WO1998048610A1

WO1998048610A1 - Method of making amylose mutant inbreds and hybrids

Info

Publication number: WO1998048610A1
Application number: PCT/US1998/008277
Authority: WO
Inventors: Peter L. Keeling; Ming-Tang Chang
Original assignee: Exseed Genetics, L.L.C.
Priority date: 1997-04-25
Filing date: 1998-04-24
Publication date: 1998-11-05
Also published as: AU7156398A

Abstract

This invention relates to a method of producing a low-amylopectin, high-apparent-amylose starch (hereinafter amylose and apparent amylose will be used interchangeable), in plants through mutagenesis. Further the invention relates to the starch from these maize plants and/or wheat plants which produce high apparent amylose containing seeds having up to 92 % amylose. Further the invention includes mutant grain having 40 % or more of amylose due to at least one specific point mutation induced by EMS.

Description

Method of Making Amylose Mutant Inbreds and

Hybrids

This application claims benefit of U.S. Application No. 08/845,319, filed April 25, 1997, the entire contents of which is hereby incorporated by reference.

Field of Invention

This invention relates to a method of producing a low-amylopectin, high-apparent-amylose starch (hereinafter amylose and apparent amylose will be used interchangeably), in plants through mutagenesis. Further the invention relates to the starch from these maize plants and or wheat plants which produce high apparent amylose containing seeds having up to 92% amylose. Further the invention includes mutant grain having 40% or more of amylose due to at least one specific point mutation induced by EMS.

Background

High amylose starches have been known for many years. There are examples of such starches in peas, maize, rice and barley. These starches are useful for such things as increasing the gel strength, improved appearance, improved adhesion to water-impermeable surfaces and improved textures. These starches also provide superior film forming ability and improved water resistance. These improved abilities are due to the molecular characteristics of the starch. Amylose is a linear or sometimes lightly-branched polymer and amylopectin is a larger, highly-branched polymer of glucose. Thus, chemically, starches can be described as a mixture of linear α-1 ,4 glucans with branch points formed from α-1 ,6-glycosidic linkages. The balance of amylose and amylopectin in normal starches isolated from storage tissues like potato tubers or cereal grain is normally about one third amylose and the remaining is described as amylopectin.

In 1993 the Journal of Biological Chemistry Vol. 268, No. 25, Issue of September 5, pp. 19084-19091 , (1993) had an article by Mizuno et al. entitled "Alteration of the Structural Properties of Starch Components by the Lack of an Isoform of Starch Branching Enzyme in Rice Seeds" in which it was reported that the rice mutants had been chemically induced and high amylose mutants of rice were achieved. The range of these high amylose mutants was narrow with approximately 7% difference between the highest level and the lowest level reported.

A 1993 publication from American Maize Products entitled " Designer Genes " discusses the development of Amylose-five and Amylose- seven material (Heightened Amylose material) formed by using contemporary breeding technology (Amylose-five is approximately 50% amylose and Amylose-seven is approximately 70% amylose). The article describes that the high amylose starches were developed and isolated by employing classical established genetics and standard agronomic techniques without resorting to genetic engineering. The amylose content of these starches range in apparent amylose content from 0 to 70% with gelatinization temperatures from 60-95°F. Specifically under the title High Amylose Starches, the article states, "that several varieties of corn plants with apparent amylose content ranging from 40-75% have been developed." The high amylose starches have apparent amylose contents ranging from 40-75% and can be cooked out at atmospheric pressure and normal cooking temperatures, have unique Brabender profiles and a range of gel strengths and film forming capabilities. Thus the market for these new starches is known in the food and starch industries.

Such high amylose starch is usually harvested from the grain of cereal crops. This starch has primarily been produced from amylose- extender (ae) mutant cereal plants, most often maize plants. Mutants of ae traditionally have produced certain expected levels of apparent amylose. These levels are the amylose-five level or fifty percent (50%) amylose or the amylose-seven level or seventy percent (70%). Levels of amylose above seventy percent (70%) have been claimed to have been developed by Goodman Fielder in PCT Application WO94/03049 by traditional recurrent breeding practices. The seed industry has attempted to increase the ae mutants production of apparent amylose through standard breeding practices. These breeding practices are time consuming and difficult as the selection is not simply for the ae mutant and increased amylose but also for ae mutants of agronomically acceptable plants as well.

