ZA200006018B - Gene combinations that alter the quality and functionality of soybean oil. - Google Patents

Description

TITLE

NOVEL GENE COMBINATIONS THAT ALTER THE

QUALITY AND FUNCTIONALITY OF SOYBEAN OIL

FIELD OF THE INVENTION

This invention relates to novel gene combinations that result in novel lipid composition of soybean seeds and the oil extracted from such soybean seeds. These novel soybean gene combinations provide an alternative to the production of more expensive oils and to the chemical modification of oil to provide specific functional qualities and health attributes.

BACKGROUND OF THE INVENTION

Plant lipids have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrial properties of the lipid. Many of these structures result either directly or indirectly from metabolic processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids. and either domestication of exotic plant species or modification of agronomically adapted species is usually required to economically produce large amounts of the desired lipid.

Plant lipids find their major use as edible oils in the form of triacylglycerols. The specific performance and health attributes of edible oils are determined largely by their fatty acid composition. Most vegetable oils derived from commercial plant varieties are composed primarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids. Palmitic and stearic acids are, respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic, linoleic. and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two. and three double bonds, respectively. Oleic acid is referred {0 as a mono-unsaturated fatty acid, while linoleic and linolenic acids are referred to as poly- unsaturated fatty acids. The relative amounts of saturated and unsaturated fatty acids in commonly used, edible vegetable oils are summarized below (Table 1).

TABLE 1

Percentages of Saturated and Unsaturated Fatty

Acids in the Oils of Selected Oil Crops

Saturated Mono-unsaturated Poly-unsaturated

Canola 6% 58% 36%

Soybean 15% 24% 61%

Corn 13% 25% 62%

Peanut 18% 48% 34%

Safflower 9% 13% 78%

Sunflower 9% 41% 51%

Cotton 30% 19% 51%

Many recent research efforts have examined the role that saturated and unsaturated fatty acids play in reducing the risk of coronary heart disease. In the past, it was believed that mono-unsaturates, in contrast to saturates and poly-unsaturates, had no effect on serum cholesterol and coronary heart disease risk. Several recent human clinical studies suggest that diets high in mono-unsaturated fat and low in saturated fat may reduce the “bad” (low- density lipoprotein) cholesterol while maintaining the “good” (high-density lipoprotein) cholesterol (Mattson et al. (1985) Journal of Lipid Research 26:194-202).

A vegetable oil low in total saturates and high in mono-unsaturates would provide significant health benefits to consumers as well as economic benefits to oil processors. As an example, canola oil is considered a very healthy oil. However, in use, the high level of poly-unsaturated fatty acids in canola oil renders the oil unstable, easily oxidized, and susceptible to development of disagreeable odors and flavors (Gailliard (1980) in The

Biochemistry of Plants Vol. 4, pp. 85-116, Stumpf, P. K., ed., Academic Press, New York).

The levels of poly-unsaturates may be reduced by hydrogenation, but the expense of this process and the concomitant production of nutritionally questionable trans isomers of the remaining unsaturated fatty acids reduces the overall desirability of the hydrogenated oil (Mensink et al. (1990) New England J. Medicine N323: 439-445). Similar problems exist with soybean oil and as noted in Table 1, commodity soybean oil typically contains over twice the saturated fat content of canola oil.

Mutation-breeding programs have met with some success in altering the levels of poly-unsaturated fatty acid levels found in the edible oils of agronomic species. Examples of commercially grown varieties are high (85%) oleic sunflower and low (2%) linolenic flax (Knowles (1980) in World Conference on Biotechnology for the Fats and Oils Industry

Proceedings, Applewhite, T. H., ed., American Oil Chemists’ Society, pp. 35-38). Similar commercial progress with the other plants shown in Table 1 has been elusive, largely due to the difficult nature of the procedure and the pleiotropic effects of the mutational regime on plant hardiness, yield potential and the environmental instability of the low poly-unsaturate trait. Above all, the inability to consistently produce an oil of defined composition from season to season and in differing locations has made the commercial production of low poly- unsaturate soybean oil infeasible.

The discovery of a method for altering the expression of the enzymes responsible for introduction of the second (international patent publication WO 94/11516) and third (international patent publication WO 93/11245) double bonds into soybean seed storage lipid in a directed manner has allowed the production of soybeans with a high mono- unsaturated, very low polyunsaturated fatty acid content and especially a very low linolenic acid content. The genetic combination of these two transgene profiles described in the instant invention leads to a soybean line with minimal poly-unsaturates and high mono- unsaturates and extreme environmental stability of the seed fatty acid profile.

WO ‘99/58689 PCT/US99/09869

S oybeans with decreased levels of saturated fatty acids have been described resulting from mu tation breeding (Erickson, E. A. et al., (1994) J. Hered. 79:465-468; Schnebly, S. R. et al. (19294) Crop Sci. 34:829-833; and Fehr, W. R. et al. (1991) Crop Sci. 31:88-89) and transgen-ic modification (U.S. Patent No. 5,530,1 86). The demonstration of the combination of these ~two traits in a single soybean line in this invention brings the health benefits of a high momo-unsaturate, low saturate oil to soybean production.

DVhile oils with low levels of saturated fatty acids are desirable from the standpoint of providinsg a healthy diet, fats that are solid at room temperature are required for their function_al properties in some foods. Such applications include the production of non-dairy margarires and spreads, and various applications in confections and in baking. Many animal and dairy fats provide the necessary physical properties, but they also contain both choleste-rol and the cholesterogenic medium chain fatty acids. An ideal triglyceride for solid fat applii cations should contain a predominance of the very high melting, long chain fatty acid stezric acid and a balance of mono-unsaturated fatty acid with very little polyuns aturated fat. Natural plant solid fat fractions typically have a triacylglyceride structuree with saturated fatty acids occupying the sn-/ and sn-3 positions of the triglycerides and an Lansaturated fatty acid at the sn-2 position. This overall fatty acid composition and triglyce-ride structure confers an optimal solid fat crystal structure and a maximum melting point w- ith minimal saturated fatty acid content. “The natural fat prototype for this high melting temperature vegetable fat is cocoa butter. The fatty acid composition of cocoa butter is 26% palmitic (16:0), 34% stearic (18:0), 35% oleic (18:1), and 3% linoleic(18:2) acids. This fatty acid profile gives cocoa butter a_ melting point range of from 25° to 36°C depending upon its precise crystal structure.

The higeh price and fluctuating supply of cocoa butter has led to several processes for the production of cocoa butter substitutes and margarine stocks by fractionating other oils with relative=ly high 18:0 content or catalytic hydrogenation of high polyunsaturated oils followed by fractionation of the product.

Oilseeds capable of producing directly high stearate, low polyunsaturate oils would be advantageous in that both the cost of hydrogenation and the undesirable side products of hydrogenation, trans monounsaturated fatty acids, could be avoided. In addition, the fractioration process could be made more cost effective or possibly eliminated if the melting temper: ature range of the vegetable fat produced were high enough.

Oil biosynthesis in plants has been fairly well-studied [see Harwood (1989) in

Critica’1 Reviews in Plant Sciences, Vol. 8 (1):1-43]. The biosyntheses of palmitic, stearic and oleic acids occur in the plastids by the interplay of three key enzymes of the “ACP track”: palmitoyl-ACP elongase, stearoyl-ACP desaturase and the acyl-ACP thioesterases.

Of these three enzyme types. the acyl-ACP thioesterases function to remove the acyl chain £rom the carrier protein (ACP) and thus away from the metabolic pathway.

Oleoyl_-ACP thioesterase catalyzes the hydrolysis of oleoyl-ACP thioesters at relatively high rates, although it also catalyzes the hydrolysis of palmitoyl-ACP and stezaroyl-ACP at much lower rates. This multiple activity leads to substrate competition between enzymes, and it is the competitions of acyl-ACP thioesterase and palmitoyl-ACP elongase for the same substrate and of acyl-ACP thioesterase and stearoyl-ACP desaturase for ®he same substrate that contributes to the production of the palmitic and stearic acids found in the triacylglycerides of vegetable oils.

Once removed from the ACP track, fatty acids are exported to the cytoplasm and there they are used to synthesize acyl-coenzyme A. These acyl-CoAs are the acyl donors for at least three different glycerol acylating enzymes (glycerol-3-P acyltran =sferase, 1-acyl- glycerol-3-P acyltransferase and diacylglycerol acyltransferase) which irmcorporate the acyl moieties into triacylglycerides during oil biosynthesis.

These acyltransferases show a strong, but not absolute, preferenc se for incorporating saturated fatty acids at sn-/ and sn-3 positions and monounsaturated fatt>y acids at the sn-2 position of the triglyceride. Thus, altering the fatty acid composition of -the acyl pool will drive a corresponding change in the fatty acid composition of the oil. Fuarthermore, there is experimental evidence that because of this specificity, plants can producee cocoa butter substitutes or other specialty fats if there is the correct composition of fa-tty acids available in the substrate pool for the acyltransferases [Bafor et al., (1990) JAOCS 67:217-225].

Based on the above discussion, one approach to changing the lev els of paimitic, stearic and oleic acids in vegetable oils is to alter their levels in the cytoplasmic acyl-CoA pool used for oil biosynthesis.

Manipulation of stearate levels has been described (Knutzon, D.S. et al., (1992) Proc.

Natl Acad. Sci. USA 89(7): 2624-2628). Seeds from both B. campestris and B. napus plants were produced by antisense expression of a cDNA encoding the B. campestris stearoyl-ACP desaturase, the enzyme responsible for introducing the first double bond into 18 carbon fatty acids in plants, using a seed specific promoter region. These seeds prodwced oils high in stearic acid, that also contained elevated levels of linolenic acid (18:3), vwhen compared to seeds from unmodified plants from the same species. Elevated levels of stearic acid have been obtained in soybean by a similar underexpression of stearoyl-ACP desaturase (U.S.

Patent No. 5,443,974) and in canola by over expression of an acyl-ACP "thioesterase (U.S.

Patent No. 5,530,186). Mutation breeding has also produced soybean limes with elevated levels of stearic acid in their seed oils (Graef, G. L. et al., (1985) JAOCSs 62:773-775;

Hammond, E. G. and W. R. Fehr, (1983) Crop Sci. 23:192-193).

Poly-unsaturated fatty acids contribute to the low melting point Of liquid vegetable oils. In high saturate oils their presence is a detriment in that they decrezase the melting point, and therefore even higher levels of undesirable saturated fatty acicd are required to achieve a plastic fat at room temperature. Additionally, when used in baaking and confectionery applications high levels of poly-unsaturates leads to oxidamtive instability as described above for liquid oils. Thus for maximum utility a high saturatte fat produced in soybean should contain saturated fatty acids, mono-unsaturated fatty acid and as little poly- unsaturated fatty acid as possible. Gene combinations discovered in this invention provide novel fatty acid profiles in soybean which meet these criteria.

SUMMARY OF THE INVENTION

The present invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of greater than 21%, a C18:1 content of greater than 60% and a combined C18:2 and C18:3 content of less than 7%.

In a second embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a C16:0 content of greater than 10%, a combined C16:0 and C18:0 content of greater than 30%, a C18:1 content of greater than 55% and a combined C18:2 and C18:3 content of less than 7%.

In a third embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of greater than 42% wherein said plant is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:0 content comprises at least 10% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a nucleic acid fragment encoding an oleoyl-ACP thioesterase enzyme with a second parent comprising a fasa allele for elevated seed stearic acid content; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

In a fourth embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of less than 7%, a C18:1 content of greater than 87% and a combined C18:2 and

C18:3 content of less than 6%.

In a fifth embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of less than 12%, a C18:1 content of greater than 84% and a combined C18:2 and

C18:3 content of less than 5%.

