JP2007267740A - New plants and methods for obtaining them - Google Patents

Abstract

The present invention provides a transgenic or mutated plant having a genomic component that alters the normal starch synthesis pathway in the plant.
A plant having a genotype that produces a significant amount of a new form of starch, particularly an embryo containing a heterozygous genotype (eg, Aa / Bb) of two or more wild type genes A grain having endosperm having a heterozygous genotype (eg, AAa / BBb or AAa / bbB or aaA / BBb or aaA / bbB), and starch produced therefrom.
[Selection figure] None

Description

  The present invention relates to transgenic or mutated plants having genomic components that alter the normal starch synthesis pathway in the plant. More particularly, the present invention relates to plants having a genotype that produces significant amounts of new forms of starch. In particular, the present invention relates to an embryo comprising a heterozygous genotype (eg, Aa / Bb) of two or more wild type genes and a heterozygous genotype (eg, AAa / BBb or AAa / bbB) of the gene. Or aaA / BBb or aaA / bbB) and the starch produced therefrom.

  Such a grain is a recessive homozygote of at least one other gene in a plant having a recessive homozygous genotype of at least one gene and a wild type of another gene (eg aa / BB). And pollen from another plant having the wild type of other genes (eg AA / bb).

  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 plant is cereal grains. These cereals include rice, corn, sorghum, barley, wheat, rye and oats. In addition, some fruits such as potatoes, including sweet potatoes, and bananas are known for starch productivity.

  Starch is an important end product of carbon fixation during leaf photosynthesis and an important storage product of seeds and fruits. Under economic conditions, starch produced by the edible portion of three grain crops, wheat, rice and corn, provides approximately two-thirds of the world's food, calculated as calories.

  Starch from plants is used in a variety of ways. For example, it can be extracted and used for cooking and food processing. Starch is in grains or plants and can be used for animal and human consumption. Starch can also be used in distillation processes that process alcohol, for example, starch can be converted to ethanol. Furthermore, starch can be converted to high fructose syrup and other industrial components.

  Starch is defined in the dictionary as a granular solid, which is a chemically complex carbohydrate used in adhesives, sizing agents, foods, cosmetics, pharmaceuticals and the like. More generally, starch consists of amylose and amylopectin. Amylose and amylopectin are synthesized in the plastid compartment (chloroplasts in photosynthetic cells or amyloplasts in non-photosynthetic cells). Different plants produce different ratios of amylopectin and amylose. Furthermore, the different branching patterns of amylopectin and the different chain lengths of amylose and amylopectin chains give rise to different starch properties. For example, because the microstructure of amylose and amylopectin is different in different plants, the branching pattern and chain length are quite different, resulting in new and novel properties that are useful in different applications. To date, four methods of producing starch with unique properties, (i) using starch extracted from various plant species, (ii) starch extracted from a mutant line of a particular plant In some cases, (iii) using chemically modified natural and mutant starches, and (iv) using physically modified natural and mutant starches. In all cases, the new starch was beneficial because of the unique properties conferred by the new starch species.

  It is known that mutant genes in plants affect the properties of starch. Various mutated genes related to starch in maize have been identified and some have been cloned. These mutant genes were named by the physical appearance (phenotype) of corn kernels or starch properties. These recessive mutant genes include waxy (wx), sugar (su) [sugar-1 (su1), sugar-2 (su2), sugar-3 (su3), Including but not limited to carbohydrate-4 (su4)], dull (du), amylose extender (ae), horny (h), shrunken (sh) ) [Including, but not limited to, shrinkage-1 (sh-1), shrinkage-2 (sh-2)]. Many of these recessive gene mutants produce isoforms of known enzymes in the starch synthesis pathway. Recessive mutant alleles of these genes, when homozygous in a plant or expressed in sufficient amounts in a transgenic plant, are full or nearly full of activity of a particular isoform of one enzyme in the pathway. Causes a complete decrease (hereinafter defined as a complete decrease in enzyme isoform activity). This change in the starch synthesis pathway causes the production of starch with various properties.

  Several crop varieties are known that produce different types of starch. High quality types of starch will be suitable for several purposes including specific processing methods or specific end uses. Naturally occurring corn mutants produce starches with different microstructures suitable for use in various foods and other applications. Although known mutants produce altered starch, many of these lines are unsuitable for crop breeding and / or farmer purposes. For example, they can be relatively low in yield and / or difficult to process and / or poorly germinate.

  Single and double mutant plants were bred to produce various starches. A single mutant is a plant that is homozygous for one recessive mutant gene. For example, waxy corn, waxy rice, waxy barley and waxy sorghum have homozygous mutant waxy (wx) genes. While starch from the waxy genotype contains no or very little amylose, another mutation known as the amylose extender (ae) results in high amylose starch. A double mutant is a type of plant that has a homozygote (or full expression) of two recessive mutant genes. For example, a wxfl1 double mutant is shown in US Pat. No. 4,789,738. A number of other novel starches are given in other starch patents where double or triple mutants have been produced (eg, different microstructures suitable for use in various foods). U.S. Pat. Nos. 4,789,557, 4,790,997, 4,774,328, 4,770,710, which describe naturally occurring corn mutants that produce starch having No. 4,798,735, US Pat. No. 4,767,849, US Pat. No. 4,801,470, US Pat. No. 4,789,838, US Pat. No. 4,792,458, and US Pat. The present invention is quite surprising in view of these applications as it produces altered starch and does not require double or triple mutants.

  Normal starch is defined as starch produced from plants that have not been (artificially) chemically modified or have the expected gene (wild type) that regulates the starch synthesis pathway. In simple terms, a double lower case letter, eg aa, means a homozygous recessive mutant gene, a double upper case letter, eg AA, means a homozygous non-mutant gene (wild type), One lowercase letter and one uppercase letter, for example, Aa, means a non-homozygous gene pair, one mutant and one non-mutant. Different letters of the same size mean different genes, “aa / bb” is a double mutant, “aa / bb” is a single homozygous mutant gene and a heterozygous mutation in the plant genome It is considered to be a mutated gene. For the purposes of this application, the order of any three letters on one side of the slash may be interchanged, and does not define the parent that gave the gene. For example, AAa / bbB is defined to be equivalent to aAA / bBb, AaA / Bbb, AaA / bBb, aAA / Bbb, etc.

