WO1995035026A1 - Novel plants and processes for obtaining them - Google Patents

Abstract

The invention relates to a transgenic or mutated plant having genomic material which alters the normal starch synthesis pathway within the plant. More specifically, the present invention relates to a plant having a genotype which creates new forms of starch in significant quantity. Particularly, the invention relates to grain having an embryo with a genotype heterozygous for two or more wild type genes (for example, Aa/Bb) and an endosperm having a genotype heterozygous for such genes (for example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced therefrom.

Description

NOVEL PLANTS AND PROCESSES FOR OBTAINING THEM

FIELD OF THE INVENΗON

The invention relates to a transgenic or mutated plant having genomic material which alters the normal starch synthesis pathway within the plant. More specifically, the present invention relates to a plant having a genotype which creates new forms of starch in significant quantity. Particularly, the invention relates to grain having an embryo with a genotype heterozygous for two or more wild type genes (for example, Aa Bb) and an endosperm having a genotype heterozygous for such genes (for example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced therefrom.

Such grain are produced by pollinating a plant having a genotype homozygous recessive for at least one gene and wild type for another gene (for example, aa BB) with pollen from another plant having a genotype homozygous recessive for at least one other gene and wild type for the other gene (for example, AA/bb).

BACKGROUND OF THE INVENΗON

Most plants produce and store starch. These plants have a starch synthesis pathway for starch production. The amount of starch produced varies with the type of plant. The most commonly known starch producing plants are the cereal grains. These cereals include rice, maize, sorghum, barley, wheat, rye, and oats. Additionally, the potato family, including the sweet potatoes and certain fruits, like the banana, are known as starch producing.

Starch is an important end-product of carbon fixation during photosynthesis in leaves and is an important storage product in seeds and fruits. In economic terms, the starch produced by the edible portions of three grain crops, wheat, rice and maize, provide approximately two-thirds of the world's food calculated as calories. Starch from plants is used in various ways. For example, it can be extracted and used for cooking and food processing. Starch can be left in the grain or plant and used for animal and human consumption. Starch can also be used in the distillation process for processing alcohols, for example, starch can be converted into ethanol. Additional starch can convert to high-fructose syrup and other industrial components.

Starch is defined in the dictionary as a granular solid which is chemically a complex carbohydrate which is used in adhesives, sizes, foods, cosmetics, medicine, etc. More generally, starch is comprised of amylose and amylopectin. Amylose and amylopectin is synthesized in the plastid compartment (the chloroplast in photosynthetic cells or the amyloplast in non-photosynthetic cells). Different plants generate differing proportions of amylopectin and amylose. Furthermore, the different branching patterns of amylopectin and different chain lengths of amylose and amylopectin chains gives rise to different starch properties. Thus, the fine structure of amylose and amylopectin is different in different plants so that the branching patterns and chain-lengths vary considerably resulting in new and novel properties which are useful in different applications. Until now there have been four ways of making starches with special properties: (i) using starches extracted from different plant species, (ii) using starches extracted from mutant lines of particular plants, (iii) using natural and mutant starches which had been chemically modified, and (iv) using natural and mutant starches which had been physically modified. In all cases the new starches were valuable because of the special properties provided for by the new starch type.

It is known that mutant genes in plants affect the properties of the starch. A variety of starch related mutant genes in maize have been identified and some have been cloned. These mutant genes were named according to the physical appearance (phenotype) of the maize kernel or the properties of the starch. These recessive mutant genes include waxy (wx), sugary (su) [which includes but is not limited to sugary- 1 (sul), sugary-2 (su2), sugary-3 (su3), sugary-4 (su4)] dull (du), amylose extender (ae), horny (h), shrunken (sh) which includes, but is not limited to, shrunken- 1 (sh-1), shrunken-2 (sh-2). Some of these recessive gene lutants produce an isoform of a known enzyme in the starch synthesis pathway. The recessive mutant alleles of these genes result in a complete or nearly complete reduction in the activity of a specific isoform of one enzyme (hereinafter defined as complete reduction of enzyme isoform activity) in the pathway when homozygous in a plant or when expressed in sufficient levels in a transgenic plant. This change in the starch synthesis pathway causes the formation of starches with different properties.

Several crop varieties are known which produce different types of starch. The type of quality of starch makes it suitable for certain purposes, including particular methods of processing or particular end-uses. Naturally-occurring maize mutants produce starches of differing fine structure suitable for use in various food products and other applications. Although known mutants produce altered starch, some of these lines are not suitable for crop breeding and/or for the farmers' purposes. For example, they can give relatively poor yields, and/or are difficult to process and/or can have poor germination.

In order to generate different starches, single and double mutant plants have been bred. A single mutant is a plant that is homozygous for one recessive mutant gene. For example, waxy maize, waxy rice, waxy barley, and waxy sorghum have the homozygous mutant waxy (wx) gene. Whilst starches from waxy genotypes have very little or no amylose, another mutation known as amylose extender (ae) results in starch with high amylose. A double mutant is a single plant that has homozygous (or full expression) of two recessive mutant genes. For example, the wxfll double mutant is taught in U.S. patent 4,789,738. Many other novel starches have been provided in other starch patents in which double or triple mutants are generated (for example, US Patent Serial Numbers: 4789557, 4790997, 4774328, 4770710, 4798735, 4767849, 4801470, 4789738, 4792458 and 5009911 which describe naturally-occurring maize mutants producing starches of differing fine structure suitable for use in various food products). The present invention is highly surprising in light of these applications because it produces altered starch and does not require double or triple mutants.

95 Normal starch is defined as starch which is not chemically modified (by people) or which is produced from a plant that has the expected genes (wild type) regulating the starch synthesis pathway. For ease of reading, double lower-case letters, for example aa, shall refer to a homozygous recessive mutant gene, double upper-case letters, for example AA, shall refer to a homozygous non-mutant gene (wild type), and

100 one upper-case and one lower-case letter, for example Aa, shall refer to a non- homozygous set of genes, one mutant, one non-mutant. Different letters in the same size shall mean different genes; "aa/bb" would be a double mutant; "aa/bB" would be a single homozygous mutant gene and a heterozygous mutant gene in the genome of the plant. For purposes of this application, the order of any three letters on one side of the

105 slash can be interchanged and will not define the parent that donated the gene. For example, AAa/bbB is defined to be equivalent to aAA/bBb, AaA/Bbb, AaA/bBb, aAA/Bbb, and the like.

Although maize plants and the embryo are diploid, maize endosperm is triploid. 110 The endosperm genotype has two gene doses which are inherited from the female plant portion and one gene dose which is inherited from the pollen or male plant portion. Thus, if a single mutant plant "aa" is used as the female and crossed to a non- mutant plant "AA" male, then the endosperm in the kernel of this female plant would be "aaA". If a non-mutant plant "AA" is crossed to a mutant plant "aa" with the non- 115 mutant as the female, the endosperm on the kernel of the female plant will be "Aaa", because two gene doses come from the female and one from the male plant. Classic teaching is that the mutant gene is recessive and the non-mutant is dominant; therefore, the starch produced by a plant having the following gene doses in the endosperm "aaA" or "AAA" or "AAa" results in the normal starch in the expected amounts. However, 120 the endosperm of a homozygous mutant plant "aa" acting as the female crossed to a homozygous mutant plant "aa" acting as the male plant results in the endosperm having the gene dosage "aaa". This endosperm gives starch with different properties from normal starch. Likewise, the starch from a double mutant having of an endosperm which is "aaa/bbb" shows differences in starch properties from normal starch. These 125 starch differences are useful in that they can replace chemically modified starches or be used with or in foodstuffs or as grain in alcohol production or in general starch industrial applications.

Clearly, it appears that production of grain having starch with different physical 130 properties of starch requires the crossing of two mutated plants to generate grain which is homozygous recessive for both genes. Mutant plants are less predictable than standard plants.

There is a recurrent problem with the production of grain and extraction of 135 starch 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 the non-mutant plant, there is also a loss in starch granule size and/or starch granule integrity. This problem with known double mutant lines which produce structurally- altered starch in which the quantity of starch produced in the crop is relatively low can 140 result in poor germinability of the seed. Furthermore, the reduced starch yield of the seed appears to be unavoidable since the mutations cause the normal starch synthesis functioning of the cells to be disrupted. There remains a need for a way to produce grain having structurally altered starch structurally altered starch or altered properties without a significant loss of yield or reduced starch granule size or integrity. 145

SUMMARY OF THE INVENΗON

A first object of the present invention is to provide a method of developing hybrid plants having altered complex carbohydrate content of the grain which does not 150 require the crossing of double mutant inbreds. An object of the present invention is to provide plants that produce grain having altered starch properties.

155 Another object of the present invention is to provide transgenic plants that produce grain having altered starch properties.

Yet another object of the present invention is to provide a maize plant that produces both altered starch and larger quantities of starch than the associated mutant 160 plants produce.