In a sixth embodiment, this invention concerns the use of high stearic, or high stearic and high oleic, oils produced from the soybean plants described herein to produce margarine and/or spread products. Also, products made from hydrogenation, fractionation, interesterification, or hydrolysis of such oils can be used to produce margarines and/or spread products whether in a blended or unblended form.

In a seventh embodiment, this invention concerns the use of the high stearic acid, or stearic and high oleic, oils produced from the soybean plants described herein to produce margarine and/or spread products from blends with other oils. Also, products made from hydrogenation, fractionation, interesterification, or hydrolysis of such oils can be used to produce margarines and/or spread products whether in a blended or unblended form.

BIOLOGICAL DEPOSIT

The following soybean seed has been deposited with the American Type Culture

Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, and bears the following designation, accession number and date of deposit.

Soybean Accession Number Date of Deposit

Soybean T1S ATCC 203033 May 14, 1998

Soybean .9216116-109 ATCC XXXXXX April 72, 1999

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detailed description, a description of the four figures, and the Sequence Descriptions which form a part of this application.

Figure 1depicts the Solid Fat Content (SFC) profiles of a high stearic, and four different high stearic and high oleic acid soybean oils compared to a soft tub vegetable oil spread. Only the 22% high stearic and high oleic (HS/HO A) performs as a spread product.

The SFC profiles of the remaining oils indicated that these oils would not perform as a spread product.

Figure 2 depicts the relative oxidative stabilities (OSI) of several unblended and blended oils. A high oleic acid content coupled with a low polyunsaturate content is necessary to achieve high oxidative stability. Blended oils such as soybean oil (SO) mixed with palm oil (PO), or high stearic (HS) oil mixed with palm oil or fully hydrogenated oil may perform well as margarine or spread products based upon SFC profile.

Figure 3 shows that blending high stearic and high oleic acid oils (HS/HO C and

HS/HO D), with hardstocks such as palm oil or fully hydrogenated soybean oils, improves their SFC profiles to conform with the characteristics of a vegetable oil spread.

Figure 4 shows that very high stearic acid oils have SFC profiles consistent with butter and hardstock oils.

The sequence descriptions summarize the Sequences Listing attached hereto. The

Sequence Listing contains one letter codes for nucleotide sequence characters and the three letter codes for amino acids as defined in the [IUPAC-IUB standards described in Nucleic

Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984), and the symbols and format used for all nucleotide and amino acid sequence data further comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825 and WIPO Standard St.25. The nucleotide sequences read from 5' to 3'.

SEQ ID NO:1 shows a 1412 base pair cDNA of an acyl-ACP thioesterase from

Brassica napus.

SEQ ID NO:2 shows the amino acid sequence of the precursor protein of a B. napus seed acyl-ACP thioesterase (the coding sequence of SEQ ID NO:1).

SEQ ID NO:3 shows the nucleotide sequence of a second acyl-ACP thioesterase cDNA from Brassica napus, corresponding to GenBank accession U17098.

SEQ ID NO:4 shows the amino acid sequence of the precursor protein of the second

B. napus seed acyl-ACP thicesterase (the coding sequence of SEQ ID NO:3).

DETAILED DESCRIPTION OF THE INVENTION

A wide variety of novel soybean genes that alter oil quality have been discovered through selection for natural variation, mutation breeding, and genetic engineering. Certain soybean lines or combinations containing those novel genes are used in this disclosure and are referred to by the “Gene Combinations or Line Name” designations described in Table 2.

TABLE 2

Typical Fatty Acid Profiles of Soybeans With Different Fatty Acid Modification Genes

Gene Combination Individual Fatty Acid Content or Line Name! (% of total seed fatty acid) Reference 16:0 18:0 18:1 18:2 18:3 wild type 12 4 25 51 7 -

T1S 16 10 19 47 7 Example 11, this application

D2T 7 3 85 1 3 WO 94/11516

D3A 10 5 32 49 3 WO 93/11245 fan 11 3 45 36 4 US 5,534,425 fap! 8 4 28 52 8 US 5,585,535 fap? 16 4 19 53 8 US 5,850,029 fap3 7 3 26 53 11 US 5,585,535 fapl x fap3 4 3 22 63 8 US 5,585,535 fasa 8 23 26 35 7 US 5,557,037

L9216116-109 9 22 15 48 5 Example 3, this application

N85-2176 11 3 42 40 4 Kuhr et al., March 26, 1987,

USDA Agric. Res. Services

HST! 9 28 15 43 5 Example 3, this application

HO2 11 3 45 37 4 Example 3, this application

HO4 11 3 45 37 4 Example 3, this application

T2T 3 2 17 65 12 WO 9606936,

Example 13, this application : 1The Gene Codes used in this Table refer to the following:

: TIS refers to a oleoyl-ACP thicesterase expression construct which is in the sense orientation and expresses a functional enzyme.

D2T refers to a delta-12 desaturase construct which is in a sense orientation, the integration of which results in a reduction of activity.

D3A refers to a delta-15 desaturase construct which is in the antisense orientation, the integration of which results in a reduction of activity. fan refers to a gene for decreased seed linolenic acid content. fap] refers to a gene for decreased seed palmitic acid content. fap? refers to a gene for elevated seed palmitic acid content. fap3 refers to a gene for decreased seed palmitic acid content. fasa refers to a gene for elevated seed stearic acid content. 1.9216116-109 refers to a line with high stearic acid content derived from pedigree (HST1*(HO2*HO4))* wild-type.

HST] refers to a high stearic acid mutant line derived from N85-2176.

HO?2 refers to a high oleic acid mutant line derived from N85-2176.

TT HOA4 refers to a high oleic acid mutant line derived from AS. :

T2T refers to a palmitoyl-ACP thioesterase construct that is in the sense orientation, the integration of which results in a reduction of activity.

In the context of this disclosure, a number of terms shall be utilized. “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory - sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene - may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. y

The expression “genetic locus”, as used herein, means the position of a.gene on a chromosome or set of chromosomes. The term “allele”, as used herein. refers to any of the alternative forms of a genetic locus. The term “expression”, as used herein, is intended to mean the production of a functional end-product. Expression or overexpression of a gene involves transcription of the gene and translation of the mRNA into precursor or mature protein. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Cosuppression” refers to the expression of a transgene which has substantial homology to an endogenous gene resulting in the suppression of expression 40 of both the ectopic and the endogenous gene. “Altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ significantly from that activity in comparable tissue (organ and of developmental type) from wild-type organisms.

The terms “environmentally stable” or “environmental stability” as used herein are used to describe a phenotype that is relatively constant, regardless of the environmental conditions in which a plant is grown. “Transformation” refers to the transfer of a nucleic acid fragment into the genome of $a host organism. resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 153:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Patent No. 4,945,050).

As used herein, “soybean” refers to the species Glycine max, Glycine soja, or any species that is sexually cross compatible with Glycine max. A “line” is a group of plants of similar parentage that display little or no genetic variation between individuals for a least one trait. Such lines may be created by one or more generations of self-pollination and selection, or vegetative propagation from a single parent including by tissue or cell culture techniques.

An “agronomically elite line” or “elite line” refers to a line with desirable agronomic performance that may or may not be used commercially. A “variety”, “cultivar”, “elite variety”, or “elite cultivar” refers to an agronomically superior elite line that has been : extensively tested and is or was being used for commercial soybean production. “Mutation” : refers to a detectable and heritable genetic change (either spontaneous or induced) not caused by segregation or genetic recombination. “Mutant” refers to an individual, or lineage of individuals, possessing a mutation. As used herein, an “F1 population” is the progeny resulting from cross-pollinating one line with another line. The format used herein to depict such a cross-pollination is “female parent*male parent”. An “F2 population” is the progeny } of the self-pollinated F1 plants. An “F2-derived line” or “F2 line” is a line resulting from the self-pollination of an individual F2 plant. An F2-derived line can be propagated through ) subsequent generations (F3, F4, FS etc.) by repeated self-pollination and bulking of seed from plants of said F2-derived line. A “segregating population” is a population of plants resulting from a cross that is at a stage of inbreeding of F2 or later.

The term “mature seed(s)” as used herein refers to a soybean that is no longer green that has, or did have, a moisture content less than 20%, and preferably less than 12%.

The term “fat product” as used herein refers to vegetable oils either in a natural (non- hydrogenated and non-chemically modified) form, or in a hydrogenated and/or chemically modified form, or fractions derived therefrom, either in a natural (non-hydrogenated and non-chemically modified) form, or in a hydrogenated and/or chemically modified form.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T.

Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold

Spring Harbor, 1989 (hereinafter “Maniatis™).

The present invention concerns soybean lines with novel fatty acid compositions.

In one embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of greater than 21%, a C18:1 content of greater than 60% and a combined C18:2 and

C18:3 content of less than 7%.

S A soybean plant having such a fatty acid profile is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a soybean nucleic acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising either an appropriate transgene, or a mutation such as the

Tr fasa allele, that confers an elevated seed stearic acid content; SER a (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

Also of interest are seeds obtained from such a plant, oil obtained from these seeds, products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil and by-products made during the production of this oil. In addition, this invention also concerns the use of such oils, and the use of products made from the hydrogenation, fractionation, interesterification, or hydrolysis of such oils, to make a margarine or spread ‘product whether in a blended or unblended form.

In a second embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a C16:0 content of greater ‘than 10%, a combined C16:0 and C18:0 content of greater than 30%, a C18:1 content of greater than 55% and a combined C18:2 and C18:3 content of less than 7%.

A soybean plant having such a fatty acid profile is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C1 8:1 content comprises at least 80% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a soybean nucleic acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising chimeric transgenes that increase the content of both palmitic and stearic acid in the seeds, or an alternative wherein the second parent comprises mutations such as fasa for elevated seed stearic acid content and fap? for elevated seed palmitic acid; : (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and

(c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

Also of interest are seeds obtained from such a plant, oil obtained from these seeds, products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil and by-products made during the production of this oil. In addition, this invention also concerns the use of such oils and the use of products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oils to make a margarine or spread product whether in a blended or unblended form.

In a third embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of greater than 42% wherein said plant is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:0 content comprises at least 10% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a nucleic acid fragment encoding an oleoyl-ACP thioesterase enzyme with a second parent comprising either an appropriate transgene, or a mutation such as the fasa allele, that confers an elevated seed stearic acid content; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, - growing and producing a segregating population of soybean plants through one or more . 20 cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

Also of interest are seeds obtained from such a plant, oil obtained from these seeds, products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil and by-products made during the production of this oil. In addition, this invention also concerns the use of such oils, products made from such oils, and blended products made from such oils that can be used to make a margarine or spread product.

In a fourth embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of less than 7% , a C18:1 content of greater than 87% and a combined C18:2 and

C18:3 content of less than 6%.

A soybean plant having this fatty acid profile is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a soybean nucleic acid fragment encoding a delta-12 desaturase enzyme with (i) a second parent comprising both a fap! allele for decreased seed palmitic acid content and a fap3 allele for decreased seed palmitic acid content, or (ii) the second parent comprises at least one transgene copy of a soybean nucleic acid fragment encoding a plant acyl-ACP thioesterase for decreased seed palmitic acid content wherein said thioesterase has a preference of at least two-fold for palmitoyl-ACP over either stearoyl-ACP or oleoyl-ACP; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

Also of interest are seeds obtained from such a plant, oil obtained from these seeds, products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil and by-products made during the production of this oil.

In a fifth embodiment, this invention concerns a soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of less than 12%, a C18:1 content of greater than 84% and a combined C18:2 and

C18:3 content of less than 5%.

A soybean plant having this fatty acid profile is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a soybean nucleic acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising either a mutation such as the fan allele for decreased seed linolenic acid content, or at least one transgene copy of a soybean nucleic acid fragment encoding a delta-15 desaturase enzyme and further wherein the C18:3 content comprises less than 4% of the total seed fatty acid, (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, - growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

Also of interest are seeds obtained from such a plant, oil obtained from these seeds, products made from the hydrogenation, fractionation, interesterification or hydrolysis of such oil and by-products made during the production of this oil.