Traditionally, a naturally occurring mutant of ae is identified and through standard breeding practices is developed to have some level of acceptable agronomic traits so that the mutant plant can be grown in farmers' fields. The known and widely used methods of increasing the levels of amylose produced by mutants at the amylose-extender, or ae, locus of maize is recurrent selection or backcrossing. Through these traditional breeding methods the level of some amylose sevens are over 15- 20 years brought to the level of 80% and up to 90%. These methods required the production of homozygous ae inbreds, followed by the production of hybrid seed for planting in open pollinated production fields. Many high amylose plants have fairly poor agronomic traits, particularly yield traits.

One method of producing mutations in corn is to use the transposon tagging to form a gene mutation. This method does not form a point mutation. This method was used by an university to produce a dominant form of ae in corn.

Chemical mutagenesis, such as EMS, produces a point mutation in the genome. A method of EMS pollen mutation was published by Nueffer as early as 1974. In a number of early references, one of which is entitled the "Allelic studies of the amylose- Extender Locus of Zea mays L: Levels of the Starch Branching Enzymes" in the Biochemical Genetics, Vol. 21 , Nos. 11/12, 1983 used EMS mutated corn in studies on the enzymes in starch. This article specifically notes that as of 1952 ae mutant maize produced by traditional breeding was associated with an increase in the amylose content of the endosperm of about 60%. The research in the article was directed to demonstrate that six independently derived ae mutants are deficient in branching enzyme I IB activity. In the methods and materials of the reference the material used include EMS (Ethyl-Methane Sulfonate) induced mutants of ae. In spite of this use of EMS to form ae mutants there does not appear to be any suggestion that amylose content of the ae mutants by EMS would contain any different levels of amylose then the expected 60% cited in the first part of the reference. In spite of the industry's knowledge of this method of making new point mutations to make an ae mutant plant, the industry has not understood nor used this method to produce a range of high amylose levels in grain plants such as corn. The industry has traditionally believed that EMS would not result in a range of amylose levels of starch, but would simply produce the same lower levels, 50-60% of amylose. The industry's approach to increasing the apparent amylose content of an ae mutant is to use recurrent selection or backcrossing. Through these traditionally breeding practices the breeders have attempted to add "ae modifying genes" that increase the amylose content of the plant.

In 1983 Charles Boyer and Karen Hedman wrote a paper entitled "Allelic Studies of the Amylose- Extender Locus ofZea mays L.: Levels of the starch Branching Enzymes" which indicated that EMS could be used to mutagenized ae corn. From the germplasm W64, ae-EMS1 and ae-EMS3 were created. The paper does not directly analyze the apparent amylose content of either of these two mutants. However, it does indicate the activity of the branching enzyme fractions isolated from the maize kernels with endosperm homozygous for differing ae alleles. Three of the mutants had no branching enzyme and the two EMS mutants and the ae-M mutant each had very low levels of enzyme. These low levels of enzyme in the branching enzyme would lead researchers to believe that the apparent amylose of the ae-EMS 1 and 3 would be lower than the apparent amylose of ae-ref which is considered to be in amylose δrange. This paper does not suggest nor indicate that in fact the use of EMS to mutagenize Ae can result in mutants that range in amylose content from 40-90's.

There are two basic drawbacks with any breeding method. One is the time element. It is usually five to nine generations of backcrossing before a elite inbred is made. This is because the native mutants are not in material that is agronomically adapted for a farmer's use in the field. These type of mutants are found in germplasm that is fairly old material with lower then desirable yield results. That means that the material has to be selected for the mutant trait and developed for the increase in yield and lower moisture and for the standability and the root strength that the farmer's require for crop production. The typical ae mutant has substantially poorer agronomics traits then does the commercially available crop.

At the University of Iowa State, researchers made a surprising discovery that is evidenced in US Patent No.5, 004, 864 that through transposon tagging technology a dominant ae gene could be created. The Iowa State researchers' dominant gene produces kernels with an apparent amylose content of up to 70%. However, it did not appear that this level of amylose production could be increased easily.

The US Patent No. 5,300,145 indicates that persons have for a number of years miscalculated the amylose contain of maize. By using a full starch dispersion followed by butanol fractionation and exclusion chromatography, a starch containing in excess of 75% optionally in excess of 85% amylose (normal amylose), together with about 8 to 25% low molecular weight amylose-like materials and less than 10% amylopectin could be extracted from a corn breeding population having a recessive amylose extender gene. The patent clearly indicates that this material came from maize germplasm selections in which the "ae modifiers genes" had been accumulated by recurrent selection. In contract to the method described in the '145 patent, the generally accepted iodine method of measuring amylose, as used herein, is a measure of apparent amylose which includes iodine- reactive amylopectin species.