  Corn plants and their embryos are diploid while maize endosperm is triploid. The endosperm genotype has two gene quantities derived from female plant parts and one gene quantity derived from pollen or male plant parts. Thus, if a single mutant plant “aa” is used as a female and crossed to a non-mutant plant “AA” male, the endosperm of the grain of this female plant will be “aaA”. When a non-mutant plant “AA” is crossed to a mutant plant “aa” using the non-mutant as a female, 2 gene amounts are derived from females and 1 gene amount is derived from male plants. The endosperm of a female plant kernel would be “Aaa”. The classical teaching is that the mutated gene is recessive and the non-mutant is dominant, and thus the following gene dosage in endosperm: “aaA” or “AAA” or “AAa” Starch produced by a plant having an expected amount of normal starch. However, the endosperm of the homozygous mutant plant “aa” acting as a female crossed to the homozygous mutant plant “aa” acting as a male becomes an endosperm having a gene dosage “aaa”. This endosperm yields starch with properties that are different from normal starch. Similarly, starch from a double mutant with endosperm that is “aaa / bbb” shows a difference in starch properties from normal starch. The difference between these starches is that they can replace chemically modified starches, or can be used with or in food or as a grain in alcohol production or in general starch industry applications. It is useful in.

  Clearly, the production of kernels with starches with different physical properties of starch requires the crossing of two mutated plants to produce a kernel that is a recessive homozygote of both genes. And Mutant plants are less predictable than standard plants.

  Problems are frequently encountered in grain production and starch extraction from double mutant hybrids and / or inbreds and some single mutants. The amount of starch produced is usually less than the amount of starch produced by non-mutant plants, and the starch granule size and / or starch granule integrity is reduced. This problem with known double mutant lines producing structurally altered starch with relatively low amounts of starch produced in crops can lead to insufficient seed germination ability. Furthermore, reduced starch yield in seeds is considered unavoidable because mutations disrupt the normal starch synthesis function of cells. There is still a need for a method of producing structurally altered starch or grains with altered properties without significant loss of yield or reduced starch grain size or integrity.

SUMMARY OF THE INVENTION A primary object of the present invention is to provide a method for generating hybrid plants having grains with altered complex carbohydrate content that do not require crossing of double mutant inbreds.

  The object of the present invention is to provide plants that produce grains with altered starch properties.

  Another object of the present invention is to provide a transgenic plant that produces grains having altered starch properties.

  Yet another object of the present invention is to provide a corn plant that produces both altered starch and a greater amount of starch than that produced by the associated mutant plant.

  Yet another object of the present invention is to provide a new plant comprising a gene that incompletely reduces the activity of at least two isoforms of a particular enzyme in the starch synthesis pathway of the plant.

  Another object of the present invention is to provide a corn plant having an endosperm having 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 endosperm genotype “wxwxWX / AeAeae”.

  Yet another object of the present invention is to provide altered starch that can be produced by plants having the genotype "AAa / BBb or AAa / bbB or aaA / BBb or aaA / bbB".

  Yet another object of the present invention is to provide a novel use of the starch obtained from the corn plant of the present invention.

  The present invention generally includes a method of producing a number of intermutants having an endosperm genotype of AAAa / bbB and endosperm that is a waxy, waxy, amylose extender (wxwxWx / AEAEae). . A method of producing a grain with altered starch characteristics involves planting a parent that acts as a female that can flower. A female parent in which at least one specific isoform enzyme is almost completely reduced in the starch synthesis pathway. This can be done either by homozygous recessive mutants or by the use of cloned genes using techniques commonly known as antisense or co-suppression or sense-downregulation. May be due to partial downregulation of type genes. Furthermore, females are incompletely reduced in at least one specific isoform enzyme in the starch synthesis pathway. This may be due to heterozygous recessive mutated genes or partial down regulation. Regardless of how the female occurs, it should simply serve as the female part. To ensure this, the process involves removing the ability of the first parent to produce pollen. The method includes pollinating pollen of a parent working as a male who is a non-mutant parent to a parent working as a female. Harvest the grain produced by the first parent. Further, the method can include extraction of starch from the grain.

  The present invention further includes a plant having a genomic component comprising a gene that incompletely reduces the activity of at least two specific isoforms of the enzyme in the starch synthesis pathway of said plant. And the starch it produces contains a genomic component that has an altered structure when compared to starch produced by plants similar to those described, but does not produce an enzyme isoform in the plant's starch synthesis pathway .

  Plants that produce said starch in grains such as cereal grains. A grain produced by a female plant having a waxy genotype (wxwx) crossed with a male plant having an amylose extender genotype (aeae) whose grain endosperm genotype is wxwxWx / AeAeae.

  In other words, the present invention comprises a starch-producing plant comprising a genomic component comprising a gene that incompletely reduces the activity of at least two specific isoform enzymes in the starch synthesis pathway of said plant, thereby A plant produces substantially greater amounts of starch than would the plant produce if the gene completely reduced the activity of two similar specific isoforms of enzymes in the starch synthesis pathway. Said starch-producing plant.

  A grain having endosperm having two genes having one gene dosage of a recessive mutant gene and two wild-type genes; and two genes having a recessive mutant and one wild-type gene. As used herein, the present invention refers to wxwxWx / AeAeue, or aaeaeAe / WxWxwx, or wxwxWx / DuDudu, or duduDu / WxWxwx, or aaeaeAe / DuDu / AduAuS Includes kernels with endosperm genotypes such as aaeaeAe / SuSusu, or susuSu / AeAaee, or duduDu / SuSusu, or susuSu / DuDudu.

  Starch from grain with genotype wxwxWx / AeAeae. Starch from kernels having the genotype Aeaeae / WxWxwx.

  has a diploid genotype of aa / BB and a triploid genotype of aaA / BBb, where a is a recessive mutant gene and A is a wild type gene, and b is recessive It is a mutated gene and B is a wild type gene, and starch appears to have changed from normal starch, where a and b are 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 according to the present invention produces a ferroelastic gel that breaks down in the mouth at a unique rate. The starch of the present invention produces a gel having a unique and special texture compared to conventional starch. The unique and specialized texture makes the starch of the present invention suitable as a substitute for conventional gelling gums such as natural gums and gelatin, in whole or in part of the food product. The starch of the present invention has also been found to produce a more elastic gel than ordinary starch. Furthermore, corn starch made from corn has been found to yield gels with improved transparency compared to gels made from ordinary starch. Such improved transparency makes the food visible to the human eye and more appetizing.

  The invention will now be described, by way of example, by the following description and examples with reference to the accompanying drawings.

Detailed description of the invention In general, the present invention relates to the manipulation of at least two starch-synthesizing enzymes that alter the amount and type of starch and, consequently, the grain produced by the plant. Improved crop line with high expression.

  A plant having at least two genes that partially down-regulate or reduce the activity of a particular isoform of an enzyme in the starch synthesis pathway surprisingly produces a significant amount of starch in the grain and It has been discovered to produce altered starch species.

  Special corn or mutant plants differ from “normal” corn because of their altered endosperm. Altered endosperm results in a high starch branching, or altered sugar content, or a different grain structure. Endosperm are, of course, produced from sperm and ovules and the choice of parents affects the composition of the endosperm.

