CA2058849A1 - 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

Sunflower Products Having Lower Levels of Saturated Fatty Acids Thomas C. Heaton Glenn S. Cole Barry A. Martin ~ackqround 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 (Helianthus annuus L.) is a major worldwide source of vegetable oil. In the United States, approximately 4 million 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 develop-ment was the discovery of cytoplasmic male sterility and genesfor fertility restoration, a discovery that allowed the pro-duction of hybrid sunflowers. The hybrids thus produced were introduced during the early 1970's.
A description of cytoplasmic male sterility (CMS1 and genetic fertility restoration in sunflowers is presented by Fic~, "Breeding and Genetics", in Sunflower Science and Tech-noloqy 279-338 (J.F. Carter ed. 1978), the contents of which are incorporated herein by reference.
Sunflower oil is comprised primarily of palmitic, stearic, oleic, linoleic and linolenic acids. While other unusual . ., fatty acids exist in plants, palmitic, stearic, oleic, lino-leic, and linolenic acids comprise about 88% of the fatty 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 cholssterol 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 accordinq to recent studies. Satu~ated 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 desirablè 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: A 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 ., sunflower has emphasized generally the alteration of the per-centage of oleic or linoleic acids. The relative proportions of oleic 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 (19643; 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 (l5~i9L~Y~ annuus L. ~ Populations in a Uniform Environment," 10th International Sunflower Conference, March 14-18, 19~2. 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," ~p Science, 23: 1063-1068 (1983); Seiler, G.J., "Interrelation of Fatty Acids in Oil of Wild Annual Sunflower ( ~ annuus L. ) " Proceedinqs 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, K.I.,nChemical Mutagenesis in Sunflower Breeding, n ~nternational 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 Biosynthe~ls ~n Sunflower Seeds," Fizioloqiya Rastenii (Rugsian) 26: 1226-1232 (1979); Fick, G.N., "Breeding and Genetics," Sun10wer Science and Technoloqy, Carter, Jack F. ~ed.). 1978. Urie, A.L., '~Inheritance of Very High Oleic Acid Content in Sunflower, n Proc. Sunflower Research Workshop. 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 Association. Bismarck, ND. p. 10; Urie, A. L. ~Inheritance of High Oleic Acid in Sunflower." Crop Science 25:986-989 (1985); Simpson, B.W. and D. L. George, ~Potential for Selection of Fatty Acids on a Single Seed Basis in Sunflower (Helianthus annuus L.)." Proceedinas of the XI International 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." Proceedings 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." Proceedings o 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.

~u~mary 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 unique determinant is described in this application (see pages 6~
To summarize, originally, sunflower line VX9G was crossed with pollen sf 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 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 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 ~tearic acid levels are reduced. Thus, male ~terility i6 a convenisnt 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 sterile, or maintainer, tall or dwarf, high-yielding or low.
Any sunflower breeder skilled in the art provided the ~; - 4B -knowledge o~ a plant incorporating this trait and the present disclosure, would be enabled to make and utilize the instant invention.

In plants, the female and male parents contribute egual 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 the seed i5 borne i5 called the maternal parent or seed 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 Nt and cytoplasm Cr, 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 C~ and nuclear genotype Nm.

~ he classical method for the determination of cytoplasmic inheritance i8 to note whether there is a difference in the 25 trait in progeny from reciprocal crosses of two plant types.
In th~s 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. ~he 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 -- ~c --,,~'.~.

maternal line regardless of the nuclear genotype, then 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 content of the progeny was either 50% or 75% distinct from that of the original inbred carrying this trail. In Table 6, particuIarly, sterile hybrids were made using a plant designated 8904W06F (carrying the determinant) by crossing it to a maintainer P~A078 (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 genetlc 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 were used as the pollen donors. In other words, three new hybrids were made using the Fl 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.
~11 of the F2 proqeny produced seeds with low total saturated 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 sunflo~er lines, having seed with high content of linoleic or oleic fatty acid, have reduced saturated fatty acid content in 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 increase in unsaturated fatty acids. Coincident with decreased levels of saturated fatty acids in oil from seeds of plants of this invention, is a further increase in unsaturated fatty acids.

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 existing and yet to be discovered nuclear genetic variants offers unigue 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.

The present invention provides a new 6unflower which is true-breeding under a wide variety of growing condit$ons 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.

. - 4E --This invention provides a new sunflower plant that can be used efficiently to produce parent lines and hybrids possessing desira~le 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 from the 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, slnce 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.

,~y.
-~' Detailed Descri~tion 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 ~unflower 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 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.
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 literature. See, for example, the Fick article identified above.

