AU642907B2 - Production of improved rapeseed exhibiting an enhanced oleic acid content - Google Patents

Description

WO 90/10380 PC/US89/00835 1 PRODUCTION OF IMPROVED RAPESEED EXHIBITING AN ENHANCED OLEIC ACID CONTENT packqround of the Invention Rape Brassica napus and Brassica campestris) is being grown as an increasingly important oilseed crop in many parts of the world. As a source of vegetable oil, it presently ranks behind only soybeans and palm and is virtually tied with sunflower for the number three position of commercial importance.

.The oil is used as both a cooking and salad oil throughout the world.

In its original form rapeseed oil was found to have deleterious effects on human health due to its relatively high level of eruqic acid which commonly is present in native cultivars in concentrations of 30 to percent by weight based upon the total fatty acid content. (In the past plant scientists identified a germplasm source of low erucic acid rapeseed oil and began incorporating this trait into commercial cultivars. See, Chapter 6 entitled "The Development of Improved Rapeseed Cultivars" by B.R. Stefansson from "High and Low Erucic Acid Rapeseed Oils" edited by John K.G. Kramer, Frank D. Sauer, and Wallace J. Pigden, Academic Press Canada (1983).

In Canada plant scientists focused their efforts on creating so-called "double-low" varieties which were low in erucic acid in the oil apd low in glucosinolates in the solid meal remaining after oil a extraction an erucic acid content of less than percent by weight baed upon the total fatty acid content, and a glucosinolate content of less than micromoles per gram of the oil-free meal). These higher quality forms of rape developed in Canada are known as canola. In contrast European scientists worked to achieve only "single-low" types which were low in erucic acid, but did not attempt to improve the WO 90/10380 PCT/US89/0035 -2quality of the solid meal which retained a glucosinolate content of about 100 micromoles per gram Of oil-free meal. The result of this major change in the fatty acid composition of rapeseed oil was to create an entirely new oil profile which often contained approximately 62 percent by weight of oleic acid based upon the total fatty acid content. Sincd the overall perceitage o oil in the seed did not change appreci1aly when the new low-erucic cultivars were developed, it appeared that the erucic acid had simply been redirected into other fatty acids with most becoming oleic acid. This\level of oleic-acid tended to vary within a fairly narrow range of approximately to 65 percent by weight based upon the total fatty acid content. See, Chapter 7 entitled "The Introduction of Low Erucic )cid Rapeseed Varieties Into Canadian Production" by J.X Daun from the previously identified Academic Press Canada (1983) publicataon.

The weight percent of other acids also varied somewhat, but not enough to define uniqe types wth the potential for distinct uses rr added commercial value. See, also "Prospects for the Development of Rapeseed naDus ith improved Linoleic and Linolenic Acid Content" by N.N. Roy and A.W. Tarr, Pant Breedin, Vol. 98, Pages 89 to 96 (1987).

At the presenttime, canola oil is being marketed by Procter Ga ble under the Puritan a trademark. Such vegetable oil typically is free of cholesterol, and the fatty acids present in it consist of approximately 6 percent saturated fatty acids in the form of stearic and palmitic acids, approximately 22 percent by weight linoleic acid which contains two double bonds per molecule of 18 carbon atoms, approximately 10 percent by weight alpha-linolenic acid WO 90/10380 PCr/US89/00835 -3which contains three double bonds per molecule of 18 carbon atoms, approximately 62 percent by weight oleic Sacid which contains a single double bond per molecule of 18 carbon atoms, and less than one percent byweight eidcic cid which contains a single double bond per molecule of/22 carbon atoms.

SOver the years scientists have attempted to improve the fatty acid profilE for canala oil. For example, the oxidative stability of tie vegetable oil is related to the number of double bonds in its fatty acids. That is molecules with sevsral double bonds are recognized to be more unstable. Thus, scientists have attempted to reduce the content of alpha-linolenic acid in order to improve shelf life-and oxidative stability, particularly under heat. This has not proved to be possible through the use of naturally occurring germplasm and the reported values for alpha-linolenic acid for such gernplasm have been greater than 6 percent by weight greater than 6 up to approximately 12 percent by weight). As reported by Gerhard R6bbelen in Chapter 10 entitled "Changes and Limitations of Breeding for Improved Polyenic Fatty Acids Content in Rapeseed" from "Biotechnology for the Oils and Fats Industry" edited by Colin Ratledge, Peter Dawson, and James Rattray, American Oil Chemists' Society (1984), a mutagenesis experiment was able to ,achieve lines with less than approximately 3.5 percent by weight of alpha-linolenic acid based upon the total, fatty acid content. The profiles of these lines indicate that nearly all of the alpha-linolenic fatty acid was being directed to linoleic acid and that the levels of oleic acid increased only one or two percent.

Nevertheless the oil appeared to offer some advantages over normal canola oil. For instance, t e raefining WO 90/10380 P~/US9/00835 -4process required less hydrogenation than normal canola oil and it exhibited a superior fry life.

In recent years studies have established the value of monounsaturated fatty acids as a dietary constituent. This has led to the popularization of the "Mediterranean Diet," with its emphasis on olive oil, a naturally occurring high source of oleic acid. Such a diet is thought to avoid the problem of arteriosclerosis that results from saturated fatty acids. Even in this diet, however, olive oil is thought to be less than ideal, due to its level of saturates. Canola oil is potentially a superior dietary oil, since it contains approximately one-half the saturated fats as olive oil, and since its relatively high levels of alpha-linolenic acid which are deleterious to shelf life and oxidative stability may be a benefit from a dietary point of view. Alphalinolenic acid is believed to be a precursor for the synthesis by the body of a family of chemicals which may reduce risk from cardiovascular diseases.

It is recognized in the literature that the oleic acid content of canola varies slightly with the environment, temperature, and the moisture availability when the rapeseed is formed. As previously indicated the oleic acid content of available canola cultivars commonly is approximatey 55 to 65 percent by weight.