  The present invention can be produced by two basic methods. The present invention can be produced in certain selected crop species through the use of mutant breeding. The present invention can then be produced in various plants by using plant transformation with genes that partially down-regulate two or more enzymes in the starch synthesis pathway. More specifically, one of the isoforms of the enzyme in the starch synthesis pathway is down-regulated to about 1/3 of the normal activity and up to 2/3 of the normal activity of other isoform enzymes or up to about 2/3 of the normal activity Down-regulation of both isoform enzymes in the starch synthesis pathway. Each of these methods has advantages.

First, the use of mutants to develop unique grains and starches in cereal crops is widely known. However, since the present invention is expected to produce grains with normal starch properties, it is very unique and surprising.
The following table illustrates how the present invention is surprising.

  Apparently, the endosperm genotype of the present invention shown in the table did not have a completely recessive gene, so starch yield and structure were expected to be normal. Indeed, according to the present invention, the grain does not show starch with altered normal starch structure. Historically, when altered starch is produced, it is usually produced in small quantities. The altered starch of the present invention was surprisingly produced in higher amounts than expected. Furthermore, this starch production is much simpler than that of double mutant crops. Traditionally, only single mutant hybrids have been widely used for large-scale starch production. Traditionally, to develop double mutants, both parents have both mutations that require considerable research and development efforts and result in poor starch yield and poor seed germination ability. I had to. Therefore, only small-scale production of double mutants was possible.

  The present invention is a method of producing a grain having altered starch properties, comprising pollinating a parent capable of flowering, wherein the parent comprises at least one enzyme (A) in the starch synthesis pathway. Including the above steps wherein the specific isoform is almost completely reduced and at least one other specific isoform of enzyme (B) is almost completely reduced in the starch synthesis pathway. Other parents have not reduced one isoform of enzyme (A) in the starch synthesis pathway and almost completely reduced at least one other specific isoform of enzyme (B). Next, it is necessary to eliminate the ability of the first parent to produce pollen and to generate pollen from the second mutant parent and finally harvest the grain produced by the first parent There is. In addition, the method can include the extraction of starch from the grain and the use of the starch as a special starch for various applications that have been found to be beneficial.

  To produce a sol according to the present invention, a slurry comprising water and an effective amount of the starch of the present invention is prepared and the sol is subjected to a boiling step to produce a paste. In general, boiling involves raising the temperature of the slurry approximately above the gelatinization temperature of starch and subjecting it to sufficient shear stress such that the granules burst and a paste is produced. It is not necessary for all the granules to burst. Preferably, the sol comprises the starch of the present invention in an amount of about 1 to about 20% of the total sol weight. The slurry is boiled at a temperature of about 90 ° C. or higher to give thickening, and then added to the food. The boiling time is about 10 minutes.

  The sol according to the invention does not need to be boiled if the starch has already been subjected to a process that makes it cold-swellable. Boiling generally involves raising the temperature of the aqueous starch slurry of the present invention to the gelatinization temperature of the starch and subjecting the starch to shear stresses that cause the starch granules to break and produce a paste.

  The starch sol or thickener composition of the present invention is added to food in a conventional manner to give the food the benefits of the starch of the present invention.

  To produce a thickened food product, the sol produced according to the present invention is mixed with the food product and the composition is boiled to the extent necessary to give a thickened food product. The sol and food are mixed using conventional mixing methods. The boiling of the sol and food composition is also done in a conventional manner.

  Alternatively, the starch of the present invention is mixed with a food product, or a slurry comprising the starch of the present invention and water is mixed with the food product, and the resulting mixture is boiled to the extent desired to obtain a thickened food product . When starch itself or a slurry containing starch itself is mixed with food, the resulting mixture needs to be boiled to give a thickened food. Mixing and boiling are performed by conventional methods. The boiling is performed at a temperature of about 90 ° C. or higher. The boiling time is about 10 minutes, but may vary depending on the amount of food present and the amount of shear stress applied to the formulation during boiling.

  Such thickener compositions can provide considerable economic benefits to the user. Those skilled in the art have long used various gelling gums for their superior breaking texture. Applications of the present invention include, but are not limited to, gum candy, gel desserts, glazes and spreads, including conventional gelling gums such as kappa carrageenan, agar, pectin or gelatin. It can be used as a substitute. These conventional gelling gums can be quite expensive, but also include poor flavor, lack of heat or acid stability, limited availability or lack of suitability Have the disadvantages. The starch of the present invention can be substituted for all or part of these conventional gelling gums.

  To replace gelled gum in foods, the starch: gelled gum of the present invention can be used in a weight ratio of about 1: 1. Further, a large amount or a small amount of starch of the present invention may be used to replace the gelled gum. Such gelling gums include gelatin, pectin, carrageenan, gum arabic, gum tragacanth, guar, locust bean, zanthan, agar, algin and carboxymethylcellulose.

  Of course, the starches of the present invention can be used in any food product that needs to provide gel properties and cleanness in the mouth. For example, when the starch of the present invention is used in foods in which ordinary starch has been conventionally used, thereby improving food properties, i.e., friability, when compared to similar foods using ordinary starch. Can give

  The friability of the gel obtained with the starch of the present invention is useful in various food applications. The friability of starch gels can be used in various confectionery applications, such as pie cream or fruit fillings, such as lemon, banana cream or bavaroa; and low or reduced fat high solids fruit cores, such as fig bars. It is useful in. The starches of the present invention also give improved texture in mousse, egg custard, franc and aspic.

  The term starch as used in the specification and claims refers not only to substantially pure starch granules extracted from starch-containing plants, but also to starch grain products, such as powders, coarse grains, hominy. And groats.

  The following examples of the invention are provided merely to illustrate. These examples do not limit the form or use of the invention. The invention or its grain or starch or sugar may be useful in food, paper, plastic, adhesive, paint production, ethanol production and corn syrup products, which may not be limited.

  Physical properties of various embodiments of the invention (similar genotypes are derived from various maize hybrids). These tables show data well known to those skilled in the art evaluating new and novel starches. Water and oil, protein, soluble and starch data are useful in assessing yield and pulverizability. Starch DSC (Differential Scanning Calorimetry) data is useful for evaluating the boiling and pasting properties of starch. Starch particle size data is useful for making decisions about starch milling and separation characteristics. Brabender and starch paste data is most important in assessing the potential of new starches primarily for improved food applications where specific starch thickening and pasting and pasting properties are desired. Such data would allow one of ordinary skill in the art to determine whether to conduct a more detailed test for starch properties and possibilities when judging the overall information gathering.