The general method used to assemble the specific genotypes responsible for the novel seed fatty acid profiles described herein is as follows: 1) Parental lines chosen for their seed fatty acid profile were cross-pollinated by manual transfer of pollen from the flowers of one parent plant to the emasculated flowers of the second parent plant. 2) Mature seeds from the cross pollinated flowers were planted to produce Fl plants which were allowed to self pollinate to produce an F2 population of seeds carrying varying doses of genes involved in producing the seed fatty acid phenotype of the two parents. 3) A small chip of the mature seed was removed from a portion of the cotyledon which is not in direct contact with the embryonic axis. The relative content of the

S five main fatty acids comprising the seed lipids was determined by gas liquid chromatography as described in WO 93/11245, and the remaining portions of seeds chosen on the basis of the fatty acid profile of the analyzed chip were planted. The chosen F2 plants were allowed to self pollinate and produce F3 seeds. The above analytical procedure was repeated on single seeds from this generation and on the bulk seed lot from this generation.

As an alternative, the F2 seed population can be grown and self pollinated without prior selection and bulk seed from individual plants in the resulting F2 plants analyzed to select F2:3 families on the basis of single plant bulk seed fatty acid analysis.

Once identified lines with desired fatty acid profiles can be advanced as self pollinated populations to maintain and test the seed fatty acid profile.

To produce lines with concomitantly high levels of oleic acid and low levels of poly- unsaturated fatty acids, a delta-12 desaturase down-regulated transgenic line described in

WO 97/40698 was chosen as one parent. To produce soybean lines low in poly-unsaturates but high in saturated fatty acids a soybean line that contained a mutation resulting in high : levels of stearic acid was chosen as the second parent. An example of such a line can be : 20 found in U.S. Patent No. 5,557,037, the disclosure of which is hereby incorporated by reference. That patent discloses the soybean line A6 which carries a mutation at a locus : designated as the fasa locus.

To produce lines with concomitantly high levels of oleic acid and very low levels of saturated fatty acids, the delta-12 desaturase down-regulated line was chosen as one parent, and a line with very low levels of palmitic acid was chosen as the second parent. One approach was to use a second parent containing a combination of genes. such a line can be found in U.S. Patent No. 5,585,535. the disclosure of which is hereby incorporated by reference. Another approach is to use the line disclosed in WO 96/06936 as the second parent wherein a palmitoyl-ACP thioesterase construct is disclosed that is in the sense orientation, the integration of which results in a reduction of activity.

To further decrease the level of the poly-unsaturated fatty acid linolenic acid and to enhance the environmental stability of the low poly-unsaturated fatty acid phenotype the delta-12 desaturase down regulated transgenic line was chosen as one parent and either a transgenic line with down regulated expression of the delta-15 desaturase or 2 mutant line with a similar linolenic acid phenotype was chosen as the second parent. An example of a mutant line with a linolenic acid phenotype similar to that produced by down regulation of the delta-15 desaturase may be found in U.S. Patent No. 5,534,425, the disclosure of which is hereby incorporated by reference.

Very high levels of saturated fatty acids in soybean seeds were produced by the combination of a mutant line producing seeds with high levels of stearic acid with a transgenic line carrying a chimeric gene for seed specific over-expression of a plant acyl-ACP thioesterase.

Methods for the extraction and processing of soybean seeds to produce soybean oil and meal are well known throughout the soybean processing industry. In general, soybean oil is produced using a series of steps which accomplish the extraction and purification of an edible oil product from the oil bearing seed. Soybean oils and soybean byproducts are produced using the generalized steps shown in the diagram below. oo Process Impurities Removed /

Byproducts Obtained

Soybean Seed

Oil Extraction —————® Meal \ _— I

Degumming Lecithin

Alkali or Physical Refining ——®= Gums, Free Fatty Acids, Pigments

Water Washing —————® Soap : Bleaching —— = (Color, Soap, Metal (Hydrogenation) (Winterization) ——————® Stearine

Deodorization ——————® FFA, Tocopherols, Sterols, Volatiles

Oil Products

Soybean seeds are cleaned, tempered, dehulled, and flaked which i-ncreases the efficiency of oil extraction. Oil extraction is usually accomplished by solvent (hexane) extraction but can also be achieved by a combination of physical pressure and/or solvent extraction. The resulting oil is called crude oil. The crude oil may be deg~ummed by hydrating phospholipids and other polar and neutral lipid complexes that facilitate their separation from the nonhydrating, triglyceride fraction (soybean oil). The resulting lecithin gums may be further processed to make commercially important lecithin products used in a variety of food and industrial products as emulsification and release (antis ticking) agents.

Degummed oil may be further refined for the removal of impurities; primarily free fatty acids, pigments, and residual gums. Refining is accomplished by the addi tion of caustic which reacts with free fatty acid to form soap and hydrates phosphatides aand proteins in the crude oil. Water is used to wash out traces of soap formed during refining. The soapstock byproduct may be used directly in animal feeds or acidulated to recover tie free fatty acids.

Color is removed through adsorption with a bleaching earth which removees most of the chlorophyll and carotenoid compounds. The refined oil can be hydrogenated resulting in fats with various melting properties and textures. Winterization (fractionation) may be used to remove stearine from the hydrogenated oil through crystallization under carefully controlled cooling conditions. Deodorization which is principally steam distillation under vacuum, is the last step and is designed to remove compounds which impart odor or flavor to the oil.

Other valuable by products such as tocopherols and sterols may be remov~ed during the deodorization process. Deodorized distillate containing these byproducts may be sold for production of natural vitamin E and other high value pharmaceutical products. Refined, bleached, (hydrogenated, fractionated) and deodorized oils and fats may toe packaged and sold directly or further processed into more specialized products. A more detailed reference to soybean seed processing, soybean oil production and byproduct utiliza tion can be found in

Erickson, 1995, Practical Handbook of Soybean Processing and Utilization. The American

Oil Chemists’ Society and United Soybean Board.

Hydrogenation is a chemical reaction in which hydrogen is added to the unsaturated fatty acid double bonds with the aid of a catalyst such as nickel. High olesic soybean oil contains unsaturated oleic, linoleic, and linolenic fatty acids and each of ®hese can be hydrogenated. Hydrogenation has two primary effects. First, the oxidative stability of the oil is increased as a result of the reduction of the unsaturated fatty acid content. Second, the physical properties of the oil are changed because the fatty acid modifica tions increase the melting point resulting in a semi-liquid or solid fat at room temperature.

There are many variables that affect the hydrogenation reaction which in turn alter the composition of the final product. Operating conditions including pre ssure, temperature, catalyst type and concentration, agitation and reactor design are among thine more important parameters which can be controlled. Selective hydrogenation conditions can be used to hydrogenate the more unsaturated fatty acids in preference to the less unssaturated ones. Very

Wa 99/58689 PCT/US99/09869 light owr brush hydrogenation is often employed to increase stability of liquid oils. Further hydrogenation converts a liquid oil to a physically solid fat. The degree of hydrogenation depeneds on the desired performance and melting characteristics designed for the particular end product. Liquid shortenings, used in the manufacture of baking products, solid fats and shorte nings used for commercial frying and roasting operations, and base stocks for margaurine manufacture are among the myriad of possible oil and fat products achieved througzh hydrogenation. A more detailed description of hydrogenation and hydrogenated produacts can be found in Patterson, H. B. W., 1994, Hydrogenation of Fats and Oils:

Theory and Practice. The American Oil Chemists’ Society.

Hydrogenated oils have also become controversial due to the presence of trans fatty

Tr acid issomers that result from the hydrogenation process. Ingestion of large amounts of trans: isome=rs has been linked with detrimental health effects including increased ratios of low density to high density lipoproteins in the blood plasma and increased risk of coronary heart disease. It would be advantageous to produce foods that currently use hydrogenated oils in a form what would be free of trans fatty acids. The term “substantially free of trans fatty acids’ * as used herein means a non-health threatening level of trans fatty acids. For example, such a level can range from 0.1% (i.e., an amount which cannot be reliably detected by currer1t methods for assessing trans fatty acid levels) to an upper limit which does not pose a health risk. In the near future, the Federal government is expected to place an upper limit on the levels of trans fatty acid isomers that can be present in foods and have the designation “tran=s fatty acid free”. It is believed that all of the oils, margarines and spread products of the in_vention are expected to conform to whatever limits are imposed by governmental autho rities. 5

The limit of detection for trans isomers of fatty acids in oils is around 0.1%. (The gas : chromatography method for detecting trans fatty acids in oils is outlined in AOCS Ce 1C-89)). Reports of “low trans isomer oils” produced by modifications of the hydrogenation method can achieve levels of 5-20% (w/w), but usually at the cost of high saturated fatty acid levels (Allen, D. A. (1998) Lipid Technology, 10(2), 29-33). It is believed that the oils, fat products, and blended fat products, that are wholly or partially non-hydrogenated and non- chem ically modified, in the instant invention, should be substantially free of trans fatty acids, i.e., they should achieve trans fatty acid concentrations of below 20% (w/w), preferably belovav 10%, more preferably below 5%, even more preferably below 3%, and again more prefe rably below 1%, and most preferably below 0.5% of the oil.

Interesterification refers to the exchange of the fatty acyl moiety between an ester and an ac-id (acidolysis), an ester and an alcohol (alcoholysis) or an ester and ester (transesteri- fication). Interesterification reactions are achieved using chemical or enzymatic processes.

Random or directed transesterification processes rearrange the fatty acids on the triglyceride mole .cule without changing the fatty acid composition. The modified triglyceride structure may result in a fat with altered physical properties. Directed interesterfication reactions using lipases are becoming of increasing interest for high value specialty products like cocoa butter substitutes. Products being commercially produced using interesterification reactions include but are not limited to shortenings, margarines, cocoa butter substitutes and structured lipids containing medium chain fatty acids and polyunsaturated fatty acids. Interesteri- fication is further discussed in Hui, Y. H., 1996, Baileys Industrial Oil and Fat Products,

Volume 4, John Wiley & Sons.

Fatty acids and fatty acid methyl esters are two of the more important oleochemicals derived from vegetables oils. Fatty acids are used for the production of many products such as soaps, medium chain triglycerides, polyol esters, alkanolamides, etc. Vegetable oils can be hydrolyzed or split into their corresponding fatty acids and glycerine. Fatty acids produced from various fat splitting processes may be used crude or more often are purified into fractions or individual fatty acids by distillation and fractionation. Purified fatty acids and fractions thereof are converted into a wide variety of oleochemicals, such as dimer and trimer acids, diacids, alcohols, amines, amides, and esters. Fatty acid methyl esters are increasingly replacing fatty acids as starting materials for many oleochemicals such as fatty alcohols, alkanolamides, a-sulfonated methyl esters, diesel oil components, etc. Glycerine is also obtained by the cleavage of triglycerides using splitting or hydrolysis of vegetable oils.

Further references on the commercial use of fatty acids and oleochemicals may be found in

Erickson, D. R., 1995, Practical Handbook of Soybean Processing and Utilization, The

American Oil Chemists’ Society, and United Soybean Board; Pryde, E.H., 1979, Fatty

Acids, The American Oil Chemists’ Society; and Hui, Y.H., 1996, Bailey’s Industrial Oil and Fat Products, Volume 4, John Wiley & Sons.

Those skilled in the art will appreciate that products obtained from the hydrogenation, fractionation, interesterification or hydrolysis of the oils described in the instant invention can be used to make a margarine or spread product. Furthermore, blended products can be made as described below utilizing fractionated or unfractionated oils of the invention, either in their natural state or modified by hydrogenation, fractionation, interesterification and/or hydrolysis, in combination with other ingredients to produce a margarine or spread product.