See, for instance, Table V at Page 171 from Chapter 7 entitled "The Introduction of Low Erucic Acid Rapeseed Varieties Into Canadian Production" by J.K. Daun appearing in "High and Low Erucic Acid Rapeseed Oils", Academic Press Canada (1983). As reported in the same article, rape varieties which possess greater concentrations of erucic acid will possess even lower oleic acid contents.

WO 90/10380 PCUS89/00835 Occasionally higher oleic acid contents have been mentioned but have not been made available to the rapeseed grower. For instance, at Page 23 of proceedings of the 7th International Rapeseed Congress held at Poznan, Poland, on May 11 to 14, 1987, passing reference is reported to a canola sample having an oleic content of /9.0 percent under a given set of growing conditions and oleic acid content of '74 percent under different growing conditions. This plant was said to be produced by recurrent selection while employing unidentified parent plants. This is a nonenabling disclosure which will not place the reader of this publication in possession of a rape plant which produces rapeseeds having the increased oleic acid content.

As reported in United States Patent Nos..

4,517,763; 4,658,084; and 4,658,085; and the publications identified therein, hybridization processes suitable for the production of rapeseed are known.

It is an object of the present invention to provide a substantially uniform assemblage of improved rapeseeds which yield a vegetable oil of increased stability, It is an object of the present invention to provide a substantially uniform assemblage of improved rapeseeds which yield a vegetable oil of increased stability at elevated temperatures which particularly is suited for the deep-frying of food products for humni consumption.

It is an object of the present invention to provide a substantially uniform assemblage of improved rapeseeds which yield a vegetable oil which possesses a WO 90/10380 PCr/US89/00835 -6higher oleic acid content than heretofore available in combination with other desirable traits.

It is an object of the present invention to provide in a preferred embodiment a substantially uniform assemblage of improved rapeseeds which yield a vegetable oil which possesses a higher oleic acid content in combination with a lower alpha-linolenic acid content than heretofore available.

It is another object of the present invention iO to provide a substantially uniform stand of rape plants capable upon self-pollination of forming rapeseeds which yield a vegetable oil of increased stability.

It is another object of the present invention to provide a substantially uniform stand of rape plants capable upon self-pollination of forming rapeseeds which yield a vegetable oil of increased stability which particularly is suited for the deap-frying of food products for human consumption.

It is another object of the present invention to provide a substantially uniform stand of rape plants capable upon self-pollination of forming rapeseeds which yield a vegetable oil which possesses a higher oleic acid content than heretofore available in combination with other desirable traits.

It is another object of the present invention to provide in a preferred embodiment a substantially uniform stand of rape plants capable upon selfpollination of forming rapeseeds which yield a vegetable oil which possesses a higher oleic acid content in combination with a lower alpha-linolenic acid content than heretofore available.

It is another object of the present invention to provide an improved vegetable oil derived from rapeseeds.

WO 90110380 PCT/US89/00835 -7- It is a further object of the present invention to provide a method for increasing the oleic acid content of rapeseeds and to thereby improve the stability of the vegetable oil derived therefrom.

These and other--bjects and advantages of the invention will be apparent to those skilled in the art from a reading of the following description and appended claims.

Summary of the Invention A substantially homogeneous assemblage of mature rapeseeds is provided, wherein the rapeseeds are capable of yielding a vegetable oil of increased stability when exposed to heat, having an unusually high oleic acid content of at least 79 percent by weight based upon the total fatty acid content, an erucic acid content of no more than 2.0 percent by weight based on the total fatty acid content, and a glucosinolate content in the solid component of less than 100 micromoles per gram.

A substantially uniform stand of rape plants is provided which upon self-pollination are capable of forming rapeseeds which yield a vegetable oil of increased stability when exposed to heat, wherein the rapeseeds have an unusually high oleic acid content of at least 79 percent by weight based upon the total fatty acid content, an erucic acid content of no more than'2.0 perent by weight based on the total fatty acid content, and a glucosinolate content in the solid component of less than 100 micromoles per gram.

An improved vegetable oil derived from rapeseeds of increased stability when exposed to heat,/- said rapeseeds having an unusually high oleic acid 1:1

Z**

WO 90/10380 PCT/US89/00835 -8content of at least 79 percent by weight based upon the total fatty acid content, an erucic acid content of no more than: 2.0 percent by weight based upon the total fatty ;acid content, and\(3) an alpha-linolenic acid content less than 5 percent by weight based upon the total fatty acid content.

It has been found that a method of enhancing the oleic acid content of rapeseeds comprises: subjecting in at least one generation cells derived from a rapeseed plant which forms rapeseeds having an oleic acid content of less than 79 percent by weight based on the total fatty acid content to a technique selected from the group consisting of gamma irradiation, contact with a chemical mutagen, and a combination of the foregoing, in order to induce mutagenesis, regenerating said cells to produce a rape plant and to form rapeseed in at least one generation subsequent to that of step selecting a rapeseed produced in step which has an oleic acid content of at least 79 percent by weight based upon the total fatty acid content, producing a rape plant on the basis of said selection of step and self-pollinating the rape plant of step (dy for a sufficient number of generations to achieve substantial genetic homogeneity and to form rapeseeds which contain at least 79 WO 90/10380 PCT/US89/00835 -9percent oleic acid by weight based upon the total fatty acid content.