Definition:
Differential scanning calorimetry (DSC)
IR indicates an initial rise.
HP indicates a thermal peak.
HF indicates the final heat.
CP indicates a cooling peak.
CF indicates final cooling.
Brookfield viscometer The Brookfield viscometer measures the shear strength (in centipoise, in cP) and the stability of the starch paste.
Brabender viscometer (BRABENDER VISCO-AMYLOGRAPH) data The pasting temperature indicates the paste forming temperature.
The peak viscosity indicates the temperature required to give a usable paste.
The viscosity at 95 ° C. indicates the ease of boiling of the starch.
Viscosity at 50 ° C. indicates a regression in paste viscosity during boiling of the hot paste.
The viscosity after 1 hour at 50 ° C. indicates the stability of the boiling paste.
Corn protein, starch, oil and moisture%
The percentage of oil, tempun and protein in corn gives an indication of starch yield how starch can be recovered.
Starch protein, starch, oil and moisture%
The percentage of oil, starch and protein in the starch gives an indication of how well the starch is refined and showing pulverizability.
% Amylose and L-Maximum These data provide a measure of the apparent amylose concentration in the starch.
Starch particle size data Starch particle size gives an indication of starch yield and recoverability by the milling process.
Aeaewx in expedient <br/> Table 2 refers to aeaeAE / wxWxWx, similarly, means duduwx = duduDU / wxWxWx. In this table, wild type is not listed.

  FIG. 1 is a graph of enzyme activity of individual gene dosages (eg, MMM, mMM, mmM, mmm) of mutated alleles of amylose extenders and matte single mutants. These data include sucrose synthase (SS), UDP-glucose pyrophosphorylase (UDPG-PP), glucokinase (GK), fructokinase (FK), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), ATP -Dependent phosphofructokinase (PFK), PPi-dependent phosphofructokinase (PFP), ADP-glucose pyrophosphorylase (ADPG-PP), soluble starch synthase (SSS), branching enzyme (BE) and binding starch synthase The enzyme activity of (BSS) is shown. Enzyme activity is shown as a percentage relative to the wild type control (MMM). In the case of the complete mutant (mmm), there is a dramatic effect on the expression levels of various enzymes in the starch synthesis pathway. In the case of partial mutants (mM and mmM), there is little detectable change in expression level. These data indicated that the change in starch properties seen in the single mutant was the result of overexpression of several enzymes as well as removal of the enzyme encoded by the mutated allele. By combining two mutant amounts (eg, wxwxWx) with other amounts of other mutations (eg, AeAeae), the two enzymes are associated with the overexpression seen in the rest of the pathway Will decrease in part without.

  FIG. 2 is a DSC scan graph of starch extracted from kernels obtained from waxy, amylose extender and normal (wild-type) corn. Such a DSC scan makes it easy for one skilled in the art to provide a large number of data (see table in text for peak temperature, ΔH, peak II temperature, start temperature and end temperature data). It is particularly noteworthy that the profile of high amylose starch is different from ordinary starch and waxy starch.

  Figure 2b is a graph of a Bunten DSC scan extracted from kernels obtained from double mutant (aeaeae / wxwxwx) corn. Such a DSC scan makes it easy for one skilled in the art to provide a large number of data (see table in text for peak temperature, ΔH, peak II temperature, start temperature and end temperature data). It is particularly noteworthy that the double mutant profile differs from the data given in FIG. 2a for ordinary starch and single mutant, waxy and high amylose.

  FIG. 2c is a DSC scan graph of starch extracted from kernels obtained from intermutant (aeaeAe / WxWxwx) corn. Such DSC scans make it easy for those skilled in the art to provide a large number of data (see table in text for peak temperature, ΔH, peak II temperature, start temperature and end temperature data). It is particularly noteworthy that the profile of the intermutant starch appears to be different from that of the double mutant starch and similar to that of waxy starch.

  FIG. 2d is a graph of a Bunten DSC scan extracted from kernels obtained from intermutant (wxwxWx / AeAeae) corn. Such a DSC scan makes it easy for one skilled in the art to provide a large number of data (see table in text for peak temperature, ΔH, peak II temperature, start temperature and end temperature data). It is particularly noteworthy that the profile of the intermutant starch appears to be different from that of the double mutant starch and similar to that of waxy starch.

  FIG. 3a is a graph of Brabender data obtained from ordinary starch in neutral or acidic conditions. Ordinary corn starch shows a substantial decrease in viscosity using acidic conditions.

  FIG. 3b is a graph of Brabender data obtained from waxy starch in neutral or acidic conditions. Waxy mutations significantly affect starch viscosity in neutral conditions.

  FIG. 3c is a graph of Brabender data obtained from amylose extender (70% amylose) starch in neutral or acidic conditions. High amylose starches increase in viscosity in acidic or neutral conditions.

  FIG. 3d is a graph of Brabender data obtained from double mutant (aeaeae / wxwxwx) starch in neutral conditions. The double mutant starch maintains viscosity despite being homozygous for the waxy mutation.

  FIG. 3e is a graph of Brabender data obtained from intermutant (aeaeAe / WxWxwx) starch in neutral conditions. From these data, it is particularly noteworthy that the new intermutant starch gives the same degree of increase in viscosity as that seen for the high amylose mutants, despite the fact that the apparent amylose content has not increased. .

  FIG. 3f is a graph of Brabender data obtained from intermutant (wxwxWx / AeAeae) starch in neutral conditions. From these data, it is particularly noteworthy that the new intermutant starch gives the same degree of increase in viscosity as that seen for the high amylose mutants, despite the fact that the apparent amylose content has not increased. .

This example illustrates the production of the modified corn kernel starch of the present invention. Corn plants of various backgrounds can be converted to mutant genotypes using traditional breeding and / or backcross techniques or otherwise using mutagenesis such as chemical treatment of pollen. Alternatively, Waxy inbreds and hybrids can be purchased from a number of suppliers and foundation-founded subsidiary companies. Any maize line with good agronomic characteristics and a relatively high yield can be used. In the present invention, a normal inbred breeding system is converted to a mutant inbred breeding system using chemical mutagenesis followed by careful selection of mutant grain types from isolated progeny. This method is well known to those skilled in the art (eg, Neufer, MG and Chang, MT, 1989, induced mutations in biology and agronomy research, Voltr. Pfalzenzuchtg. 16, 165-178). Any commercially available inbred breeding system can be used for this purpose. The line was confirmed to have the mutation of interest by allelicity testing, a method well known to those skilled in the art that can cross the line with known mutation lines. In addition, the grains from the line will have an appearance and iodine staining characteristic specific to the selected mutation. Methods well known to those skilled in the art. In order to obtain the highest yield from a plant, it is possible to have a hybrid between two inbreds that both have the same mutation (for example, both inbreds are waxy or amylose extender species). the nearest. It is preferable to produce two hybrids, one being male and homozygous for one mutation, and the other being female and homozygous for the other mutation. Male and female hybrids can be composed of the same or different genetic backgrounds, and the two lines mature similarly in the field (ie require similar heat units from germination to flowering and pollen dropping). It is simply important. In order to cross intermutants on site, it is necessary to remove pollen production from females. This includes, but is not limited to, artificial pollination, manual and mechanical removal of male flower ears, introgression of genes or cytoplasmic male sterility into female plants, genetic transformation and chemistry In particular, it can be performed by various methods including the introduction of male sterility by the use of a drug that removes male flower spikes. The grain from this cross contains the invention where the endosperm genotype is aaA / BBb, and the starch from this genotype is referred to as intermutant starch. It is well known to those skilled in the art that the genetic background can be optimized for the best starch properties.

