SEEDS, PLANTS AND OILS WITH ALTERED FATTY ACID PROFILES

Abstract: Seeds, plants and oils are provided having low FDA saturates; high oleic acid; low linoleic acid; high or low palmitic acid; low stearic acid; and low linoleic acid plus linolenic acid; and advantageous functional or nutritional properties.

Description: TITLE SEEDS, PLANTS AND OILS WITH ALTERED FATTY ACID PROFILES CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of U.S. Serial No. 07/575,542, filed August 30, 1990. TECHNICAL FIELD This invention relates to improved Brassica seeds, plants and oils having altered fatty acid profiles which provide advantageous nutritional or manufacturing properties. BACKGROUND OF THE INVENTION Diets high in saturated fats increase low density Hpoproteins (LDL) which mediate the deposition of cholesterol on blood vessels. High plasma levels of serum cholesterol are closely correlated with atherosclerosis and coronary heart disease (Conner et al., Coronary Heart Disease: Prevention, Complications, and Treatment, pp. 43-64, 1985). By producing oilseed Brassica varieties with reduced levels of individual and total saturated fats in the seed oil, oil-based food products which contain less saturated fats can be produced. Such products will benefit public health by reducing the incidence of atherosclerosis and coronary hear disease. The dietary effects of monounsaturated fats have also been shown to have dramatic effects on health. Oleic acid, the only monounsaturated fat in most edible vegetable oils, lowers LDL as effectively as Iinoleic acid, but does not affect high density Hpoproteins (HDL) levels (Mattson, F. H., J. Am. Diet. Assoc, ≤£:387-391, 1989; Mensin et al., New England J. Med., 321:436-441. 1989). Oleic acid is at least as effective in lowering plasma cholesterol as a diet low in fat an high in carbohydrates (Grundy, S. M., New England J. Med., 214:745-748, 1986; Mensink et al., New England J. Med., 221:436-441, 1989). In fact, a high oleic acid diet is preferable to low fat, high carbohydrate diets for diabetics (Garg et al., New England J. Med., 212:829-834, 1988). Diets high in monounsaturated fats are also correlated with reduced systoHc blood pressure (WilHams et al., J. Am. Med. Assoc., 252:3251-3256, 1987). Epidemiological studies have demonstrated that the "Mediterranean" diet, which is high in fat and monounsaturates, is not associated with coronary heart disease (Keys, A., Circulation, 44(Suppl):l. 1970). Many breeding studies have been conducted to improve the fatty acid profile of Brassica varieties. Pleines and Friedt, Fat Sci. Technol., 90(5), 167-171 (1988) describe plant lines with reduced Cιs : 3 levels (2.5- 5.8%) combined with high oleic content (73-79%). Rakow and McGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (Oct. 1973) discuss problems associated with selecting mutants for Iinoleic and linolenic acids. In Can. J. Plant Sci, 68, 509-511 (Apr. 1988) Stellar summer rape producing seed oil with 3% linolenic acid and 28% Iinoleic acid is disclosed. Roy and Tarr, Z. Pflanzenzuchtg, 95(3), 201-209 (1985) teaches transfer of genes through an interspecific cross from Brassica πran into Brassica napus resulting in a reconstituted line combining high Iinoleic with low linolenic acid content. Roy and Tarr, Plant Breeding, 98, 89-96 (1987) discuss prospects for development ofB. napus L. having improved linolenic and linolenic acid content. European Patent appHcation 323,751 pubHshed July 12, 1989 discloses seeds and oils having greater than 79% oleic acid combined with less than 3.5% linolenic acid. Canvin, Can. J. Botany, 43, 63-69 (1965) discusses the effect of temperature on the fatty acid composition of oils from several seed crops including rapeseed. Canola-quaHty oilseed Brassica varieties with reduced levels of saturated fatty acids in the seed oil could be used to produce food products which promote cardiovascular health. Canola lines which are individually low in palmitic and stearic acid content or low in combination will reduce the levels of saturated fatty acids. Similarly, Brassica varieties with increased monounsaturate levels in the seed oil, and products derived from such oil, would improve Hpid nutrition. Canola lines which are low in Iinoleic acid tend to have high oleic acid content, and can be used in the development of varieties having even higher oleic acid content. Increased palmitic acid content provides a functional improvement in food appHcations. Oils high in palmitic acid content are particularly useful in the formulation of margarines. Thus, there is a need for manufacturing purposes for oils high in palmitic acid content. Decreased alpha linolenic acid content provides a functional improvement in food appHcations. Oils which are low in linolenic acid have increased stabifity. The rate of oxidation of Hpid fatty acids increases with higher levels of linolenic acid leading to off-flavors and off- odor in foods. There is a need in the food industry for oils low in alpha Hnolenic acid. SUMMARY OF THE INVENTION The present invention comprises canola seeds, plant lines producing seeds, and plants producing seed, said seeds having a maximum content of FDA saturates of about 5.9% and a maximum erucic acid content of about 2% based upon total extractable oil and belonging to a line in which said saturates content has been stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds, canola oil having a maximum erucic acid content of about 2%, based upon total extractable oil, and a method of producing this canola oil comprising extraction from said seeds are additional aspects of this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having an FDA saturates content of from about 4.2% to about 5.9% based upon total extractable oil. The present invention further comprises Brassica seeds, plant Hnes producing seeds, and plants producing seeds, said seeds having a minimum oleic acid content of about 69% based upon total extractable oil and belonging to a line in which said oleic acid content has been stabilized for both the generation to which the seed belongs and its parent generation. A further aspect of this invention is such high oleic acid seeds additionally having a maximum