Plant Breedina To produce the novel sunflower of the present invention 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 USS~ in the 1970's (Soldatov, 19763 with high levels of oleic acid. Similar breeding methods are described in Fernandez-Martinez, J., Dominguez-,~

Giminez, J. and Jiminez-Ramirez, A., Breeding for High Content of Oleic Acid in Sunflower (Helianthus annuus L.) Oil, Helia Nr. Scientific Bulletin of the F . A. O . Research Network on Sunflower 11-15 1988); Fick, G.N., Sunflower, Oil CroPs of S the World Ch. 14 pp 301-318 ~1989); Knowles, P. F. Genetics and Breeding of Oil Crops, Oil Cro~s 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 (F6) 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 ~ac~ground 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 sees 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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Table l.
Ungaturates Satur~te6 EntrY N~e Source % 18:2 % 18:1 ~ 16:0 ~ 18:0 2B 8904W04F BWB9-3 4-1 4.0 89.9 3.9 2.2 29 8904W04G 8WB9-4 4-1 3.9 ~6.2 3.5 6.4 32 8904W06F ~W~9-3 6-1 3.7 90.4 2.0 1.9 33 B904W06G 8W89-4 6-1 3.2 86.6 2.2 5.9 Pairs of sterile and maintainer ~ister lines from the Woodland nursery were fient to Kekaha, Hawaii for winter nursery breedi~g. Additional pairs of maintaincr and CMS
counterparts were crossed. Seeds from those crosses were analyzed. Once aqain the sterile forms of the maintainer 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 B904W03G (a sister line sclection of a904W05G and a904W06G) 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 ~O forms were less than that of the corresponding ma$ntainers 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 a~id synthesis such that the total ~tearic and palmitic acid content is less in seed of the ~terlle form (F) than in the paired maintainer line (G). The result is less total saturated fatty ac~ds in the sterile line. This trait is inherited from generation to generation across different environments demonstrating that low total content of saturated fatty acids in these sunflower lines is genetically determined in a predictable and herita~le manner.

2~.~8~9 Sable 2.
Entry N~-e Source Unsaturate~ 8atur~tec%
lB:2 18:3 18:116:0 18:0 B904W03F L8-9lBllkl4.3 0.6 88.1 4.8 2.2 61 8904W03G L8-92811kl3.0 0.6 87.9 3.5 5.0 62 B904W03F L8-91811k24.2 0.5 BB.6 4.9 1.8 63 B904W03G L8-92811k22.9 0.6 88.2 3.8 4.5 64 8904W03F L8-91811k34.6 0.5 B8.1 4.9 2.0 8904W03G L8-92Bllk33.0 0.6 87.4 3.7 5.3 66 8904W06F L8-91812kl0.2 0.2 88.9 4.6 2.1 67 8904W06G L8-92B12kl0.4 0.1 86.1 3.~ 6.9 68 8904W06F LB-9lBl2k34.0 1.4 88.1 4.4 2.1 6~ B904W06G L8-92012k33.2 0.3 80.~ 5.1 5.1 In the ~oodland nursery, the cterile counterpartg of ~904W03G and 8904W06G were cro6sed with ~c~torer lines. That hybrid 6eed was distributed to ~unflower re~e~rch ~tat$0n6 for te6ting in California, France, and Argentlna. Hybrlds were grown, and bags were placed on heads prior to flowering to force ~elf pollinatlon of the hybrlt and prevent contamlna-tion. Seeds from individual heads of tho-e hybrlds were analyzed for fatty acid composltion. ~t was found that oil in bulked seed~ of lndividual heads from hybrid~ made with the female (male ~terilc) inbreds B9W04W03F and B9W04W06F was low ln tot~l satur~ted fatty acids ~Table 3.). The fact that individual heads had seed6 who6e oil ~n a ~ulk ~ample had low levels of saturated fatty acid temonstrated that 6uch levels can be produced in a hybrid as well a6 parental inbred lines.
Moreover, the low total saturates were obtaincd in three dlfferent sunflower growing envlronments. It w~6 demon~trated that these male sterile lines when used a~ female parents 20.38~ ~9 produced low ~aturated fatty acids in hybrids grown ~n typical ~unflower crop production ~reas.
~able 3.
Re~ult~ of f~tty ncid analy~e~ of co-po~ite ~eed c~ple~ from ~ndividual ~elf poll~nated hend~ fro~ hybr~d~ ~nde u~ng B904~06r ~nd 8904wo3r ~ the fon~le paront gro~n ln three env~ron-ent~.