Description of Preferred Embodiments Heretofore available rapeseed plants, whether Brassica napus or Brassica camDestris, have formed rapeseeds which possess an oleic acid content of well under 79 percent by weight based upon the total fatty acid content. For the purposes of the present invention the oleic acid content of a given rapeseed is determined by a standard procedure wherein the oil is removed from the rapeseeds by crushing the seeds and is extracted as a methyl ester following reaction with methanol and sodium hydroxide. Next the resulting ester is a7alyzed for fatty acid content by gas liquid chromatography using a capillary column which allows separatio on the basis of the degree of unsaturation and chain length. This analysis procedure is described in the work of J.K. Daun et al Amer. Oil Chem. Soc. 60: 1751-1754 (1983) which is herein incorporated by reference. The higher quality canola varieties of rapeseed which \re available for commercial planting commonly possess an oleic acid content of no more than 65 percent by weight based upon Sthe total fatty acid content. Accordingly, a need has persisted in the past for improved canola varieties which exhibit a significantly higher oleic aid content.

In accordance with the concept of the present invention one preferably selects plant cells capable of regeneration seeds, microspores, ovules, vegetative parts) from any of the canola varieties which ara re &gnized to have superior agronomic characteristics. Such plant cells may be derived from "T WO 90/10380 PC/US89/00835 Brassica naPus or Brassica campestris plants. The Brassica napus plants may be of either the summer or winter yes. The plant cells derived from a rapeseed plant Wich forms rapeseeds which possess an oleic acid content of less than 79 percent by weight based upon the total fatty acid content next are subjected in at least one generation to mutagenesis, a rape plant is regenerated from the cells to produce a rape plant and to form rapeseed in at least one subsequent generation, rapeseed is selected having an oleic acid content of at least 79 percent by weight based upon the total fatty acid content, and agape plant is produced on the basis of this selction which is self-pollinated for a sufficient number of generations 2 to 8 additional generations) to achieve substantial genetic homogeneity and to form rapeseeds thereon which contain at least' 79 percent oleic acid by weight based upon the total weight of fatty acids present. The plant cells which are subjected to mutagenesis also commonly are from plants which form rapeseeds having an alphalinolenic content of greater than 5.0 percent by weight greater than 3.5 percent by weight), and selection concurrently is made for a reduced alphalinolenic acid content.

The mutagenesis preferably is carried out by subjecting the plant cells a rapeseed) to a technique selected from the group consisting of gamma irradiation, contact with a chemical mutagen, and a combination of the foregoing, for a sufficient duration to accomplish the desired increase in oleic acid content (and preferably also the desired decrease in alpha-linolenic acid content) via a genetic modification but insufficient to destroy the viability of the cells and their ability to be regenerated into a WO 90/10380 PC/US89/00835 -11plant. A rapeseed preferably possesses a moisture content of approximately 5 to 6 percent by weight at the time of such mutagenesis. The mutagenesis "preferably is carried out by gamma radiation, such as that supplied by a Cesium 137 source. The gamma radiation preferably is supplied to the plant cells q a rapeseed) in a dosage of approximately 60 to 200 K ad., and most preferably in a dosage of approximately 60 to 90 Krad. It should be understood that ev n when operating at radiation dosages within the ranges specified, some plant cells rapeseeds\) will lose their viability and must be discarded\ The desired mutagenesis may be accomplished by use of chemical means such as by contact with ethylmethylsulfonate, ethyinitrosourea, etc., and by the use of physiail means rsch as x-ray, etc.

It will be appreciated that the mutagenesis treatment will result in a wide variety of genetic changes within the rape plants which are produced.

Many of these changes ill be deleterious to the Sviability of the resuliing plant over an extended period of time. Spme changes also will produce viable plants which possess deficient agronomic characteristics. Sich off-types may be simply discarded. Howaver if desired planth which have undergone mutation wibh respect to oleic acid production coupled with undesirable agronomic traits can be retained and used as breeding or source material from which plants having the targeted trait coupled with satisfactory agronomic characteristics are derived.

Following mutagenesis rape plants are regenerated from the treated cells using known techniques. For instance, the resulting rapeseeds may WO 900380 PCT/US89/00835 -12be, planted in accordaice with conventional rape growing procedures and following self-pollination rapeseed is formed thereon. Alternatively, doubled haploid piantlets may be extracted. The planting of the treated rapeseed preferably is carried out in a greenhouse in which the pollination is carefully controlled and monitored. Additional rapeseed which is formed as a result of such self-pollination in the present or a stbsequent generation is harvested and is subjected to analysis for oleic acid content. Since Brassica naus and Brassica eampestris are dicotyledons, the analysis for oleic acid can be carried out on a halfseed, i nd the remaining halfseec-, can be retained for possible future ger*ination if the oleic acid content is found to be favorable as a result of the mutagenesis. The rapeseeds can be carefully separated into two halfseeds using known techniques.

When a mature halfseed is found to possess an oleic content of at least 79 percent by weight (preferably at least 80 percent by weight), it is selected and retained. The oleic acid content of such selection preferably will be 79 to 90 percent by weight 80 to 85 percent by weight).

The other halfseed fi.e., cotyledon) which will be ge etically the same as the halfseed which was subjected to halfseed analysis can next be caused to germinate and a rape plant is formed and allowed to undergo self-pollination. Such planting of the halfseed preferably also is carried out in a greenhouse in which the pollination is carefully controlled and.monitored. The resulting rapeseed is harvested, planted, and is self-pollinated for a sufficient number of generations to achieve substantial genetic homogeneity. The genetic WO 90/10380 P(rUS9/00835 0 -13stabilization of the rape plant material enables the creation of plants having a reasonably predictable genotype which can be used as breeding or source material for the production of other improved rape varieties, as a finished variety for use by the rpeseed grower, or as a parent in the production of hybrid rapeseed with the high oleic iAlid content being transferred to the progeny.