Margarine is a flavored food product, commonly used as a dairy butter substitute for table or cooking purposes such as frying, baking, etc. The composition of margarine is typically 80% fat, made by blending selected fats and oils with other ingredients and fortified with Vitamin A. Examples of ingredients that can be used to make a blended fat include. but are not limited to. at least one component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, partially hydrogenated soybean oil, partially hydrogenated cottonseed oil, partially hydrogenated palm oil, soybean oil, corn oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil or mixtures thereof.

The process of blending, and the ultimate product, can be varied for different applications. All products containing less than 80% fat require labeling as “spreads”. The terms “spread” or “spread products” are used interchangeably herein. Margarine and spreads may contain one or more aqueous-phase ingredients, as well as other optional ingredients with specific functions. There are over 10 different types of margarine and spreads produced today including regular, whipped, soft-tub, liquid, diet, spreads, no-fat, restaurant, bakers, and specialty types that are all packaged as different products.

In the early 1950’s almost all consumer margarine was a stick variety. Sales and per capita consumption rose and surpassed that of butter in 1957. Sales increases stimulated new technological progress and new product development. Spreadable margarine, oo polyunsaturated margarine, and low—fat products were developed to satisfy the desire for - : - convenience and nutritional awareness, and weight consciousness of the consumer.

The first major departure from the past was the introduction of soft margarine spreadable from the refrigerator and packaged in tubs. This soft, full-fat product captured one-fourth of the margarine market by 1973. In recent years, stick table spreads account for slightly less than half of the total table spread market. The most significant recent trend is away from margarine (80%) fat to spreads containing lower fat levels. Originally introduced at 60% fat, since 1980 spread containing from 75% to less than 5% fat have appeared in the marketplace. The products are sold in stick, liquid, and soft whipped forms. Low calorie, soft and stick spreads containing 40-75% fat are usually formulated from the same oil blends as those used for the manufacture of soft and stick margarines, respectively. A detailed : description of margarine and spread manufacturing practices and product characteristics can be found in Bailey’s Industrial Oil and Fat Products, Fifth Edition, Volume 3, Y.H. Hui, Ed,, -

John Wiley & Sons, Inc, New York, 1996, pp 65-114; and Bailey's Industrial Oil and Fat

Products, Fifth Edition, Volume 4. Y.H. Hui, Ed,, John Wiley & Sons, Inc, New York, 1996, pp 491-568.

Consumer margarines are formulated by blending two or more oil basestocks with different degrees of hardness. This permits the margarines to be spreadable directly out of the refrigerator and to maintain a solid consistency at room temperature. The properties of a good performing margarine can be profiled by measuring the Solid Fat Content (SFC, see below) of the fat blend. This profile can then be used to predict the performance of other oil blends as margarine products. A detailed description of margarine formulation can be found in: Fats and Oils, Formulation and Processing for Applications, ed. R. D. O’Brien,

Technomic Publishing Co, Lancaster, PA, 1998, pp 437-457.

The melting characteristics and plasticity of a fat can be measured by industry standard techniques. The most commonly used methods are Solid Fat Index (SFI, AOCS

Cd10-57-93) and Solid Fat Content (SFC, AOCS Cd16b-93(97)). SFI determinations are based on dilatometry which measures the amount of solid or liquid in a fat based on volumetric changes resulting from a fat melting to the liquid state. This technique is gradually being replaced by SFC. This method is basecd on pulsed, low resolution nuclear magnetic resonance (NMR) to measure the relative amounts of solid and liquid in a sample based on the difference in the rates of relaxation of protons in the two phases after the sample has been pulsed. Conversion of SFI to SFC data has not always been reliable. For a given sample, the percentage of solids is measured across termperatures generally ranging between 10° and 40°C. The entire SFC curve is required in order to understand the properties of the fat at different temperatures. The functionality of the fat is based on both the solids content and on the slope of the SFC curve at critical temperatures, for example, between room and body temperature. In this way, the plasticity of that fat can be predicted for temperatures critical to performance.

For margarine or conventional oils and fats, SFC is determined using the direct, serial, non-stabilizing method. The SFC NMR direct mmethod measures and compares signals from the solid and liquid phases. SFC is defined as the- percent ratio between the NMR response obtained from the hydrogen nuclei in the solicl phase versus the response from nuclei in both the solid and the liquid phases of the sarmple. The serial, non-stabilizing method utilizes a single set of samples that are tempere=d by melting and storage at 100°C for 15 minutes, holding at 60°C for 5 minutes, and at 0°C or | hour. The samples are then held at each recording temperature for 30 minutes and move=d to the next higher temperature immediately after determination of SFC. It is believed that high stearic with high oleic oils presented here, as well as those oils with similar compositions, will be capable of producing fat products with SFC profiles of less than 20 at 25°C, preferably less than 15 at 25°C, more preferably less than 10 at 25°C, and most preferably lesss than 5 at 25°C, and greater than 20 at 10°C, preferably between 20 and 50 at 10°C, and most preferably between 20 and 35 at 10°C.

The melting point of a fat is also an important rmeasurement. Since fats are made up of a mixture of triglycerides which have different melting points, a sharp determination is not always possible. There are several methods common teo the industry which measure melting point including capillary melting point (AOCS Ccl1-25 -93), Wiley melting point (AOCS

Cc2-38-91), slip point (AOCS Cc3-25-93), and droppimg point (AOCS Cc18-80-95).

Typical solid fat index values of US margarine oils are given in Table 3. The numbers are representative of commercial products. Feor a given type of margarine, however, specifications may vary considerably between manufacturers depending on: 1) the organoleptic characteristics desired, 2) compositional requirements to meet nutrient content claims or other information on the nutrition panel, 3) wvhether the product will be marketed using unrefrigerated display, and 4) the type of packag ing equipment available. The solids values are indicative of finished product spreadability aat refrigerator temperatures (10°C), resistance to oil-off at room temperature (21.1°C), and melt in the mouth qualities (33.3°C).

TABLE3

Typical Solid Fat Indices of U.S. Table Spreads

Solid Fat Index 10° 21.1° 26.7° 33.3° 37.7°

Stick 28 16 10 2 0

Soft stick 20 13 9 2.5 0

Soft tub 11 7 5 2 0.5

Liquid 3 2.5 2.5 2 1.5

Butter 32 12 7 2 0 _ The physical and function al aspects of a margarine product are primarily dependent upon the characteristics of its oil pinase. A direct relationship exists between the fat solids content and the structure, consistency, and plasticity of the finished margarine. Margarine consistency, flavor and emulsion stabil ity depends upon crystallized fat. In the United States, hydrogenation is the prefesrred process for changing the solids/liquid relationship of margarine basestocks. However, hydrogenated oils have become controversial due to the presence of trans fatty acied isomers that result from the hydrogenation process. Ingestion of large amounts of trans iso mers has been linked with detrimental health effects including increased ratios of low density to high density lipoproteins in the blood plasma and increased risk of coronary heart disssase. From an environmental standpoint, the use of metal catalysts in the hydrogenation proc=ess has raised issues regarding the reclamation and disposal ofa toxic metal waste product. Increased public awareness of the negative aspects of hydrogenation will likely lead to significant changes in margarine formulation to reduce or : eliminate the presence of industrially-modified oils. K

A number of methmods are well known to those skilled in the art for determining oxidative stability. A stamdard method now commonly used to evaluate the stability of commercial cooking oils ds the Oxidative Stability Index (OSI) which is measured automatically using a machine manufactured by Ominion, Inc. of Rockland, MA, USA.

The OSI machine works by bubbling air through oil heated to 110°C. As the oil oxidizes, volatile organic acids, primarily formic acid, is formed which can be collected in distilled water in a cell. The machine constantly measures the conductivity of the distilled water and the induction p eriod is determined as the time it takes for this conductivity to begin a rapid rise. It is believed that the high stearic with high oleic oils presented in the invention, in their unblen ded and blended forms, as well as those oils with similar compositions, will be capable of producing fat products with OSI(110) greater than 15, preferably greater than 25, and most preferably greater than 35.

EXAMPLES

The following Examples are intended to illustrate the present invention and do not constitute a limitation the=reon. All temperatures are given in Celsius unless indicated otherwise. Solid Fat Content (SFC) profiles listed in these Examples report the percentage of the total fat moieties that were determined to be in the solid phase at the indicated temperature.

EXAMPLE 1

Preparation of oils from soybeans and analyses of fatty acid compositions

All of the oils used in these examples were prepared according to the following laboratory scale method. Harvested soybeans were heated in the microwave to 180°F, cooled to room temperature and cracked using a Roskamp TRC 650-6 Crack and Roll.

Soybean hulls were removed using a Kice Aspirator and the remaining meats were heated to 180°F and flaked in a Roskamp TRC 912 Flake and Roll. Crude oil was extracted in a glass, water-jacketed extraction vessel heated to 60° for 45 minutes using a solvent to solids ratio : of approximately 4:1. The hexane/oil miscella was collected and the extraction repeated.

The miscella was desolventized using a rotary evaporator leaving crude oil.

A volume of an 85% phosphoric acid solution equal to 0.1% (v/v) of the crude oil was added and the solution heated to 65°-70° for 10 minutes while stirring. Warm (60°)

NaOH (8% aqueous solution) was added dropwise to the oil to neutralize the free fatty acids and the H3PO, with an additional 0.2% wt/wt excess. The solution was stirred for five minutes and the solids separated by centrifugation. The oil was water washed by adding hot water to 20% (v/v) as the sample was heated to 90° with rapid agitation. The oil and water were allowed to cool at room temperature for 10 minutes and then separated by centrifugation. The oil was dehydrated using very rapid agitation under vacuum at 85°-95° for 30 minutes or until all moisture (bubbles, condensation) had been removed. The vacuum was then broken with nitrogen. The oil was bleached by adding 2% (wt/wt) Activated

Bleaching Earth (AOCS #21077) and the solution mixed under vacuum for 30 minutes at 85°-95° before cooling to 80°. The vacuum was broken with nitrogen and 1% (wt/wt) of diatomaceous earth was added and the mixture filtered through a prepared bed of diatomaceous earth.

Citric acid was added to approximately 50 ppm, and the oil was deodorized at 240° with steam (4 mL water per 100 g oil) in a glass deodorizer for approximately 1 hour. The oil was cooled to 80° with sparging, and it was further cooled to 40° under nitrogen. The refined, bleached, and deodorized oil was stored frozen under a nitrogen atmosphere.

All of the fatty acid composition analyses described in these examples were determined essentially by the methods described in AOCS Ce 1¢-89. Fatty acid methyl esters were prepared as follows. Ten pL oil or liquefied fat was mixed with 1 mL hexane and 0.25 mL of a 3% sodium methoxide solution for 30 minutes. Acetic acid (0.1 mL of a 10% solution) was added, the sample was mixed and the layers separated by centrifugation.

The resulting fatty acid methyl esters extracted in the hexane layer were resolved by gas chromatography (GC). Hewlett Packard 5890GC (Wilmington, DE) equipped with a

SP2340 column (60 m, 0.25 mm ID, 0.20 micron film thickness) (Supelco, Bellefonte, PA).

Column temperature was 150° at injection and the temperature programmed from 150° to 200° at 2°C/min over 40 minutes. Injector and detector temperatures were 215° and 230°, respectively. All compositional values reported are relative values calculated from the integrated areas measured by the GC detector.

All of the SFC determinations were done essentially by the methods described in

AOQOCS Cd16b-93.

All of the oxidative stability measurements were determined by OSI essentially by the methods described in AOCS Cd12b-92(93).

EXAMPLE 2

Solid Fat Content of Commercial Margarine

TT Samples of commercial margarine with varying degrees of solidity were analyzed for -

SFC. Samples were prepared by melting the product at 100 degrees and removing the aqueous layer. The resulting fat fraction was analyzed for SFC as a function of temperature and the results are listed in Table 4. The table shows the range of solids which can be used to produce different types of margarine and spread products depending on the fat plasticity and the product form desired (e.g., soft tub, stick, low fat spread).