Loc~t~on Hybrid Bead Sterile Vn~aturnte~% S~turate~%
lB:2 18:116:018:0 ~ontech, France 8W1070 8ulk 8904WOÇF 2.194.12. B 1.0 Woodl and, C~lifornia 8W1070 18904W06F 4.487.65.4 2.3 2 4.2 B9.55.31.1 3 4,5 91.13.50.9 4 7.0 88.23.61.2 3.8 90.84.50.9 BW1075 18904W03F 3.591.63.9 1.0 2 8.6 86.43.71.3 3 4,0 90.64.40.9 4 4.0 91.04.20.9 Venado Tuerto, Argentina 8W1070 18904W06F 10.480.04.1 5.1 2 1.7 91.43.83.1 3 1.6 93.53.31.5 4 1.9 93.63.70.7 BW1075 18904W03F 1.992.83.6 1.7 2 1.7 94.23.2O.g 3 2.2 93.43.41.0 4 2.3 93.53.41.0 Standard Comparison 66.220.8 7.0 5.8 2Q~8~9 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).

T~ble 4 Su-mary of restorer lines found in fatty acid analy~is s~reen-~ng that have le~6 than 5% total 8aturated fatty ~c~d levels.
LineSelection ~16:0 ~18:0 ~otal Sat ~lB:l %1~:2 9A4W005M 1 3.01.6 4.6 90. a 4.5 9A4W005M 6 ~.21.4 4.6 91.4 4.0 9A4W005M 7 3. 2 .6 4.~ 9~.9 2.0 9A4WOOSM11 3.1~ 4.6 91.7 3.8 9~3W006M 1 2.'~ 4. 6 92. 7 2.7 9B3W006M 3 2. ) 4.9 93.2 1.8 9B3W006M11 2. 0 3. 6 94.4 1.9 9B3w006M12 3.~1.5 4.7 92.4 2.8 9E- J737 2 2.31.~ 4.1 95.0 1.0 in the Hawaiian winter nursery hy~rids 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-served heads were analyzed for fatty acid composition. The results are presented in Table 5.
Table 5 ~ ummary of total saturated fatty acid ~SFA) levels in seed~ of indivi~ual self pollinated head~ of hybrid~ grown in Woodland, CA and Moorhea~, MN.
Lowe~t 8FA% Observation Heads ~eed~ ~FA%
~vbrid~ocation no. no. m~an sinqle seed~ingle head 9W1204Moorhead 5 25 4.8 3.9 4.1 Wsodland 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 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 lO.S 8.8 10.5 The data in Table 5 demonstrate that inbred sunflower lines having less than 6% total saturated fatty acids combined to produce hybrids whose seed had less that 6% total ~aturated fatty acid~ 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 thi~ discovery, female line 8904W06F with cytoplasm conferring low saturated fatty acid totals was crossed with another sunflower maintainer line PHA078. P~A078 is a proprietary inbred line having high oleic acid content. As expected, the progeny from the cross of . ~

8904W06F/PHA078 were sterile hybrids since PHA078 is a main-tainer line and lacks genes for male fertility restoration of 8904W06F. These male sterile plants were crossed with pollen from three other lines: PHA076, 9B3W006M, and PHA061. PHA076 S 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.
The~e seeds were analyzed for fatty acid composition. The data are presented in Table 6.

~able 6 ~ummary of total saturated fatty a¢i~ level~ ~8FA%) in seeds wlth cytoplasmic backgroun~ conferring low s~turated f~tty acid tr~its w~th nuclear parentage 75~ di~tinct from 8904W06F.

8ee~ ~FA~ ~F~tty ACi~9 Compo~it~on 20Cross no. mean 16:0 18:0 18sl ~8:2 8904W06F*PHA078/PHA076 10 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*PHA078/9B3W006M10 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/PHA061 10 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 ~our 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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20~8~A9 Thus, while not intending to be limited by thcory, it appears that the cytoplasm contains the factors wh$ch eontrol for reduced ~aturated fatty acid levelr. There cytopla6mic factor6 can be transferred to off6pr$ng when the plant~
according to thi- $nv-ntion ar- ured ac the femal- par-nt in a cr9C~ W~ th other runflower cultlvarr, providing a aethod of r-ducing the content of ~aturated fatty acid moietlec ln any cunflower cultivar by convent$onal cro~ing and backcroc6ing 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 (ATCC), Rockville, MD 20852, W.S.A., ATCC Deposit No.
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.

Claims (68)

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

2. The cytoplasmically - inherited trait of claim 1 in which the percentage of total saturated fatty acids of an aggregate of the mature seeds is less than 10%.

3. The cytoplasmically - inherited trait of claim 1 in which the percentage of total saturated fatty acids of an aggregate of the mature seeds is less than 5%.