erucic acid content of about 2% based upon total extractable oil. Progeny of said seeds; Brassica oil having 1) a minimum oleic acid content of about 69% or 2) a minimum oleic acid content of about 69% and a maximum erucic content of about 2%; and a method of producing these oils comprising extraction from said seeds are also included in this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having an oleic acid content of from about 69% to about 80%, and most preferred a minimum oleic acid content of about 71% based upon total extractable oil. The present invention further comprises canola seeds, plant lines producing seeds, and plants producing seeds, said seeds having a maximu Iinoleic acid content of about 14% and a maximum erucic acid content of about 2% based upon total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds, canola oil having a maximum Iinoleic acid content of about 14% and a maximum erucic acid content of about 2%, and a method of producing this canola oil comprising extraction from said seeds are additional aspects of this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having a Hnoleic acid content of from about 8.4% to about 14% based upon total extractable oil. The present invention further comprises Brassica seeds, plant lines producing seeds, and plants producing seeds, said seeds having a maximum palmitic acid content of about 3.5% and a maximum erucic acid content of about 2% based on total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds, canola having a maximum palmitic acid content of about 3.5% and a maximum erucic acid content of about 2%, and a method of producing this canola oil comprising extraction from said seeds are additional aspects of this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having a palmitic acid content of from about 2.7% to about 3.5% based upon total extractable oil. The present invention further comprises Brassica seeds, plant Hnes producing seeds, and plants producing seeds, said seeds having a mini u palmitic acid content of about 6.0% based upon total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. A further aspect of this invention is such high palmitic acid seeds additionally having a maximum erucic acid content of about 2% based upon total extractable oil. Progeny of said seeds; Brassica oil having 1) a minimum palmitic acid content of about 6.0%, or 2) a minimum palmitic acid content of about 6.0% and a maximum erucic acid content of about 2%; and methods of producing these oils comprising extraction from said seeds are also included in this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having a palmitic acid content of from about 6.0% to about 12.0% based upon total extractable oil. The present invention further comprises Brassica seeds, plant lines producing seeds, and plants producing seeds, said seeds having a maximum stearic acid content of about 1.1% based upon total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds have a canola oil having a maximum stearic acid content of about 1.1% and maximum erucic acid content of about 2%. Preferred are seeds, plant Hnes producing seeds, and plants producing seeds having a palmitic acid content of from about 0.8% to about 1.1% based on total extractable oil. The present invention further comprises Brassica seeds, plant Hnes producing seeds, and plants producing seeds, said seeds having a sum of Hnoleic acid content and linolenic acid content of a maximum of about 14% based upon total extractable oil and belonging to a line in which said acid content is stabiHzed for both the generation to which the seed belongs and its parent generation. Progeny of said seeds have a canola oil having a sum of Hnoleic acid content and linolenic acid content of a maximum of about 14% and a maximum erucic acid content of about 2%. Preferred are seeds, plant lines producing seeds, and plants producing seeds having a sum of Hnoleic acid content and linolenic acid content of about 12.5% based on total extractable oil. DETAILED DESCRIPTION OF THE INVENTION The U.S. Food and Drug Administration defines saturated fatty acids as the sum of lauric (Ci2:θ) > myristic (Ci4 ; o), palmitic (CIQ-Q) and stearic (Cιβ:θ) acids. The term "FDA saturates" as used herein means this above-defined sum. Unless total saturate content is specified, the saturated fatty acid values expressed here include only "FDA saturates". All percent fatty acids herein are percent by weight of the oil of which the fatty acid is a component. As used herein, a "line" is a group of plants that display Httle or no genetic variation between individuals for at least one trait. Such Hnes may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the term "variety" refers to a line which is used for commercial production. As used herein, "mutation" refers to a detectable and heritable genetic change not caused by segregation or genetic recombination. "Mutant" refers to an individual, or lineage of individuals, possessing a genetic mutation. The term "Mutagenesis" refers to the use of a mutagenic agent to induce random genetic mutations within a population of individuals. The treated population, or a subsequent generation of that population, is then screened for usable trait(s) that result from the mutations. A "population" is any group of individuals that share a common gene pool. As used herein "MQ" is untreated seed. As used herein, "Mj" is the seed {and resulting plants) exposed to a mutagenic agent, while "M2" is the progeny (seeds and plants) of self-pollinated Mj plants, "M3" is the progeny of self-pollinated M2 plants, and "M4" is the progeny of self-pollinated M3 plants. "M5" is the progeny of self- pollinated M4 plants. "Mg", "M7", etc. are each the progeny of self- pollinated plants of the previous generation. The term "progeny" as used herein means the plants and seeds of all subsequent generations resulting from a particular designated generation. The term "selfed" as used herein means pelf-pollinated. "StabiHty" or "stable" as used herein means that with respect to a given fatty add component, the component is maintained from generation to generation for at least two generations and preferably at least three generations at substantially