TABLE 4

Solid fat content of commercial margarine at various temperatures

Functionality Product Form 10 15 - 20 25 30 35 40 68% vegetable oil spread stick 24.2 23.5 16.2 8.5 4.1 09 . 05 40% vegetable oil spread stick 222 21.7 15 8 3.6 1.1 0.0 70% corn oil spread stick 31.0 25.8 17.6 10.9 5 0.7 0.1 40% vegetable oil spread soft tub 16.4 11.9 7.2 4.1 1.7 0.6 0.3 ; 24% vegetable oil spread soft tub 18.5 13.9 9.4 5.8 353 1.3 0.6 70% vegetable oil spread stick 40.3 32.1 22.7 12.6 5.1 1.3 0.1 corn oil margarine stick 35.9 nd 22.1 13.8 6.7 1 nd soybean oil margarine stick 36.5 nd 23 15.4 8.9 3 nd soft margarine soft tub 19.1 nd 9 44 1.5 -0.1 nd butter stick 473 349 20 11.8 5.1 1.2 0.0

EXAMPLE 3

Solid Fat Content of a High Stearic Soybean Oils

High stearic acid soybeans from the line designated L9216116-109 were developed at

DuPont from a pedigree (HST1*(HO2*HO4))* A2506 (which means the progeny from a cross between HO?2 and HO4, was crossed to HST1, and the resulting progeny was crossed to a wild-type line, A2506 to produce L9216116-109). The seeds produced have a higher stearic acid content and lower linolenic acid content than conventional soybeans. The parents HST1, HO2, and HO4 (see Table 2) are mutant lines selected by the mutagenesis protocols outlined in U.S. Paten.t No. 5,710,365, except different soybean lines were used as starting materials for the mutagesnesis, and selection was based on variation in fatty acid content instead of carbohydrate content. HST1 is a mutant of line N85-2176 which is a high oleic, low linolenic line developed at North Carolina State University by J. W. Burton (Kuhr etal, March 26, 1987 Release Notice for N85-2124, N85-2131, and N85-2176. USDA

Agric Res. Services). HST! dif fers from its parent N85-2176 by virtue of its abnormally high stearic acid content (HST=high stearic), while retaining the lower linolenic acid content of N85-2176. The high stearic mmutation in HST! suppresses (is epistatic to) the high oleic gene(s) that are present in the genetic background from N85-2176. Therefore, HST1 is not high in oleic acid like N85-2176. The stearic mutation in HST] is allelic to fasa (see

Table 2) in line A6 (W. Fehr from Iowa State) and results in a similar phenotype when crossed into similar backgroundks as the fasa allele. Crosses between N85-2176 and A5 (W. Fehr from Iowa State) confirm that N85-2176 contains an allele of the fan gene (see

Table 2) present in AS5 that confers a similar low linolenic phenotype. Therefore, HST! contains both a high stearic mutation (allelic to fusa) and a low linolenic mutation (allelic to fan).

HO?2 is a DuPont proprisetary mutant line selected from mutagenesis of the line

N85-2176. The mutagenesis pr-otocol was essentially the same as the one described in U.S. : Patent No. 5,710,365, except th at N85-2176 was used as the starting material for x 20 mutagenesis, and selection was based upon variation in fatty acid content instead of carbohydrate content. HO2 differs from N85-2176 (fan) in that it has a slightly higher oleic acid content (HO=high oleic), but retains the low linolenic acid content of N85-2176.

Therefore, HO2 contains an unrmamed mutation that confers higher oleic acid content than

N85-2176 in addition to the fare gene. .

HO4 is a DuPont propri-etary mutant line selected from mutagenesis of the line AS.

The mutagenesis protocol was essentially the same as the one described in U.S. Patent

No. 5,710,365, except that AS vas used as the starting material for the mutagenesis, and selection was based upon variattion in fatty acid content instead of carbohydrate content.

HO4 differs from AS (fan) in thaat it has a higher oleic acid content (HO=high oleic), but retains the low linolenic acid content of A5. Therefore, HO4 contains an unnamed mutation that confers higher oleic acid content than AS in addition to the fan gene.

It is believed that a deriwvative of A6 (fasa), or similar plant yielding seeds with an oil composition comprising a high stearic and low linolenic acid phenotype similar to that disclosed in the instant invention, would be useful for the methods described herein.

High stearic and high ol eic acid soybeans were developed as outlined in Example 5.

All soybean oils were prepared as described in Example 1 and analyzed for fatty acid composition and solid fat conte=nt at a variety of temperatures.

Tables 5 and 6 show thes compositional and functional characteristics of high stearic soybean oils ranging from 15% to 22% stearic acid. In addition, four of the oils have elevated levels of oleic acid. The oil containing 22% stearic acid and a normal oleic content did not have enough solidity to meet the specific-ations required for a soft tub margarine (see

Figure 1). However, the SFC profile of the 22% stearic acid, high oleic acid combination fell within the ranges published for commercial soft tub margarine. It is believed that high stearic combined with high oleic oils presented kere, as well as those oils with similar compositions, will be capable of producing fat products with SFC profiles of less than 20 at 25°C, preferably less than 15 at 25°C, more preferably less than 10 at 25°C, and most preferably less than 5 at 25°C, and greater than 20 at 10°C, preferably between 20 and 50 at 10°C, and most preferably between 20 and 35 at 10°C.

Oils with less than 22% stearic acid did rot have an SFC profile consistent with ; margarine and spread products. Therefore, SFC profiles consistent with margarine and = spread products could be obtained from unblend ed oils with high oleic acid concentrations combined with stearic acid concentrations of at Beast 22%. ~

TABLE 5

Fatty acid compos ition

Chain length: unsaturation 16:0 18:0 18:1 18:2 18:3 20:0 high stearic 88 222 14.6 48.1 4.9 1.5 high stearic/high oleic oil A 59 219 62.8 2.6 3.6 1.8 : high stearic/high oleic oil B 6.0 18.6 66.8 2.3 3.6 1.5 high stearic/high oleic oil C 5.5 17.6 67.6 34 3.7 1.4 high stearic/high oleic oil D 5.9 15.1 69.2 2.6 3.6 1.2

TABLE 6

Percent SFC as a function of temperature

Temperature 10° 15° 20° 25° 30° 35° 40° high stearic 13.9 6.8 46 0.1 04 0 0 high stearic/high oleic oil A 21.2 16.4 7.1 4.5 0 0 0 high stearic/high oleic oil B 29.9 19.8 43 3.1 0.3 06 0.5 high stearic/high oleic oil C ~~ 28.4 17.6 2.9 14 05 0 0.3 high stearic/high oleicoil D 23.6 11.5 1.0 07 02 0.4 0.3

EXAMPLE 4

Base Oil Blend to Produce a Margarin -€ Lacking Trans Fatty Acid Isomers

High stearic oils were blended (as indicated in Table 7) with either palm oil or fully hydrogenated soybean flakes (Dritex PST Hydrogenated Soybean Flakes, AC HUMKO, 525 W. 1% Avenue, Columbus, OH) to produce fats that met the SFC requirements for a margarine and would not contain trans fatty acicl isomers. The resulting blends were analyzed for fatty acid composition, solid fat co mtent at a variety of temperatures, and oxidative stability. Table 7 shows the fatty acid compositions and OSI values of each of these blends. A graphical representation is shown in Figure 2. Blends made from high stearic/high oleic oils show oxidative stability values 4-7 fold greater than blends made from high stearic oils with normal oleic levels. It is believed that the high stearic with high oleic oils presented here, in their unblended and blended forms, as well as those oils with similar compositions, will be capable of producing fat products with OSI(110) greater than 15, preferably greater than 25, and most preferably greater than 35. Table 8 shows the solid fat content for each of these blends. The blended forms of high stearic/ high oleic oils demonstrating high oxidative stability (HSHO C + palm oil, and HSHO D + fully hydrogenated soybean oil) have also improved their SFC profiles as shown in Figure 3, and now conform with the curve for soft-tub margarine. It is believed that high stearic with high oleic oils presented here, in their unblended and blended forms, as well as those oils with similar compositions, will be capable of producing fat products with SFC profiles of less than 20 at 25°C, preferably less than 15 at 25°C, more preferably less than 10 at 25°C, and most preferably less than 5 at 25°C. and greater than 20 at 10°C. preferably between 20 and 50 at 10°C, and most preferably between 20 and 35 at 10°C.

TABLE 7

Fatty acid composition of blended oils (%) 14:0 16:0 18:0 18:1 18:2 18:3 20:0 OSI(110) high stearic/high oleic oil C nd 5.5 17.6 67.6 3.4 37 14 41.2 high stearic/high oleic oil D 5.9 15.1 69.2 2.6 36 1.2 nd palm oil 1.2 44.7 4.5 39.0 8.6 0.1 04 25.5 fully hydrogenated soybean {.3 55.2 41.2 0.3 0.1 0 0.5 nd oil (FHS) normal soybean oil 0.1 10.9 4.0 246 51.4 6.7 0.3 6.1 - blend of HSHO oil C + palm 0.6 24.0 11.0 53.8 6.5 19 09 35.7 oil (50%) blend of high stearic + palm 04 222 15.1 24.6 324 26 1.0 8.5 oil (40%) blend of HSHO oil D + FHS nd 7.3 15.8 68.2 2.8 34 11 433 (3%) blend of high stearic + FHS 0.1 10.3 22.4 14.2 45.6 40 1.4 5.8 (3%) normal soybean + palm oil 04 22.1 4.1 30.1 36.7 45 03 74

TABLES

SFC as a function of temperature

Temperature 10° 20° 25° 30° 35° high stearic/high oleic oil C 28.4 2.9 1.4 0.5 0 high stearic/high oleic oil D 24.2 1.8 0.6 0 0 palm oil 57.2 31.6 19.7 10.9 5.0 blend of HSHO oil C + palm oil (50%) 25.8 10.4 6.1 3.7 1.7 blend of high stearic + palm oil (40%) ~~ 22.7 7.4 4.8 2.5 0.7 h blend of HSHO oil D + FHS (3%) 244 4.6 3.5 2.0 0.9 blend of high stearic + FHS (3%) 17.3 8.2 3.6 23 1.2 oo normal soybean + palm oil (35%) 12.8 6.3 42 33° 15

EXAMPLE 5

Soybeans with High Stearic and High Oleic Content

Crosses were performed between a soybean line which has elevated levels of oleic acid in its seed fatty acids and a line which has elevated levels of stearic acid in its seed fatty acids. The high oleic line contains a transgene copy of the soybean fatty acid desaturase gene, gmFAD2-1 (Heppard, E.P. et al. (1996) Plant Physiol. 110:311-319), that results in co- suppression and therefore down regulation of the gmF AD2-1 message level and is described in WO 97/40698. Decreased expression of the FAD2-1 function leads to a decrease in activity of delta-12 desaturase, and a decrease in the accumulation of poly-unsaturated fatty acids. The high oleic line is designated D2T and the typical fatty acid profile of its seed lipid is given in Table 2. The high stearate parent is a fatty acid synthesis mutant isolated from a mutagenized soybean seed population (U.S. Patent No. 5,557,037), designated A6, and containing a fatty acid mutant fasa allele. Its typical seed lipid fatty acid profile is given in

Table 2.

F1 seeds obtained from the crosses were planted to obtain F1 plants. The F1 plants were then self-pollinated to obtain F2 seeds that were segregating for both of the loci affecting the seed fatty acid profile. These F2 seeds were planted and the plants were self- pollinated as in the previous generation. The relative content of the five main fatty acids in bulked seed samples from individual F2 plants was determined by gas liquid chromatography as described in WO 94/11516. Remaining seed from F2 plants containing maximum stearic and oleic acid content were selected, planted and allowed to self pollinate in order to obtain F3:4 seed. A sample of F4 seed from each F3 plant (F3:4 seed) was then submitted for GC analysis so that individual F3:4 phenotypes could be determined. F3:4 plant phenotypes tracing back to a common F2 plant ancestor were then averaged to obtain mean phenotypes for F2-derived families (F2:4 family means). Single plants and family means that were highest in oleic and stearic acid are shown in Table 9. The individual plant lines ancy the family means are presented in order of decreasing stearic acid in the seed fatty acids.