The resulting rapeseeds also are selected so that they possess the erucic acid and glucosinolate contents of canola. More specifically, the erucic acid content is no more than 2.0 percent by weight based upon the total fatty acid content, and preferably less than 0.1 percent by weight less than 0.05 perct by weight) -based on the total fatty acid content, and the gluconsinolate content in the solid component is less than 100 micromoles per gram (preferably less than 30 micromoles per gram). The glucosinolate content may be any one or a mixture of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2hydroxy-3-butenyl glucosinolate, and 2-hydroxy-4pentenyl gluconsinolate. The gluconsinolate determination preferably is made on the air-dry-oilfree solid as measured by the gas liquid chromatograph method of the Canadian Grain Commission. The erucic acid and glucosinolate levels commonly are made possible by selecting starting materials which already possess highly desirable levels of these components.

In a preferred embodiment wherein the vegetable oil is intended for frying applications the resulting rapeseeds also are selected which have an alphalinolenic acid content less than 5 percent by weight based upon the total fatty acid content preferably no more than 3.5 percent by weight based WO 90/10380 PCr/US89/0035 -14upon the total fatty acid content). Also, in a preferred embodiment the vegetable oil contains no more than 7 percent by weight of saturated fatty acids in the form of stearic and palmitic acids based upon the total fatty acid content 6 to 7 percent by weight).

The desired traits described herein unusually high oleic acid content) once established can be readily transferred into other plants within the same Brassica nanus or Brassica campestris species by conventional plant breeding techniques involving crosspollination and selection of the progeny.. It has been demonstrated that the characteristics are highly heritable, cankbe-transmitted to their progeny, and can be recovered in segregating progeny in subsequent generations following crossing. Also, once established the desired traits can be transferred between the ~aus.

and campestris species using the same conventional plant breeding techniques involving pollen transfer and selection. The transfer of other traits, such as low erucic cid content, between the napus and camDestris species by standard plant breeding techniques is already well documented in the technical literature.

See, for instance, Brassica Crops and Wild Allies Biology and Breeding, edited by S. Tsunada, K. Hinata, and Gomez Campo, Japan Scientific Press, Tokyo (1980).

As an example of the transfer of thedesired traits described herein unusually high oleic acid content) from napus to campetris, one may select a commercially available camestris variety such as Tobin, Horizon, or Colt and carry out an interspecific cross with an appropriate plant of the napus breeding lines discussed hereafter FA677-39, Topas H6-90 and FA677M5-132). Alternatively, other naus breeding WO 90/10380 PCT/US89/00835 lines may be reliably and independently developed when following the mutagenesis techniques described herein.

The Tobin variety is available from Agriculture Canada, Saskatoon, Saskatchewan, and other distributors. The Horizon and Colt varieties are available from Bonis Company Ltd. of Lindsay, Ontario, Canada. Following the interspecific cross, members of the F 1 generation are self-pollinated to produce F 2 seed. Selection for the desired traits unusually high oleic acid content) is then conducted on single F 2 seeds which are Stheri backcrossed with the camDestris parent through the number of generations required to obtain a euploid campestris line exhibiting the desired traits unusually high oleic acid content).

In accordance with the concept of the present invention the rapeseeds possessing the specified combination of characteristics are multiplied to form a substantially uniform assemblage of such seeds a bag of such seeds) which can be used to produce a substantially uniform stand of such rape plants. The rapeseeds present in such assemblage number at least 250 seeds, and the resulting substantially uniform stand of rape plants numbers at least 250 plants.

The improved vegetable oil of the present invention may be formed by simple extraction in a direct manner from the mature rapeseeds such as by crushing and extraction in accordance with known techniques. See, for example, Chapter 8 entitled "Rapeseed Crushing and Extraction" by D.H.C. Beach appearing in "High and Low Erucic Acid Rapeseed Oils," Academic Press Canada (1983) which is herein incorporated by reference. In a preferred embodiment the vegetable oil is present in a quantity convenient WO 90/10380 PCF/US89/00835 -16for commercial or domestic use a quantity of at least one liter).

The theory whereby the mutagenesis has been found to be capable of increasing the oleic acid content to such high levelsiIn rape is considered to be complex and incapable of simple explanation. For instance, the mutation may adversely impact upon the formation of one or more enzymes which normally would function in dehydrogenation of the fatty acids as the seeds mature. The following Examples are presented as a specific illustrations of the claimed invention. It should be understood, however, that the invention is not limited to the specific details set forth in the Examples.

Example' I Seeds of the Regent variety of Brassica napus were selected as the starting material. This variety of canola is of th summer type and is suitable to produce vegetable oil when grown in the north central region of the United States, the western prairie area of Canada, and other areas where summer rape is S adapted. The Regent variety was first introduced in 1977 by the University of Manitoba. Planting seed for the Regent variety is-available from the Department of Plant Science of the University of Manitoba. A representative sample (ija., 2.0 grams) of the mature seeds of the starting material prior to subjection to gamma radiation (as described hereafter) contained the following fatty acids in the approximate concentrations indicated based upon the total weight of the fatty acids present while using gas liquid chromatography analysis technique previously described: WO 90/10380 PCiUS89/00835 i -17- Number of Number of Carbon Atoms Double Bonds Weight SFatty Acid Per Molecule Per Molecule Percent Palmitic 16 0 4.8 Palmitoleic 16 1 0.1 Stearic 18 0 1.6 Oleic 18 1 65.4 Linoleic 18 2 19.3 Alpha-Linolenic 18 3 6.9 Arachidic 20 0 0.6 Eicoseroic 20 1 Behenic 22 0 0.3 Erucic 22 1 nondetectable The gluconsinolate content in the solid component was 13.44 micromoles per gram as determined by the gas liquid chromatograph method of the Canadian Grain Commission.

Prior to gamma irradiation the seeds of the Regent variety of canola were stored under conditions so as to maintain viability. More specifically, the seeds were stored in a cold storage room maintained at approximately 10'C. and 40 percent relative humidity, and contained a moisture content of approximately percent by weight following air drying.