the same level, e.g., preferably ± 15%, more preferably ± 10%, most preferably ± 5%. The method of invention is capable of creating Hnes with improved fatty add compositions stable up to ± 5% from generation to generation. The above stabiHty may be affected by temperature, location, stress and time of planting. Thus, comparison of fatty acid profiles should be made from seeds produced under similar growing conditions. StabiHty may be measured based on knowledge of prior generation. Intensive breeding has produced Brassica plants whose seed oil contains less than 2% erucic add. The same varieties have also been bred so that the defatted meal contains less than 30 μmol glucosinolates gram. "Canola" as used herein refers to plant variety seed or oil which contains less than 2% eruric add (C22:l » and meal with less than 30 μmol glucosinolates/gram. Seeds of Westar, a Canadian (Brassica napus) spring canola variety, were subject to chemical mutagenesis. Mutagenized seeds wer* » planted in the greenhouse and the plants were self-pollinated. The progeny plants were individually analyzed for fatty add composition, and regrown either in the greenhouse or in the field. After four successive generations of self-pollinations, foUowed by chemical analysis of the seed oil at each cycle, several Hnes were shown to carry stably inherited mutations in specific fatty acid components, including reduced palmitic add ( IQ- ), increased palmitic add, reduced stearic add (Ci8:θ) > increased oleic add (Ci8;l), reduced Hnoleic add (Ci8:2) and reduced linolenic add (Cιg : 3), in the seed oil. The general experimental scheme for developing Hnes with stable fatty add mutations is shown in Scheme I hereinafter. SCHEME I < Chemical analysis < Select mutants based on statistical analysis of control population < Grow out select mutants in greenhouse < Self-pollination < Chemical analysis < Select mutants based on statistical analysis of control population < Grow out select mutants in nursery < Self-pollination < Chemical analysis < Confirm altered fatty add < composition in selected lines STABLE FATTY ACID MUTANTS Westar seeds (MQ) were mutagenized with ethylmethanesulfonate (EMS). Westar is a registered Canadian spring variety with canola quaHty. The fatty add composition of field-grown Westar, 3.9% Cig.Q, 1.9% Cι 8:0 , 67.5% Cι 8: ι, 17.6% Cι 8: 2, 7.4% Cι 8: 3. <2% C20:l + C 2 2:l, has remained stable under commerrial production, with < ± 10% deviation, since 1982. The disclosed method may be appHed to all oilseed Brassica spedes, and to both Spring and Winter maturing types within each spedes. Physical mutagens, including but not limited to X-rays, UV rays, and other physical treatments which cause chromosome damage, and other chemical mutagens, including but not limited to ethidium bromide, nitrosoguanidine, diepoxybutane etc. may also be used to induce mutations. The mutagenesis treatment may also be appHed to other stages of plant development, including but not Hmited to cell cultures, embryos, microspores and shoot spices. The M seeds were planted in the greenhouse and Mi plants were individually self-pollinated. M£ seed was harvested from the greenhouse and planted in the field in a plant-to-row design. Each plot contained six rows, and five M2 Hnes were planted in each plot. Every other plot contained a row of non- mutagenized Westar as a control. Based on gas chromatographic analysis of M2 seed, those lines which had altered fatty add composition were self-pollinated and individuaUy harvested. M3 seeds were evaluated for mutations on the basis of a Z- distribution. An extremely stringent 1 in 10,000 rejection rate was employed to estabHsh statistical thresholds to distinguish mutation events from existing variation. Mean and standard deviation values were determined from the non-mutagenized Westar control population in the field. The upper and lower statistical thresholds for each fatty add were determined from the mean value of the population ± the standard deviation, multiplied by the Z-distribution. Based on a population size of 10,000, the confidence interval is 99.99%. Seeds (M3) from those M2 lines which exceeded either the upper or lower statistical thresholds were replanted in the greenhouse and self- pollinated. This planting also included Westar controls. The M4 seed was re-analyzed using new statistical thresholds estabHshed with a new control population. Those M4 Hnes which exceeded the new statistical thresholds for selected fatty add compositions were advanced to the nursery. FoUowing self-pollination, M5 seed from the field were re¬ analyzed once again for fatty add composition. Those lines which remained stable for the selected fatty adds were considered stable mutations. "Stable mutations" as used herein are defined as M5 or more advanced Hnes which maintain a selected altered fatty add profile for a minimum of three generations, including a minimum of two generations under field conditions, and exceeding estabHshed statistical thresholds for a minimum of two generations, as determined by gas chromatographic analysis of a minimum of 10 randomly selected seeds. Alternatively, stabiHty may be measured in the same way by comparing to subsequent generations. In subsequent generations, stability is defined as having similar fatty add profiles in the seed as that of the prior or subsequent generation when grown under substantially similar conditions. Mutation breeding has traditionally produced plants carrying, in addition to the trait of interest, multiple, deleterious traits, e.g., reduced plant vigor and reduced fertility. Such traits may indirectly affect fatty add composition, producing an unstable mutation; and/or reduce yield, thereby reducing the commerdal utiHty of the invention. A low mutagen treatment was selected so as to reduce the occurrence of deleterious traits. Other than changes in the fatty add composition of the seed oil, the mutant Hnes described here have normal plant phenotype when grown under field conditions, and are commercially useful. "Commerdal utiHty" is defined as having good plant vigor and high fertility, such that the crop can be produced by farmers using conventional farming equipment, and the oil with altered fatty acid composition can be extracted using conventional crushing and extraction equipment. To be commerrially useful, the yield, as measured by both seed weight, oil content, and total oil produced per acre, is within 15% of the average yield of the starting (MQ) canola variety (Westar) grown in the same region. The seeds of several different fatty add lines have been deposited with the American Type Culture Collection and have the following accession numbers. While the invention is susceptible to various modifications and alternative forms, certain specific embodiments thereof are described in the general methods and examples set forth below. For example the invention may be appHed to all Brassica spedes, including £. rapa. £. j-- ~ - - > ->.