TABLE 9

Seed fatty acid profiles from individual plants and F2:4 family means arising from a cross of the high oleic parent D2T and the high stearic parent fasa

Individual Fatty Acid Content _ Line ID (%oftotalseed fattyacid) ~~ generationandfype 16:0 18:0 18:1 18:32 18:3 7S0-2334-1 7 26 61 1 3 F3:4 single plant 7S0-2293-2 6 26 61 2 3 F3:4 single plant 7S0-22*93-1 6 25 62 2 3 F3:4 single plant 7S0-23:03-5 6 24 66 0 2 F3:4 single plant 7S0-23 39-1 6 24 64 1 3 F3:4 single plant 7S0-22 95-1 6 24 64 1 3 F3:4 single plant 7S0-23 31-3 7 24 63 1 3 F3:4 single plant 750-50 97-3 6 20 67 2 3 F3:4 single plant 750-50 97-1 6 20 66 2 3 F3:4 single plant 750-23 56-1 6 18 72 0 3 F3:4 single plant 780-23 06-2 6 18 71 2 3 F3:4 single plant 780-23 05-3 6 18 70 2 2 F3:4 single plant 780-23 10-3 6 18 70 2 3 F3:4 single plant ) 7S0-22.93 6 24 63 2 3 F2:4 family mean of 9 plants : 7S0-23 34 6 23 65 1 3 F2:4 family mean of 8 plants 780-23 39 6 23 65 1 3 F2:4 family mean of 7 plants : 7580-23-79 6 19 70 1 2 F2:4 family mean of 3 plants -. 750-2305 6 18 71 2 5 F2:4 family mean of 10 plants

The very low poly-unsaturated fatty acid phenotype arising from the D2T parent is maintai ned in selected progeny from the cross as is the very high level of stearic acid coming from th e fasa containing parent. The increased level of stearic acid in the selected progeny from th_e cross relative to the D2T parent causes a decrease in the oleic acid content.

EXAMPLE 6

Soybeans with High Oleic Acid and High Palmitic Acid and High Stearic Acid Content

Crosses were made between soybean lines containing the D2T gene (for high oleic acid content) and soybean lines containing both a fap? allele (for high palmitic acid content) and a fezasa allele (for high stearic acid content) to obtain F1 progeny. F1 progeny were then selfed in subsequent generations to obtain F2 seeds and F2:3 families. F2:3 families containing maximum palmitic + stearic + oleic acid content were selected and planted and allowed to self pollinate in order to obtain F3:4 seed. A sample of F4 see=d from each F3 plant (F3:4 seed) was then submitted for GC analysis so that individual 3:4 phenotypes could be determined. F3:4 plant phenotypes tracing back to a common 2 plant ancestor were then averaged to obtain mean phenotypes for F2-derived families (F=2:4 family means).

Single plants and family means that were highest in palmitic + stearic + oleic acid content are shown in Table 10, and are presented in order of decreasing total seecd saturated fatty acids.

TABLE 10 :

SE Individual Plant and Family Mean Phenotypes With a Combi nation of

High Oleic Acid and High Palmitic Acid and High Stearic Acid Content (D2T + fap? + fasa genes)

Individual Fatty Acid Content

Line ID (% of total seed fatty acid) generation and type 16:0 18:0 18:1 18:2 183 7HSO-5076-5 12 21 58 3 3 F3:4 single plant 7HSO-2201-2 12 19 62 2 3 F3:4 single plant

THSO-2229-1 12 18 65 1 3 F3:4 single plant 7HSO-2201-1 11 19 62 2 3 F3:4 single plant

THSO-2227-5 11 18 66 1 3 F3:4 single plant

THSO-2207-5 11 18 65 1 3 F3:4 single plant 7HSO-5074-3 11 17 63 1 3 F3:4 single plant 7HSO-2201-0 11 18 63 2 3 F2:4 family mean of 3 plants 7HSO-2208-0 11 16 68 1 3 F2:4 family mean of 3 plants 7HSO-2228-0 11 16 68 1 3 F2:4 family mean of 4 plants

THS0-2207-0 11 15 68 1 3 F2:4 family mean of 6 plants

EXAMPLE 7

Soybeans with High Oleic Acid and Low Paimitic Acid Content

Crosses were made between soybean lines containing the D2T gezne (for high oleic acid content) and soybean lines containing both a fap! allele and a fap3 allele (for low palmitic acid content) to obtain F1 progeny. F1 progeny were then selfe d in subsequent generations to obtain F2 seeds and F2:3 families. F2:3 families containi mg both minimum palmitic acid content and maximum oleic acid content were then selected and planted and allowed to self pollinate in order to obtain F3:4 progenies. A sample of F4 seed from each

F3 plant (F3:4 seed) was then submitted for GC analysis so that individuaal F3:4 phenotypes could be determined. F3:4 plant phenotypes tracing back to a common E72 plant ancestor were then averaged to obtain mean phenotypes for F2-derived families ( F2:4 family means).

Single plants and family means that were both lowest in palmitic acid ccontent and highest in oleic acid content are shown in Table 11, and are presented in order of increasing palmitic acid in total seed fatty acids. Since selection for low palmitic acid content requires precision to a tenth of a percent, palmitic values in Table 11 are shown to that level of precision while values for the other fatty acids are rounded to the nearest whole percent.

TABLE 11

Individual Plants and F2:4 Family Mean Phenotypes With a Combination of

High Oleic and Low Palmitic Acid Content (D2T + fap] + fap3 genes)

Individual Fatty Acid Content

Line ID (% of total seed fatty acid) generation and type 16:0 18:0 18:1 18:2 18:3 7A0-2079-12 27 3 89 1 3 F3:4 single plant 7A0-2083-1 28 3 90 1 2 F3:4 single plant 7A0O-2072-4 29 3 89 1 2 F3:4 single plant 7A0-2065-1 29 3 89 1 3 F3:4 single plant 7A0-2083-3 30 3 89 1 2 F3:4 single plant 7A0-2075-3 3.0 3 89 1 3 F3:4 single plant 7A0-2079-1 30 3 88 2 3 F3:4 single plant 7A0-2072-1 3.1 3 88 1 3 F3:4 single plant 7A0-2085-5 32 3 89 1 2 F3:4 single plant 7A0-2084-1 32 3 88 2 2 F3:4 single plant 7A0-2072-6 32 3 88 1 3 F3:4 single plant 7A0-2081-6 33 3 90 1 2 F3:4 single plant 7A0-2074-7 33 3 89 1 3 F3:4 single plant 7A0-2081-3 34 3 89 1 2 F3:4 single plant 7A0-2087-1 34 3 88 3 3 F3:4 single plant 7A0-2072-0 3.0 3 89 ] 3 F2:4 family mean of 6 plants 7A0-2083-0 3. 3 89 ] 2 F2:4 family mean of 4 plants 7A0-2074-0 35 3 88 1 3 F2:4 family mean of 7 plants 7A0-2080-0 36 3 88 1 3 F2:4 family mean of 5 plants 7A0-2065-0 36 3 88 1 3 F2:4 family mean of 3 plants 7A0-2082-0 36 3 88 2 3 F2:4 family mean of 5 plants 7A0-2068-0 36 3 88 1 3 F2:4 family mean of 4 plants 7A0-2079-0 36 3 88 1 3 F2:4 family mean of 12 plants

EXAMPLE 8

Sovbeans with High Oleic Acid and Low Linolenic Acid Content

Crosses were made between soybean lines containing the D2T gene for high oleic acid content and soybean lines containing either a fan allele or the D3A gene for low linolenic acid content to obtain F1 progeny. F1 progeny were then selfed in subsequent generations to obtain F2 seeds and F2:3 families. F2:3 families containing both minimum linolenic acid content and maximum oleic acid content were then selected and planted and allowed to self pollinate in order to obtain F3:4 progenies. A sample of F4 seed from each

F3 plant (F3:4 seed) was then submitted for GC analysis so that individual F3:4 phenotypes could be determined. F3:4 plant phenotypes tracing back to a common F2 plant ancestor were then averaged to obtain mean phenotypes for F2-derived families (F2:4 family means).

Single plants and family means that were both lowest in linolenic acid content and highest in oleic acid content are shown in Table 12, and are presented in order of increasing linolenic acid. Since selection for low linolenic acid content requires precision to a tenth of a percent, linolenic acid vatues in Table 12 are shown to that level of precision while values for the

To other fatty acids are rounded to the nearest whole percent. : SEE

TABLE 12

Individual Plants and F2:4 Family Mean Phenotypes

With a Combination of High Oleic and Low Linolenic Acid Content

Individual Fatty Acid Content

Line ID (% of total seed fatty acid) generation and type 16:0 18:0 18:1 18:2 18:3 70L-2703-6 5 3 87 2 1.7 F3:4 single plant 70L-2708-1 7 4 85 1 1.7 F3:4 single plant 70L-2703-7 6 3 87 1 1.8 F3:4 single plant 70L-2706-6 6 4 86 1 1.8 F3:4 single plant 70L-2703-5 5 3 88 1 1.9 F3:4 single plant . 70L-2703-2 6 3 87 1 1.9 F3:4 single plant ~ 70L-5180-7 6 3 87 2 1.9 F3:4 single plant ~ 70L-51817 6 3 8 2 19 F3:4singleplant : 70L-2706-7 6 4 86 1 1.9 F3:4 single plant 70L-2707-5 6 4 86 1 1.9 F3:4 single plant 70L-2705-10 7 4 85 1 1.9 F3:4 single plant 70L-2711-4 7 4 85 2 1.9 F3:4 single plant 70L-2712-5 7 4 84 2 1.9 F3:4 single plant 70L-2708-0 7 5 84 1 1.8 F2:4 family mean of 4 plants 70L-2703-0 6 3 87 I 1.9 F2:4 family mean of 7 plants 70L-2707-0 6 4 86 1 2.1 F2:4 family mean of 5 plants 70L-2705-0 6 4 85 1 2.1 F2:4 family mean of 12 plants 70L-2710-0 7 4 85 2 2.2 F2:4 family mean of 9 plants

Individual Fatty Acid Content

Line ID (% of total seed fatty acid) generation and type 70L-2711-0 7 4 84 2 2.2 F2:4 family mean of 5 plants 70L-2712-0 7 5 82 3 2.3 F2:4 family mean of 11 plants 70L-2713-0 6 4 85 2 2.4 F2:4 family mean of 11 plants 70L-2709-0 7 4 83 2 2.5 F2:4 family mean of 11 plants

EXAMPLE 9

Isolation of cDNA from Brassica napus Encoding acyl-ACP Thioesterase

A cDNA library was prepared from mRNA isolated from developing seeds of

Brassica napus harvested from pods that were between 20 and 26 days post pollination.

Total RNA was extracted and mRNA purified by oligo dT chromatography. The library was made by the technique described by Ray and Ray (1991, Nucleic Acids Research 19: 4559) using reagents purchased from Pharmacia, with some modifications. The resulting cDNA was cloned into the Lambda ZAP vector (Stratagene, La Jolla, CA) as per the manufacturer's instructions. The primary library contained approximately 1x100 individuals and was amplified once.

Cloning techniques used were from Sambrook et al. [(1989) Molecular Cloning: A

Laboratory Manual, 2" Edition, Cold Spring Harbor Laboratory Press]. Approximately 153x105 plaques from the amplified library were developed by infection of E. coli plated on a total of six plates and duplicate nitrocellulose lifts were taken from each developed plate.