Starch can be extracted from the grain by a number of different methods. The most commonly used methods require the “wet grinding” method known and used throughout the world. The basic principle requires soaking and starch separation. An essential step in this method required the grain to be softened in a dip tank, and the method was optimized so that the corn kernel components could be optimally separated. Using this method, starch wxwxWx / AeAeae was extracted from the grain of the intermutant produced by Example 2. Embryos were easily released as they were and were separated from adhering endosperm and rind. Endosperm is macerated with water, starch is easily separated as white aggregates, and gluten protein is obtained as yellow aggregates. The kernels were soaked at 48-52 ° C. for 30-40 hours in a tank usually containing 50-90 metric tons of kernels. The immersion water contains 0.2% sulfur dioxide (aerated with SO ₂ gas) and is therefore weakly acidic (pH 4.0). Sulfur dioxide accelerates the degradation of the protein substrate and degrades the endosperm substrate into granules. After soaking, the grain was coarsened or made into pulp. Oily embryos float on the surface and dense starchy endosperm sinks. Separation is achieved by hydroclone (continuous separation). Starch was pulverized by impact with an external impact ring, followed by an “impact pulverizer known as an Entleter pulverizer” that pulverizes the slurry at high speed with a reverse grooved plate made of hardened steel alloy It was further purified later. Defibrillated starch was separated from gluten by centrifugation to give two fractions, protein (70% protein) and starch (2% protein). The processing temperature was maintained above 45 ° C. to prevent microbial growth. The starch was dried by flash drying by pouring into a stream of air heated to 200-260 ° F.

  Various intermutant kernels can have starch purified and manufactured in this way that is suitable for various food, feed and industrial applications. It can be used directly as unmodified corn starch. It may be modified by chemical or physical treatment that preserves the granule structure, and the granules may be washed to remove residual reactants. Sometimes bleach is used to produce ultra white starch. Starch can be gelatinized using high temperature processing and sold directly as gelatinized starch. Such starch can be chemically modified and dried. The polymer itself can be partially or fully hydrolyzed to produce maltodextrin or glucose. Such products can be further modified by fermentation to produce ethanol for the gasoline manufacturing industry or to convert glucose into high fructose corn syrup for the sweetener manufacturing industry.

  Mutations referred to as shrink-2 (sh2), brittle-2 (bt2), dull (du), carbohydrate (su), waxy (wx) and amylose extender (ae) It encodes isoforms of pyrophosphorylase, debranching enzyme, soluble starch synthase, binding starch synthase and branching enzyme.

Shrink-2 encodes one subunit of ADP glucose pyrophosphorylase,
Fragile-2 encodes one subunit of ADP glucose pyrophosphorylase,
Waxy encodes granule-bound starch synthase,
Amylose extender encodes an isoform of branching enzyme,
Matte changes the expression of soluble starch synthase and branching enzyme isoforms,
Sugars alter the expression and activity of soluble starch synthase and debranching enzymes.

  Using known mutants and genetic hybridization methods, the inventors examined the effect of altered gene expression on starch storage in the grain (see figure). For the bt2 mutant, the inventors have found a measurable progressive decrease in ADP Glc pyrophosphorylase activity that correlates well with a decrease in starch synthesis in the grain. The regulatory strength imparted by this enzyme on the flux to starch cannot be quantified from these data. Indeed, this study shows that this enzyme is one of the main determinants during starch synthesis, but may hardly regulate starch synthesis rate. This mutation hardly changes the starch structure. If the mutation is due to sugar, dullness, waxy and amylose extenders, we now detect changes in starch microstructure (branch chain length changes as well as amylose / amylopectin ratio changes). In these cases, there is even less regulation of flux to starch (except for the sugar mutants used to make the sweet corn genotype). In either of these cases, it is a change in the ratio of starch synthase and branching enzyme that has altered the starch microstructure. A dramatic new finding in these studies was the discovery that mutations not only reduced the expression of key enzymes, but also induced overexpression of other enzymes in the pathway. Furthermore, it is structural only in the complete mutant (mmm) genotype that we have found changes in starch microstructure, only when there is a decrease in enzyme isoform along with enzyme isoform overexpression. It has been demonstrated that changes occur. Although not wishing to be bound by this suggestion, these data indicate that starch structure can only be affected by reducing expression (eg, using an antisense construct), as well as the enzyme ( Illustrates means by which expression can be increased simultaneously (eg, using a sense construct).

  Plant transformation vectors for use in the methods of the invention can be constructed using standard techniques. Since these enzymes are located in the amyloplast compartment of the cell, the genetic construct requires the presence of an amyloplast transit peptide to confirm its exact location in the amyloplast. The transformation construct can have genes in a partial sense orientation or an antisense orientation. Expression of the gene in plants causes a decrease in the expression of the enzyme by an action known in the art as “sense co-suppression or antisense”. If only a decrease in expression is required, the transit peptide is not required. However, if enzyme overexpression is required, the correct plastid concentrating sequence is required in the construct. Key enzymes required for the present invention include branching enzymes and soluble and bound starch synthases. Branching enzyme [1,4-α-D-glucan: 1,4-α-D-glucan 6-α-D- (1,4-α-D-glucano) transferase] converts amylose to amylopectin, Often referred to as Q-enzyme (transferring a segment of 1,4-α-D-glucan chain to the primary hydroxyl group in a similar glucan chain). Soluble starch synthase [ADP glucose: 1,4-α-D-glucan 4-α-D-glucosyltransferase] extends the chain length of amylopectin and possibly further amylose. Bound starch synthase [ADP glucose: 1,4-α-D-glucan 4-α-D-glucosyltransferase] extends the chain length of amylose and possibly further amylopectin.

For any antisense or sense co-suppression construct, only a partial cDNA clone needs to be expressed in the transgenic plant. If enzyme overexpression is required, a full length cDNA clone is required. The sequence of maize branching enzyme-I is described in Baba, T .; Nishihara, M .; Mizuno, K, Kawasaki, T .; Shimada, H .; Kobayashi, E .; Ohnishi, S .; Tanaka, K .; And Arai, Y. et al. (Rice (Oryza-sativa)
L) Identification of Soluble Starch Synthase, cDNA Cloning and Gene Expression) ImmatureSeeds. Plant Physiology. 103: 565-573, 1993). Starch branching enzyme-11 from maize endosperm has been described by Fisher, D. et al. K. Boyer, C .; D. And Hanna, L .; C. (Starch branching enzyme from maize endosperm-II Plant Physiology. 102: 1045-1046, 1993). Mu, C.I. Harn, C .; Ko, Y .; Singletary, G .; W. Keeling, P.A. L. And Wasserman, B .; P. The argument by shows the relationship between the 76 kDa polypeptide and soluble starch synthase I activity in maize (cv B73) endosperm. Plant Journal 6, 151-159 (1994). The corn waxy locus of UDP-glucose starch glycosyltransferase was found in 1986 by Kloesgen, R .; B. Gier, A .; Schwarz-Sommer, Z .; And Saedler, H .; ( Mole. Gene . Genet . 203, 237-244). Recently, the sequence of the corn sugar locus has been described by James, M .; And Wright, A.M. ( ThePlant Journal ) was observed using transposon mutagenesis to locate the gene. The gene for any such protein is considered a debranching enzyme and can be used in constructs according to the present invention.

