CA2129621A1 - Sunflower products having lower levels of saturated fatty acids - Google Patents

Abstract

SUNFLOWER PRODUCTS HAVING LOWER LEVELS
OF SATURATED FATTY ACIDS

Thomas C. Heaton Glenn S. Cole Barry A. Martin Abstract of the Disclosure Sunflower seed, plants and oil are described which have a total level of saturated fatty acids equal to 10% or less relative to the total fatty acid content, a total level of stearic acid less than 1% of the total fatty acid content, and a total level of palmitic acid less than 3% of the total fatty acid content.

Description

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Sunflower Products Having Lower Levels ~;
of Saturated Fatty Acids Thomas C. Heaton Glenn S. Cole ;~
Barry A. Martin Background of the Invention This invention relates to a novel sunflower (Helianthus spp.) plant, to products obtained from the novel plant and to methods of producing the sunflower products.
The cultivated sunflower (genus Helianthus annuus L.) is a major worldwide source of vegetable oil. In the United States, approximately 4 ~illion acres are planted in sunflowers annually, primarily in the Dakotas and Minnesota.
The very rapid expansion over the last decade of acreage planted in sunflower in the United States is due in part to several important developments in the field of sunflower breeding and varietal improvement. One significant dev~lop~
ment was the discovery of cytoplasmic male sterility and genes for fertility restoration, a discovery that allowed the pro-duction of hybrid sunflowers. ~he hybrids thus produced were introduced during the early 1970's.
A description of cytoplasmic male sterility (CMS) and genetic fertility restoration in sunflowers is presented by Fick, "Breeding and Genetics", in Sunflower Science and Tech- -`~
25 noloqv Z79-338 (J.F. Carter ed. 1978).

Sunflower oil is comprised primarily of palmitic, stearic, oleic, linoleic and linolenic acids. While other unusual ~ ~

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fatty acids exist in plants, palmitic, stearic, oleic, lino leic, and linolenic acids comprise about 88% of the fatt~
acids present in the world production of vegetable oils.
(Harwood, J.L. Plant Acyl Lipids: Structure, Distribution and Analysis, 4 Lipids: Structure and Function, P.K. Stumpf and E.E. Conn ed. (1988)). Palmitic and stearic acids are saturated fatty acids that have been demonstrated in certain studies to contribute to an increase in the plasma cholesterol level, a factor in coronary heart disease. Vegetable oils high in unsaturated fatty acids, such as oleic and linoleic acids, may have the ability to lower plasma cholesterol according to recent studies. Saturated fatty acids also have higher melting points in general than unsaturated fatty acids of the same carbon number, which contributes to cold tolerance problems in foodstuffs and can contribute to a waxy or greasy mouthfeel. It is also known that food products made from fats and oils having less than 3% saturated fatty acids will typically contain less than 0.5 gram saturated fat per serving and as a result can be labeled as containing "zero saturated fat" under current labelling regulations. Thus, for a number of reasons it is desirable to produce a sunflower oil having low levels of palmitic and stearic acids and high levels of oleic or linoleic acids.
Prior to the present invention there were no naturally occurring sources of low saturated fatty acid sunflower oils.
The generally accepted pathway of fatty acid biosynthesis in plants is that palmitic and stearic acids are the products of the beta ketoacyl-ACP synthetase system which includes three isozymes which are referred to as KAS I, II and III.
Stearoyl-ACP acid is desaturated to oleic acid which is esterified to phosphatidylcholine then sequentially desatu-rated to linoleic and linolenic acids (Stymne, S and Stobart,A.K. Triacylglycerol Biosynthesis, 9 The Biochemistry of Plants: _ Comprehensive Treatise 175-214 (1987) and Stumpf, P.K., Biosynthesis of Fatty Acids in Higher Plants, Oil Crops of the World 38-62 (1989), Previously published research in .. ~-: .. .. ,. ~, -. 2 ~