-. and B- Ϊ———1, to produce substantially similar results. It should be understood, however, that these examples are not intended to Hmit the invention to the particular forms disclosed but, instead the invention is to cover all modifications, equivalents and alternatives falHng within the scope of the invention. This includes the use of somaclonal variation; physical or chemical mutagenesis of plant parts; anther, microspore or ovary culture followed by chromosome doubling; or self- or cross- pollination to transmit the fatty add trait, alone in combination with other traits, to develop new Brassica Hnes. EXAMPLE 1 Selection pf FDA Satwates Prior to mutagenesis, 30,000 seeds of __[. napus cv. Westar seeds were preimbibed in 300-seed lots for two hours on wet filter paper to soften the seed coat. The preimbibed seeds were placed in 80 mM ethylmethanesulfoante (EMS) for four hours. FoUowing mutagenesis, the seeds were rinsed three times in distiHed water. The seeds were sown in 48-weU flats containing Pro-Mix. Sixty-eight percent of the mutagenized seed germinated. The plants were maintained at 25 β C/15°C, 14/10 hr day/night conditions in the greenhouse. At flowering, each plant was individually self-pollinated. M2 seed from individual plants were individually catalogued and stored, approximately 15,000 M2 lines was planted in a summer nursery in Carman, Manitoba. The seed from each selfed plant were planted in 3- meter rows with 6-inch row spacing. Westar was planted as the check variety. Selected lines in the field were selfed by bagging the main raceme of each plant. At maturity, the selfed plants were individuaHy harvested and seeds were catalogued and stored to ensure that the source of the seed was known. Self-pollinated M3 seed and Westar controls were analyzed in 10- seed bulk samples for fatty acid composition via gas chromatography. Statistical thresholds for each fatty add component were estabHshed using a Z-distribution with a stringency level of 1 in 10,000.. The selected M3 seeds were planted in the greenhouse along with Westar controls. The seed was sown in 4-inch pots containing Pro-Mix soil and the plants were maintained at 25°C/15°C, 14 10 hr day/night cycle in the greenhouse. At flowering, the terminal raceme was self-pollinated by bagging. At maturity, selfed M4 seed was individually harvested from each plant, labeHed, and stored to ensure that the source of the seed was known. The M4 seed was analyzed in 10-seed bulk samples. Statistical thresholds for each fatty add component were estabHshed from 259 control samples using a Z-distribution of 1 in 800. Selected M4 lines were planted in a field trial in Carman, Manitoba in 3-meter rows with 6- inch spacing. Ten M4 plants in each row were bagged for self-pollination. At maturity, the selfed plants were individually harvested and the open pollinated plants in the row were bulk harvested. The M5 seed from single plant selections was analyzed in 10-seed samples and the bulk row harvest in 50-seed samples. Selected M5 lines were planted in the greenhouse along with Westar controls. The seed was grown as previously described. At flowering the terminal raceme was self-pollinated by bagging. At maturity, selfed Mg seed was individually harvested from each plant and analyzed in 10 seed samples for fatty add composition. Selected Mg lines were entered into field trials in Eastern Idaho. The four trial locations were selected for the wide variabiHty in growing conditions. The locations included Burley, Tetonia, Lamont and SheUey (Table I). The lines were planted in four 3-meter rows with an 8-inch spacing, each plot was repHcated four times. The planting design was determined using a Randomized Complete Block Design. The commerdal cultivar Westar was used as a check cultivar. To determine the fatty acid profile of entries plants in each plot were bagged for self-polHnation. At maturity, the selfed plants were bulk harvested to determine yield. The M7 seed from single plants was analyzed in 10-seed samples. TABLE I Trial Locations for Selected Fatty Add Mutants. Yield of the entries in the trial was determined by taking the statistical average of the four repHcations. The Least Significant Difference Test was used to rank the entries in the randomized complete block design. For chemical analysis, 10-seed samples were hand ground with a glass rod in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 N KOH in 1:1 ether/methanol. The sample was vortexed for 30 sec and heated for 60 sec in a 60°C water bath. Four τάL of saturated NaCl and 2.4 ΠT. of iso-octane were added, and the mixture was vortexed again. After phase separation, 600 μL of the upper organic phase were pipetted into individual vials and stored under nitrogen at -5°C. One μL samples were injected into a Supelco SP-2330 fused siHca capiUary column (0.25 mm ID, 30 M length, 0.20 μm df). The gas chromatograph was set at 180°C for 5.5 minutes, then programmed for a 2°C/minute increase to 212°C, and held at this temperature for 1.5 minutes. Total run time was 23 minutes. Chromatography settings were: Column head pressure - 15 psi, Column flow (He) - 0.7 ml min, AuxiHary and Column flow - 33 mL min, Hydrogen flow -33 mL min, Air flow - 400 mL min, Injector temperature - 250°C, Detector temperature - 300°C, SpHt vent - 1/15. Table II describes the upper and lower statistical thresholds for each fatty add of interest. __________]! Statistical Thresholds