Still another object of the present invention is to provide new plants which contain genes which produce incomplete reduction of the activity of at least two isoforms of the specific enzymes in the starch synthesis pathway of the plant. 165

A further object of the present invention is to provide maize plants which have endosperms with the genotype "AAa/BBb or AAa/bbB or aaA BBb or aaA/bbB".

Yet another object of the present invention is a plant producing the following 170 endosperm genotype "wxwxWX/AeAeae".

Still an additional object of the present invention is to provide the altered starch which can be produced by plants having the genotype "AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB". 175

Yet another object of the present invention is to provide novel uses of the starch obtained from the Maize plants of the present invention.

The present invention broadly covers a method of producing intermutants

180 generally which have the endosperm genotype of AAa/bbB and certain intermutants including endosperm which is waxy, waxy, amylose extender (wxwxWx/AEAEae). The method of producing grain with altered starch qualities includes the steps of planting the female acting parent which is capable of flowering. The female parent having substantially complete reduction of at least one specific isoform enzyme in the

185 starch synthesis pathway. This can be by a homozygous recessive mutant or by the partial down regulation of the wild type gene through the use of a cloned gene using techniques generally known as antisense or co-suppression or sense-down regulation. Additionally the female has incomplete reduction of at least one specific isoform enzyme in starch synthesis pathway. This can be by a heterozygous recessive mutant

190 gene or partial down regulation. Regardless of how the female is produced it should only act as the female portion. To assure this a step includes eliminating the first parent's capability to produce pollen. The method includes the step of pollinating the female acting parent with the pollen of the male acting parent which is a non-mutant parent. Harvesting the grain produced by said first parent. Additionally, the method can

195 include the extraction of starch from the grain.

This invention also encompasses a plant having genomic material which includes genes which give incomplete reduction of the activity of at least two specific isoforms of the enzymes in the starch synthesis pathway of said plant. And the starch it 200 produces which has altered structure when compared with the starch formed by a similar plant as described but which comprises genomic material which does not form isoforms of the enzymes in the starch synthesis pathway of the plant.

A plant which forms said starch in grain such as cereal grains. Grain produced 205 by a female plant having for example a waxy genotype (wxwx) crossed with a male plant having for example an amylose extender genotype (aeae) in which the genotype of the endosperm of the grain is wxwxWx/AeAeae.

In other words, the present invention is a starch producing plant comprising

210 genomic material which includes genes which give incomplete reduction of the activity of at least two specific isoforms enzymes in the starch synthesis pathway of said plant whereby said plant produces substantially more starch than said plant would produce if said genes gave complete reduction of the activity of the same two specific isoforms of the enzymes within the starch synthesis pathway.

215

Grain having an endosperm that has two genes which contain one gene dose of recessive mutant gene and two doses of wild type; and, having two gene doses of recessive mutant and one dose of wild type. Within this description the invention encompasses grain having endosperm genotypes of wxwxWx/AeAeae, or

220 aeaeAe/WxWxwx, or wxwxWx/DuDudu, or duduDu/WxWxwx, or aeaeAe/DuDudu, or duduDu/AeAeae, or wxwxWx/SuSusu, or susuSu/WxWxwx, or aeaeAe/SuSusu, or susuSu/AeAeae, or duduDu/SuSusu, or susuSu/DuDudu and the like.

The starch from a grain having a genotype of wxwxWx/AeAeae. The starch 225 from a grain having a genotype of Aeaeae/WxWxwx.

A female plant having a diploid genotype of aa/BB and having a triploid genotype of aaA/BBb where a is a recessive mutant gene and A is the wild type gene, and b is a recessive mutant gene and B is the wild type gene such that the starch is

230 altered from the normal starch where a and b can be selected from ae, wx, sh, bt, h, su, fl, op and B and A can be selected from Ae, Wx, Sh, Bt, H, Su, Fl, Op.

The starch obtained in accordance with the present invention produces a strong resilient gel which clears from the mouth uniquely fast. The starch of the present

235 invention produces a gel with a unique and distinctive texture compared to conventional starches. The unique and distinctive texture makes the starch of the present invention suitable as a replacement for conventional gelling gums such as natural gums and gelatin, in whole or in part in food formulations. The starch of the present invention has also been found to produce a more resilient gel than common

240 starch. Furthermore, it has been found that cornstarch produced from maize produces a gel which has improved clarity compared to a gel made from a common starch. Such improved clarity is visible to the human eye and lends itself to a more appetizing foodstuff.

245 BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described, by way of illustration, by the following description and examples with reference to the accompanying drawings of which: 250

FIG 1 is a graph of enzyme activity for different gene dosages of a single mutant;

FIG 2a is a graph of the DSC scan of waxy, amylose extender and common

255 maize;

FIG 2b is a graph of the DSC scan of a double mutant (aeaeae/wxwxwx);

FIG 2c is a graph of the DSC scan of starch from an intermutant

260 (aeaeAe/wxwxwx).

FIG 2d is a graph of the DSC scan of starch from another intermutant

(wxwxWx/AeAeae).

265 FIG 3a is a graph of Brabender data of common starch in various pH.

FIG 3b is a graph of Brabender data of waxy starch in various pH.

FIG 3c is a graph of Brabender data of 70% amylose starch in various pH.

270

FIG 3d is a graph of Brabender data of double mutant starch in various pH. FIG 3e is a graph of Brabender data of a first intermutant starch in various pH.

275 FIG 3f is a graph of Brabender data of a second intermutant starch in various pH.

FIG 4a is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels of branching 280 enzyme I.

FIG 4b is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels of branching enzyme IL 285

FIG 4c is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels of bound starch synthase (waxy).

290 FIG 4d is a schematic showing the design and restriction enzyme sites of plant transformation vectors used to alter gene expression levels soluble starch synthase.

FIG 5 is a plot of the elastic modulus (G') over time comparing a gel made

295 from the starch of the present invention to a gel made from an aewx starch and a gel made from a waxy (wx) starch; and

FIG 6 is a plot of the elastic modulus (G') plotted against strain for both the present invention and an aewx starch.

300 DETAILED DESCRIPTION OF THE INVENΗON

Broadly then the present invention is an improved crop line which has manipulated expression of at least two starch-synthesizing enzymes which alter the 305 amount and type of starch, and, consequently, alters the grain produced by the plant.

It has been discovered that plants which contain at least two genes which partially down regulated or reduce the activity of specific isoforms of enzymes in the starch synthesis pathway will surprisingly produce significant amounts of starch in the 310 grain and will produce altered starch types.

Specialty maize or mutant plants differ from "normal" maize because of its altered endosperm. The changed endosperm gives rise to a high degree of starch branching, or changed sugar content, or different kernel structure. The endosperm of

315 course is formed by the sperm and ovule, and the selection of both parents effects the endosperm's makeup.

The present invention can be formed by two principle methods. The invention can be formed within a selected crop species by the use of mutant breeding. And the

320 invention can be formed in various plants by the use of transformation of the plants with genes which partially down regulate two or more enzymes in the starch synthesis pathway. More particularly, down regulation of one of the isoforms of the enzyme in the starch synthesis pathway to approximately 1/3 of the normal activity and 2/3 of the normal activity in the other isoform enzyme or down regulation of both isoform

325 enzymes in the starch synthesis pathway approximately 2/3 of the normal activity. Each of these methods has its own advantages.

First, the use of mutants to develop unique grain and starches in cereal crops is widely known. However, the present invention is highly unique and surprising because 330 it was expected to produce grain having normal starch characteristics. The following table explains how unexpected the present invention is.

TABLE 1

335 Genotype of Parents Genotype Type of Starch

(female 1st) of endosperm Starch Yield

Wild-Type AA*AA AAA Normal Normal

340 Gene dosage AA*aa AAa Normal Normal aa*AA aaA Normal Normal

Single mutant aa*aa aaa Altered Lowerei

345 Double mutant aa bb*aa/bb aaa bbb Altered Lowered

PRESENT INVENTION - EXPECTED RESULTS

Intermutant aa/BB*AA/bb aaA/BBb Normal Normal

AA BB*aa bb AAa BBb Normal Normal

350 aa/bb*AA/BB aaA/bbB Normal Normal aa/bb*aa/BB aaa/bbB Normal Normal aa/BB*aa/bb aaa/BBb Normal Normal

PRESENT INVENTION - ACTUAL RESULTS

355 Intermutant aalBB*AAlbb aaA/BBb Altered Medium to

AAIBB*aalbb AAa/BBb Altered high - 70% aalbb*AAIBB aaA/bbB Altered of normal aa/bb*aa/BB aaa/bbB Altered aalBB*aalbb aaa/BBb Altered

360 aa = mutant gene (homozygous) AA = wild type (or non-mutant homozygous gene) * = signifies a cross-pollination between two varieties

365 aa/bb = two mutant genes (both homozygous) Clearly, since the genotype of the endosperm of the present invention shown in the table did not have completely recessive genes, the starch yield and structure was expected to be normal. In fact, according to the present invention, the grain does not

370 evidence normal starch structure altered starch. Historically, when altered starch is produced only small quantities are usually produced. The altered starch of the present invention was surprisingly produced in larger than expected quantities. Additionally, production of this starch is much simpler than the production of double mutant crops. Previously only single mutant hybrids have been used extensively for large-scale starch

375 production. Previously, to develop the double mutants, both parents had to carry both mutations which requires significant research and development efforts and results in poor starch yield and poor seed germinability. Heretofore, only small-scale production of the double mutants has been possible.