sunflower has e~phasized generally the alteration of the per-centage of oleic or linoleic acids. The relative proporti ;
of oleîc and linoleic acids can be environmentally infiuenced (Kinman, M.L., and F.R. Earle., "Agronomic Performance and Chemical Composition of the Seed of Sunflower Hybrids and Introduced Varieties," Crop Science 4:417-420 (1964~; Putt, .
E.D., B.M. Craig, and R.B. Carson. "Variation in Composition of Sunflower Oil from Composite Samples and Single Seeds of Varieties and Inbred Lines, J. Am. Oil Chem. Soc. 46:126-129 (1969); Seiler, G.J., "Variation in Oil and Oil Quality of Wild Annual Sunflower (~ _S ~ L.) Populations in a Uniform Environment," 10th International Sunflower Conference, March 14-18, 1982. Surfers' Paradise, Australia. p. 212-215;
Seiler, G.J., "Effect of Genotype, Flowering Date, and Environment on Oil Content and Oil Quality of Wild Sunflower Seed," Crop Science, 23: 1063-1068 (1983); Seiler, G.J., "Interrelation of Fatty Acids in Oil of Wild Annual Sunflower (Helianthus annuus L.)" Proceedings of the XI International Sunflower Conference._ March 10-13, 1985. Mar del Plata, Argentina. p. 529- 534; or have been shown in certain geno-types to be inherited in stable manner (Soldatov, R.I., ~Chemical Mutagenesis in Sunflower Breeding, n International Proceedinqs, 7th International Sunflower Conference, Krasnodar, U.S.S.R., 27 June - 3 July, 1976. International Sunflower Association Vlaardingen, p. 352-357, The Netherlands; Karachenko, L.N., "Genotypic and Phenotypic Mechanisms Ensuring Regulation of Fatty Acid Biosynthesis in Sunflower Seeds," Fiziologiva Rastenii (Russian) 26: 1226-1232 (1979); Fick, G.N., "Breeding and Genetics," Sunflower Science and Technoloqy, Carter, Jack F. (ed.). 1978. Urie, A.L., "Inheritance of Very High Oleic Acid Content in Sunflower,"
Proc. Sunflower Research Wockshop. Bismarck, ND. 1 February, ~.. ~,. . .
1984. National Sunflower Association. Bismarck, ND. p.9-10; ~-Miller, J.F. and D.C. zimmerman. "Inheritance of High Oleic ,~

Fatty Acid Content in Sunflower." Proc. Sunflower Research Workshop. Fargo, ND. 26 January, 1983. National Sunflower ; -- 3 ~

' ~ ' '; . . ' . ' ' ' Association. Bismarck, ND. p. 10; Urie, A. L. "Inheritanc-of High Oleic Acid in Sunflower." CroP Science 25:986-98 (1985); Simpson, B.W. and D. L. George, "Potential for Selection of Fatty Acids on a Single Seed Basis in Sunflower (Helianthus annuus L.)." Proceedinqs of the XI Int$rnational Sunflower Conference, March 10-13, 1985. Mar del Plata, Argentina. p 791-796; Miller, J.F. D.C. Zimmerman, and B.A.
Vick, "Genetic Control of High Oleic Acid Content in Sunflower Oil," Crop Science 27:923-926 (1987); George, D.L., B.W.
Simpson, and C.M. McLeod. "Proposed Development of a High Linoleic Acid Sunflower Hybrid." Proceedinqs of the 12th International Sunflower Conference. July 25-29, 1988. Novi Sad, Yugoslavia. p 448-543; Simpson B.W., C.M. McLeod and D.L.
George. "Selections for High Linoleic Acid Content in Sunflower (Helianthus annus L.)." Aust. J. of Ex~er. Aaric.
29:233-239 (1989). Recent research has claimed that the level of palmitic acid in sunflower oil can be increased to as high as 40.2% of the total oil (Ivanov, P, D. Petakov, V. Nikolova, and E. Pentchev, "Sunflower Breeding for High Palmitic Acid Content in the Oil." Proceedinqs of the 12th International Sunflower Conference. Vol II. July 25-29, 1988. Novi Sad, Yugoslavia. p 463-465). The invention disclosed here pertains to the proportion of palmitic and stearic acids relative to the other major fatty acids in sunflower oil: oleic and linoleic acids.

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SummarY of the Invention This invention relates to the use of a cytoplasmically~
inherited trait that lowers sunflower seed saturated fatty ;
acid content. This trait was isolated in specific sunflower -~-cultivars and is easily transferable to any other sunflower line when the plant carrying the trait is used as the female parent in a sunflower cross so that the progeny inherit the maternal cytoplasm of the plant carrying the trait.