The duplicate nitrocellulose lifts were probed with the 32P labeled (random primer labeling kit, Life Technologies, Gaithersburg, MD) sequence from the Brassica genomic clone (SEQ

ID NO:20 in WO 92/11373) and annealed at 63° in hybridization buffer (6 X SSC [0.9 M

NaCl, 0.09 M sodium citrate, pH 7], 5X Denhardts’s solution [0.5 g Ficoll (type 400,

Pharmacia), 0.5 g polyvinylpyrrolidone, 0.5 g bovine serum albumin (fraction V, Sigma), 1 mM EDTA, 1% SDS and 100 micrograms denatured salmon sperm DNA/mI (Sigma). The lifts were annealed for 18 hr, then washed at 63° in 0.2 X SSC and placed on photographic film. Sixteen plaques were scored as hybridizing to the probe, and of these seven were purified and excised according to directions in the Lambda ZAP Cloning Instruction Kit

Manual (Stratagene). The resulting phagemids were used to infect £. coli XL-1 Blue cells, and resulted in double stranded plasmids containing the selected cDNA inserts. Two classes of clones were indicated by restriction analysis of the purified plasmids. One class was represented by a clone designated pSC. The insert in p5C was sequenced by dideoxy sequencing and was confirmed as encoding the Brassica napus oleoyl-ACP thioesterase.

The sequence of this cDNA in shown in SEQ ID NO:1. The sequence of the encoded protein is shown in SEQ ID NO:2. The predicted translation product is 98.6% identical to the protein encoded by another cDNA reported by Jones etal. (1995, Plant Cell 7:359-371) mand submitted to GenBank with accession number U17098. The sequences of this second thioesterase clone and the protein are presented in SEQ ID NO:3 and 4, respectively.

EXAMPLE 10

Expression of the Oleoyl-ACP Thioesterase from

Brassica napus in Soybean Seeds

The entire cDNA insert in pSC was released from pBluescript as an Xmn I fragment and cloned into a soybean seed expression vector for use in biolistic transformation of elite soybean cultivar A2396.

A plasmid was constructed in which the acyl-ACP (oleoyl-ACP) thioesterase fromm

TT Brassica napus cDNA sequence was placed under the control of the soybean beta-conglycinin promoter (Beachy R. N. et al. (1985) EMBO J. 4:3047-3053). The construction of this vector was facilitated by the use of plasmid pCW109, described in

WO 94/11516. Vector pPCW109 contains an 830 base DNA segment which includes the promoter sequences for the alpha subunit of the soybean seed storage protein beta conglycinin, a region with maltiple restriction sites, and 1080 bases of 3' regulatory sequmence from the common bean seed storage protein, phaseolin. Vector pPCW109 was modified to contain a Sma I site in the multiple cloning region as well. The cDNA insert of p5C was removed by digestion with Xmn I, isolated, and ligated into the modified pCW109 whickh had been digested with Smal and an event in which the cDNA insert from pSC was orierted in the sense direction was chosen and designated pST14.

A plasmid designated pKS18HH was constructed to allow conferred resistance tO . hygromycin B to either plants or bacteria transformed with the plasmid. The plasmid was . constructed using the following genetic elements: 1) plasmid vector pSP72 (Promega, oo

Madison, WI), which was modified by removal of the -lactamase coding region, 2) a plant - selectable marker cassette consisting of the 35S promoter from cauliflower mosaic virus (CaMV) linked to the hygromycin B phosphotransferase (HPT) and the nopaline synthase 3' regulatory sequence from Agrobacterium tumefaciens, and 3) a bacterial selectable markzer cassette consisting of the promoter from bacteriophage T7, a Shine-Dalgarno sequence upstream of the HPT coding sequence, and the T7 transcription terminator sequence.

The hygromycin B phosphotransferase gene was obtained from £. coli strain W6e77 which contained a Klebsiella-derive plasmid (pJR225, Gritz, L. and Davies, J. (1983) GENE 25:179-188). The T7 promoter:HPT:T7 terminator cassette for expression of the HPT enzyme in certain strains of E. coli, such as NovaBlue (Novagen, Madison, WI) was obtained from a derivative of a pET vector. The origin of the 35S:HPT:Nopaline synthase 3' end cassette for expression of HPT in plant is described in detail WO 94/11 516.

One skilled in the art can incorporate these elements into a single plasmid using “the usual protocols of molecular cloning.

: .

To allow use of the vector in the transformation protocol described by Christou et al. (1990, Trends Biotech 8:145) the HPT encoding sequence in the 35S:HPT:Nopaline synthase cassette was removed by restriction endonuclease digestion and replaced by the correctly oriented coding region for the bacterial B-glucuronidase gene (Jefferson et al. (1987) EMBO

J.6:3901-3907). The resulting plasmid was designated pKS18.

The B-conglycinin:p5C:phaseolin-3' expression cassette in pST14 was removed by a partial HindIII digestion, isolated and ligated into pKS18 that had also been digested with

HindIII to give the final transformation vector designated pRB19.

The transformation technique gives plants which set seeds that may be segregating for the introduced transgene. The bulked seed from the plants may also be chimeric for the transgene.

Three fertile plants were recovered from the transformation and the seeds were analyzed for their fatty acid phenotype by a partial seed analysis technique. A small chip of cotyledon oriented away from the embryonic axis was cut from the seed with a razor blade.

The chip was digested in methanol containing 1% sodium methoxide and the resulting fatty acid methyl esters were extracted into hexane prior to separation and quantification by gas liquid chromatography. Seeds with altered fatty acid profiles were thus identified for subsequent germination and growth.

Of the three soybean plants transformed with the seed expression vector containing : 20 the Brassica acyl-ACP thioesterase, one had no alteration in seed fatty acid phenotype (32 seeds analyzed with a mean of 3.9% stearic acid and an observed range of 3.2% to 4.6%). The two remaining plants had an increased stearic acid content in their seeds.

Twenty-nine seeds were analyzed from the second plant, and were determined to have a mean stearic acid content of 5.4% with an observed range from 3.8% to 7.5%, while forty seeds were analyzed from the third plant and were determined to have a mean stearic acid content of 4.85 and an observed range from 3.5 to 6.7%.

In seeds selected from the initial transformant on the basis of elevated levels of 16:0 and 18:0, progeny were selected in subsequent generations that had consistently elevated levels of the saturated fatty acids stearic acid and palmitic acid. Stearic acid level was increased by about two- to three-fold while the palmitic acid level increased by about 25 to 40% over typical, non-transformed elite lines. The stable line that was obtained from this transformation was designated T1S. It has been deposited with the American Type Culture

Collection, Manassas, VA.

EXAMPLE 11

Soybean with Very High Stearic Acid Content Resulting from Combined Alterations of

Stearoyl-ACP Desaturase and Oleoyl-ACP Thioesterase

Crosses were made between soybean lines containing a fasa allele and soybean lines containing the T1S gene (increasing oleoyl ACP thioesterase) to obtain F1 progeny. The intent is to accumulate stearoyl-ACP which is subsequently cleaved by the thioesterase resulting in the accumulation of stearic acid in the seed. F1 progeny were then selfed in subsequent generations to obtain F2 seeds and F2:3 families. F2:3 families containing elevated levels of both palmitic and stearic acid were then selected and planted and allowed to self pollinate in order to obtain F3:4 progenies. A sample of F4 seed from each F3 plant (F3:4 seed) was then submitted for GC analysis so that individual F3:4 phenotypes could be determined. Single plant fatty acid profiles are shown in Table 13, and are presented in order of increasing total seed saturated fatty acids.

TABLE 13

Fatty acid profiles of individual plants with high saturated fatty acid content due to increased oleoyl-ACP thioesterase and decreased stearoyl-ACP desaturase

Individual Fatty Acid Content

Line ID (% of total seed fatty acid) generation and type 16:0 18:0 18:1 182 183 7S-2244-6 16 36 7 33 7 F3:4 single plant 7S-2244-7 15 35 8 34 7 F3:4 single plant 7S-2244-3 15 35 7 34 7 F3:4 single plant 7S-2236-4 13 35 15 30 6 F3:4 single plant 7S-2236-2 14 33 9 37 7 F3:4 single plant 7S-2236-6 13 34 9 36 6 F3:4 single plant 7S-2236-7 13 34 9 36 6 F3:4 single plant 7S-2244-1 14 32 9 37 7 F3:4 single plant : 7S-2244-4 14 32 9 37 7 F3:4 single plant 7S-2244-5 14 32 9 37 7 F3:4 single plant 7S-2236-3 15 30 11 37 6 F3:4 single plant 7S-2240-3 12 33 13 35 6 F3:4 single plant = 75-2240-2 12 32 11 37 6 F3:4 single plant 7S-2240-4 12 31 13 37 6 F3:4 single plant 75-2240-5 12 31 11 38 6 F3:4 single plant 7S-2240-1 12 29 13 39 6 F3:4 single plant 7S-2236-1 13 25 11 42 7 F3:4 single plant

EXAMPLE 12

Solid Fat Content of a Very High Stearic Soybean Oils

Very high stearic acid soybeans were obtained from plants described in Example 11.

Oils were prepared from the seeds, as described in Example 1, and analyzed for fatty acid composition and solid fat content at a variety of temperatures.

Tables 14 and 15 show the compositional and functional characteristics of very high stearic soybean oils ranging from 33% to 38% stearic acid. All of the oils met the solidity requirements for a margarine (for comparison see curves in Figure 1). The SFC profiles for these high stearic oils are consistent with those obtained from stick margarines or butter (see

Figure 4). It is believed that the oils derived from Example 11, as well as oils with similar compositions, will be capable of producing fat products with SFC profiles of less than 20 at 25°C, preferably less than 15 at 25°C, more preferably less than 10 at 25°C, and most preferably less than 5 at 25°C, and greater than 20 at 10°C, preferably between 20 and 50 at 10°C, and most preferably between 35 and 50 at 10°C. Therefore, these oils could be useful for producing stick margarines that are substantially free of trans fatty acids, oras a hardstock additive in blends to produce margarines or oil-based spreads.

TABLE 14 :

Fatty acid composition 16:0 18:0 18:1 18:2 18:3 20:0

Very high stearic A 14.4 35.4 8.8 33.2 5.0 1.7

Very high stearic B 14.0 33.6 9.1 34.8 5.4 1.6

Very high stearic C 13.5 38.2 83 30.9 3.5 1.9

TABLE 15

SEC as a function of temperature

SFC % Temperature: 10° 15° 20° 25° 30° 35° 40°

Very high stearic A 44.7 31.5 204 8.9 0 0.3 0

Very high stearic B 36.3 23.9 156 42 0 0.1 0

Very high stearic C 48.2 36.8 26.6 13.8 0.5 0.2 0

EXAMPLE 13

Alternative Method to Produce High Oleic and Low Saturated Fatty Acid Soybeans

The promoter:palmitoyl-ACP thioesterase coding region plant transformation vector described in WO9606936 was uscd to transform soybean somatic embryo producing cultures and transformed, fertile soybean plants were obtained again using the methods detailed in

W09606936. The fatty acid profile of seed chips removed from mature cotyledons of the seed born on the transgenic plants was determined. A small fraction of the plants had seeds that appeared to be segregating for a very low saturated fatty acid phenotype. Three such plants were identified; the low saturate class for all three transgenic events averaged a combined 16:0+18:0 content of 5.6% of the total fatty acids while the normal saturated fatty acid classes averaged 14.9%.

The total fatty acid profile averaged from 228 seed chips from the low saturated fatty acid class of seeds from three plants combined from the best event is given in Table 16.