for Specific Fatty Adds Derived From Control Westar Plantings Perςent Fatty Adds Genotype Cι : o Cig Ci8:l Cιs : 2 Ci8;3 Sats* M3 Generation (1 in 10,000 rejection rate) Lower 3.3 1.4 Upper 4.3 2.5 71.0 M4 Generation (1 in 800 rejection rate) Lower 3.6 0.8 Upper 6.3 3.1 76.0 M5 Generation (1 in 755 rejection rate) .wer 2.7 0.9 Upper 5.7 2.7 80.3 *Sats ■ Total Saturate Content At the M3 generation, twelve Hnes exceeded the lower statistical threshold for palmitic acid (≤3.3%). Line W13097.4 had 3.1% palmitic add and an FDA saturate content of 4.5%. After a cycle in the greenhouse, M4 seed from line W13097.4 (designated line A144) was analyzed. line W13097.4.1 (A144.1) had 3.1% C : Q, exceeding the lower statistical threshold of 3.6%. The FDA saturate content for A144.1 was 4.5%. The fatty add compositions for the M3, M4 and M5 generations of this family are summarized in Table IE. TAE E-IH Fatty Acid Composition of a Low Palmitic Acid/Low FDA Saturate Canola Line Produced bv Seed MutagreneBis Percent Fattv Acids Genotype 6 C 16:0 C 18:0 c 18:l c 18:2 c 18:3 Sats Tot Sat c Westar 3.9 1.9 67.5 17.6 7.4 5.9 7.0 W13097.4 • M 3 3.1 1.4 63.9 18.6 9.5 4.5 5.6 W13097.4 Q-4- 3.1 1.4 66.2 19.9 6.0 4.5 5.5 A144.1.9 Q-δ ) 2.9 1.4 64.3 20.7 7.3 4.4 5.3 a Letter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates and individual plant. b Sat B FDA Saturates °Tot Sat = Total Saturate Content The M5 seed often self-pollinated A144.1 (ATCC 40813) plants averaged 3.1% palmitic add and 4.7% FDA saturates. One selfed plant (A144.1.9) contained 2.9% palmitic acid and FDA saturates of 4.4%. Bulk seed analysis from open-pollinated (A144.1) plants at the M5 generation averaged 3.1% palmitic acid and 4.7% FDA saturates. The fatty add composition of the bulked and individual A144.1 lines are summarized in Table IV. Average of Individually Self-Pollinated Plants A144.1.1-10 3.1 1.6 64.8 20.1 6.9 4.7 5.7 Bulk Analysis of Open-Pollinated Plants A144.1B 3.1 1.6 64.8 19.4 7.8 4.7 5.7 a Letter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates and individual plant. b Sat = FDA Saturates C Tot Sat = Total Saturate Content These reduced levels have remained stable to the Mγ generations in both greenhouse and field conditions. These reduced levels have remained stable to the M7 generation in multiple location field trails. Over aU locations, the self-pollinated plants (A144) averaged 2.9% palmitic add and FDA saturates of 4.6%. The fatty add composition of the A144 Hnes for each Idaho location are summarized in Table V. In the multiple location repHcated trial the yield of A144 was not significantly different in yield from the parent cultivar Westar. By means of seed mutagenesis, the level of saturated fatty adds of canola (β. napus) was reduced from 5.9% to 4.6%. The palmitic add content was reduced from 3.9% to 2.9%. TABLE V Fatty ... d Composition of a Mutant Low Palmitic Acid/Low FDA Saturate Canola Line at Different Field Locations in Idaho Percent Fattv Acids Trial Location 16:0 C 18:0 C 18:l c 18:2 18:3 Satβ Tot Sats Burley 2.9 1.3 62.3 20.6 10.3 4.2 5.0 Tetonia 2.9 1.7 59.7 21.0 11.2 4.6 5.7 Lamont 3.1 1.8 63.2 19.5 9.0 4.9 5.9 Shelley 2.8 1.9 64.5 18.8 8.8 4.7 5.9 EXAMPLE 2 An additional low FDA saturate line, designated A149.3 (ATCC 40814), was also produced by the method of Example 1. A 50-seed bulk analysis of this line showed the foUowing fatty add composition: Cl6:0 " 3.6%, C 18; θ - 1.4%, Cι 8: l - 65.5%, Cι 8: 2 - 18.3%, c 18:3 - 8 - 2% » FDA Sats - 5.0%, Total Sats - 5.9%. This line has also stably maintained its mutant fatty acid composition to the M5 generation. In a multiple location repHcated trial the yield of A149 was not significantly different in yield from the parent cultivar Westar. EXAMPLE 3 An additional low palmitic add and low FDA saturate line, designated M3094.4 (ATCC 75023), was also produced by the method of Example 1. A 10-seed bulk analysis of this line showed the foUowing fatty add composition: Cιg :0 - 2.7%, Ci8:0 - 1-6%, Cι 8: ι - 66.6%, Cι 8: 2 - 20.0%, c 18:3 " 6 -l % » c 20:l " 1-4%, C22;l - 0.0%, FDA Saturate - 4.3%, Total Saturates - 5.2%. This line has stably maintained its mutant fatty add composition to the M5 generation. In a single repHcated trial the yield of M3094 was not significantly different in yield from the parent cultivar. EXAMPLE 4 In the studies of Example 1, at the M3 generation, 470 Hnes exceed the upper statistical threshold for palmitic acid (_> 4.3%). One M3 line, W14538.6, contained 9.2% palmitic add. Selfed progenies of this line, since designated M3007.4 (ATCC 75022), continued to exceed to the upper statistical threshold for high palmitic acid at both the M4 and M5 generations with palmitic add levels of 11.7% and 9.1%, respectively. The fatty add composition of this high palmitic add mutant, which was stable to the M7 generation under both field and greenhouse conditions, is summarized in Table VI. Fatty Add Composition of a High Palmitic Add Canola Line Produced bv Seed Mutagenesis Percent Fattv Adds Genotype Cιg : o Cιg :0 Ci8;i Cιg : 2 Cιg : 3 Sats Westar 3.9 1.9 67.5 17.6 7.4 7.0 W14538.6 8.6 1.6 56.4 20.3 9.5 10.2 (M3) M3007.2 11.7 2.1 57.2 18.2 5.1 13.9 (M 4 ) M3007.4 9.1 1.4 63.3 13.7 5.5 12.7 (M 5 ) SATS - Total Saturate Content An additional M3 line, W4773.7, contained 4.5% palmitic add. Selfed progenies of this line, since designated A200.7 (ATCC 40816), continued to exceed the upper statistical threshold for high palmitic add in both the M4 and M5 generations with palmitic add levels of 6.3% and 6.0%, respectively. The fatty acid composition of this high palmitic add mutant, which was stable to the M7 generation under both field and greenhouse conditions, is summarized in Table VII. TABLE Vπ Fatty Add Composition of a High Palmitic Add Canola Line Produced bv Seed Mutagenesis Percent Fattv Adds Genotype Ci6 : Q Ci8;0 Ci8;i Ci8 : 2 Cιs : 3 Sats* Westar 3.9 1.9 67.5 17.6 7.4 7.0 W4773.7 4.5 2.9 63.5 19.9 7.1 9.3 (M 3 ) W4773.7.7 6.3 2.6 59.3 20.5 5.6 10.8 (M ) A200.7.7 6.0 1.9 60.2 20.4 7.3 9.4 (M 5 ) *Sats = Total Saturate Content EXA PLE ? Selection of Low Stearic Acid Canola Lines In the studies of Example 1, at the M3 generation, 42 Hnes exceeded the lower statistical