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~ ., The history of the isolation of this unknown and uniqu~
determinant is described in this application (see pages 6-8).
To summarize, originally, sunflower line VK9G was crossed with pollen of variety "Pervenets" (page 6, lines 29-30). Inbred selection was carried out for six generations and maintainer as well as cytoplasmically male sterile counterparts were developed. The trait was first noted in the CMS counterparts, 8904W04F and 8904W06F (Table 1). Sister lines grown in Hawaii ~ ri,,~
demonstrated the same unique results. The male sterile forms produced seed oil with considerably reduced levels of saturated fatty acids independent of environmental effects.

Because the invention relates to the use of a cytoplasmic trait, its effects are inherited only when the plant carrying this trait is used as the female parent. However, this novel '~
15 invention is not linked to male sterility in sunflowers. ;~
Table 3 shows that hybrids as well and inbreds can be produced with low total saturated fatty acids, and when restorers are `
utilized so that the hybrids thus produced are self~
pollinating, seeds of the F2 generation produce oils with low ~ - -saturated fatty acids when self-pollinated. Compared to the standard (normal plants lacking the trait), both palmitic and stearic acid levels are reduced. Thus, male sterility is a convenient but entirely unnecessary feature of this invention.

Furthermore, Table 3 shows the same results occur across different environments, demonstrating that there are few, if any, environmental effects on expression of this trait. This phenomenon is unlike any known nuclear trait affecting fatty acid composition of seed oil, including the high-oleic ;~
varieties. ~-The cytoplasm carrying this trait can be incorporated into any sunflower variety or cultivar, whether fertile, male i~
sterile, or maintainer, tall or dwarf, high-yielding or low.
Any sunflower breeder skilled in the art provided the ~ ~ ;

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~ :' , ~. ' ''' . '. : ' . ' knowledge of a plant incorporating this trait and the presen' disclosure, would be enabled to make and utilize the instan~
invention.

In plants, the female and male parents contribute equal amount of chromosonal DNA to the zygote. In addition, it is ;~ -~
generally accepted that the female parent contributes the cytoplasm, including all of the DNA-bearing cytoplasmic organelles to the zygote. The pollen parent does not ~--contribute to the cytoplasm of the zygote. The plant on which 10 the seed is borne is called the maternal parent or seed '''~;''`r''`''~'"'`parent. Therefore, all cells of the plant derived from seeds ~ -of a cross have the cytoplasm of the maternal parent and equal amounts of nuclear DNA from the female and male parents. In - -plants where self pollination can occur, inbred lines can be made such that plants have the cytoplasm of the maternal parent as well as 100% of the nuclear DNA of the maternal parent. Conversely, a breeder can start with a female parental line having nuclear genotype Nf and cytoplasm Cf, a .
male parental line having nuclear genotype Nm and cytoplasm 20 Cm~ and using backcross techniques in which pollen from the ;~
male is used as the recurrent parent, produce a line having ~ ~
cytoplasm Cf and nuclear genotype Nm. : ~ ~.
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The classical method for the determination of cytoplasmic inheritance is to note whether there is a difference in the trait in progeny from reciprocal crosses of two plant types.
In this case, pollen from plant A is used as the male parent on plant B, and in the reciprocal cross, pollen from plant B
is used on plant A. The result is seed in which the cytoplasm is from either A or B depending on which was used as the maternal parent, and the DNA in the nucleus is identical. If the inheritance of a trait continually follows a particular ~ :...' _ ac ~
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maternal line regardless of the nuclear genotype, the~
cytoplasmic inheritance is shown in that maternal parent line. ;

The present invention is the first report that a cytoplasmic trait~s) can affect fatty acid composition in sunflower and may be the first report of such for any oilseed.
To demonstrate this, applicant points to Tables 3, 5, and 6 which show the reduction of total saturated fatty acids in hybrids wherein inbreds or hybrids carrying this novel cytoplasmic trait were used as female parents and the nuclear 10 content of the progeny was either 50% or 75% distinct from -s that of the original inbred carrying this trail. In Table 6, particularly, sterile hybrids were made using a plant designated 8904W06F (carrying the determinant) by crossing it to a maintainer PHA078 (lacking the determinant), used as a male parent. The entire cytoplasm of the hybrid, but only one-half of the nuclear material, was inherited from 8904W06F.
The genetic content of the F1 nucleus was further diluted by crossing this hybrid with either of three other varieties `~
(lacking the cytoplasmic trait of this invention) all of which 20 were used as the pollen donors. In other words, three new ~ ~
hybrids were made using the F1 hybrid as the female plant. ` -Thus the nucleus of any of these F2 progeny was 75~ distinct from 8904W06F but the cytoplasm was genetically identical. ~ ;~
~1~ of the F2 progeny produced seeds with low total saturated 25 fatty acids demonstrating the presence of a ~ -~
heritablecytoplasmic determinant. No segregation of this `
trait was observed in any of the crosses.