4. The cytoplasmically - inherited trait of claim 1 in which the percentage of total saturated fatty acids of an aggregate of the mature seeds is less than 3%.

5. The cytoplasmically - inherited trait of claim 1 in which the percentage of total palmitic fatty acid of an aggregate of the mature seeds is less than 5%.

6. The cytoplasmically - inherited trait of claim 1 in which the percentage of total palmitic fatty acid of an aggregate of the mature seeds is less than 3%.

7. The cytoplasmically - inherited trait of claim 1 in which the percentage of total stearic acid of an aggregate of the mature seeds is less than 6%.

8. The cytoplasmically - inherited trait of claim 1 in which the percentage of total stearic acid of an aggregate of the mature seeds is less than 3%.

9. The cytoplasmically - inherited trait of claim 1 in which the percentage of total stearic acid of an aggregate of the mature seeds is less than 1%.

10. The use of a cytoplasmically - inherited trait to reduce the percentage of total saturated fatty acids in an aggregate of mature seeds of a sunflower plant.

11. The use of a cytoplasmically - inherited trait of claim 10 in which the percentage of total saturated fatty acids of an aggregate of mature seeds is less than 10%.

12. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total saturated fatty acids of an aggregate of the mature seeds is less than 5%.

13. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total saturated fatty acids of an aggregate of the mature seeds is less than 3%.

14. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total palmitic fatty acid of an aggregate of the mature seeds is less than 5%.

15. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total palmitic fatty acid of an aggregate of the mature seeds is less than 3%.

16. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total stearic acid of an aggregate of the mature seeds is less than 6%.

17. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total stearic acid of an aggregate of the mature seeds is less than 3%.

18. The use of the cytoplasmically - inherited trait of claim 10 in which the percentage of total stearic acid of an aggregate of the mature seeds is less than 1%.

19. A 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.

20. A 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.

21. A 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.

22. Sunflower oil produced by crushing mature seed of 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.

23. Sunflower oil produced by crushing mature seed of a sunflower plant 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.

24. Sunflower oil produced by crushing 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.

25. Sunflower oil of claim 22 in which the percentage of total saturated fatty acids in an aggregate of the mature seeds is less than 10%.

26. Sunflower oil of claim 22 in which the percentage of total saturated fatty acids in an aggregate of the mature seeds is less than 5%.

27. Sunflower oil of claim 22 in which the percentage of total saturated fatty acids in an aggregate of the mature seeds is less than 3%.

28. Sunflower oil of claim 22 in which the percentage of total palmitic fatty acid in an aggregate of the mature seeds is less than 5%,

29. Sunflower oil of claim 22 in which the percentage of total palmitic fatty acid in an aggregate of the mature seeds is less than 3%.

30. Sunflower oil of claim 22 in which the percentage of total stearic acid in an aggregate of the mature seeds is less than 6%.

31. Sunflower oil of claim 22 in which the percentage of total stearic acid in an aggregate of the mature seeds is less than 3%.

32. Sunflower oil of claim 22 in which the percentage of total stearic acid in an aggregate of the mature seeds is less than 1%.

33. Sunflower oil of claim 22 in which the percentage of total palmitic acid of an aggregate of the mature seeds is less than 5% when the percentage of total stearic acid of an aggregate of the mature seeds is less than 3%.

34. 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.

35. The sunflower plant of claim 34 in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 10%.

36. The sunflower plant of claim 34 in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 5%.

37. The sunflower plant of claim 34 in which the percentage of total saturated fatty acids of an aggregate of its mature seeds is less than 3%.

38. The sunflower plant of claim 34 in which the percentage of total palmitic fatty acid of an aggregate of its mature seeds is less than 5%.

39. The sunflower plant of claim 34 in which the percentage of total palmitic fatty acid of an aggregate of its mature seeds is less than 3%.

40. The sunflower plant of claim 34 in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 6%.

41. The sunflower plant of claim 34 in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 3%.

42. The sunflower plant of claim 34 in which the percentage of total stearic acid of an aggregate of its mature seeds is less than 1%.

43. A mature seed of the plant of claim 34.

44. 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.

45. 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.

46. 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%.

47. 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%.

48. 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%.

49. 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%.

50. 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%.

51. 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.

52. 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.

53. 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.

54. A seed of the sunflower plant of claim 51.

55. A seed of the sunflower plant of claim 52.

56. A seed of the sunflower plant of claim 53.

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

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

59. 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%.

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

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

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

63. A seed of the plant of claim 57.

64. A seed of the plant of claim 58.

65. A seed of the plant of claim 59.

66. A seed of the plant of claim 60.

67. A seed of the plant of claim 61.

68. A seed of the plant of claim 62.

0969b/1-9