Thus even in light of the dominant gene and in light of the practice of recurrent selection there remains a need for the development of method of making an ae corn plant with a procedure that substantially decreases the time and energy necessary to make an elite inbred that evidences higher than 50% amylose content without the time and effort of recurrent selection. There remains a need for a grain that has high amylose content that grows from a plant having a good agronomic package and which does not require recurrent selection or backcrossing for its development.

Summary of the Invention

Accordingly, the objects and advantages of the present invention are to provide a method of forming an ae mutant from commercial inbred lines which can be crossed to a second recessive ae line to form a hybrid that can produce amylose at about 80% or above in an elite germplasm base.

Providing an elite inbred having the characteristic of containing a point mutation that is less active in the formation of amylopectin when compared to the wildtype and resulting in higher levels of amylose in the grain.

Providing a method of forming cereal grain such as wheat, barley or maize that has substantially higher levels of amylose then has been developed before without the use of extended plant breeding practices. In a preferred embodiment, the starch bearing plant is a maize plant.

Still further objects and advantages will become apparent from a consideration of the ensuing description.

Broadly the present invention is a method of producing high amylose starch comprising the steps of :applying EMS to pollen of plants, forming treated pollen; self-pollinating plants with the treated pollen; selecting kernels with at least one point mutation which appear to have a high-amylose phenotype; planting said selected kernels and self- pollinating to produce more seed; selecting kernels which appear to have a high-amylose phenotype; repeating this cycle of planting and selection to increase seed volume; optionally, said plants may be backcrossed to ensure purity; extracting starch wherein said starch has above normal levels of amylose. This method can include the step of increasing the number of said kernels.

Additional method steps can be steps of planting said kernels to form high amylose seeds on starch bearing plants, or the step of harvesting the seeds, or the step of crossing the starch bearing plant with a second high amylose plant wherein hybrid seeds are formed on at least one of the plants and then the additional step of harvesting the seeds.

The present inventions scope also encompasses the step of harvesting the grain for the extraction of starch from the grain.

The present invention can also be described as a method of producing plants having above normal levels of amylose content including the steps of: inducing at least one point mutation in a starch affecting locus of the seed bearing plants; selecting seed from the plant, having the point mutation;-growing plants from the seeds; forming grain on the plants; and extracting high amylose starch from the grain. More particularly the point mutation is located in the starch affecting locus selected from a group of loci consisting of ae locus, or su2 locus in the plants genome. The method of the present invention includes, in one embodiment, the selection of mutated material, preferably EMS mutated material, which has or produces a high amylose content. The present invention also provides high amylose content materials so selected.

The present invention also includes a product which is a high amylose starch extracted from grain of a starch bearing plant comprising starch produced by the plant having at least one point mutation originally induced into the genomic ancestry of the plant by EMS and at least one of the point mutations wherein the grain of the plant produces high levels of amylose starch relative to normal amylose levels of standard yellow dent grain by the iodine method disclosed herein.

The grain from the mutants of the present invention do not appear to lack the Branching enzyme (BE) protein but the naturally occurring mutants in the commercial market do lack such protein. Accordingly, in one embodiment, the present invention provides methods to produce, and cells, seeds and plants produced by this method, seed and plants, such as maize and wheat, which contain an elevated amylose content, branching enzyme protein (active or inactive) and optionally, agronomically preferable additional traits as can be found in inbreds available from commercially sources (for example, from any one of Pioneer, Dekalb, Novartis, Cargill, Advanta, Mycogen, Golden Harvest, NC+ Hybrids, Asgrow, Agripro, Wyffels, Growmark, Limagrain, Curry, Barrus, Kaltenberg, Cenex, Dairyland, GreatLakes, Tarra, Becks, Fontanelle, and Fielders Choice). Detailed Description of the Invention

Normal Amylose Content is the average of the amylose content of B73 and Mo17 grain without mutations and is in the range of 22-27% apparent amylose as measured by the iodine method described herein.

Heightened Normal Amylose content is any mutagenized corn plant's grain that consistently averages a higher amylose contain than Normal Amylose Content.