Chloroplast transit peptides are thought to have similar sequences (Heijne et al. Described a database of chloroplast transit peptides in Plant Mol Biol Reporter , 9 (2): 104-126 in 1991. ) Other possible transit peptides are those of ADPG pyrophosphorylase (1991, Plant Mol Bio Reporter , 9: 104-126), small subunit RUBSCO, acetolactate synthase, glyceraldehyde-3P-dehydrogenase and nitrite reductase. For example, the consensus sequence for the transport peptide of the small subunit RUBISCO from multiple genotypes is the sequence MASMSMLSSAAVARTNPAQASM VAPFTGLKSAAFPVSRKQNLDSIASNGGRVQC
Have The RUBISCO maize small subunit transport peptide has the sequence MAPVMMASSATARTNPAQAS AVAPFQGLKSTASLPVARRSSSRSLGNVASNGGRIRC
Have The transport peptide of leaf starch synthase from corn has the sequence MAALATSQLVATRALGLGVPDAS TFRRGAAQGLRGARASAAAADTLSMRTANASAAPRHQQQARRGGR FPSLVC
Have

  Production of dwarf transgenic corn plants has been carried out since 1990. A number of DNA delivery systems are known, but the choice is particle bombardment. As mentioned above, various maize mutant gene constructs are available from US and European contractors. Listed are a few examples of many of these constructs shown in FIGS. FIG. 4c shows a promoter that is CaMv (cauliflower mosaic virus), Adh1, a waxy gene, nos (noporin), and a pat gene and amp useful as selectable markers.

  FIG. 4d is similar but shows the soluble starch synthase first isoform gene in the construct. FIG. 4a also has a similar construct but shows the branching enzyme first isoform. FIG. 4b shows the second branching enzyme second isoform. Of course, other constructs linked to gene mutants used in maize breeding are also available.

For the purposes of this example, FIG. 4c, a waxy construct, is discussed. The purpose of this experiment is to create inbreds with partial downregulation of the Waxy gene. If the selected inbred is already a mutant of ae, the kernel produced by crossing with the non-mutant inbred would be an intermutant kernel. Depending on the strength of the down regulation, female inbred breeding kernels will resemble mm ^* / mm ^* or mm ^* / m ^* types of starch and kernels. Clearly, transformation allows for a more accurate method of down-regulating starch synthesis activity so that starch changes can be finely tuned.

  To ensure an appropriate level of down-regulation of the waxy gene, the transformed target tissue is an immature zygote and can also be used by embryogenic callus. An immature zygote from plant A188 12 days after pollination with the inbred line B73ae can be selected. The callus medium is 6 mM L-proline, 2% (w / v) sucrose, 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg / l and 0.3% (w / v) Gelrite. (Caroline Biological Supply) (pH 6.0). Callus was grown and suspension culture started.

  Myo-inositol 100 mg / l, 2,4-D 2 mg / l, 1-naphthaleneacetic acid (NAA) 2 mg / l, 6 mM proline, casein hydrolyzate (Difco Laboratories) 200 mg / l, 3% (w / v) MS-based liquid medium containing sucrose and 5% (v / v) coconut water (Difco Laboratories) (pH 6.0). The cell suspension was maintained on a rotary shaker at 125 rpm in the dark at 28 ° C. in this medium in a 125 ml Erlenmeyer flask.

  Select the transformation vector of FIG. 4c. This plasmid contains a 355-1adh-pat nos 3 'selectable gene expression cassette.

  After sieving the cell suspension, it is suspended in 5 ml of suspension medium and placed on filter paper by vacuum. The construct was coated into particles as is known in the art. Next, the plate was impacted. The cells are then transferred to N-6 medium and after 14 days transformed cells are selected with 1 mg / l bialaphos. The cells were then suspended in medium containing 0.6% (w / v) (Sea-Plaque; FMC) and kept at 37 ° C.

  After 2-5 weeks, the grown calli were removed and transferred to a fresh selective medium surface. Plants were regenerated in MS base medium containing 6% sucrose, 1 g / l myo-inositol, 1 mg / l NAA (34) and 0.3% (w / v) gellite (pH 6.0). Next, embryo germination occurred in MS medium with NAA 0.25 mg / l and 3% (w / v) sucrose and in the light. Plants are grown and transferred to the greenhouse. The expression level of the plant can then be evaluated.

  Plants were bred and developed into inbred breeds with mutant and down-regulated pathways. Alternatively, the selected inbred breed can have a mutant that has been crossed transgenically after transformation to produce the desired starch in the grain when the transgenic plant is used as a female.

  This example illustrates the gelatinization temperature of the starch of the present invention compared to other starches. The gelatinization temperatures are shown in Table 3 below.

  Sample 1 was a commercial product sold by American Maize-Products Company of Hammond, Indiana. The amylose% and gelatinization temperature of Sample 1 above are average values determined by random sampling of the product. The 99% confidence levels for% amylose and gelatinization temperature are 25.9 to 29.3 and 68.7 to 72.9, respectively.

  AMY V and AMY VII are commercial high amylose corn starch sold by American Maze Products Company of Hammond, Indiana. The% amylose and gelatinization temperature in Table 3 above are average values determined by random sampling of the product. The 99% confidence intervals for% amylose in AMYV and AMY VII were 53.4-62.5 and 65.5-73.8, respectively. The 99% confidence intervals for the gelatinization temperatures of AMYV and AMY VII were 72.8-84.4 and 83.1-90.8, respectively. Both AMYV and AMY VII were produced in natural corn.

  Starch sample 4 corresponds to the present invention, while sample 5 corresponds to the average value from Example 1 of US Pat. No. 5,009,911. Sample 6 corresponds to a commercially available waxy starch sold by American Maze Products Company.

  The method for determining both amylose% and gelatinization temperature was as follows.

  % Amylose was first gelatinized with sodium hydroxide, then reacted with iodine solution, and the resulting sample was measured using a spectrophotometer at 600 nm in a 1 cm cell against a plank of 2% iodine solution. Determined using a standard calorimetric iodine method.