Seeds of the Regent variety approximately 10 grams) next were placed in a Gammacell 1000 gamma irradiation apparatus manufactured by Atomic Energy of Canada, Ltd. where they were subjected Krad. of irradiation produced by a Cesium 137 source at WO 90/10380 PCT/US89/IU835 -18a rate of 26.61 Krad. per hour in order induce mutagenesis. These seeds can be termed Ml seeds.

The Ml seeds following subjection to gamma irradiation were planted in a greenhouse at Georgetown,) Ontario, Canada, having a day temperature of approximately 25 and a night temperature of approximately 18*C. Approximately 40 percent of the gamma irradiated seeds produced fertile rape plants which upon self-pollination yielded M2 seeds. The M2 seeds were next planted in the field at the same location to produce plants which following pollination produced M3 seeds.

Representative M3 seeds produced on the M2 plants next were soaked in water and one cotyledon from each seed was carefully removed for the analysis of its fatty acid composition using the gas liquid chromatography analysis technique previously described, Such halfseed analysis was carried out in accordance with the procedure of "Methods for Breeding for Oil Quality in Rape" by R.K. Downey and B.L. Harvey reported in Canadian Journal _of Plant Science, Vol. 43, Pages 271 to 275 (1963) which is herein incorporated by reference. From a total of 4490 cotyledon analyses from M3 plants, 37 cotyledons were determined to contain an increased oleic acid content within the range of 70.2 to 76 percent based upon the total fatty acid content, and an alpha-linolenic acid content within the range of 5.4 to 13.1 percent based upon the total fatty acid content.

An M3 halfseed designated as FA 677 was selected which was found to contain thi following fatty acids in the concentratiins indicated based upon the total weight of fatty acids present: WO 90/10380 PCT/US89/00835 -19- Number of Number of Carbon Atoms Double Bonds Weight Fatty Acid Per Molecule Per Molecule Percent -almitic 16 0 Palmitoleic 16 1 nondetectable Stearic 18 0 1.4 Oleic 18 1 70.9 Linoleic 18 2 10.7 Alpha- 18 3 11.0 Linolenic Arachidic 20 0 Eicosenoic 20 1 1.2 Behenic 22 0 0.3 Erucic 22 1 nondetectable All the M3 halfseeds including FA 677 were planted in the greenhouse and were caused to undergo self-pollination and to form the M4 generation. Each of these plants produced sufficient seed for random 57 seed samples from each plant t.9' crushed and analyzed by gas liquid chromatography. iWvan these representativ 50 seed samples from the M4 generation were analyzed, it was found that the oleic acid content ranged from 63 to 80 percent by weight based upon the total fatty acid content, and the alpha-linolenic acid content ranged from 3.2 to 7.7 percent by weight based upon the total fatty acid content. The single plant designated FA 677 was found to have the highest oleic WO 90/10380 PCT/US89/00835 acid content 80 percent). Sixty-five seeds from this plant were planted to grow the M5 generation. For reference purposes, 50 of these 65 seeds were also subjected to cotyledon analysis, which revealed oleic acid levels ranging from 74.0 to 85.0 percent by weight based upon the total fatty acid content. The cotyledon analysis-derived profile of the best plant (85.0 percent oleic acid) is shown in the table below: Number of Number of Carbon Atoms Double Bonds Weight Fatty Acid per Molecule Per Molecule Percent Palmitic 16 0 3.2 Palmitoleic 16 1 0.2 Stearic 18 0 2.4 Oleic 18 1 85.0 Linoleic 18 2 4.4 Alpha- 18 3 2.7 Linolenic Arachidic I\20 0 Eicosenoic 20 1 1.2 Behenic 22 0 non- ,detectable Erucic 2 1 nondetectable The specific FA 677 plant identified above which produced the oleic acid content of 85 percent b )weight based upon the total fatty acid content was Zost in the next generation through raceme break"z in the greenhouse. However, when a 50 seed sample from a W 'e 90110380 PC/US89/00835 i j; -21sister plant in the M5 generation, designated FA 677- 39, was analyzed after crushing, the fbllowing fatty acids were observed in the concentrations indicated based upon the total weight of the fatty acids present.

The plants resulting from the germination of the seeds exhibited a substantially uniform phenotype.

Number of Carbon Atoms Per Molecule Number of Double Bonds Per Molecule Fatty Acid Wet;et Myristic Palmitic Palmitoleic Stearic Oleic Linoleic Alpha- Linolenic Arachidic Eicosenoic Behenic Erucic Lignoceric 4.1 0.3 1.6 79.2 7.1 4.6 0.7 0.06 0.3 The gluconsinolate content in the solid component was 10.94 micromoles per gram. The plants produced from the seed are true-breeding upon self-pollination and exhibit a substantially uniform phenotype.

Significantly, the average oleic acid content of all of the samples analyzed (65 samples, 50 seeds per sample) NO 90/10380 PCT/US89/00835 -22was 77.1 percent by weight based upon the total fatty acid content indicating a stably high oleic acid level in all descendents of the plant designated FA 677.

Further selections using the FA677-39 breeding line can result in the identification of ,1_plants exhibiting even higher oleic acid contents.

These plants can be preserved and multiplied using conventional techniques.

The increased oleic acid content renders the rapeseeds capable of providing a vegetable oil of increased stability when exposed to heat. Accordingly, the resulting oil can reliably be used for food-frying applications for a more extended period of time without deleterious results when compared to the canola oil of the prior art. Also, the reduced alpha-linolenic acid content of the resulting vegetable oil imparts enhanced oxidative stability to the same.

Comparable rapeseed seeds of the generation designated FA 677-39 have been deposited under the Budapest Treaty in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, on December 31, 1987. This seed deposit has received Accession No. 40409, and will be made available upon the maturation of this application into a patent. However, the avaiiibility of these seeds is not to be construed as a icense to practice this invention in contravention of the rights granted under the authority of any government in accordance with its patent or breeder's rights laws.