The action of the cytoplasmically-inherited trait of this invention is independent of the nuclear genotype. Both normal sunflower lines, having seed with high content of linoleic or oleic fatty acid, have reduced saturated fatty acid content in ~ ~`p~
their seed oil when crossed into a SFPET1 cytoplasm. Since fatty acid content is the arithmetic total of all fatty acids, a decrease in saturated acids would result in a proportional :: ~
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decreased levels of saturated fatty acids in oil from seeds OL
plants of this invention, is a further increase in unsaturated : -fatty acids.

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The cytoplasmic trait affecting saturated fatty acids in seeds of this invention can be used in conjunction with different nuclear genotypes that also control fatty acid synthesis. The combination of this cytoplasmic trait with exîsting and yet to be discovered nuclear genetic variants offers unique fatty acid compositions in sunflower seed oil.

The present invention provides a sunflower seed that has a total saturated fatty acid content of approximately 6% or less.
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The present invention provides a new sunflower which is true-breeding under a wide variety of growing conditions for the trait of low saturated fatty acid content. This trait can be identified and characterized by restriction fragment length polymorphism analysis of the genome of the sunflower varieties provided by this invention.

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_ aE - '; -' ~: "' - , This invention provides a new sunflower plant that can b~
used efficiently to produce parent lines and hybrid~
possessing desirable agronomic traits in combination with a low content of saturated fatty acids.
This invention also provides a method for producing a hybrid sunflower that has seed which has a total saturated fatty acid content of approximately 10% or less.
This invention provides a novel sunflower oil that has a total level of saturated fatty acids of 10% or less.
In accomplishing the foregoing, there has been provided, in accordance with the present invention, a sunflower seed having a total saturated fatty acid content of 10% or less.
There has also been provided, in accordance with another aspect of the present invention, a hybrid sunflower which is cytoplasmic male sterile, or alternatively, which comprises a genetic determinant encoding fertility restoration, and which, in addition, produces seed the triglycerides of which have a total saturated fatty acid content of 10% or less.
In accordance with yet another aspect of the present invention, there has been provided a sunflower oil which as obtained fromethe sunflower seed contains approximately 10%
total saturated fatty acids or less relative to its total fatty acid content.
Other features, and advantages of the present invention will become apparent from the following detailed description.
It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the area from this detailed description.

: , .. . ,, ~, Detailed Description --In the description and examples that follow, a number of terms are used herein. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following ~;~
definitions are provided~
Sunflower Seed: Botanically referred to as an "achene", comprised of the pericarp and embryo.
Maintainer Line: A male fertile version of an inbred 10 sunflower line.
Cytoplasmic male sterile (CMS) plant or inbred line: A
sunflower line that produces no viable pollen is called male sterile. Male sterility is inherited maternally, ie. the male sterile plant is used as the female parent in a cross 15 with pollen from another sunflower. CMS lines are produced by crossing a maintainer line with a sunflower plant with the cytoplasmic male sterility trait and then backcrossing to the maintainer line until a male sterile line that is homologous -to the maintainer line in all other respects is developed. ;~
20 CMS lines are also referred to as female lines.
Restorer Line: A line possessing the gene or genes to restore male fertility or viable pollen to a sunflower hybrid or inbred line and progeny having a maternal cytoplasm that ~ ~
conditions male sterility. This term is also discussed in the h.. , 25 literature. See, for example, the Fick article identified above. :

Plant Breedinq To produce the novel sunflower of the present invention 30 sunflower line VK9G was emasculated and crossed with pollen of variety 'Pervenets.' Inbred line VK9G is a male~fertile sun~
flower line that produces seeds which have a normal fatty acid composition. 'Pervenets' is an open-pollinated (heterogenous) `
variety introduced from the USSR in the 1970's (Soldatov, 35 1976) with high levels of oleic acid. Similar breeding ~- ~ F
methods are described in Fernandez-Martinez, J., Dominguez-- G