380 The present invention encompasses a method of producing grain with altered starch qualities which includes the steps of planting a parent which is capable of flowering, this parent having substantially complete reduction of at least one specific isoform of an enzyme (A) in the starch synthesis pathway and having no reduction of at least one other specific isoform of an enzyme (B) in the starch synthesis pathway. The

385 other parent has no reduction of one isoform of an enzyme (A) and substantially complete reduction of at least one other specific isoform of an enzyme (B) in the starch synthesis pathway. It is then necessary to eliminate said first parents capability to produce pollen and allow pollination to proceed from said second mutant parent, and finally harvesting the grain produced by said first parent. Additionally, the method can include the

390 extraction of starch from the grain and using said starch as a specialty starch for a variety of uses for which it is shown to be valuable.

In order to prepare a sol in accordance with the present invention, a slurry is prepared which comprises water and an effective amount of starch of the present invention

395 and the sol subject to a cooking step to form a paste. Generally, cooking entails raising the temperature of the slurry to above about the gelatinization temperature of the starch and subjecting the starch to enough shear such that the granules rupture and a paste is formed. It is not necessary that all the granules rupture. Preferably, the sol contains the starch of the present invention in the amount of about 1 to about 20% by weight total sol.

400 The slurry is cooked at a temperature of about 90° C and above to provide thickening characteristics prior to adding to the foodstuff. Cooking time is about 10 minutes. The sol in accordance with the present invention need not be cooked if the starch has already been subjected to a process which makes it cold water swellable. Cooking generally comprises raising the temperature of an aqueous slurry of the starch of the present

405 invention to the gelatinization temperature of the starch and subjecting the starch to shear such that the starch granules rupture and form a paste.

A sol or a thickener composition of the starch of the present invention is added to a foodstuff in a conventional manner in order to provide the benefits of the starch of the 410 present invention to the foodstuff.

In order to prepare the thickened foodstuff, a sol made in accordance with the present invention is combined with a foodstuff and the composition is cooked to the necessary degree to provide a thickened foodstuff. Conventional mixing is employed to

415 combine the sol with the foodstuff. Cooking of the sol and foodstuff composition is also carried out in a conventional manner.

Alternatively, starch of the present invention is mixed with the foodstuff or a slurry comprising the starch of the present invention and water is mixed with a foodstuff 420 and the resulting mixture is cooked to the desired degree to obtain a thickened foodstuff. When the starch itself or a slurry containing the starch itself is mixed with a foodstuff, the resulting mixture must be cooked in order to provide a thickened foodstuff. The mixing as well as the cooking is accomplished in a conventional manner. Cooking is carried out at a temperature of about 90° C and above. Cooking time is about 10 minutes but may 425 vary depending on the amount of foodstuff present and the amount of shear that the mix is subject to during cooking.

Such a thickener composition can provide considerable economic advantage to the user. Those familiar with the art have long used a variety of gelling gums for their clean

430 breaking texture. Application of the present invention have included but are not limited to gum candies, gelled desserts, glazes and spreads and can be used to replace conventional gelling gums such as kappa carrageenin, agar, pectin, or gelatin. These conventional gelling gums can be quite expensive however, and have other disadvantages including the presence of off-flavors, lack of heat or acid stability, limited availability, or lack of Kosher

435 approval. It has been found that the starch of the present invention can replace all or a portion of these conventional gelling gums.

In order to replace a gelling gum in food formulations, a weight ratio of about 1:1, starch of the present inventio gelling gum, can be employed. Larger or smaller amounts

440 of the starch of the present invention may be used to replace a gelling gum. Such gelling gums include gelatin, pectin, carrageenin, gum arabic, tragacanth, guar, locust bean, zanthan, agar, algin and carboxymethyl cellulose.

Naturally, the starch of the present invention can be used in any food formulation, 445 where there is a need to provide gel characteristics and a clean break from the mouth. For example, the starch of the present invention can be used in a food formulation which had heretofore employed a common starch, thereby providing the food with improved properties, i.e. clean break when compared to the same food formulation using a common starch. 450

The clean break of a gel made with the starch of the present invention is useful in a variety of food applications. The clean break of the starch gel has value in a variety of bakery applications, for example cream or fruit fillings for pies such as lemon, banana cream or Bavarian cream; and in low or reduced fat high solids fruit centers for cookies, 455 for example, in fig bars. The starch of the present invention also creates an improved texture in mousses, egg custards, flans and aspics.

The term starch as used in the specification and claims means not only the substantially pure starch granules as extracted from a starch bearing plant but also grain 460 products of the starch granule such as flour, grit, hominy and meal.

The following examples of the present invention are given for illustrative purposes only. These examples are not intended to limit the type or the uses of the present invention. The present invention or the grain or starch or sugar thereof can be useful in,

465 but may not be limited to, the preparation of foodstuff, paper, plastics, adhesives, paints, production of ethanol and co syrup products.

Example 1

470 The physical properties of various embodiments of the present invention (similar genotypes are from different maize crosses). These tables display data well known to people skilled in the arts of evaluating new and novel starches. Data on moistures, and percent oil, protein, solubles and starch are

475 useful in evaluating yield and milling potential. Starch DSC

(Differential Scanning Calorimetry) data are valuable for evaluating starch cooking and gelatinization properties. Starch particle size data are valuable for making decisions on starch milling and separation properties. Brabender and starch paste

480 data are most important for evaluating a new starch's potential for improved food applications where particular starch thickening and pasting and gelatinization properties are mostly desired. Such data when interpreted as a whole collection of information enable one skilled in the art to decide whether to conduct further more detailed tests of the starches properties and potential.

Genotype %Moisture %Starch %Protein %Oil %Solubles

aeae/wx 8.01 72.12 11.30 3.63 6.51

aeae/wx 8.00 71.34 11.74 3.79 6.41

aeae/wx 8.03 67.52 12.61 3.67 7.01

aeae/ae 8.13 60.11 14.59 6.60 10.14

sulsul/wx 8.18 69.66 12.09 4.03 6.91

dudu/wx 7.98 71.10 11.57 3.92 6.61

dudu/sul 7.90 70.53 11.67 3.93 6.81

flfl o 7.78 70.23 11.60 4.23 6.59

sulsul/du 7.81 69.00 12.20 4.77 7.98

sulsul/ae 7.88 69.03 12.27 4.88 8.02

sulsul/su2 7.95 69.67 12.28 4.55 7.49

Table 2

- * ,υπι udia

S93 % % % % %

Entry Background Row Moisture Starch Protein Oil Solubles

1 aeaewx 4660 9.15 64.80 14.58 4.47 7.64

2 aeaesu 4636 9.27 66.25 14.77 4.54 7.40

3 aeaedu 4612 9.24 66.01 14.65 4.73 7.80

4 wxwxae 4600 9.07 65.72 13.91 4.38 8.14

5 xwxsu 4648 9.06 66.03 14.79 4.08 7.83

6 wxwxdu 4624 8.77 65.32 15.62 4.27 8.23

7 susuae 4594 8.85 64.83 13.11 4.71 8.49

8 susudu 4672 8.73 65.39 13.20 5.01 8.35

9 susudu 4618 8.77 65.15 14.52 4.35 8.26

10 single ae 4582 9.14 60.52 15.19 5.95 9.29

11 single wx 4654 8.83 65.44 14.04 4.77 9.43

12 single su 4630 7.75 54.98 14.25 6.50 9.78

13 duduae 4588 8.97 69.26 12.30 4.89 6.85

14 duduwx 4666 8.88 70.43 11.25 4.40 6.96

15 dudusu 4642 9.48 70.43 13.48 4.67 6.92

16 single du 4606 8.50 65.54 12.02 6.03 9.33

- Starch Data

S93 % % % % %

Entry Background Row Moisture Starch Protein on Amvlose L-max

1 aeaewx 4660 3.82 86.44 1.68 0.18 31.60 597.1

2 aeaesu 4636 6.51 86.95 1.56 0.20 31.74 603.9

3 aeaedu 4612 5.22 86.44 1.70 0.11 33.38 598.8

4 wxwxae 4600 7.33 87.89 0.39 0.09 21.10 601.1

5 wxwxsu 4648 7.27 85.87 1.32 0.09 24.68 591.7

6 wxwxdu 4624 7.31 86.07 1.12 0.10 24.57 595.2

7 susuae 4594 8.19 86.32 0.93 0.07 29.00 604.5

8 susudu 4672 8.09 87.16 0.52 0.08 27.86 599.6

9 susudu 4618 7.67 84.20 0.60 0.12 30.82 601.1

10 single ae 4582 7.16 83.31 1.37 0.15 65.55 602.1

11 single wx 4654 8.30 85.16 0.51 0.14 0.43 XX

12 single su 4630 6.82 80.92 5.08 XX 29.32 598.4

13 duduae 4588 6.58 84.31 0.62 0.17 29.86 XX

14 duduwx 4666 11.08 84.77 0.48 0.16 28.74 XX

15 dudusu 4642 8.48 87.34 0.62 0.16 26.07 XX

16 single du 4606 8.36 80.70 0.46 0.08 37.88 608.9 Table 2

(Starch i Data)