threshold for stearic add (< 1.4%). Line W14859.6 had 1.3% stearic add. At the M5 generation, its selfed progeny (M3052.1) continued to faU within the lower statistical threshold for Ci8 ; o with 0.9% stearic add. The fatty add composition of this low stearic add mutant, which was stable under both field and greenhouse conditions is summarized in Table VIII. TABLE Vm Fatty Add Composition of a Low Stearic Acid Canola line Produced bv Seed Mutagenesis Percent Fattv Adds Genotype Cι : Q Ci8:0 Cl8:l Cιs : 2 Ci8:3 Sats Westar 3.9 1.9 67.5 17.6 7.4 5.9 W14859.6 5.3 1.3 56.1 23.7 9.6 7.5 (M 3 ) M3052.1 (M 4 ) 4.9 0.9 58.9 22.7 9.3 5.8 M3052.6 (M 5 ) 4.4 0.8 62.1 21.2 7.9 5.2 EXAMPLE 6 High Oteic Acid C w?la Lfoes In the studies of Example 1, at the M3 generation, 31 Hnes exceede- 4 the upper statistical threshold for oleic acid _* 71.0%). Line W7608.b had 71.2% oleic acid. At the M4 generation, its selfed progeny (W7608.3.5, since designated A129. - ) continued to exceed the upper statistical threshold for Cιg ; ι with 78.8% oleic add. M5 seed of five self- pollinated plants of line A129.5 (ATCC 40811) averaged 75.0% oleic add. A single plant selection, A129.5.3 had 75.6% oleic add. The fatty add composition of this high oleic add mutant, which was stable under both field and greenhouse conditions to the M7 gereration, is summarized in Table IX his line also stably maintained its mutant fatty add composition to the M7 generation in field trials in multiple locations. Over all locations the self-poUinated plants (A129) averaged 78.3% oleic add. The fatty add composition of the A129 for each Idaho trial location are summarized in Table X. In multiple location repHcated yield trials, A129 was not significantly different in yield from the parent cultivar Westar. An additional high oleic add line, designated A128.3, was also produced by the disclosed method. A 50-seed bulk analysis of this line showed the foUowing fatty add composition: C : Q - 3.5%, Ci8;0 - 1.8%, Ci8;i - 77.3%, Ci8 : 2 - 9.0%, Cιs : 3 - 5.6%, FDA Sats - 5.3%, Total Sats - 6.4%. This line also stably maintained its mutant fatty acid composition to the M7 generation. In multiple locations repHcated yield trials, A128 was not significantly different in yield from the parent cultivar Westar. TABLE IX Fatty Add Composition of a High Oleic Add Canola Line Produced by Seed Mutagenesis Percent Fattv Add Genotype Cιg : o Ci8;0 18:l c 18:2 Ci8;3 Sats Westar 3.9 1.9 67.5 17.6 7.4 7.0 2.4 71.2 12.7 6.1 7.6 2.0 78.8 7.7 3.9 7.3 2.3 75.6 9.5 4.9 7.6 Sats = Total Saturate Content TAB ED Fatty Add Composition of a Mutant High Oleic A d Line at Different Field Locations in Idaho Percent Fattv Adds Location Cι :0 Cι 8: o Cι 8: i Cι 8: 2 Cι 8: 3 Sats Burley 3.3 2.1 77.5 8.1 6.0 6.5 Tetonia 3.5 3.4 77.8 6.5 4.7 8.5 Lamont 3.4 1.9 77.8 7.4 6.5 6.3 Shelley 3.3 2.6 80.0 5.7 4.5 7.7 Sats - Total Saturate Content An additional high oleic add Hne, designated M3028.-10 (ATCC 75026), was also produced by the disclosed method in Example 1. A 10- seed analysis of this Hne showed the following fatty add composition: Cl6:0 - 3.5%, Cι 8: θ - 1.8%, Cι 8: ι - 77.3%, Cι 8: 2 - 9.0%, Cι 8: 3 - 5.6%, FDA Saturates - 5.3%, Total Saturates - 6.4%. In a single location repHcated yield trial M3028.10 was not significantly different in yield from the parent cultivar Westar. EXAMPLE 7 Low Linoleic Acid Canola In the studies of Example 1, at the M3 generation, 80 lines exceeded the lower statistical threshold for linoleic acid (≤ 13.2%). line W12638.8 had 9.4% Hnoleic acid. At the M4 and M5 generations, its selfed progenies [W12638.8, since designated A133.1 (ATCC 40812)3 continued to exceed the statistical threshold for low Ci8;2 with Hnoleic add levels of 10.2% and 8.4%, respectively. The fatty add composition of this low linoleic add mutant, which was stable to the M7 generation under both field and greenhouse conditions, is summarized in Table XI. In multiple location repHcated yield trials, A133 was not significantly different in yield from the parent cultivar Westar. An additional low linoleic add line, designated M3062.8 (ATCC 75025), was also produced by the disclosed method. A 10-seed analysis of this Hne showed the foUowing fatty add composition: Cι : o - 3.8%, Ci8:0 - 2.3%, C18.1 - 77.1%, Ci8:2 - 8.9%, Cιs : 3 - 4.3%, FDA Sats - 6.1%. This line has also stably maintained its mutant fatty add composition in the field and greenhouse. TABLE XI Fatty Add Composition of a Low Linoleic Add Canola Line Produced bv Seed Mutagenesis Percent Fatty Adds Genotype* Cι :0 Ci8 : 0 Cι 8: i Cι 8:2 Ci8:3 Sats* Westar 3.9 1.9 67.5 17.6 7.4 7.0 W12638.8 3.9 2.3 75.0 9.4 6.1 7.5 (M 3 ) W12638.8.1 4.1 . 1.7 74.6 10.2 5.9 7.1 (M 4 ) A133.1.8 3.8 2.0 77.7 8.4 5.0 7.0 (M 5 ) a Letter and numbers up to second decimal point indicate the plant Hne. Number after second decimal point indicates an individual plant. D Sats = Total Saturate Content EXAMPLE S Low Linolenic and Linoleic Add Canola In the studies of Example 1, at the M3 generation, 57 lines exceeded the lower statistical -reshold for linolenic acid (≤ 5.3%). line W14749.8 had 5.3% linolenic add and 15.0% Hnoleic add. At the M4 and M5 generations, its selfed progenies [W14749.8, since designated M3032 (ATCC 75021)] continued to exceed the statistical threshold for low Ci8:3 with linolenic add levels of 2.7% and 2.3%, respectively, and for a low sum of linolenic and Hnoleic adds with totals of 11.8% and 12.5% respectively. The fatty add composition of this low linolenic add plus Hnoleic acid mutant, which was stable to the M5 generation under both field and greenhouse conditions, is summarized in Table XII. TABLE Xπ Fatty Add Composition of a Low Percent Fattv Adds Genotype Ci6 : o Cιg : o Ci8;l Ci8;2 Ci8:3 Sats Westar 3.9 1.9 67.5 17.6 7.4 7.0 2.5 69.4 15.0 5.3 6.5 2.4 77.9 9.1 2.7 6.4 2.8 80.0 10.2 2.3 6.5 Sats = Total Saturate Content