High Amylose Content is any EMS mutagenized corn plant that consistently averages a higher level of amylose content than is averaged by a commercially available amylose hybrid which has an apparent amylose content of about 54%. When the method of the present invention is employed the results are mutant EMS plants with grain having a range of amylose content. The range of high amylose mutagenized corn carrying the ae mutant ranges above 50%. The range of high amylose mutagenized corn carrying the su2 mutation ranges from above 40%. Likewise other cereals are expected to show similar variations in the range of amylose due to the use of the EMS chemical to induce point mutations. Prior to the present invention, this range of amylose contents in starch grains were previously only developed by recurrent selection and breeding by backcrossing of ae mutants. It is a new and surprising discovery that point mutations can make such a range of levels of amylose in cereal crops. This invention relates to a method of producing low-amylopectin starch-bearing plants through EMS mutagenesis. Further the invention relates to the starch from these cereal plants such as maize plants and wheat plants which produce high apparent amylose containing seeds having up to 92% amylose. And a mutant grain having 40% or more of apparent amylose due to a specific point mutation which causes the production of RNA and inactive protein, such as branching enzyme, but not the production of an enzyme that is active in the starch pathway.

The present invention relates to the discovery that these starches can be made by pollen mutagenesis and an embodiment of the present invention is mutagenized pollen. This process results in the creation of point mutants within the maize genome. The ae gene produced by EMS are allelic mutant genes having at least one point mutation at the ae locus of the cereals genome. The locus is an amylose extender locus. This gene when mutagenized gives a distinctive phenotype in that it is characterized by a glassy, tarnished endosperm; high amylose content, and as a wild type it encodes branching enzyme (1 ,4- -D- glucan:1 ,4- -D-giucan 6- -D-(1 ,4- -D-glucano)-transferase, E.C. 2.4.1.18) which is located on the long arm of the 5 chromosome. To- date commercially available high amylose starches have been marketed as either 50% amylose or 70% amylose starch. A range of about 40-75% amylose has been reported for commercial products. The existence of high amylose corn starches of up to 88% amylose has been disclosed in scientific publications.

Prior to the present invention it was not understood that point mutations to form su2 mutants from EMS treatment also give a range of amylose contents that are in the 40-65% range. The su2 mutant simply has not been the subject of many studies. The su2 locus is located on the long arm of chromosome 6 at position 54. This gene when mutagenized gives a distinctive phenotype in that it is characterized by a glassy, translucent sometimes wrinkled endosperm; semi-high amylose content.

It is described in US Patent No. 5,300,145 that 85% amylose may be extracted from a corn breeding population having a recessive amylose extender gene. The corn population contains the ae gene and selections of modified germplasm which are homozygous for the ae gene and contains these modifier genes which have been accumulated in the germplasm by recurrent selection.

A surprising and unique aspect of the present invention, heretofore undiscovered is that with the use of mutagenesis an elite inbred can be converted into a high amylose mutant. In fact, prior to our invention high levels of amylose were only produced by years of recurrent selection or to backcross for successive generations. The present invention allow the production of corn plants capable of producing higher then normal levels of amylose including levels that reach 70% amylose or more in elite commercially useable inbreds or varieties. Although there may be a yield penalty in any production of high amylose grain (including the present invention), when the starting material is a present day elite line mutated for amylose and thereafter selected for wildtype agronomic traits and not a line that was elite years ago and has been selected over years primarily for its starch traits, the yield penalty is lessened. The improved crops of the present invention having the above described characteristics may be produced by using the following pollen mutagenesis procedure on elite maize inbreds or on that variety of plant species.

The method of producing these elite, agronomically sound and fairly high yielding mutants is a known method called mutagenesis. The process is outlined in the Neuffer paper Maize Genetic Newsletter 45:146. Point mutations produced by Nueffer's method produces any single point mutation in any single kernel at a frequency of less than 1 in 5,000 to10,000 kernels. It should be noted that EMS is a mutation process. Like all mutation processes the act of mutation can adversely effect the agronomic traits especially yields of the plant. However, the starting germplasm of the present invention is superior to that in which the ae mutant is usually formed. Thus the overall agronomic traits of the plant of the present invention are more easily preserved and selected for then the industries approach of recurrent selection or backcrossing. Mutations were induced in the inbred line by treating pollen with ethyl methane sulfonate in paraffin oil according to the procedure described by Neuffer (1974). This treatment was performed on a number of inbreds from the various plant genotypes of cereal. This example will focus on the development of maize ae mutants by this process. This mutagenesis process has been used to make a number of cereal mutants. However it was surprising and unique to discover that this process could be used to form ae mutants that have above 70% apparent amylose. Prior to this discovery the only method of developing plants having high levels of amylose has been through the use of recurrent selection and backcrossing. Both of these breeding techniques require a number of generations to developed the desired plants.