Giminez, J. and Jiminez-Ramirez, A., Breeding for High Content of Oleic Acid in Sunflower ~Helianthus annuus L.) Oil, Helia Nr. Scientific sulletin- of the F.A.O. Research Network on -~
Sunflower 11-15 1988); Fick, G.N., Sunflower, Oil cro~s of the World Ch. 14 pp 301-318 (1989); Knowles, P.F. Genetics and Breeding of Oil Crops, Oil crops of the World Ch. 12 pp. 260-282 (1989). Pedigree selection was made for six generations.
A fatty acid analysis was done on the seeds of maintainer lines that had been inbred for six generations (~) from the pedigree VK9GXPervenets. Also the CMS counterparts of the maintainer lines were analyzed for fatty acid composition. At that time, the maintainer lines had been backcrossed into the CMS background three times so the CMS counterpart was 87.5%
homologous to the maintainer lines.
Breeding with the lines was continued in Hawaii by self pollinating heads (capitulae) of the maintainer lines and crossing pollen from individual heads of the maintainer lines to their respective sterile counterparts so the identity of each crossed pair was preserved. Fatty acid analyses were done on seeds of the maintainer lines and selected seeds were planted again in Woodland, CA. Crosses between the maintainer line and the male sterile counterpart were made in pairs and selections were made for good agronomic plant traits. Fatty acid analysis were performed on a bulk of five seeds from each pair of maintainer and sterile lines derived from that cross.
The maintainer selections were (VK9G/PERV)XC111211 and (VK9G/PERV)XC111121. Those lines were the F7 generation of inbreeding and were referred to as 8904W04G and 8904W06G. The male sterile counterpart was greater than 97~ homologous to its respective maintainer, having been crossed 5 times. At that time it was noted for the first time that the seed from the male sterile form of each maintainer had lower total saturated fatty acids than the maintainer. The results of this analysis are shown in Table 1.

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Unsaturates Saturates Entry Name Source % 18:2 % 18:1 % 16:0 % 18:0 28 8904W04F 8WB9-3 4-1 4.0 89.9 3.-9 2.2 29 8904W04G 8WB9-4 4-1 3.9 86.2 3.5 6.4 32 8904W06F 8WB9-3 6-1 3.7 90.4 - 2.0 1.9 33 8904W06G 8WB9-4 6-1 3.2 86.6 2.2 5.9 Pairs of sterile and maintainer sister lines from the Woodland nursery were sent to Kekaha, Hawaii for winter nursery breeding. Additional pairs of maintainer and CMS
counterparts were crossed. Seeds from those crosses were analyzed. Once again the sterile forms of the maintainer 15 lines had lower total saturated fatty acids. Saturated fatty `
acids of the sterile lines comprised less than 7% of the total oil content. Maintainer line selections of 8904W03G (a sister -line selection of 8904W05G and 8904W06G) and 8904W06G had total saturated fatty acid amounts between 8.3 and 10.2% of the total oil content which was higher than their sterile counterparts ~Table 2.).
The fact that total saturated fatty acids in the sterile forms were less than that of the corresponding maintainers in the Hawaii test as well as at the Woodland test demonstrated that a nucleo-cytoplasmic interaction in those lines affected fatty acid synthesis. Specifically, there was a demonstrated cytoplasmic influence on fatty acid synthesis such that the total stearic and palmitic acid content is less in seed of the sterile form (F) than in the paired maintainer line (G). The result is less total saturated fatty acids in the sterile line. This trait is inherited from generation to generation across different environments demonstrating that low total h `~
content of saturated fatty acids in these sunflower lines is genetically determined in a predictable and heritable manner.
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Table 2.
Entry Name Source Unsaturates% Saturates%
18:2 18:3 18~116:0 18:0 ;-60 8904W03F L8-91811kl4.3 0.6 88.1 4.8 2.2 61 8904W03G L8-92811kl3.0 0.6 87.9 3.5 5.0 62 8904W03F L8-91811k24.2 0.5 88.6 4.9 1.8 63 8904W03G L8-92811k22.9 0.6 88.2 3.8 4.5 ;
64 8904W03F L8-91811k34.6 0.5 88.1 4.9 2.0 65 8904W03G L8-92811k33.0 0.6 8~.4 3.7 5.3 66 8904W06F L8-91812kl0.2 0.2 88.9 4.6 2.1 67 8904W06G L8-92812kl0.4 0.1 86.1 3.8 6.9 68 890~W06F L8-91812k34.0 1.4 88.1 4.4 2.1 69 8904W06G L8-92812k33.2 0.3 80.7 5.1 5.1 ~ *
In the Woodland nursery, the sterile counterparts of 8904W03G and 8904W06G were crossed with restorer lines. That -hybrid seed was distributed to sunflower research stations for testing in California, France, and Argentina. Hybrids were grown, and bags were placed on heads prior to flowering to force self pollination of the hybrid and prevent contamina~
tion. Seeds from individual heads of those hybrids were analyzed for fatty acid composition. It was found that oil in ;
bulked seeds of individual heads from hybrids made with the female (male sterile) inbreds 89W04W03F and 89W04W06F was low ,J~ ' .
in total saturated fatty acids (Table 3.). The fact that individual heads had seeds whose oil in a bulk sample had low levels of saturated fatty acid demonstrated that such levels can be produced in a hybrid as well as parental inbred lines.
Moreover, the low total saturates were obtained in three different sunflower growing environments. It was demonstrated that these male sterile lines when used as female parents 2 1 2 ~ 6 2 ~