Claims: CL MS What is claimed is: 1. A seed comprising a canola variety having a maximum content of FDA saturates of about 5.9% based upon total extractable oU and belonging to a line in which said saturates content has been stabilized for both the generation to which the seed belongs and its parent generation. 2. A progeny of a seed of Claim 1. 3. A seed of Claim 1 in which the FDA saturates content is from about 4.2% to about 5.9% based upon total extractable oU. 4. A seed of Claim 1 which is Brassica napus. 5. A seed of Claim 1 deposited with the American T pe Culture CoUection and bearing accession number 40813 or 75023. 6. A plant Hne comprising a canola variety which produces seeds having a maximum content of FDA saturates of about 5.9% based upon total extractable oil and in which said saturates content is stabilized for both the generation to which the seed belongs and its parent generation. 7. A plant Hne of Claim 6 which produces seeds having an FDA saturates content of from about 4.2% to about 5.9% based upon total extractable oil. 8. A plant Hne of Claim 6 which is a species of Brassica napus. 9. A plant of the Hne of Claim 6. 10. A plant of the Hne of Claim 8. 11. An oil comprising a canola oil having a maximum content of FDA saturates of about 5.9%. 12. A canola oU derived from the seed of Claim 1. 13. A canola oil derived from the seed of Claim 4. 14. A method of producing a canola oil having a maximum content of about 5.9% FLA saturates comprising extracting oil from the seed of Claim 1. 15. A seed comprising the Brassica variety having a minimum oleic add content of about 69% based upon total extractable oil and belonging to a Hne in which said oleic acid content has been stabUized for both the generation to which the seed belongs and its parent generation. 16. A progeny of a seed of Claim 15. 17. A seed of Claim 15 in which the oleic add content is from about 69% to about 80% based upon total extractable oil. 18. A seed of Claim 15 having a minimum oleic add content of about 71%, based upon total extractable oU. 19. A seed of Claim 15 having a maximum erudc acid content of about 2% based upon total extractable oU. 20. A seed of Claim 15 which is Brassica napus. 21. A seed of Claim 15 deposited with the American Type Culture CoUection and bearing accession number 40811 or 75026. 22. A plant Hne comprising a Brassica variety which produces seeds hving a minimum oleic add content of about 69% based upon total extractable oU and in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 23. A plant line of Claim 22 which produces seeds having a minimum oleic add content of about 71%. 24. A plant Hne of Claim 22 which produces seeds having an oleic add content of from about 69% to about 80%. 25. A plant Hne of Claim 22 which produces seeds having a maximum erucic acid content of about 2% based upon total extractable oU. 26. A plant Hne of Claim 22 which is a species of Brassica ______l_. 27. A Brassica plant of the Hne of Claim 22. 28. A Brassica plant of the Hne of Claim 26. 29. An oU comprising a Brassica oU having a minimum oleic add content of about 69%. 30. A Brassica oU of Clai 29 having a maximum erudc add content of about 2%. 31. A Brassica oil derived from the seed of Claim 15. 32. A Brassica oil derived from the seed of Claim 19. 33. A Brassica oU derived from the seed of Claim 20. 34. A method of producing a Brassica oU having a minimum oleic add content of about 69% comprising extracting the oU from a seed of Claim 15. 35. A seed comprising a canola variety having a maximum linoleic add content of about 14% based upon total extractable oU and belonging to a line in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 36. A progeny of the seed of Claim 35. 37. A seed of Claim 35 in which the linoleic acid content is from about 8.4% to about 14% based upon total extractable oU. 38. A seed of Claim 35 which is a Brassica napus. 39. A seed of Claim 35 deposited with the American Type Culture CoUection bearing accession number 40812 or 75025. 40. A plant Hne comprising a canola variety which produces seeds having a linoleic add content of from about 8.4% to about 14% based upon total extractable oU and in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 41. A plant Hne of Claim 40 which produces seeds having a linoleic add content of from about 8.4% to about 14%. 42. A plant Hne of Claim 40 which is a species of Brassica ____jΩ__i. 43. A plant of the line of Claim 40. 44. A plant of the line of Claim 42. 45. An oU comprising a canola oil having a maximum linoleic add content of about 14%. 46. A canola oU derived from the seed of Claim 35. 47. A canola oU derived from the seed of Claim 38. 48. A method of producing a canola oU having a maximum linoleic add content of about 14% comprising extracting oU from a seed of Claim 35. 49. A seed comprising a Brassica variety having a minimum palmitic add content of about 6.0% based upon total extractable oU and belonging to a Hne in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 50. A progeny of the seed of Claim 49. 51. A seed of Claim 49 in which the palmitic add content is from about 6.0% to about 12.0% based upon total extractable oU. 52. A seed of Claim 49 having a maximum erudc acid content of about 2%, based upon total extractable oU. 53. A seed of Claim 49 which is a species Brassica napus. 54. A seed of Claim 49 deposited with the American Type Culture CoUection and bearing accession number 40816 or 75022. 55. A plant Hne comprising a Brassica variety which produces seeds having a minimum palmitic acid content of about 6.0% based upon total extractable oU and in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 56. A plant Hne of Claim 55 which produces seeds having a palmitic add content of from about 6.0% to about 12.0% based upon total extractable oU. 57. A plant Hne of Claim 55 which produces seeds having a maximum erucic acid content of about 2%, based upon total extractable oil. 58. A plant Hne of Claim 55 which is a species of Brassica BSΦW?- 59. A Brassica plant of the line of Claim 55. 60. A Brassica plant of the Hne of Claim 57. 61. A Brassica plant of the Hne of Claim 58. 62. An oU comprising a Brassica oU having a minimum palmitic add content of about 6.0%. 63. A Brassica oil of Claim 62 having a maximum erudc add content of about 2%. 64. A Brassica oU derived from the seed of Claim 49. 65. A Brassica oU derived from the seed of Claim 52. 66. A Brassica oU derived from the seed of Claim 53. 