The general steps of the process of the present invention include treating inbred pollen (in this case maize) with ethyl methane sulfonate hereinafter "EMS". Inbred pollen is placed in EMS in oil for 45 minutes. A paint brush is used and the pollen is brushed on to the silks of a receptive corn ear. This forms the Mutant-1(M1) seed. Such seed are grown and self-pollinated to produce the Mutant-2 (M2) kernels. The resulting M2 kernels are examined visually for the ae phenotype. This is classically a glassy, tarnished endosperm that is dented or wrinkled. Apparent amylose contents are measured using the iodine-binding method described below.

The next step is to increase of the number of kernels by growing the plant followed by self pollination. When sufficient seeds are developed the next step is a crude verification that in fact the kernel is the ae mutant. A standard ae mutant inbred is selected in this instance the standard was A632 ae. The mutant plant is grown and crossed to the standard and the hybrid seed is once again visually examined for phenotype. If the mutant is the same as the standard then the kernels on the hybrid should be consistent with one another for the phenotype. This test is used because the mutant-gene is recessive.

As noted earlier the use of transposon tagging led Iowa State University researchers to the discovery of a dominant ae mutant. It has not however led to a mutant plant that could produce amylose at high levels nor a commercially viable plant for field production on a large scale. This previous disclosure of the dominant Ae is different from the present invention which produces a recessive high amylose mutant.

The inbred mutant of the present invention carries a recessive ae gene. The recessive nature of the gene makes it necessary in the production of hybrid crops such as maize and even conventional non- hybrid plants to produce the event in two inbreds. These two inbreds should be suitably crossed to get a hybrid that is reasonably well yielding and has acceptable commercial agronomic characteristics. For example, but not as a limitation, in maize one inbred could be from the stiff stalk family such as B73 and the other could be a Mo17 or other Lancaster type. Likewise breeders with ordinary skill in the art of plant breeding can select the elite lines that should be mutagenized to make an acceptable hybrid cross. Alternatively the inbreds in existing commercial hybrid lines can be used to form two new elite inbreds each containing the desired mutant.

The next step is to determine what the level of apparent amylose in the grain is by the following procedure. The method used is an adaptation of the method in the paper "An Improved Colohmetnc Procedure for Determining Apparent and Total Amylose in Cereal and Other Starches." by W.R. Morrison and B. Laignelet, 1983 Academic Press Inc. (London) in American Association of Cereal chemists(AACC) Approved methods 8th Ed. The test is based on the fact that two polysaccharide components exist in starch. The linear amylose which adsorbs iodine to form a deep blue complex and branched fraction of amylopectin which has only a weak affinity for iodine and gives a red coloration. The solutions used in this test are urea-dimethyl sulfoxide (UDMSO)- 1 :19 dilution of 6M urea in dimethyl sulfoxide. Iodine Solution - 2 g iodine+ 20 g potassium iodide, qs to 11 with dH₂O. There was approximately 6 grams of grain ground in a coffee grinder and placed in tubes with -20 ml dH₂O. The material is homogenized in a Sorvall SS 100 mm x 25 i.d. and then filtered through cheese cloth to remove the chunks. Step one requires the centrifuge in GPR centrifuge at -3800 rpm for 10 minutes at 4°C. The second step is pouring off the supernatant and scraping off the top yellow covering. The third step is to add 15ml of water and vortex. The first three steps are repeated then the first and second steps are repeated. The starch is then dried in the a speed -vac for 45 minutes . The pellet is then broken up and dried for 45 more minutes. The starch should be very dried. The material is then placed in micro tubes.

The samples of about 20-30 mg of starch are placed in the 5 ml tubes. The weight is measured and recorded. The control of normal starch is also prepared. The standard used was Sigma's Amylose from Corn (#A-7043) and amylopectin from CORN (#A-7780) at 0, 20, 40, 60, 80, 100%.