produced low saturated fatty acids in hybrids grown in typical sunflower crop production areas.
Table 3.
Result~ of fatty acid analyses of co~posite seed samples from individual self pollinated heads from hybrids ~ade using 8904W06F and 8904w03F ac the female parent grown in three environments.

Location Hybrid Head Sterile Unsaturate~% Saturates%
18:218:116:0 18:0 ~ ..
Montech, France 8W1070 Bulk 8904W06F 2.194.1 2.8 1.0 Woodland, 15 California :~
8W1070 1 8904W06F4.4 87.6 S .4 2.3 2 4.2 89.5 S.3 1.1 3 4.5 91.1 3.5 0.9 -4 7.0 88.2 3.6 1.2 3.8 90.8 4.5 0.9 ``
8W1075 1 8904W03F3.5 91.6 3.9 1.0 2 8.6 86.4 3.7 1.3 3 4.0 90.6 4.4 0.9 4 4.0 91.0 4.2 0.9 `~
25 Venado Tuerto, Argentina 8W1070 1 8904W06F10.4 80.04.1 5.1 ~ ` i 2 1.7 91.43.8 3.1 3 1.6 93.53.3 1.5 4 1.9 93.63.7 0.7 8W1075 1 8904W03F1.9 92.83.6 1.7 -~
2 1.7 94.23.2 0.9 3 2.2 93.43.4 1.0 4 2.3 93.53.4 1.0 ; ';

Standard Comparison 66.220.8 7.0 5.8 ~ ~

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Data from other fatty acid analyses of sunflower seed were reviewed. A number of restorer lines were found that had total saturated fatty acid levels of less than 5% (Table 4).

Table 4 -~
Summary of restorer lines found in fatty acid analysis screen~
ing that have less than 5% total saturated fatty acid levels.
LineSelection %16:0 ~18:0Total Sat %18:1 %18:2 9A4W005M 1 3.0 1.6 4.6 90.8 4.5 9A4W005M 6 3.2 1.4 4.6 91.4 4.0 9A4W005M 7 3.2 1.6 4.8 94.9 2.0 9A4W005M11 3.1 1.5 4.6 91.7 3.8 9B3W006M 1 2.8 1.8 4.6 92.7 2.7 9~3W006M 3 2.9 2.0 4.9 93.2 1.8 9B3W006M11 2.6 l.0 3.6 94.4 1.9 9B3W006M12 3.2 1.5 4.7 92.4 2.8 9E-RUN737 2 2.3 1.8 4.1 95.0 1.0 - ' ' "' S`~,,J,~' In the Hawaiian winter nursery hybrids were synthesized by crossing female sunflower lines having seeds with less than 6%
total saturated fatty acids with male (restorer) lines having seed with less than 6% total saturated fatty acids. Seeds ;
from the plants resulting from those crosses were planted at Woodland, CA and Moorhead, MN. At flowering, heads of those hybrids were bagged to assure self pollination. Heads were harvested and individual seeds from separate identity pre- :