Example I Twelve selections from the M5 generation (discussed with respect to EIxample I) having the 0 highest oleic acid contents were subjected to further WO 90/10380 PCr/US89/00835 -23mutagenesis while employing a chemical mutagen. More specifically, two composite seed lots r fthese selections were formed consisting of 1,000 seeds each and were treated with ethylnitrosourea. The ethylnitrosourea was present in a dimethylsulfoxide solvent at a concentration of 8 mM. (millimoles).

During the preparation of the ethylnitrosourea solution 25 ml. of dimethylsulfoxide were added to one gram of ethylnitrosourea and the resulting solution was buffered at a pH of 5.5 with 5mM. (millimoles) of morpholinoethanesufonic acid. Each seed lot was placed in a large petri dish and 30 ml. of the resulting solution were added. The seeds while in contact with the ethylnitrosourea solution were incubated in the dark at 20*C. for 18 hours, were rinsed three times with distilled water, and were planted in flats present in a greenhouse containing a soilless greenhouse, growing media. 500 seeds were planted per flat.

Approximately 30 percent of the seeds which were treated with the ethylnitrosourea solution grew into plants, the plants were transplanted into pots, the potted plants were grown in a greenhouse, and approximately 25 percent of these exhibited sufficient fertility to undergo self-pollination and to produce seed the M2 generation following mutagenesis f while employing a chemical mutagen).

Seeds (A 2.he M2 generation) were next harvested from 153 plants (jiL., the M1 plants). Ten seeds from each of the plants were individually analyzed by the halfseed analysis previously described.

A total of 276 cotyledon selections were obtained having an oleic acid level of 77 percent by weight or W)ghrz based upon the total fatty acid content. Three o these selections were found to contain an ,leic acid WO 90/10380 PCT/US89/00835 -24content of 84 percent by weight based upon the total fatty acid content.

The remaining cotyledons from-the 276 selections were planted in a greenhouse at Georgetown, Ontario/ Canada having a day temperature of =--appro;mately 25 3C. and a night temperature of approximately 18"C., plants were formed, and seeds were formed as the result of self-pollination the M3 generation following mutagenesis while employing a chemical mutagen). A selection designated FA677M5- 132 in the M3 generation was found to exhibit while using two random 50 secd bulk analyses an oleic acid content of 81.9 percent by weight based upon the total fatty acid content, an alpha-linolenic acid content of 4.03 percent by weight based upon the total acid content, a nor.detectible erucic acid content, a saturated fatty acid content of 6.59 percent by weight in the form of stearic and palmitic acids based upon the total fatty acid content, and a glucosinolate content in the solid component of less than micromoles er gram. The plants resulting from the germination of the seeds exhibited a substantially uniform phenotype. Further selections from within the FA677 -132 breeding line will result in the identification of plants exhibiting even higher oleic acid contents. These plants can be preserved and multi'lied using conventional techniques. For instace, whn analyzing single cotyledons of this breed.g lin, oleic acid contents higher than percen~by weight based upon the total fatty-acid content have been observed.

Comparable rapeseeds of the M3 generation designated FA677M5-132 have been deposited under the Bj-dapest Treaty in the American Type Culture WO 90/10380 PCT/US89/00835 Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, on December 13, 1988. This seed deposit has received Accession No. 40523, and will be made available upon the maturation of this application into a patent. However, the availability of these seeds is not to be construed as a license to practice this invention in contravention of the eights granted under the authority of any government in accordance with its patent or breeder's rights laws.

Example ilI Seeds of Topas variety of Brassic nDu uR were selected as the starting material. This variety of canola-is of the summer type and is suitable to produce vegetable oil when grown in the United States, Canada, Sweden, and other areas where summer -ape is adapted. The Topas variety was registered in I)87 by Svaldof AB of Sweden. Planting seed for this variety is available from Bonis Company Ltd. _f Lindsay, Ontario, Canada. This variety typiclly exhibits an 2) oleic acid content of approximately 65 percent by weight based upon ?he total fatty acid content and an alpha-linolenic acid content of appro imately 8 percent by weight based upon thr fotal fatty acid content.

Ten thousand seeds of the Topas variety were subjected to mutagenesls hle employing a chemical mutagen. More specifically, seed lots were formed consisting of 1,000 seeds eaih and were treated with ethylnitrosourea as previously described. The resulting seeds were planted in flats present in a greenhouse containing a silcless greenhouse growing media. 500 seeds were planted in each flat and can be termed Ml seeds.

WO 90/10380 PCT/US89/00835 -26- Seeds the M2 generation) were formed as the result of self-pollination on 111 surviving fertile plants the Ml plants). One of the M2 seeds when subjected to cotyledon analysis exhibited on oleic acid content of 82.07 percent by weight and an alpha-linoleniic acid content of 5.12 percent by weight based,upon the total fatty acid content. The remaining M2 cotyledon was planted in a greenhouse at Georgetown, Ontario, Canada, having a day temperature of approximately 25 3'C. and a night temperature of approximately 18*C., a plant was formed, and seeds were formed as the result of self-pollination the M3 generation). This M3 generation was designated Topas H6-90 and was found to exhibit while using two random 50 seed bulk analyses an oleic acid content of 81.17 percent by weight based upon the total fatty acid content, an alpha-linolenic acid content of percent by weight based upon the total fatty acid content, a non-detectible erucic acid content, a saturated fatty acid content of 6.17 percent by weight in the form of stearic and palmitic acids based upon the total fatty acid content, and a glucosinolate content in the solid component of less than micromoles per gram. The plants rosulting from the germination of the seeds exhibited a substantially uniform phenotype. Further selections from within the Topas H6-90 breeding line (as indicated hereafter) will result in the identification of plants exhibiting even higher oleic acid contents. These plants can be preserved and multiplied using conventional techniques.