  The DSC gelatinization temperature was measured using a scanning calorimeter Model 300 from Mettler using 30% starch solids according to the procedure outlined in the manufacturer's manual for that type.

  It is readily apparent from Table 3 above that the starching temperature of the starch of the present invention is comparable to ordinary corn starch.

  This example illustrates the gel strength of a sol made from corn starch of the present invention compared to a sol made from aewx corn starch, a sol made from regular corn starch and a sol made from wxwxwx starch. The results of the test are reported in Table 4 below.

  To perform the gel strength test reported in Table 4 above, a sol was prepared by mixing water and starch, and subjecting the slurry to the Brabender viscometer quench mode to heat the sample to 50 ° C. did. Once 50 ° C was reached, the apparatus was set at a constant heating rate of 1.5 ° C / min until a temperature of 95 ° C was reached. The sample was then held at 95 ° C. for 30 minutes. The sample was then cooled at 1.5 ° C. to a temperature of 50 ° C. for 30 minutes. A portion of these sols were added separately to a 4 ounce jar containing a plunger. The sol was then left at ambient conditions for 24 hours. Gel strength was measured by determining the force required to remove the plunger from the sol.

  This example illustrates that the gel strength of the sol made according to the present invention is comparable to ordinary corn starch sol.

  This example illustrates the difference between aewx starch, waxy starch in which the plant had three times the amount of waxy gene, and starch of the present invention. All starches were obtained from corn.

  All starches were tested for rheological properties. Each starch was subjected to a similar test method using a similar method. The starch granules were made into a paste with a cold probe down and a 750 g cm cartridge using a Brabender viscometer. A starch slurry of 5.5% initial solids was rapidly heated in a Brabender cup at 60 ° C. and then made into a paste while increasing the temperature to 95 ° C. at 1.5 ° C./min. The starch paste was held at this temperature for 20 minutes and then immediately loaded onto a rheometer measuring arrangement preheated to 70 ° C. Four-part rheological characterization was performed. Gel-cured segments that monitored the formation of structures at 0.2 hertz and 0.2% strain (well within the linear viscoelastic range) were measured while the sample was cooled to 70 ° C. to 25 ° C. and held for 4 hours It was. Also measured was a strain curve that measured the rheological response of the paste or gel to an increasing amount of strain, again at 0.2 Hertz at 25 ° C.

  Using this technique, a clear difference was found between aewx starch and the starch of the present invention. FIG. 5 shows the results of gel cure analysis. The starch of the present invention formed a structure or gelled more rapidly as indicated by the rapid rise in G ′ while having a lower initial modulus (G ′) than aewx starch. Thus, the starch of the present invention differed from the aewx starch in the rate of gel formation, despite a similar apparent iodine binding content. FIG. 6 shows the results of strain curve analysis for the starch of the present invention and aewx starch only. Here, the starch of the present invention exhibited a dilatant action indicated by an increase in G ′ as the amount of strain increased. Under the given amount of strain, the structure was not destroyed. In contrast, the structure of aewx starch was rapidly destroyed when the applied strain was greater than 4%. Thus, when compared to aewx starch, the starch of the present invention produced a gel that did not break under the strain given in this test. In contrast, aewx starch showed much less time-dependent structure formation and was “crushed” or destroyed by strain imparted by this technique.

  This example illustrates the preparation of a thickener composition according to the present invention.

  The starch of the present invention is mixed with an amount of water that produces a slurry having 10% by weight starch. The sol has a friable texture and a non-irritating flavor. The sol, when boiled at about 90 ° C. for 10 minutes, produces a texture thickener composition that is clearer and more friable than a similar thickener composition made from ordinary corn starch.

  This example compares the texture of a gel made from the starch of the present invention with a gel made from ordinary starch.

  Regular starch and starch of the present invention were made into a paste using a Brabender viscometer. The starch was made into a 5.5% solids slurry and then heated to 50 ° C. using a rapid heating mode. The slurry was heated to 95 ° C. using a controlled heat of 1.5 ° C./min and then held at this temperature for 30 minutes. The final solid was 5.9%. The sample starch paste was then poured into a small jelly jar, covered with cellophane and aged for 24 hours before analysis. The samples were then graded for the following attributes by interrogating the flavor panel.

  First, they were classified into two according to the relative stiffness with which they were touched by hand.

  The samples were then graded for relative friability, firmness and clarity when chewed.

  These results indicate that the hardness of these samples is similar. However, the starch of the present invention is much more friable when chewed.

  In addition, it tends to disappear from the mouth faster than ordinary starch gels. The panels all agreed that the starch of the present invention produced a “fresh” texture gel similar to that of gelatin or pectin.

  This example illustrates the production of gum candy using the starch of the present invention.

  The following ingredients and procedures are used.

Procedure After mixing all ingredients, boil to 340 <0> F using conventional equipment such as a jet cooker. Next, the boiled slurry is poured into a candy mold and solidified.

  This example illustrates the production of a bavaria pie using the starch of the present invention.

  The following ingredients and procedures are used.

Procedure Mix all pie filling ingredients except egg yolk and boil at 195 <0> F for 3-5 minutes.
The ingredients are then cooled to 120 ° F. with constant stirring. The egg yolk is then added and the additives are thoroughly blended. The mixture is then added to conventional pie skin and allowed to cool to room temperature before serving.

  This example illustrates the production of a lemon pie filling using the starch of the present invention.

  The following ingredients and procedures are used.

Procedure Mix half water with sugar and bring to a boil. All the remaining ingredients are slurried together and then added to boiling sugar and water. The temperature of the mixture is then adjusted to 200 ° F. and held there for 2 minutes. The mixture is then poured into the prepared pie skin and allowed to cool and solidify.

  This example illustrates the production of a chocolate mousse using the starch of the present invention. A mousse mix is produced using the formulation in Table 8.

Procedure Mix the ingredients to produce a uniform blend.
Use Mix 200g of mousse mix with 1 cup of milk (250g). Mix for 1 minute at low speed using an electric mixer. Scrap the bowl. Mix for 3 minutes at high speed until light and fluffy. Scoop it into a serving dish with a spoon and refrigerate for 1 hour before serving.

  To make a mousse mix, mix all ingredients. To cook the mousse itself, mix 200 grams of mousse mix with 250 grams of milk and mix at low speed. The mixture is then stirred at high speed to make it light and fluffy and the mixture is refrigerated for 1 hour. In this way, a light and fluffy mousse is cooked.

  Accordingly, the present invention has been described in some detail with respect to preferred embodiments of the invention. However, since the present invention is defined by the claims to be interpreted in light of the prior art, modifications may be made to the preferred embodiments of the present invention without departing from the inventive concepts encompassed herein. Or it should be understood that changes can be made.