% % % % %

Entry Genotype Moisture Starch Protein on Amylose L-max

1 ae ae/wx 9.56 83.84 0.63 0.05 29.44 593.1

2 ae ae / wx 7.96 85.94 0.70 0.05 27.89 596.6

3 ae ae / wx 11.71 82.54 0.82 0.05 42.54 595.1

4 ae ae/ae 6.09 84.17 1.24 0.14 63.60 602.4

5 sul sul /wx 4.37 90.00 0.56 0.09 28.36 600.0

6 du du / wx 5.95 88.50 0.61 0.09 27.98 596.9

7 du du/sul 6.93 82.66 0.69 0.10 29.98 600.8

8 fl fl /0 9.24 82.72 0.93 0.14 29.29 596.8

9 sul sul /du 9.01 84.82 0.71 0.02 29.57 600.8

10 sul sul /ae 7.78 84.81 0.63 0.03 29.90 600.8

11 sul sul /su2 7.61 85.02 0.49 0.02 29.68 602.3

12 White waxy 18.34 94.94db 0.77 0.09 2.74 526.9

Starch DSC Data

Peak ] Delta H Peek H Onset Endset

Entry Genotype !C IlE °C £ !C

1 ae ae / wx 67.7 11.67 100.0 62.6 76.6

2 ae ae / wx 66.5 10.33 96.8 61.9 74.8

3 ae ae/wx 74.6 10.00 100.8 68.8 83.4

4 ae ae/ae 81.0 11.83 XX 66.0 107.4

5 sul sul /wx 69.7 12.00 98.3 64.2 77.7

6 du du/wx 72.2 11.50 99.5 66.6 82.3

7 dudu/sul 70.2 9.67 98.4 65.7 78.3

8 fl fl /0 71.4 12.33 100.9 67.1 80.7

9 sul sul /du 68.8 7.83 99.5 63.2 79.1

10 sul sul /ae 68.1 10.33 99.8 61.9 78.3

11 sul sul /su2 67.4 12.00 96.1 61.3 79.5

12 White waxy 72.7 15.33 XX 66.1 82.6

Starch DSC Data

S93 Peak Delta H Peek II Onset Endset

Entry Background Row fC J/g £ !C !C

1 aeaewx 4660 73.8 11.17 99.4 67.7 82.8

2 aeaewx 4636 73.0 11.50 101.0 66.6 83.4

3 aeaewx 4612 73.3 10.83 98.6 66.7 83.0

4 wxwsae 4600 72.9 12.83 99.9 67.4 81.7

5 wxwssu 4648 73.7 12.17 98.2 68.2 81.6

6 wxwsdu 4624 73.6 11.33 99.6 69.3 80.5

7 susuae 4594 72.0 10.33 97.7 67.2 79.7

8 susudu 4672 71.9 9.83 98.9 67.0 79.1

9 susudu 4618 72.5 10.33 97.0 68.4 79.8

10 single ae 4582 84.6 15.67 XX 68.7 106.9

11 single wx 4654 72.5 16.17 XX 68.1 81.4

12 single su 4630 70.9 15.50 100.8 63.6 77.2

13 duduae 4588 71.8 13.50 100.5 67.1 81.1

14 duduwx 4666 71.9 10.17 98.8 67.2 79.5

15 dudusu 4642 71.7 10.17 97.2 66.8 79.0

16 single du 4606 71.6 9.50 100.1 65.8 79.8 Table 2

500 - Brabender Data

S93 IR HP HF CP CF

Entry Background Row Brabender £ BU BU BU BU

1 aeaewx 4660 460g/5.5% 65.0 315 285 940 940

2 aeaewx 4636 460g/5.5 81.5 225 225 470 430

3 aeaewx 4612 460g 5.5% 84.5 180 180 410 380

4 wxwsae 4600 460g 5.5% 74.0 360 355 590 460

5 wxwssu 4648 460g 5.5% 74.0 325 325 540 470

6 wxwsdu 4624 460g/5.5% 80.0 315 270 480 425

7 susuae 4594 460g 5.5% 77.0 270 270 610 585

8 susudu 4672 460g/5.5 77.0 295 295 785 610

9 susudu 4618 460g/5.5% 77.0 295 295 620 620

10 single ae 4582 460g/12% 90.5 170 170 240 240

11 single wx 4654 460g/5.5 68.0 750 360 415 405

12 single su 4630 90g/5.5% 93.5 35 35 40 40

13 duduae 4588 460g 5.5% 83.0 225 225 545 520

14 duduwx 4666 460g/5.5% 86.0 245 245 600 550

15 dudusu 4642 460g 5.5% 84.5 270 270 620 585

16 single du 4606 460g/5.5% 89.0 70 70 160 160 IR = initial rise

CP = cooling peak

CF = cooling final

505

- Brabender Data

IR HP HF CP CF

Entry Genotype Brabender °C BU BU BU BU

1 ae ae / wx 460g/5.5 83.0 220 220 520 460

2 ae ae /wx 460g 5.5% 89.0 220 220 540 510

3 ae ae / wx 460g/5.5% 84.5 270 270 510 450

4 ae ae / ae 460g/12% 90.5 490 490 1220 845

5 sul sul / wx 460g/5.5% 80.0 250 240 595 535

6 du du / wx 460g/5.5% 83.0 280 250 560 470

7 du du /sul 460g/5.5% 50.0/83.0 230 230 575 535

8 fl fl /0 460g/5.5% 81.5 280 255 630 560

9 sul sul /du 460g 5.5% 84.5 200 200 500 460

10 sul sul /ae 460g/5.5% 86.0 205 205 455 415

11 sul sul / su2 460g5.5% 84.5 180 180 420 385

12 White waxy 90g 5.5 68.0 830 220 310 270 Table 2

Starch Particle Size Data

(Volume Distribution)

S93 % Starch

Entry Background Row Mode μm Mean μm Median μm Recovery

1 aeaewx 4660 16.90 12.57 16.04 45.9

2 aeaewx 4636 16.63 11.82 15.23 57.9

3 aeaewx 4612 16.36 11.48 15.20 52.4

4 wxwsae 4600 17.43 11.62 16.53 41.5

5 wxwssu 4648 17.72 12.43 16.59 58.8

6 wxwsdu 4624 17.48 12.22 16.26 54.1

7 susuae 4594 16.36 11.96 15.66 55.5

8 susudu 4672 16.65 9.83 15.29 58.7

9 susudu 4618 16.36 11.82 15.61 55.9

10 single ae 4582 13.09 9.07 12.25 64.0

11 single wx 4654 18.03 11.99 16.85 55.7

12 single su 4630 7.43 5.16 7.95 5.6

13 duduae 4588 16.90 11.01 15.37 50.9

14 duduwx 4666 17.19 11.85 16.21 52.5

15 dudusu 4642 17.17 12.22 16.01 44.9

16 single du 4606 14.41 9.77 13.19 58.9

- Starch Particle Size Data

(Volume Distribution)

% Starch

Entry Genotype Mode μm Mean μm Median μm Recovery

1 ae ae / wx 16.63 10.90 15.70 51.4

2 ae ae /wx 16.12 10.44 14.92 52.8

3 ae ae /wx 15.60 10.55 14.60 59.4

4 ae ae / ae 11.53 8.40 11.30 63.7

5 sul sul /wx 14.88 10.24 14.21 52.5

6 du du / wx 16.38 10.73 15.12 52.0

7 du du / sul 15.87 11.03 14.98 53.2

8 fl fl /O 16.12 10.73 15.35 69.3

9 sul sul /du 15.87 10.73 14.92 53.7

10 sul sul /ae 15.11 7.85 14.35 48.5

11 sul sul /su2 15.85 10.33 14.20 58.4

12 White waxy 17.19 8.45 14.81 71.5 Table 2

- Starch Paste Data

Brookfield Freeze

Viscosity Gel-24hr Thaw

Entry Genotype CPS.20 rpm (grams) Cycles

1 ae ae / wx 6,200 166.6 0

2 ae ae / wx 6,700 227.2 0

3 ae ae / wx 8,000 256.3 0

4 ae ae / ae 14,250 111.1 0

5 sul sul /wx 6,800 173.4 0

6 du du / wx 6,000 141.4 0

7 dudu/sul 7,100 215.4 0

8 flfl/0 7,500 209.4 0

9 sul sul /du 6,300 219.7 0

10 sul sul /ae 6,000 172.2 0

11 sul sul /su2 5,500 158.8 0

12 White waxy 1.900 15.3 3

Starch Paste Data

Brookfield Freeze

S93 Viscosity Thaw

Entry Genotype Row CPS.20 rpm Gel-24hr Cycles

1 aeae/wx 4660 25,000 151.8 0

2 aeae/wx 4636 6,500 240.1 0

3 aeae/wx 4612 3,150 216.6 0

4 aeae/ae 4600 5,600 159.5 0

5 sul sul /wx 4648 6,100 126.4 0

6 du du / wx 4624 5,700 114.4 0

7 dudu/sul 4594 8,700 254.8 0

8 flfl/0 4672 12,000 272.8 0

9 sul sul /du 4618 10,250 233.6 0

10 sul sul /ae 4582 2,500 53.7* 0

11 sul sul /su2 4654 3,100 13.3 0

12 White waxy 4630 275 29.0 3

13 duduae 4588 7,200 226.5 0

14 duduwx 4666 7,300 233.4 0

15 dudusu 4642 5,100 332.9 0

16 single du 4606 1,650 99.3 0

* came out as a plug DEFINΓΠONS:

DIFFERENTIAL SCANNING CALORIMETRY (DSC)

IR denotes initial rise 525 HP denotes heat peak

HF denotes heat final CP denotes cooling peak CF denotes cooling final.