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served heads were analyzed for fatty acid composition. T~
results are presented in Table 5.
Table 5 ~ ummary of total saturated fatty acid (SFA) levels in seeds of indivi~ual ~elf pollinated heads of hybrids grown in Woodl~nd, CA and Noorhea~, MN.
Lowest SFA~ Observation Heads ~eeds SFA~
~ybridLocation no. no. mean sinale seeds~nale head 9W1204Moorhead 5 25 4.8 3.9 4.1 Woodland 5 25 5.2 4.0 4.3 ~ ~:
9W1224Moorhead 5 25 4.5 4.0 4.2 Woodland 5 25 5.3 4.5 4.8 9W1234Moorhead 5 25 4.6 3.8 4.2 Woodland 5 23 5.6 4.8 5.2 9W1244Moorhead 3 15 4.4 4.0 4.1 ~ ` -Woodland 5 25 5.4 4.3 5.1 i 9W1284Moorhead 5 25 4.7 4.4 4.6 Woodland 5 24 5.9 3.9 4.8 6440Moorhead 5 25 10.4 9.0 9.8 Standard Woodland 5 25 10.5 8.8 10.5 The data in Table 5 demonstrate that inbred sunflower lines having less than 6% total saturated fatty acids combined ~-25 to produce hybrids whose seed had less that 6% total saturated -fatty acids in both the Woodland and Moorhead environments.
Individual heads were produced in both environments that had less than 5% total saturated fatty acids. In both environments individual seeds were also produced that had less -than 4~ total saturated fatty acids.
In order to validate the maternaL cytoplasmic effect on the level of saturated fatty acids in this discovery, female line 8904W06F with cytoplasm conferring low saturated fatty acid totals was crossed with another sunflower maintainer line PHA078. PHA078 is a proprietary inbred line having high oleic ~ --acid content. As expected, the progeny from the cross of - 12 ~
,., ~, ,. ~ ;,' ~., 8904W06F/PHA078 were sterile hybrids since PHA078 is a main-tainer line and lacks genes for male fertility restoration c 8904Wo6F. These male sterile plants were crossed with pollen from three other lines: PHA076, 9B3W006M, and PHA061. PHA076 and 9B3W006M are inbred lines having high oleic acid content. -~
PHA061 is an inbred line with normal fatty acid composition.
As a result of this cross, each seed from plants of 8904W06F/PHA078 could be classified as having the cytoplasm of 8904W06F and a nuclear genotype which is contributed 1/4 by 8904W06F, 1/4 PHA078, and 1/2 PHA076, 9B3W006M, or PHA061.
These seeds were analyzed for fatty acid composition. The data are presented in Table 6. ~;
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Table 6 8ummary of total saturated fatty acid levels tSFA%) in seeds with cytoplasmic background conferring low saturated fatty acid traits with nuclear parentage 75~ distinct from 8904W06F.

Seeds ~F~% %Fatty A¢ids Composition 20Cross no. mean 16:0 18:0 18:1 18:2 8904W06F*PHA078/PHA07610 3.80 2.91 0.89 93.78 2.77 Lowest SFA of cross 1 3.30 2.70 0.60 94.40 2.30 8904W06F*PHA078/9B3W006M 10 3.97 3.10 0.87 94.09 1.91 Lowest SFA of cross 1 3.40 2.70 0.70 95.30 1.40 8904W06F*PHA078/PHA06110 4.03 3.07 0.96 91.46 4.47 Lowest SFA of cross 1 3.40 2.50 0.90 93.90 2.70 .
The data from Table 6 demonstrate that even when the nuclear component of 8904W06F is diluted to 75% by four other lines (PHA078, PHA076, 9B3W006M, and PHA061) having either normal oil composition or higher oleic fatty acid composition, the cytoplasmic effect caused by the maternal parent 8904W06F
is maternally transmitted and the progeny have seeds with lower total saturated fatty acid content.

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Thus, while not intending to be limited by theory, it .
~ appears that the cytoplasm contains the factors which control for reduced saturated fatty acid levels. These cytoplasmic factors can be transferred to offspring when the plants .
according to this invention are used as the female parent in a cross with other sunflower cultivars, providing a method of ;~
reducing the content of saturated fatty acid moieties in any .
sunflower cultivar by conventional crossing and backcrossing.