Comparable rapeseeds of the M3 generation designated Topas H6-90 have been deposited under the Budapest Treaty in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland WO 90/10380 PCUS89/00835 -27- 20852, on December 13, 1988. This seed deposit has received Accession No. 40524, and will be made available upon the maturation of this application into a patent. However, the availability of these seeds is not to be construed as a license to practice this invention in contravention of the rights granted under the authority of any government in accordance with its patent or breeder's rights law. Further selection within the M3 generation of Topas H6-90 identified a selection identified as Topas H6-90-99 containing the following fatty acids in the approximate concentrations indicated based upon the total fatty acids present while using the same gas liquid chromatography analysis technique previously described: WO 90/10380 PCT/US89!00835 -28- Number of Number of Carbon Atoms Double Bonds Weight Fatty Acid Per Molecule Per Molecule Percent Palmitic 16 0 3.57 Palmitoleic 16 1 0.31 Stearic 18 0 1.87 Oleic 18 1 85.84 Linoleic 18 2 3.54 Alpha- 18 3 2.68 Linolenic Arachidic 20 0 0.49 Eicosenoic 20 1 1.29 Behenic 22 0 0.32 Erucic 22 1 nondetectable Lignoceric 24 0 0.07 Seeds produced from H6-90-99 will continue to exhibit a glucosinolate content in the solid component of less than 30 micromoles per gram.

Although the invention has been described with preferred embodiments, it is to be understood that variations and modifications may be resorted to as will be apparent to those skilled in the art. Such variations and modifications are to be corsidered within the purview and scope of the claims appended hereto.

Claims (14)