530 BROOKFIELD VISCOMETER

The Brookfield Viscometer measures shear-strength (in centipoise, cP) and stability of starch pastes.

BRABENDER VISCO-AMYLOGRAPH DATA 535 Pasting temperature denotes the temperature of paste formation.

Peak Viscosity denotes the temperature needed to provide a useable paste. Viscosity at 95C denotes the ease of cooking of the starch. Viscosity at 50C denotes the setback in paste viscosity during cooling of a hot paste. 540 Viscosity after 1 hour at 50C denotes the stability of the cooked paste.

CORN PERCENT PROTEIN, STARCH, OIL AND MOISTURE

Percentages of oil starch and protein in co give a measure of starch yield how recoverable the starch is. 545

STARCH PERCENT PROTEIN, STARCH, OIL AND MOISTURE

Percentages of oil starch and protein in starch give a measure of how well purified the starch is and indicates millability.

550 PERCENT AMYLOSE AND L-MAX

These data provide a measure of apparent amylose levels in starch. STARCH PARTICLE SIZE DATA

Starch particle size gives an indication of starch yield and recoverability through

555 the milling process.

SHORT HAND aeaewx in table 2 refers to aeaeAE/wxWxWx, likewise duduwx=duduDU/wxWxWx Throughout this table the wild type is not listed. Figure 1 is a graph of enzyme activities for individual gene-dosages (e.g., MMM,

560 mMM, mmM, mmm) of mutant alleles of the single mutants of amylose extender and dull. These data show the enzyme activities of 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-

565 glucose pyrophosphorylase (ADPG-PP), soluble starch synthase (SSS), branching enzyme (BE) and bound starch synthase (BSS). Enzyme activities are presented as percentages relative to the wild-type control (MMM). In the case of the full mutants (mmm) there is a dramatic effect on expression levels of various enzymes in the pathway of starch synthesis. In the case of the partial mutants (mMM and mmM)

570 there is almost no detectable change in expression levels. These data indicated that the alteration in starch quality observed with the single mutants is a consequence of the overexpression of several enzymes as well as elimination of the enzyme coded for by the mutated allele. By combining two mutant doses (e.g., wxwxWx) with other doses of another mutation (e.g., AeAeae) there would be partial reduction in two

575 enzymes without the overexpression seen in the rest of the pathway.

Figure 2 is a graph of the DSC scan of starches extracted from grain taken from waxy, amylose extender and common (wild type) com. Such DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta H, Peak π Temperature, Onset Temperature and Endset

580 Temperature). It is particularly noteworthy that the profile of the high amylose starch is different from the common starch and waxy starch. Figure 2b is a graph of the DSC scan of starches extracted from grain taken from the double mutant (aeaeae/wxwxwx) com. Such DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta 585 H, Peak II Temperature, Onset Temperature and Endset Temperature). It is particularly noteworthy that the profile of the double mutants is different from the data provided in Figure 2a on common starch and the single mutants, waxy and high amylose.

590 Figure 2c is a graph of the DSC scan of starches extracted from grain taken from intermutant (aeaeAe/WxWxwx) com. Such DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta H, Peak II Temperature, Onset Temperature and Endset Temperature). It is particularly noteworthy that the profile of the intermutant starch is different from the starch of the

595 double mutant and appears to be similar to that of waxy starch.

Figure 2d is a graph of the DSC scan of starches extracted from grain taken from intermutant (wxwxWx/AeAeae) com. Such DSC scans enable one skilled in the art to provide numerical data (see tables in text for data on Peak Temperature, Delta H, 600 Peak π Temperature, Onset Temperature and Endset Temperature). It is particularly noteworthy that the profile of the intermutant starch is different from the starch of the double mutant and appears to be similar to that of waxy starch.

Figure 3a is a graph of Brabender data taken from common starch in either neutral 605 or acid conditions. Common com starch shows substantial breakdown in viscosity using acid conditions.

Figure 3b is a graph of Brabender data taken from waxy starch in either neutral or acid conditions. The waxy mutation most particularly affects viscosity of the starch 610 in neutral conditions.

25

SUBSTITUTE SHEET (PULE 26) Figure 3c is a graph of Brabender data taken from amylose extender (70% amylose) starch in either neutral or acid conditions. High amylose starches increase in viscosity in either acid or neutral conditions. 615

Figure 3d is a graph of Brabender data taken from double mutant (aeaeae/wxwxwx) starch in neutral conditions. Double mutant starches maintain viscosity despite being homozygous for the waxy mutation.

620 Figure 3e is a graph of Brabender data taken from intermutant (aeaeAe/WxWxwx) starch in neutral conditions. It is particularly noteworthy from these data that the new intermutant starches provide an increasing strength of viscosity similar to that seen with high amylose mutants, despite containing no increase in apparent amylose content.

625

Figure 3f is a graph of Brabender data taken from intermutant (wxwxWx/AeAeae) starch in neutral conditions. It is particularly noteworthy from these data that the new intermutant starches provide an increasing strength of viscosity similar to that seen with high amylose mutants, despite containing no increase in apparent amylose

630 content.

Example 2

This example illustrates the production of maize grain 635 possessing starch of the present invention. Maize plants of various backgrounds can be converted to mutant genotypes using either traditional breeding and/or backcrossing techniques or else using mutagenesis such as chemical treatments of pollen. In the alternative, waxy inbreds and 640 hybrids can also be purchased from a number of suppliers and foundation seed companies. Any maize line with good agronomic traits and relatively high yield can be employed. In the present invention, normal inbred lines are converted to mutant inbred lines using chemical mutagenesis followed by

645 careful selection of the mutant grain type from the segregating offspring. This method is well known to those skilled in the art (see for example, Neuffer, M.G. and Chang, M.T. 1989. Induced mutations in biological and agronomic research. Vortr. Pfalzenzuchtg. 16, 165-178). Any commercially

650 valuable inbred line may be used for this process. The lines were confirmed to carry the mutation of interest by an allelism test in which the line may be crossed with a known mutant line, a process well known to those skilled in the art. Furthermore, the kernels from the line will have the

655 appearance and iodine-staining characteristics typical of the mutation selected, a method well known to those skilled in the art. In order to obtain the highest yields from the plants it is best next to produce a hybrid cross between two inbreds, both inbreds carrying the same mutation (e.g., both inbreds being

660 waxy or amylose extender types). It is preferred to produce two hybrids, one being the male and being homozygous for one mutation and the other being the female and being homozygous for the other mutation. The male and female hybrids can be made up of the same or different genetic

665 backgrounds, it is merely important for the two lines to have similar maturities in the field (i.e., require similar heat units from germination to silking and pollen shed). i order to make the intermutant cross in the field it is necessary to eliminate pollen production from the female. This can be

670 done by a variety of methods including, but not limited to, hand pollination, hand and mechanical detasseling, introgressing genetic or cytoplasmic male sterility into the female plants, introducing male sterility through genetic transformation and use of chemical detasseling agents. The 675 grain of this cross contains the present invention with a genotype in the endosperm of aaA/BBb, with the starch from this genotype called intermutant starch. It is well known to those skilled in the art that the genetic background can be optimized for best starch qualities.

680

Example 3

Starch may be extracted from grain by a number of different methods. The most commonly used method involves a "wet

685 milling" procedure known and used throughout the world.

The basic principle involves steeping and starch separation. The key step in this process involves softening the grain in a steep tank a process which has been optimized to permit optimal separation of the com grain components. This method

690 was employed to extract the starch, wxwxWx/AeAeae from the grain of an intermutant developed in accordance with Example 2. The germ was easily liberated intact and freed from adhering endosperm and hull. The endosperm is macerated under water, the starch was easily separated as a

695 white floe and gluten proteins are obtained as a yellow floe.