TABLE 16 :

Fatty Acid Profiles for Three Palmitoyl-ACP Thioesterase Co-Suppressed

Soybean Plants Useful as One of the Parents in the Production of High Oleic and Low Saturated Fatty Acid Soybeans

Each Class, % of Total Fatty Acids

To produce a high oleic, low saturated fatty acid seed phenotype using the above described plants as the low saturate parent, seed remaining from the seed chip analysis is planted and taken to flowering. Reciprocal crosses are made between the palmitoyl-ACP thioesterase co-suppression plants and the fatty acid delta-12 desaturase co-suppression ~~~ - plants. Since, at planting, the low saturate seed could have been from either, a seed homozygous for the transgene, or heterozygous for the transgene, remaining flowers on the parental plants are allowed to self-pollinate. The segregation pattern in the self-pollination produced seed is determined to identify plants that are homozygous for the low saturated fatty acid transgene as indicated by the absence of normal saturated fatty acid seeds. Seed from crosses, that are made to plants homozygous for the low saturated fatty acid transgene, are planted. The flowers are allowed to self pollinate and the seed, segregating for both the high oleic and low saturated fatty acid phenotypes is analyzed by the seed chip method.

Remaining seed from seed chips showing the desired phenotype is planted, the plants are self-pollinated and bulked seed from individual plants is analyzed to confirm the desired phenotype. :

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred lo in the description onpage ____ 6 ime _ 11 0000000000

B. IDENTIFICATION OF DEPOSIT Further deposits ase identified on an additional sheet [

Name of depositary institution

AMERICAN TYPE CULTURE COLLECTION

Address of depositary institution (including postal code and country) 10801 University Blvd.

Manassas, Virginia 20110-2209 usa

Date of deposit Accession Number 14 May 1998 ATCC 203033

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet OJ : In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until : the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or. 1s deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4) EPC)

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if ihc indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the Intemational Bureau later (specify the general nature of the indications e.g., “Accession

Number of Depasit”) ’

For receiving Office use only For international Bureau use only

CF is sheet was received with the international application O This shect was received by the International Bureau on:

Authorigbd Hite UVC Authorized officer

PCT/internat’l Appl Processing Div. (703) 305-3639

Form PCT/RO/134 (July 1992)

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM ( (PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the description on page 6 Jee 12 0000 8. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet ]

Name of depositary institution

AMERICAN TYPE CULTURE COLLECTION i

CT Address of depositary institution (including postal code and country) ) } - 10801 University Blvd.

Manassas, Virginia 20110-2209 :

USA

Dale of deposit Accession Number{ IN THE PROCESS OF DEPOSITING ?? April 1999 ATCC XXXXXX

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet J

In respect of those designations in which a European patent is sought, ’ a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or. is deemed to be withdrawn, only by the issue of such a sample to an - expert nominated by the person requesting the sample. (Rule 28(4) EPC) C

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) =

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the Intemational Bureau later (specify the general nature of the indications e.g.. "Accession

Number of Depasit”) ‘

For receiving Office use only For International Bureau use only [1 This sheet was received with the intemational application OJ This sheet was received by the Intemational Bureau on:

PIT RAC DOW

Authorized oll Authorized officer

BCT /internat’t Appl Processing Div. (703) 305-3639

Form PCT/RO/134 (July 1992)

Claims (65)

CLAIMS What is claimed is:

1. A soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of greater than 21%, a C18:1 content S of greater than 60% and a combined C18:2 and C18:3 content of less than 7%.

2. A soybean plant of Claim | wherein said plant is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a soybean nucleic acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising a fasa allele for elevated seed stearic acid content; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds. growing and producing a segregating population of soybean plants through one or more cycles of self-pollination: and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

3. Seeds obtained from the plant of Claim 1.

4. Oil obtained from the seeds of the plant of Claim 1.

5. The oil of Claim 4 wherein said oil has an OSI (110) greater than 15.

6. The oil of claim 4 wherein said oil has a SFC greater than 20 at 10°C and less than 20 at 25°C.

7. A margarine or spread product which comprises the oil of Claim 4, 5 or 6.

8. The margarine or spread product of Claim 7 wherein said margarine is substantially free of trans fatty acids.

9. A blended fat product suitable for making margarine or spread products which comprises the oil of Claim 4, 5 or 6.

10. The blended fat product of Claim 9 wherein said blended fat product has an OSI(110) greater than 15.

11. The blended fat product of Claim 9 which further comprises at least one component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, partially hydrogenated soybean oil, partially hydrogenated cottonseed oil, partially hydrogenated palm oil, soybean oil. corn oil, palm oil, canola oil. sunflower oil, peanut oil. safflower oil or mixtures thereof.

12. The blended fat product of Claim 10 which further comprises at least one component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, partially hydrogenated soybean oil, partially hydrogenated cottonseed oil, partially hydrogenated palm oil, soybean oil. corn oil, palm oil. canola oil, sunflower oil, peanut oil, safflower oil or mixtures thereof.

13. The blended fat product of Claim 9 wherein said blended fat product is substantially free of trans fatty acids.

14. The blended fat product of Claim 10 wherein said fat product is substantially free of trans fatty acids.

15. The blended fat product of Claim 9 which further comprises at least one component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, soybean oil, corn oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil or mixtures thereof and wherein said blended fat product is substantially free of trans fatty acids.

16. The blended fat product of Claim 10 which further comprises at least one — component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, soybean oil, corn oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil or mixtures thereof and wherein said blended fat product is substantially free of trans fatty acids.

17. A margarine or spread product which comprises the blended fat product of Claim 9.

18. A margarine or spread product which comprises the blended fat product of Claim 10.

19. A margarine or spread product which comprises the blended fat product of Claim 11.

20. A margarine or spread product which comprises the blended fat product of Claim 12. B

21. A margarine or spread product which comprises the blended fat product of Claim 13. E

22. A margarine or spread product which comprises the blended fat product of - Claim 14.

23. A margarine or spread product which comprises the blended fat product of Claim 15.

24. A margarine or spread product which comprises the blended fat product of Claim 16.

25. Products made from the hydrogenation, fractionation, interesterification or hydrolysis of the oil of Claim 4, 5, or 6.

26. A margarine or spread product which comprises the product of Claim 25.

27. A blended fat product suitable for making margarine or spread products which comprises the product of Claim 25.

28. The blended fat product of Claim 27 which further comprises at least one component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, partially hydrogenated soybean oil, partially hydrogenated cottonseed oil, partially hydrogenated palm oil, soybean oil. com oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil or mixtures thereof.

29. By-products made during the production of the oil of Claim 4, 5, or 6.

30. A soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a C16:0 content of greater than 10%. a combined C16:0 and C18:0 content of greater than 30%, a C18:1 content of greater than 55% and a combined C18:2 and C18:3 content of less than 7%.

31. A soybean plant of Claim 30 wherein said plant is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a soybean nucleic acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising a fasa allele for elevated seed stearic acid content and a fap? allele for elevated seed palmitic acid; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds. growing and producing a segregating population of soybean plants through one or more cycles of self-pollination: and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

32. Seeds obtained from the plant of Claim 30.

33. Oil obtained from the seeds of the plant of Claim 30.

34. Products made from the hydrogenation. fractionation, interesterification or hydrolysis of the oil of Claim 33.

35. By-products made during the production of the oil of Claim 33.

36. A soybean plant that produces mature seeds in which the total seed fatty acid profile comprises a combined C16:0 and C18:0 content of greater than 42% wherein said plant is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:0 content comprises at least 10% of the total seed fatty acid and the first parent further comprises at least one transgene copy of a nucleic acid fragment encoding an oleoyl-ACP thioesterase enzyme with a second parent comprising a fasa allele for elevated seed stearic acid content; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds. growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

37. Seeds obtained from the plant of Claim 36.

38. Oil obtained from the seeds of the plant of Claim 36.

39. The oil of Claim 38 wherein said oil has an SFC greater than 20 at 10°C and less than 20 at 25°C.

40. A margarine or spread product which comprises the oil of Claim 38.

41. The margarine or spread product of Claim 40 which is substantially free of trans fatty acids.

42. A blended fat product suitable for making margarine or a spread product which comprises the oil of Claim 38 or 39.

43. The blended fat product of Claim 42 which further comprises at least one other component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, partially hydrogenated soybean . oo oil, partially hydrogenated cottonseed oil, partially hydrogenated palm oil, soybean oil, corn oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil, and mixtures thereof.

44. The blended fat product of Claim 42 wherein said fat product is substantially free of trans fatty acids.

45. The blended fat product of Claim 44 which further comprises at least one other component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, soybean oil, corn oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil, and mixtures thereof wherein said blended fat product is substantially free of trans fatty acids.

46. A margarine or spread product which comprises the blended fat product of Claim 42 incorporated therein.

47. A margarine or spread product which comprises the blended fat product of - Claim 43 incorporated therein. -

48. A margarine or spread product which comprises the blended fat product of o Claim 44. a

49. A margarine or spread product which comprises the blended fat product of Claim 45.

50. Products made from the hydrogenation, fractionation. interesterification or hydrolysis of the oil of Claim 38 or 39.

51. A margarine or spread product which comprises the product of Claim 50.

52. A blended fat product suitable for making margarine or spread products which comprises the product of Claim 50.

53. The blended fat product of Claim 52 which further comprises at least one component selected from the group consisting of fully hydrogenated soybean oil, fully hydrogenated cottonseed oil, fully hydrogenated palm oil, partially hydrogenated soybean oil, partially hydrogenated cottonseed oil, partially hydrogenated palm oil, soybean oil, com oil, palm oil, canola oil, sunflower oil, peanut oil, safflower oil or mixtures thereof.

54. By-products made during the production of the oil of Claim 38 or 39.

55. A soybean plant that pr-oduces mature seeds in which the total seed fatty acid profile comprises a combined C16: 0 and C18:0 content of less than 7%, a C18:1 content of greater than 87% and a combined (CC18:2 and C18:3 content of less than 6%.

56. A soybean plant of Claim 55 wherein said plant is produced by the following S steps which comprise: (a) crossing a first paremt soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty aci.d and the first parent further comprises at least one transgene copy of a soybean nuclei c acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising both a fap! allele for decreased seed palmitic acid content and a fap3 allele for decreased seecd palmitic acid content or the second parent comprises at least one transgene copy of a soybean nucleic acid fragment encoding a plant acyl-ACP thioesterase for decreased seed palmitic acid content wherein said thioesterase has a preference of at least two-fold for foalmitoyl-ACP over either stearoyl-ACP or oleoyl-ACP; (b) obtaining hybrid se eds from the cross of step (a), germinating said seeds, growing and producing a segregati ng population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

57. Seeds obtained from the plant of Claim 55. . 20

58. Oil obtained from the seeds of the plant of Claim 35.

59. Products made from thee hydrogenation, fractionation, interesterification or . hydrolysis of the oil of Claim 358.

60. By-products made during the production of the oil of Claim 38.

61. A soybean plant that p-roduces mature seeds in which the total seed fatty acid profile comprises a combined C16 :0 and C18:0 content of less than 12%, a C18:1 content of greater than 84% and a combined &C18:2 and C18:3 content of less than 5% wherein said plant is produced by the following steps which comprise: (a) crossing a first parent soybean plant wherein the C18:1 content comprises at least 80% of the total seed fatty ac id and the first parent further comprises at least one transgene copy of a soybean nucle ic acid fragment encoding a delta-12 desaturase enzyme with a second parent comprising e ither a fan allele for decreased seed linolenic acid content or at least one transgene copy of a soybean nucleic acid fragment encoding a delta-15 desaturase enzyme and further wh erein the C18:3 content comprises less than 4% of the total seed fatty acid; (b) obtaining hybrid seeds from the cross of step (a), germinating said seeds, growing and producing a segregating population of soybean plants through one or more cycles of self-pollination; and (c) selecting the plants from the segregating population of step (b) which produce seeds having said fatty acid profile.

62. Seeds obtained from the plant of «Claim 61.

63. Oil obtained from the seeds of the plant of Claim 61.

64. Products made from the hydrogeration, fractionation, interesterification or hydrolysis of oil of Claim 63.

65. By-products made during the proeduction of the oil of Claim 63.