The standards are placed in the tubes 60mg of 20%, 50 mg 40% and 40 mg of 60% and so on. The controls and the standards and the grain are then tested by adding 4 ml of UDMSO to the tube and vortexing the tubes. The tubes are then capped with foil and boiled for 5 minutes and then mixed. Then the tubes are boiled for 1 hour and 45 minutes more. Check if all the starch is in solution if so add 500 micro liters dH₂O and vortexed on a clear 50ml conical tube. A blank is made with distilled water for nm absorbance background measurement. 24 ml. of distilled water is added and vortexed and then 500 micro liters of iodine solution is added and vortexed. The spectrophotometer was calibrated at 635 nm with water. The absorbance is read and the results are calculated Blue value = the absorbance/cm at 635nm of 10 mg anhydrous starch in 100 ml dilute iodine solution at 20° C. The absorbance needs to be calculated to the BV by translating it to 10 mg starch per 100ml. A=Subtract the background from the absorbance and calculate B which is by adjusting the absorbance to 100 ml by dividing A by four. Then calculate C which is the mg of the starch in 100ml solution (mg starch weighed and divided by 8) times four. BV= adjust the absorbance to 10mg of starch (B/C) times 10. The standards are used to calculate the slope and the intercept. % amylose= (BV-intercept)/slope.

The results of these tests clearly show that the point mutations are generating alleles that are different then the alleles present in the ae mutant gene pool that had been bred with recurrent selection for the modifier genes which increase amylose content. The plants treated by this method generates mutants that provide levels of amylose that are new and surprising. Additionally, the present invention has also developed high levels of amylose which are unexpected in both the ae and the su2. Although it is known that the su2 evidenced higher levels of amylose then did the wild type it is only upon the advent of this invention that the large increased range of amylose content was discovered in the alleles produced herein.

The following table exemplifies the various levels of amylose generated by the use of EMS in the ae mutant location. The control was a commercially available amylose starch with an apparent amylose content of 54% and the wildtype standard used as a control was starch from the inbred line B73 (Iowa State). One skilled in the art may use other available controls. The table evidences a range of results from this test. Clearly the levels are unexpected for apparent amylose content that previously were believed to be achievable only by extensive crossing or recurrent selection.

The ability to produce varying levels of amylose was evidenced in the mutant ae and mutant su2. The method of using EMS has surprisingly produced mutants that contain high levels of apparent amylose and lowers levels according to the point mutant. The presently exemplified range is from the level of 22% to the high 72% (rounded to the nearest percentage point).

The inbreds containing the desired high amylose trait can be selected and crossed with another inbred having the same mutations or additional mutations to make the hybrid. Other alternative breeding methods can be used with the inbred to form hybrids or breeding populations.

Although the description above contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. Various other embodiments and ramifications are possible within it's scope.

All references cited herein are incorporated, in their entirety, by reference.

Claims

We Claim:

1. A method of producing high amylose starch comprising the steps of :

-applying EMS to pollen of plants, forming treated pollen;

-pollinating plants with the treated pollen;

-harvesting the M1 seed produced with the treated pollen,

-planting the M1 seed, and

-harvesting the M2 kernels

-selecting kernels which appear to have a high-amylose phenotype

-extracting starch wherein said starch has above normal levels of amylose.

2. A method according to claim 1 including the step of planting said selected high-amylose phenotype kernels and self-pollinating to produce more mutant seed, and optionally repeating this cycle of planting and selection to increase mutant seed volume.

3. A method according to claim 2 including the steps of planting said kernels to form high amylose seeds on starch bearing plants.

4. A method according to claim 3 including the step of harvesting said seeds.

5. A method according to claim 3 including the step of crossing said starch bearing plant with a second high amylose plant wherein hybrid seeds are formed on at least one of the plants.

6. A method according to claim 5 including the step of harvesting said seeds.

7. A method according to claim 3 including the step of planting said seeds to form high amylose grain on starch bearing plants.

8. A method according to claim 7 including the step of harvesting said grain for the extraction of starch.

9. A method of producing plants having above normal levels of amylose content including the steps of:

- inducing at least one point mutation in a starch affecting locus of the seed bearing plants; selecting seed from said plant, having said point mutation;

-growing plants from said seeds;

-forming grain on said plants; and

-extracting high amylose starch from said grain.

10. A method according to claim 9 wherein said point mutation is located in said starch affecting locus selected from a group of loci consisting of ae locus, su2 locus.

11. A high amylose starch extracted from grain of a starch bearing plant comprising starch produced by said plant having at least one point mutation originally induced into the genomic ancestry of the plant by EMS and at least one of said point mutations wherein the grain of said plant produces high levels of amylose starch relative to normal amylose levels of standard yellow dent grain

12. A method according to claim 1 wherein said point mutation is located in a locus selected from a group consisting of ae locus, su2 locus.

13. A method according to claim 11 being located in a location selected from a group consisting of the ae locus, su2 locus.