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- 14 - ~ .`
~""'.'`''.' Deposits ~ ~-Applicants have made available to the public without restriction a deposit of at least 2500 seeds of a sunflower designated 890403F with the American Type Culture Collection ~ ~r~
(ATCC), Rockville, MD 20852, W.S.A., ATCC Deposit No.75180 .
The seeds deposited with the ATCC are taken from the same deposit maintained by Pioneer Hi-Bred International, Inc., 700 Capital Square, 400 Locust Street, Des Moines, Iowa, 50309, since prior to the filing date of this application. The deposit will be maintained without restrictions, at the ATCC
Depository, which is a public depository, for a period of 30 years, or five years after the most recent request, or for the effective life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period.

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Claims (35)

1. A sunflower plant having a cytoplasmically-inherited trait that reduces the percentage of total saturated fatty acids of the oil in an aggregate of its mature seeds.

2. The sunflower plant of claim 1 in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 10%.

3. The sunflower plant of claim 1 in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 5%.

4. The sunflower plant of claim 1 in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 3%.

5. The sunflower plant of claim 1 in which the percentage of total palmitic fatty acid of an aggregate of its mature seeds is less than 5%.

6. The sunflower plant of claim 1 in which the percentage of total palmitic fatty acid of an aggregate of its mature seeds is less than 3%.

7. The sunflower plant of claim 1 in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 6%.

8. The sunflower plant of claim 1 in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 3%.

9. The sunflower plant of claim 1 in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 1%.

10. A mature seed of the plant of claim 1.

11. A mature sunflower seed or sunflower seeds in which the percentage of total saturated fatty acids is reduced due to the presence of a cytoplasmically-inherited trait in the seed inherited from the plant producing such seed.

12. A sunflower seed product consisting of a substantially homogeneous assemblage of mature sunflower seeds in which the percentage of total saturated fatty acids is reduced due to a cytoplasmically-inherited trait.

13. A sunflower plant in which a cytoplasmically-inherited trait is present and causes formation of oil in its mature seeds, whereby the percentage of total saturated fatty acids of the oil is less than 10%.

14. A sunflower plant in which a cytoplasmically-inherited trait causes formation of oil in its mature seeds, whereby the percentage of total palmitic acid of the oil is less than 6%.

15. A sunflower plant in which a cytoplasmically-inherited trait causes formation of oil in its mature seeds, whereby the percentage of total stearic acid of the oil is less than 6%.

16. A sunflower plant in which a cytoplasmically-inherited trait causes formation of oil in its mature seeds, whereby the percentage of total stearic acid of the oil is less than 3%.

17. A sunflower plant in which a cytoplasmically-inherited trait causes formation of oil in its mature seeds, whereby the percentage of total stearic acid of the oil is less than 1%.

18. A sunflower plant produced by the method of sexually producing sunflower plants that form mature seeds having a percentage of total saturated fatty acids of less than 10%, comprising the step of growing a seed harvested from a pollinated sunflower plant having a cytoplasmically-inherited trait that reduces the percentage of total saturated fatty acids of the oil in an aggregate of its mature seeds.

19. A sunflower plant produced by the method of sexually producing sunflower plants that form mature seeds having a percentage of total palmitic acid of less than 6%, comprising the step of growing a seed harvested from a pollinated sunflower plant having a cytoplasmically-inherited trait that reduces the percentage of total saturated fatty acids of the oil in an aggregate of its mature seeds.

20. A sunflower plant produced by the method of sexually producing sunflower plants that form mature seeds having a percentage of total stearic acid of less than 6%, comprising the step of growing a seed harvested from a pollinated sunflower plant having a cytoplasmically-inherited trait that reduces the percentage of total saturated fatty acids of the oil in an aggregate of its mature seeds.

21. A seed of the sunflower plant of claim 18.

22. A seed of the sunflower plant of claim 19.

23. A seed of the sunflower plant of claim 20.

24. A sunflower plant in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 6%.

25. A sunflower plant in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 3%.

26. A sunflower plant in which the percentage of total palmitic acid of an aggregate of its mature seeds is less than 5% when the percentage of total stearic acid of an aggregate of its mature seeds is less than 3%.

27. A sunflower plant in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 6%.

28. A sunflower plant in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 3%.

29. A sunflower plant in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 1%.

30. A seed of the plant of claim 24.

31. A seed of the plant of claim 25.

32. A seed of the plant of claim 26.

33. A seed of the plant of claim 27.

34. A seed of the plant of claim 28.

35. A seed of the plant of claim 29.