The grain was steeped for 30-40 hrs at 48-52°C in tanks usually holding 50-90 metric tons of grain. The steep water contains 0.2% sulphur dioxide (SO₂ gas is bubbled-in) and so is mildly acidic (pH 4.0). The sulphur dioxide helps break-up

700 the protein matrix permitting the endosperm matrix to break¬ up into granules. After steeping, the grain were coarsely ground or pulped. The oil-rich embryo floats to the surface and dense starchy endosperm sinks. Separation is achieved through hydroclones (continuous separation). The starch was

705 purified further after being milled through "an impact mill known as Entoleter mill" which smashes the slurry at high speed through counter-rotating grooved plates made of hardened steel alloy, followed by impact with an outer impact ring. The defibered starch was separated from gluten by

710 centrifugation to give two fractions: protein (70% protein) and starch (2% protein). Processing temperature was maintained above 45°C to prevent microbial growth. The starch was dried by flash-drying by injection into an air stream heated to 200- 260°F.

715

Various intermutant grains can have the starch purified and prepared in this manner is suitable for a variety of food, feed and industrial uses. It may be used directly as unmodified com starch. It may be modified by chemical or physical treatments

720 that preserve granule structure and granules may be washed to remove residual reactants. Bleaching is sometimes used to create super-white starches. The starch can be gelatinized using a high temperature treatment and sold directly as gelatinized starch. Such starch may be chemically modified

725 and dried. The polymer itself may be hydrolysed partially or completely to produce maltodextrins or glucose. Such products can be further modified by fermentation to produce ethanol for the gasoline industry, or the glucose can be converted to high-fructose co syrup for the sweetener

730 industry. Example 4

The mutations called shrunken-2 (sh2), brittle-2 (bt2), dull 735 (du), sugary (su), waxy (wx) and amylose extender (ae) encode isoforms of ADP glucose pyrophosphorylase, debranching enzyme, soluble starch synthase, bound starch synthase and branching enzymes:

740 Shrunken-2 encodes one subunit of ADP glucose pyrophosphorylase,

Brittle-2 encodes one subunit of ADP glucose pyrophosphorylase,

Waxy encodes granule bound starch synthase, 745 Amylose Extender encodes an isoform of branching enzyme,

Dull alters expression of an isoforms of soluble starch synthase and branching enzyme,

Sugary alters expression and activities of soluble starch synthases and debranching enzyme. 750

Using known mutants and the gene-dosage crossing regimes we have examined the effects of altered gene expression on starch deposition in grain (See figures). With the bt2 mutant we see a progressive loss in measurable ADP 755 Glc pyrophosphorylase activity which correlates well with a loss in starch synthesis in the grain. The control strength exerted by this enzyme over flux to starch cannot be quantified from these data. In fact our studies indicate that this enzyme is one of the major determinants of the duration of 760 starch synthesis and may have little control over rate of starch synthesis. This mutation does not appreciably alter starch structure. When the mutations are with sugary, dull, waxy and amylose extender we now do detect changes in starch fine structure (branched chain length changes as well as changes in

765 amylose/amylopectin ratios). In these cases there is more minor control of flux to starch (except with the sugary mutant which is used to make sweet-com genotypes). In all of these cases it is the changes in ratios of the starch synthases and branching enzymes which have resulted in alterations in starch

770 fine structure. A dramatic new finding in these studies was the discovery that not only does the mutation reduce expression of key enzymes, but also it induces an overexpression of other enzymes in the pathway. Furthermore, it is only in the full mutant (mmm) genotypes where we see changes in starch fine

775 structure demonstrating that the structural changes occur only when there is an enzyme isoform loss in combination with an enzyme isoform overexpression. Whilst not wishing to be bound by this proposal, these data illustrate the means by which starch structure may be influenced by not only reducing

780 expression (eg using antisense constructs) an enzyme but also be simultaneously increasing expression (eg using sense construct).

Plant transformation vectors for use in the method of the invention may be 785 constructed using standard techniques. Since these enzymes are localized in the amyloplast compartment of the cell, the gene construct requires the presence of an amyloplast transit peptide to ensure its correct localization in the amyloplast. The transformation construct may carry the gene either in the partial sense orientation or in the antisense orientation. Expression of said gene in the plant results in a reduction 790 in expression of the enzyme by effects well known in the art as "sense cosuppression or antisense". When only a reduction in expression is needed the transit peptide is not required. However, when enzyme overexpression is required then a correct plastid targeting sequence is needed in the construct. Key enzymes required for this invention include branching enzyme and soluble and bound starch synthase.

795 Branching enzyme [1,4-α -D-glucan: 1,4-α -D-glucan 6-α -D-(l,4-α-D-glucano ) transferase] converts amylose to amylopectin, (a segment of a 1,4-α-D-glucan chain is transferred to a primary hydroxyl group in a similar glucan chain) sometimes called Q-enzyme. Soluble starch synthase [ADPglucose: 1 ,4-α-D-glucan 4-α-D- glucosyltransferase] extends the chain-length of amylopectin and perhaps also

800 amylose. Bound starch synthase [ADPglucose: 1 ,4-α-D-glucan 4-α-D- glucosyltransferase] extends the chain length of amylose and perhaps also amylopectin.

For any antisense or sense-cosuppression construct only a partial cDNA clone is

805 required to be expressed in a transgenic plant. Where enzyme overexpression is required, then a full length cDNA clone is needed. The sequence of maize branching enzyme-I was investigated by Baba, T., Nishihara, M., Mizuno, K., Kawasaki, T., Shimada, H., Kobayashi, E., Ohnishi, S., Tanaka, K., and Arai, Y. (Identification, cDNA Cloning, and Gene Expression of Soluble Starch Synthase in Rice (Oryza-

810 sativa L) Immature Seeds. Plant Physiology. 103:565-573, 1993). Starch branching enzyme-II from maize endosperm was investigated by Fisher, D.K., Boyer, CD., and Hannah, L.C. (Starch Branching Enzyme-II from Maize Endosperm. Plant Physiology. 102:1045-1046, 1993). The article by Mu, C, Ham, C, Ko, Y., Singletary, G. W., Keeling, P. L. and Wasserman, B. P. shows an association of a

815 76kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm. Plant Journal 6, 151-159 (1994). The maize waxy locus for UDP-glucose starch glycosyl transferase was cloned in 1986 by Kloesgen, R.B., Gierl, A., Schwarz-Sommer, Z. and Saedler, H (Molecular analysis of the waxy locus of Zea. Mol. Gen. Genet. 203, 237-244). Recently, the sequence for the maize sugary

820 locus was observed by James, M. and Wright, A. (The Plant Journal) using transposon mutagenesis to locate the gene. The gene for any such protein is thought to be a debranching enzyme and may be used in constructs according to this invention.

825 It is believed that the chloroplast transit peptides have similar sequences (Heijne et al describe a database of chloroplast transit peptides in 1991, Plant Mol Biol Reporter, 9(2): 104- 126). Other potential transit peptides are those of ADPG pyrophosphorylase (1991, Plant Mol Biol Reporter, 9:104-126), small subunit RUBISCO, acetolactate synthase, glyceraldehyde-3P-dehydrogenase and nitrite 830 reductase. For example, the consensus sequence of the transit peptide of small subunit RUBISCO from many genotypes has the sequence:

MASSMLSSAAVATRTNPAQASM VAPFTGLKSAAFPVSRKQNLD1TSIAS NGGRVQC The co small subunit transit peptide of RUBISCO has the sequence: 835 MAPTVMMASSATATRTNPAQAS AVAPFQGLKSTASLPVARRSSRSLGN

VASNGGRIRC. The transit peptide of leaf starch synthase from com has the sequence:

MAALATSQLVATRAGLGVPDAS TFRRGAAQGLRGARASAAADTL SMRTASARAAPRHQQQARRGGR FPSLWC.

840

Example 5

Production of fertile transgenic maize plants has been done since 1990. Although a number of DNA delivery systems are

845 known, the selection is a particle bombardment. As noted above, constructs of the various maize mutant genes are available from depositories in the U.S. and Europe. Attached are a few examples of some of these constructs as shown in Figures 4a - d. Figure 4c shows a promoter, which is CaMv

850 (cauliflower mosaic virus), an Adhl, the waxy gene, the nos(nopoline), and the pat gene which is useful as a selectable marker and amp. Figure 4d is similar but shows the soluble starch syntheses first isoform gene in the construct. Figure 4a again has the same construct but shows the branching enzyme 855 first isoform. Figure 4b shows the second branching enzyme second isoform. Of course, other constructs associated with the gene mutants used in maize breeding are also available.

For purposes of this example reference is made to Figure 4c, the waxy construct.

860 The purpose of this experiment is to form an inbred that has partial down regulation of the waxy gene. If the inbred selected is already a mutant for ae, then the grain produced by crossing with a non-mutant inbred will be the grain of an intermutant. Depending on the strength of the down regulation, the female inbreds grain will resemble the mm*/mm* or the mm*/m** type of starch and grain. Clearly, the

865 transformation allows a more precise way of down regulation of the starch synthesis activity such that the alteration of the starch can be finely tuned.