2 is a graph of enzyme activity of various gene dosages of a single mutant. Figure 2 is a DSC scan graph of a waxy, amylose extender and normal corn. Figure 2 is a DSC scan graph of a double mutant (aeaeae / wxwxwx). Figure 2 is a DSC scan graph of starch from an intermutant (aeaeAe / wxwxwx). FIG. 3 is a DSC scan graph of starch from another intermutant (wxwxWx / AeAeae). Figure 2 is a graph of Brabender data for ordinary starch at various pHs. Figure 2 is a graph of brabender data for waxy starch at various pHs. Figure 2 is a graph of Brabender data for 70% amylose starch at various pHs. Figure 2 is a graph of Brabender data for double mutant starch at various pHs. 2 is a graph of Brabender data for first intermutant starch at various pHs. 2 is a graph of Brabender data for second intermutant starch at various pHs. It is a figure which shows the design of a plant transformation vector used for changing the gene expression level of branching enzyme I, and a restriction site. It is a figure which shows the design of a plant transformation vector used for changing the gene expression level of branching enzyme II, and a restriction site. FIG. 2 shows the design and restriction sites of a plant transformation vector used to change the gene expression level of conjugated starch synthase (Waxy). FIG. 3 shows the design and restriction sites of a plant transformation vector used to change the gene expression level of soluble starch synthase. Figure 2 is a plot of modulus of elasticity (G ') versus time for gels made from starch of the present invention compared to gels made from aewx starch and gel made from waxy (wx) starch. Figure 5 is a plot of modulus of elasticity (G ') plotted against strain for both the inventive and aewx starches.

Claims (35)

  A plant comprising a genomic component comprising a gene that incompletely reduces the activity of at least two specific isoforms of the enzyme in the starch synthesis pathway of the plant.   A genomic component that produces starch having a branched structure different from that produced by a plant similar to that described in claim 1 and that does not produce an isoform of the enzyme in the starch synthesis pathway of the plant. Plant described in 1.   The plant according to claim 2, wherein the starch is produced in a grain. A grain produced by a plant where the grain genotype is mm ^* / n ^** , where m = first mutant, n = second mutant, and ^* is wild type. A starch-producing plant,
A genomic component comprising a gene that incompletely reduces the activity of at least two specific isoform enzymes in the starch synthesis pathway of the plant, whereby the plant has the same two types of genes in the starch synthesis pathway Such plants that produce substantially greater amounts of starch than would be produced by the plant when the activity of a particular isoform enzyme is completely reduced.   From wxwxWx / AeAaee, aaeaeAe / WxWxwx, wxwxwx / SuSusu, susuSu / wxWxwx, ae from AeaeAe / SuSudu, suduSu / AeAaeae, wxwxWx / DuDuDu .   A grain having an endosperm genotype that contains two first-mutation alleles of a gene that affects starch structure and one second-mutation allele of a second gene that affects starch structure. The above grain, wherein the gene can be selected from waxy, amylose extender, matte, keratin, sugar, shrinkage, brittleness, flour, and opacity.   The starch extracted from the grain according to claim 7, wherein the genotype is wxwxWx / AeAeae.   The starch extracted from the grain according to claim 7, wherein the genotype is Aeaeae / WxWxwx.   Aa / BB diploid genotype with starch and aaA / BBb triploid genotype, where a is a recessive mutant gene and A is a wild type gene, and b is recessive A mutated gene and B is a wild type gene and the starch is altered from normal starch and a and b are from ae, du, wx, sh, bt, h, su, fl, op Plants that can be selected and B and A can be selected from Ae, Du, Wx, Sh, Bt, H, Su, Fl, Op.   It has a diploid genotype of aA / Bb / Cc and a triploid endosperm genotype of aaA / BBb / CCc (and other combinations thereof), where a is a recessive mutant gene and A Is a wild type gene, and b is a recessive mutant gene and B is a wild type gene, c is a recessive mutant gene and C is a wild type gene, and starch is derived from normal starch. Where a and b can be selected from ae, wx, sh, bt, h, su, fl, op, du and B and A are Ae, Wx, Sh, Bt, Plants that can be selected from H, Su, Fl, Op, Du. A method for producing a grain with a variable amount of starch,
(A) planting a parent capable of flowering and having at least one specific isoform enzyme fully reduced in the starch synthesis pathway;
(B) planting a second parent in which at least one other specific isoform enzyme is completely reduced in the starch synthesis pathway;
(C) removing the ability to produce the first parent pollen;
(D) the method comprising the step of pollinating the first parent to sow the second parent's pollen; and (e) harvesting the grain produced by the first parent.   13. The method of claim 12, comprising extracting the altered starch from the grain.   Starch extracted from the grain of claim 4 wherein a and b represent the same mutant and B and A represent the same wild type.   Starch extracted from a grain having at least 4 gene dosage mutants and 2 gene dosage wild types so that the genotype has wild type on both sides.   Starch extracted from grains having at least 3 gene dosage mutants and 3 gene dosage wild type so that the genotype has mutations on both sides.   A single mutant male sterile plant wherein the mutant is selected from ae, wx, sh, bt, h, su, fl, op, du.   A sol comprising an effective amount of starch and water extracted from a starch-containing plant having a waxy, waxy, amylose extender genotype.   The sol of claim 18, wherein the starch is present in an amount of about 1 wt% to about 20 wt%.   The sol according to claim 18, wherein the plant is corn.   The sol according to claim 18, wherein the starch is soluble in cold water.   The sol according to claim 18, wherein the starch is granular.   The sol of claim 21, wherein the starch is extracted from corn.   A food product comprising an effective amount of starch extracted from a starch-containing plant comprising a food stub and having, as an essential ingredient, a waxy, waxy, amylose extender genotype.   25. The food product of claim 24, wherein the starch is present in an amount from about 0.1% to about 10% by weight of the food product.   25. A food product according to claim 24, wherein the starch is extracted from corn. A method for producing a sol containing starch comprising:
Producing a slurry comprising an effective amount of starch and water extracted from a starch-containing plant having a waxy, waxy, amylose genotype; and boiling the starch to gelatinize the starch.   28. The method of claim 27, wherein the effective amount is from about 1% to about 20% by weight of the slurry.   28. The method of claim 27, wherein the starch is extracted from corn. A method for thickening food,
The above method comprising the steps of: mixing an effective amount of starch extracted from a starch-containing plant having a waxy, waxy, amylose extender genotype with a food; and boiling the food to thicken the food.   32. The method of claim 30, wherein the starch is extracted from corn.   32. The method of claim 30, wherein the starch is present in an amount from about 0.1% to about 10% by weight of the food product.   19. An improved method of producing a food product comprising gelatin, the improvement comprising replacing at least a portion of the gelatin in the food product with a sol according to claim 18.   19. An improved method of producing a food product comprising natural gum, wherein the improvement comprises replacing at least a portion of the natural gum with a sol according to claim 18.   35. The method of claim 34, wherein the food is a gum candy, a gelled dessert, a glaze or a spread.

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