waxy starch wherein the plant had a triple dose of the 975 waxy gene, and the starch of the present invention. All starches were obtained from maize.
• Figure 5 shows the results of the gel cure analysis.
• the starch of the present invention while having an initial Modulus (G') lower than 995 aewx starch, more quickly formed structure or gelled as evidenced by the rapid rise in
• the starch of the present invention formed a gel which didn't break under the applied strain of this test.
• the starch of the present invention is mixed with water in an amount to produce a slurry having 10% by weight starch.
• the sol has a short texture and a bland taste. 1015
• the sol when cooked at about 90° C for 10 minutes produces a thickener composition which had better clarity than a similar thickener composition made from common co starch and a shorter texture.
• This example compares the mouth feel of a gel made from the starch of the present invention to a gel made with a common starch.
• the starch was slurried at 5.5% solids, and then heated using the rapid heat mode to 50° C. Using controlled heat of 1.5° C per minute the slurries were heated to 95° C, and then held at this temperature for 30 minutes. The final solids was 5.9%.
• the sample starch pastes were then poured into small jelly
• This example illustrates making a gum candy using the starch of the present invention.
• This example illustrates making a Bavarian cream pie using starch of the present invention.
• This example illustrates preparing a lemon pie filling with the

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