To assure reasonable levels of down regulation of the waxy gene, the transformation target tissue is immature zygotic embryos, through embryogenic

870 callus can also be employed. Immature zygotic embryos from A 188 plants 12 days after pollinated with the B73 ae inbred can be selected. The medium for the callus was 6 mM L-proline, 2% (w/v) sucrose, 2 mg/1 2,4- dichlorophenoxyacetic acid (2,4- D) and 0.3% (w/v) Gelrite (Caroline Biological Supply) (pH 6.0). Callus is grown and suspension cultures were initiated.

875

A MS-based liquid medium containing 100 mg 1 myo-inositol, 2 mg 1 2,4-D, 2 mg/1 1-naphthalenacetic acid (NAA), 6 mM proline, 200 mg/1 casein hydrolysate (Difco Laboratories), 3% (w/v) sucrose, and 5% (v/v) coconut water (Difco Laboratories) (pH 6.0). Cell suspensions were maintained in this medium in 125 ml

880 Erlenmeyer flasks at 28°C in the dark on a gyrating shaker at 125 rpm. The transformation vector in Figure 4c is selected. This plasmid contains a 355- ladh-pat nos 3' selectable gene expression cassette.

885 The cell suspensions are sieved and then suspended in 5 ml of suspension medium and placed on filter paper through vacuum. The constmct was coated into particles as is know in the art. The plates were then bombarded. The cells are then transferred to a N-6 medium and after 14 days transformed cells are selected by 1 mg/1 bialaphos. The cells are then suspended in a medium containing .6% (w/v) (Sea-

890 Plaque; FMC) and held at 37°C.

Two to five weeks later, growing calli are removed and transferred to the surface of fresh selection medium. Plants were regenerated in a MS based medium having 6% sucrose lg/1 myo-inositol, 1 mg/1 NAA (34), and 0.3% (w/v) Gelrite (pH 6.0). 895 Next the embryo germination occurred in a MS media of 0.25 mg/1 NAA and 3%

(w/v) sucrose and light. Plants are grown and transferred to the greenhouse. The expression levels in the plant can then be evaluated.

The plant is bred and developed to an inbred having the mutant and the down 900 regulated pathway. Alternatively, the selected inbred can have the mutant crossed onto a transgenic after transformation to form the desired starch in the grain when the transgenic plant is employed as the female.

Example 6

905

This example illustrates the gelatinization temperature of the starch of present invention compared to other starches. The gelatinization temperatures are listed in Table 3 below. 910 Table 3

Gelatinization*

Starch Samples % Amvlose* Temperature °C

1 Native common maize 28 71

2 AMY V native 57 80

3 AMY Vπ native 73 90

4 Present Invention 21 73

5 Native aewx 25 80

6 Native wxwxwx 3 72

^♦Values rounded to a whole number

Sample 1 was a commercial product sold by American Maize-Products Company of Hammond, Indiana. The percent amylose and the gelatinization temperature for

915 Sample 1 above are mean values determined by random sampling of product. The

99% confidence level for percent 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 co starches sold by 920 American Maize-Products Company of Hammond, Indiana. The percent amylose and the gelatinization temperatures in Table 3 above are mean values determined from a random sampling of product. The 99% confidence interval for the percent amylose in AMY V and AMY VU was 53.4 to 62.5 and 65.5 to 73.8, respectively. The 99% confidence interval for the gelatinization temperature for AMY V and 925 AMY Vπ was 72.8 to 84.4 and 83.1 to 90.8, respectively. Both AMY V and AMY

Vπ were grown in native maize.

Starch Sample 4 corresponds to the Present Invention, while Sample 5 corresponds to the average values from Example 1 of U. S. Patent No. 5,009,911. 930 Sample 6 corresponds to a commercial waxy starch sold by American Maize-

Products Company. The method for determining both the percent amylose and the gelatinization temperature was:

935 The percent amylose was determined using standard calorimetric iodine procedures wherein the starch is first gelatinized with sodium hydroxide and then reacted with an iodine solution and the resulting sample measured using a spectrophotometer in a 1 cm cell at 600 nm against a blank of 2% iodine solution.

940 The DSC gelatinization temperature was measured using a scanning calorimeter manufactured by Mettler Moddle No. 300 using 30% starch solids following the procedure outlined in the owner's manual for that model.

It is readily apparent from Table 3 above that the gelatinization temperature of the 945 starch of the present invention is comparable to common com starch.

Example 7

This example illustrates the gel strength of a sol made from

950 the co starch of the present invention compared to a sol made from aewx com starch, a sol made from common com starch, and a sol made from wxwxwx starch. The results of the test are reported in Table 4 below.

955 Table 4

Sample Strength (grams) Present Invention 159.5

Common Com Starch 225.0 aewx Co Starch 55.0

Waxy Co Starch 16.0 In order to perform the gel strength test reported in Table 4 above, sols were prepared by mixing water with starch and subjecting the slurry to a rapid heat mode in the Brabender Visco-Amylograph to heat the sample to 50° C. Once 50° C was

960 reached, the instrument was set at a controlled rate of heating, 1.5°C/minute, until a temperature of 95° C was reached. The sample was then held at 95° C for 30 minutes. Next, the sample was cooled at 1.5° C to a temperature of 50° C for 30 minutes. Portions of these sols were added separately to 4 ounce jars into which a plumger was placed. The sols were then allowed to stand at ambient conditions for

965 24 hours. Gel strength was measured by determining the force needed to remove the plunger from the sol.

This example illustrates that the gel strength of a sol made in accordance with the present invention is comparable to common com starch sols.

970

Example 8

This example illustrates the difference between aewx starch, a waxy starch wherein the plant had a triple dose of the 975 waxy gene, and the starch of the present invention. All starches were obtained from maize.

All starches were tested for their rheological properties. Each starch was subjected to the same test procedure using the same method. The starch granules were pasting

980 using a Brabender Visco-Amylograph with the cooling probe down and with the 750 g cm cartridge. The starch slurry, 5.5% initial solids, was rapidly heated to 60° C in the Brabender cup, and then pasted while increasing the temperature to 95° C at 1.5° C per minute. The starch paste was held at this temperature for 20 minutes, and then immediately loaded onto the measuring geometry of the rheometer which had been

985 preheated to 70° C. A four-part rheological characterization was performed. The gel cure segment which monitored the formation of structure at .2 hertz and .2% strain (well within the linear viscoelastic region) was measured as the sample was cooled from 70° C to 25° C and held for 4 hours. Also measured was the strain sweep, which measures the rheological response of the paste or gel to increasing levels of 990 strain, also at 0.2 hertz, at 25° C.

Using this technique, distinct differences between aewx starch and the starch of the present invention were found. Figure 5 shows the results of the gel cure analysis. The starch of the present invention, while having an initial Modulus (G') lower than 995 aewx starch, more quickly formed structure or gelled as evidenced by the rapid rise in

G'. Thus the starch of the Present Invention was distinct from aewx starch in the rate of gel formation despite similar apparent iodine binding contents. Figure 6 shows the results of the strain sweep analysis only for the starch of the present invention and the aewx starch. Here the starch of the present invention showed

1000 dilatant behavior as evidenced by the increase in G' as the strain level was increased.

Under the applied levels of strain, the structure was not destroyed. In contrast, the structure of the aewx starch was rapidly destroyed when the applied strain became greater than 4%. Thus, when compared to aewx starch, the starch of the present invention formed a gel which didn't break under the applied strain of this test. In

1005 contract, aewx starch showed much less time-dependent structure formation and did

"break" or was destroyed by the strains applied in this technique.

Example 9

1010 This example illustrates preparing a thickener composition in accordance with the present invention.

The starch of the present invention is mixed with water in an amount to produce a slurry having 10% by weight starch. The sol has a short texture and a bland taste. 1015 The sol when cooked at about 90° C for 10 minutes produces a thickener composition which had better clarity than a similar thickener composition made from common co starch and a shorter texture.

Example 10

1020

This example compares the mouth feel of a gel made from the starch of the present invention to a gel made with a common starch.

1025 The common starch and starch of the present invention were pasted using a

Brabender visco-amylograph. The starch was slurried at 5.5% solids, and then heated using the rapid heat mode to 50° C. Using controlled heat of 1.5° C per minute the slurries were heated to 95° C, and then held at this temperature for 30 minutes. The final solids was 5.9%. The sample starch pastes were then poured into small jelly

1030 jars, covered with cellophane, and allowed to age 24 hours before analysis. A taste panel was then asked to rank the samples for the following attributes.

First they ranked the two for relative firmness to the touch.

Common Present Invention 5.1 5.6

1035

Next, they ranked the relative break, firmness, and clearing of this sample while being masticated.

Common Invention

Degree of clean break 3.2 8.5

Firmness 4.4 4.5

Rate of clearing 2.5 4.6 These results show that the firmness of these samples is similar. However the

1040 starch of the present invention has a much cleaner break while being masticated.

Furthermore, it tends to clear from the mouth faster than a common based starch gel. The panel all agreed that the starch of the present invention produced a gel which had a "clean" mouth feel similar to that of a gelatin or a pectin.