67. A method of producing a Brassica oil having a minimum palmitic add content of about 6.0% based upon total extractable oU comprising extracting oU from theseed of Claim 49. 68. A seed comprising the Brassica variety having a maximum palmitic add content of about 3.5% based upon total extractable oU and belonging to a line in which said palmitic add content has been stabilized for both the generation to which the seed belongs and its parent generation. 69. A progeny of a seed of Claim 68. 70. A seed of Claim 68 in which the palmitic add content is from about 2.7% to about 3.5% based upon total extractable oU. 71. A seed of Claim 68 having a maximum erudc acid content of about 2% based upon total extractable oU. 72. A seed of Claim 68 which is Brassica napus. 73. A seed of Claim 68 deposited with the American Type Culture CoUection and bearing accession number 40813 or 75023. 74. A plant Hne comprising a Brassica variety which produces seeds having a maximum palmitic add content of about 3.5% based upon total extractable oU and in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 75. A plant Hne of Claim 74 which produces seeds having a maximum palmitic add content of about 3.5%. 76. A plant Hne of Claim 74 which produces seeds having a palmitic add content of from about 2.7% to about 3.5%. 77. A plant Hne of Claim 74 which produces seeds having a maximum erudc acid content of about 2% based upon total extractable oil. 78. A plant line of Claim 74 which is a species of Brassica 79. A Brassica plant of the line of Claim 74. 80. A Brassica plant of the line of Claim 78. 81. An oU comprising a Brassica oU having a maximum palmitic add content of about 3.5%. 82. A Brassica oU of Claim 81 having a maximum erudc add content of about 2%. 83. A Brassica oU derived from the seed of Claim 68. 84. A Brassica oU derived from the seed of Claim 71. 85. A Brassica oil derived from the seed of Claim 72. 86. A method of producing a Brassica oil having a maximum pak tic add content of about 3.5% comprising extracting the oil from a seed of Claim 68. 87. A seed comprising the Brassica variety having a maximum stearic add content of about 1.1% based upon total extractable oU and belonging to a line in which said stearic add content has been stabilized for both the generation to which the seed belongs and its parent generation. 88. A progeny of a seed of Claim 87. 89. A seed of Claim 87 in which the stearic add content is from about 0.6% to about 1.1% based upon total extractable oU. • 90. A seed of Claim 87 having a maximum erudc acid content of about 2% based upon total extractable oU. 91. A seed of Claim 87 which is Brassica napus. 92. A seed of Claim 87 deposited with the American Type Culture CoUection and bearing accession number 75024. 93. A plant Hne comprising a Brassica variety which produces seeds hving a maximum stearic add content of about 1.1% based upon total extractable oU and in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 94. A plant Hne of Claim 93 which produces seeds having a maximum stearic add content of about 1.1%. 95. A plant Hne of Claim 93 which produces seeds having a stearic acid content of from about 0.8% to about 1.1%. 96. A plant Hne of Claim 93 which produces seeds having a maxim im erucic add content of about 2% based upon total extractable oU. 97. A plant Hne of Claim 93 which is a species of Brassica napus. 98. A Brassica plant of the Hne of Claim 93. 99. A Brassica plant of the Hne of Claim 97. 100. An oU comprising a Brassica oU having a maximum stearic add content of about 1.1%. 101. A Brassica oU of Claim 100 having a maximum erudc add content of about 2%. 102. A Brassica oU derived from the seed of Claim 87. 103. A Brassica oU derived from the seed of Claim 90. 104. A Brassica oU derived from the seed of Claim 91. 105. A method of producing a Brassica oU having a maximum stearic acid content of about 1.1% comprising extracting the oU from a seed of Claim 87. 106. A seed comprising the Brassica variety having a sum of linoleic add content and linolenic acid content of a maximum of about 14% based upon total extractable oil and belonging to a Hne in which said palmitic add content has been stabilized for both the generation to which the seed belongs and its parent generation. 107. A progeny of a seed of Claim 106. 108. A seed of Claim 106 in which the sum of Hnoleic add content and the linolenic add content is a Tnavi n of about 12.5% based upon total extractable oU. 109. A seed of Claim 106 having a maximum erudc acid content of about 2% based upon total extractable oU. 110. A seed of Claim 106 which is Brassica napus. 111. A seed of Claim 106 deposited with the American Type Culture CoUection and bearing accession number 75021. 112 A plant Hne comprising a Brassica variety which produces seeds having a sum of Hnoleic acid content and linolenic add content of a maximum of about 14% based upon total extractable oU and in which said add content is stabilized for both the generation to which the seed belongs and its parent generation. 113. A plant line of Claim 112 which produces seeds having a sum of Hnoleic acid content and linolenic add content of a maximum of about 12.5%. 114. A plant Hne of Claim 112 which produces seeds having a maximum erudc acid content of about 2% based upon total extractable oU. 115. A plant Hne of Claim 112 which is a species of Brassica napus. 116. A Brassica plant of the Hne of Claim 112. 117. A Brassica plant of the Hne of Claim 115. 118. An oU comprising a Brassica oU having a sum of Hnoleic add content and linolenic add content of a maximum of about 14%. 119. A Brassica oU of Claim 118 having a maximum erudc add content of about 2%. 120. A Brassica oU derived from the seed of Claim 106. 121. A Brassica oU derived from the seed of Claim 109. 122. A Brassica oU derived from the seed of Claim 110. 123. A method of producing a Brassica oil having a sum of Hnoleic add content and linolenic acid content of a maximum of about 14% comprising extracting the oU from a seed of Claim 106.

Inventors: DEBONTE LORIN ROGER, FAN ZHEGONG, LOTT WILLIE HSIAO-TSU, DEBONTE, LORIN, ROGER, FAN, ZHEGONG, LOTT, WILLIE, HSIAO-TSU
Applicants: DU PONT, E.I. DU PONT DE NEMOURS AND COMPANY
Application year: 1991
Main classification: A01H5
IPC codes: A01H5, A23D9, C11B1