1045

Example 11

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

1050 The following ingredients and procedure are used:

Table 5

% by Weight

Ingredients Present Invention

44/62 Com Syrup Unmixed 56.34

Sugar, fine granular 23.98

Water 7.73

Present Invention Starch 11.80

Citric Acid 0.07

Sodium Citrate 0.08

100.00

Procedure

1055 All ingredients are mixed and then cooked to 340° F using conventional equipment such as a jet cooker. The cooked slurry is then poured into candy molds and allowed to solidify. Example 12

1060

This example illustrates making a Bavarian cream pie using starch of the present invention.

The following ingredients and procedure are used:

1065

Table 6

% by Weight

Ingredients Present Invention

Whole milk, fresh 3.5% 72.794

Sugar, fine grain 17.586

Salt, Flour 0.101

Present Invention 5.410

Banana Flavoring 0.300

Egg Yolk, fresh 3.809

100.000

Procedure

1070 All of the pie filling ingredients except for egg yolks are combined and cooked at 195° F for 3 to 5 minutes. Then the ingredients are cooled to 120°F with constant stirring. Next, egg yolks are added and the admixture well blended. This mixture is then added to a conventional pie crust and allowed

1075 to cool to room temperature before serving. Example 13

This example illustrates preparing a lemon pie filling with the

1080 starch of the present invention.

The following ingredients and procedure are used:

Table 7

% By Weight

Ingredients Present Invention

Water 62.94

Sugar 19.30

Maltodextrin 6.67

Present Invention 4.50

Com Syrup Solids 2.50

Lemon Juice 2.50

Vegetable Shortening 1.03

Salt 0.23

Citric Acid 0.20

Emulsifier 0.10

Lemon Oil (2x) 0.03

100.00

1085

Procedure

Half of the water is combined with the sugar and brought to a boil. All of the remaining ingredients are slurried together and then added to the boiling sugar and water. The

1090 temperature of this mixture is then adjusted to 200° F. and held there for two minutes. The mixture is then poured into prepared pie crusts and allowed to cool and solidify. Example 14

1095 This example illustrates making a chocolate mousse using the starch of the present invention. The formulation in Table 8 is employed to prepare a mousse mix.

Table 8

1100

%

Frodex 24-924 39.20

Sugar (Baker's) 30.75

Whiptreme 3554 (Kerry Ingredients) 12.88

Starch of Present Invention 9.80

Cocoa, Dutch Red (Gill & Duffus Products) 7.17

Leceitreme 40 (Kerry Ingredients) 0.20

Flavor as desired

100.00

Procedure

Combine ingredients to form a uniform blend._.

1105 Use

Combine 200 gms. of mousse mix with 1 cup (250 grs.) milk. Using an electric mixer, combine on low speed for 1 minute. Scrape bowl. Mix on high speed for 3 minutes, until light and fluffy. Spoon into serving dishes and refrigerate for

1110 1 hour before serving.

To prepare the mousse mix, all the ingredients are mixed. To prepare the mousse itself, 200 grams of mousse mix are combined with 250 grams of milk and combined at a low 1115 speed. Then the mixture is stirred at a high speed to make it light and fluffy and the mixture is refrigerated for one hour. In this way, a light, fluffy mousse is prepared.

Accordingly, the present invention has been described with some degree of 1120 particularity directed to the preferred embodiment of the present invention. It should be appreciated, though, that the present invention is defined by the following claims construed in light of the prior art so that modifications or changes may be made to the preferred embodiment of the present invention without departing from the inventive concepts contained herein. 1125

Claims

We claim:

1. A plant comprising:

1130 genomic material which includes genes which give incomplete reduction of the activity of at least two specific isoforms of the enzymes in the starch synthesis pathway of said plant.

2. A plant according to Claim 1 which forms starch which has different branching 1135 structure than the starch formed by a similar plant as described in Claim 1 which comprises genomic material which does not form isoforms of the enzymes in the starch synthesis pathway of the plant.

3. A plant according to Claim 2 in which said starch forms in grain. 1140

4. Grain produced by a plant in which the genotype of the grain is mm*/n**, where m = first mutant n=second mutant and * equals wild type.

5. A starch producing plant comprising:

1145 genomic material which includes genes which give incomplete reduction of the activity of at least two specific isoform enzymes in the starch synthesis pathway of said plant whereby said plant produces substantially more starch than said plant would produce if said genes gave complete reduction of the activity of the same two specific isoform enzymes within the starch pathway.

1150

Grain having an endosperm genotype selected from the group consisting of: wxwxWx/AeAeae, aeaeAe/WxWxwx, wxwxWx/SuSusu, susuSu/wxWxwx, aeaeAe/SuSusu, susuSu/AeAeae, wxwxWx/DuDudu, aeaeAe/DuDudu, susuSu/DuDu du.

1155 7. Grain having an endosperm genotype comprising two doses of a first mutant allele of a gene which affects starch structure and one dose of a second mutant allele of a second gene which affects starch structure, said genes can be selected from waxy, amylose extender, dull, homy, sugary, shrunken, brittle, floury,

1160 opaque.

8. The starch extracted from a grain according to claim 7 having a genotype of wxwxWx/AeAeae.

1165 9. The starch extracted from a grain according to claim 7 having a genotype of Aeaeae/Wx Wx wx .

10. A plant having a diploid genotype of aa/BB and a triploid endosperm genotype of aaA/BBb which has starch where a is a recessive mutant gene and A is the 1170 wild type gene, and b is a recessive mutant gene and B is the wild type gene and the starch is altered from the normal starch, a and b can be selected from ae, du, wx, sh, bt, h, su, fl, op and B and A can be selected from Ae, Du, Wx, Sh, Bt, H, Su, Fl, Op.

1175 11. A plant having 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 the wild type gene, and b is a recessive mutant gene and B is the wild type gene, and c is a recessive mutant gene and C is the wild type gene and the starch is altered from the normal starch where a and b

1180 can be selected from ae, wx, sh, bt, h, su, fl, op, du and B and A can be selected from Ae, Wx, Sh, Bt, H, Su, Fl, Op, Du.

12. A method of producing grain with altered starch qualities which includes the steps of: 1185 a) planting a parent which is capable of flowering and has complete reduction of at least one specific isoform enzyme in the starch synthesis pathway; b) planting a second parent having a complete reduction of at least one other specific isoform enzyme in the starch synthesis pathway;

1190 c) eliminating said first parent's capability to produce pollen; d) pollinating said flowering first parent with said second parent's pollen; and e) harvesting the grain produced by said first parent.

1195 13. The method according to claim 12 including the step of extracting said altered starch from said grain.

14. The starch extracted from the grain according to claim 4 wherein a and b designate the same mutant and B and A designate the same wild type.

1200

15. The starch extracted from grain having at least four doses of mutant and two doses of wild type, such that the genotype has wild type on each side.

16. The starch extracted from grain having at least three doses of mutant and three 1205 doses of wild type, such that the genotype has mutation on each side.

17. A single mutant male sterile plant where the mutant is selected from ae, wx, sh, bt, h, su, fl, op, du.

1210

18. A sol comprising water and an effective amount of a starch extracted from a starch bearing plant having a waxy, waxy, amylose extender genotype. 19. The sol of claim 18 wherein the starch is present in an amount of about 1 % to 1215 about 20% by weight.

20. The sol of claim 18 wherein the plant is maize.

21. The sol of claim 18 wherein the starch is cold water soluble. 1220

22. The sol of claim 18 wherein said starch is in granular form.

23. The sol of claim 21 wherein said starch is extracted from maize.

1225 24. A foodstuff comprising a foodstubb and having as an essential ingredient therein an effective amount of a starch extracted from a starch bearing plant having a waxy, waxy, amylose extender genotype.

25. The foodstuff of claim 24 wherein said starch is present in an amount of about 1230 0.1 % to about 10% by weight foodstuff.

26. The foodstuff of claim 24 wherein said starch is extracted from maize.

27. A method for making a sol containing starch comprising the steps of: 1235 forming a slurry comprising water and an effective amount of a starch extracted from a starch bearing plant having waxy, waxy, amylose genotype; and

cooking said starch to gelatinize said starch. 1240

28. The method of claim 27 wherein said effective amount is about 1 % to about 20% by weight slurry. 29. The method of claim 27 wherein said starch is extracted from maize. 1245

30. A method for thickening a foodstuff comprising the steps of:

combining with a foodstuff an effective amount of a starch extracted from a starch bearing plant having a waxy, waxy, amylose extender genotype; and 1250 cooking said foodstuff to thicken said foodstuff.

31. The method of claim 30 wherein said starch is extracted from maize.

1255 32. The method of claim 30 wherein said starch is present in an amount of from about 0.1 % to about 10% by weight of said foodstuff.

33. An improved method for making a foodstuff which contains a gelatin said improvement comprising replacing at least a portion of the gelatin in said

1260 foodstuff with the sol of claim 18.

34. An improved method for making a foodstuff which contains a natural gum, said improvement comprising replacing at least a portion of the natural gum with the sol of claim 18.

1265

35. The method of claim 34 wherein said foodstuff is a gum candy, a gelled dessert, a glaze, or a spread.