1. 2. A substantially homogeneous assemblage of seeds according'to Claim 1 wherein said rapeseeds were formed on Brassica napus plants.
2. 3. A substantially homogeneous assemblage of rapeseeds according to Claim 1 which have an endogenous oil which exhibits following crushing and extraction an unusually high oleic acid content of to 90 percent by weight based upon the total fatty acid content.
3. 4. A substantially homogeneous assemblage of rapeseeds according to Claim 1 which additionally 0 have an endogenous oil which exhibits following crushing and extraction an alpha-linolenic acid S) SUBSTUTOtiEE ^yi iKfAbS PFCT/ 89/00835 ,.Io 3 MAY 9 content of less than 5 percent by weight based upon the total fatty acid content. A substantially homogeneous assemblage of rapeseeds according to Claim 1 which additionally have an endogenous oil which exhibits following crushing and extraction an alpha-linolenic acid content of no more than 3.5 percent by weight based upon the total fatty acid content.
4. 6. A substantially uniform stand of rape plants which upon self-pollination are capable of forming rapeseeds which yield an endogenous vegetable oil of increased stability when exposed to heat, said rapeseeds having an oil which exhibits following crushing and extraction an unusually high oleic acid content of at least 80 percent by weight based upon the total fatty acid content wherein said oleic acid content is reliably controlled by genetic means for the expression of suc, trait resulting from a mutation induced by man followed by selection, and an oil which exhibits following crushing and extraction a erucic acid content of no more than percent by weight based on the total fatty acid content, and a glucosinolate content in the solid component following crushing and extraction of less than 100 micromoles per gram.
5. 7. A substantially uniform stand of rape plants according to Claim 6 wherein said plants are Brassica naDus.
6. 8. A substantially uniform stand of rape Splants according to Claim 6 wherein said rapeseeds SSUBSTUTSHEET AoUFiS PC>. 89/0 083 -31- have an endogenous-oil which exhibits following crushing and extraction an unusually high oleic acid content of 80 to 90 percent by weight based upon the total fatty acid content.
7. 9. A substantially uniform stand of rape plants according to Claim 6 wherein said rapeseeds additionally have an endogenous oil which exhibits following crushing an extraction an alpha-linolenic acid content of less than 5 percent by weight based upon the total fatty acid content. A substantially uniform stand of rape plants according to Claim 6 wherein said rapeseeds additionally have an endogenous oil which exhibits following crushing and extraction an alpha-linolenic acid content of no more than 3.5 percent by weight based upon the total fatty acid content.
8. 11. A substantially uniform stand of pe plants according to Claim 6 wherein said unu ally high oleic acid content of said rapeseeds s the result of a mutation induced by man f owed by selection.
9. 12. An improved dogenous vegetable oil of increased stability in exposed to heat extracted from rapeseeds forme on said rape plants of step (d) of Claim 16, sai rapeseeds having and oil which exhibits fol ing crushing and extraction an unusually igh oleic acid content of a least perce by weight based upon the total fatty acid co ent, an oil which exhibits following crushIng and extraction a erucic acid content of no more tlban tIMiM I .AU 11. An improved endogenous vegetable oil of increased stability when exposed to heat extracted from rapeseeds formed on said rape plants of step (d) of claim 16 (hereinbelow), said rapeseeds having an oil which exhibits following crushing and extraction an unusually high oleic acid content of at least percent by weight based upon the total fatty acid content, an oil which exhibits following crushing and extraction a erucic acid content of no more than percent by weight based on the total fatty acid content, and an oil which exhibits following crushing and extraction an alpha-linolenic acid content of less than 5 percent by weight based upon the total fatty acid content. 12. Ah improved vegetable oil extracted from rapeseeds according to claim 11 wherein said rapeseeds were formed on Brassica napus plants.
10. 13. An improved vegetable oil extracted from rapeseeds according to claim 11 wherein said rapeseeds have an endogenous oil which exhibits following crushing and extraction an unusually high oleic acid content of 80 to 90 percent by weight based upon the total fatty acid content.
11. 14. An improved vegetable oil extracted from rapeseeds according to claim 11 wherein said rapeseeds have an endogenous oil which exhibits following crushing and extraction an alpha-linolenic acid content of no more than percent by weight based upon the total fatty acid content.
12. 15. A method for enhancing the oleic acid content of rapeseeds comprising: subjecting in at least one generation cells d7rived from a rapeseed plant which forms rapeseeds having an endogenous oleic acid content in the oil of less than 80 percent by weight based on the total fatty acid content and an erucic acid content of no more than 2.0 percent by weight based on the total fatty acid content to a technique selected from the group consisting of gamma irradiation, contact with a chemical mutagen, and a combination of the foregoing, in order to induce mutagenesis with respect to the increased production of oleic acid, regenerating said cells to produce a rape plant and to form rapeseeds in at least one generation subsequent to that of step i(c) selecting a rapeseed produced in step which has an endogenous oleic acid content in the oil of at least 80 percent by weight based upon the total fatty acid content, and an erucic acid content of no more than percent by weight based on the total fatty acid content, and producing rape plants in a subsequent generation derived from said selection of step having substantial genetic homogeneity and forming rapeseeds thereon which contain an endogenous oil which exhibits following crushing and extraction an oleic acid content of at least percent by weight based upon the total fatty acid content wherein said oleic acid content and an erucic acid content of no more than 2.0 percent by weight based on the total fatty acid content is reliably controlled by genetic means for the expression of such trait resulting from such mutagenesis.
13. 16. A method for enhancing the oleic acid content of rapeseeds according to Sclaim 15 wherein said cells derived from a rapeseed plant in step are present S-in the form of a rapeseed seed. S S SCTI 89/0083 IP"' n 3 MAYM -34- A method for enhancing the oleic acid ,ontent of rapeseeds according to Claim 16 wherein during step said cells are present in the form of a rapeseed seed having a moisture content of approximately 5 to 6 percent by weight and are subjected to approximately 60 to 200 Krad. of gamma irradiation. t. A method for enhancing the oleic acid content of rapeseeds according to Claim 16 wherein during step said cells are present in the form of a rapeseed seed having a moisture content of approximately 5 to 6 percent by weight and are subjected to approximately 60 to 90 Krad. of gamma irradiation. A method for enhancing the oleic acid content of rapeseeds according to Claim 16 wherein said mutagenesis is carried out at least in part by contact with a chemical mutagen. o A method for enhancing the oleic acid content of rapeseeds according to Claim 16 wherein said starting material of step is present in the form of a rapeseed seed which additionally has an endogenous alpha-linplenic acid content in the oil greater than 5 percent by weight following crushing and extraction based upon the total fatty acid content, said step includes a selection which additionally has in the oil an alpha-linolenic acid content following crushing and extraction of less than 5 percent by weight, and the product of step (d) t A t II.tWOS I PCTI 89/0083 0 3 MAYS" nV wir additionally has in the oil an content following crushing and than 5 percent by weight. alpha-linolenic acid extraction of less 0. A method for enhancing the oleic acid content of rapeseeds according to Claim 16 wherein said starting material of step is present in the form of a rapeseed seed which additionally has an endogenbus alpha-linolenic acid content in the oil greater than 3.5 percent by weight following crushing and extraction based upon the total fatty acid content, said step includes a selection which additionally has in the oil an alpha-linolenic acid content of no more than 3.5 percent by weight following crushing and extraction, and the product of step additionally has in the oil an alpha- linolenic acid content of no more than 3.5 percent by weight following crushing and extraction. IRA/s 2 ,2 INTEf NATIONAL SEARCH REPORT International Appiicationl *9CT/UfO 9 0 083 1. CLA' SIlFlCATION OF SUBJECT MATTER (it several classification symbols apply. indicate all) AccofI! dingto International Patent Classification (IPC) orto both National Classification Jind I 0F /0 U.S. CL.: 80,17/1; 47/58; 435/172.1;260/4165.5 Oocunintatiol' Searched 7 Claisficaton Sytem 'tassificat ion ymbals U.S.800Y 4 DIG t.5/172.1 260/405.5 Doculmentation Si ed other than Minimum Documentation to the Eittent that such ~)cuments are Included f i the Fiolds Searched 11 ,COMPUTER SEARCH~ CAS, BIOSIS,<-\AGRI CO'LA 1969 To Peet BRASSICA, OLEIC ACID, LINO-LENIC ACID, ERUCIC ACID, MUTANT Itt. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, It with indication. where spproprinte, oiir relevant passages 11 Relevant to claim No. 1 3 X _FAT SCIENCE TECHNOLOGY, Volume 90, issued 1,2,5,8, T' MAY 198S.,(Berlin, \West Germany), Pleines, 9,12,15, "Breeding for improved C18-fatty acid 16 composition in rapeseed," pages 167-171, 3,4.67-,,7T see pages 168 and 170 in particular. 10,11,13, 14,17-27 THE INDIAN4 JOURN4A L OF NUTRITION AND Y DIETETICS,~ Volume 13,,issued October 1976, I1-20 (Coimbatore,, India), Srivastava, "Comparison of some chemical characteristics of Indian and Canadian.Brassica seeds," pages 336-342t, see pages 33 and 338 in particular. Y PLANT BREEDING, Volume 98, issued 1,2,5-9, 4February 1987, (Berlin, West Germany), 12-16, ROY, "Prospects for development of rapeseed 19-27 VI-th improved linoleic and linolenic acid content," pages 89-96, see pages 89, 94 and in particulaz. Speciat cateoroies eot cited dr, umenta: 0 IT* later document oublishod after the internttaional filinjN date A doumet dfinng he eneal sateof he rt hic isnot or priority date and not in conflict with the spolscstio, \but '"dcunsidefi to e ll*ae of thrtacular relevanct cited to understand the principle or theory undoriyin;\he consderd t beof sirtculr rlevnceinvention ,rI earlier document but published on or alter the International document of particular relevance-, the citimed invention,- filing datecantb osotsnvlocantbcoidrdo IVdocument which MAy throw doubts on orioritir claim(s) or cnote n ientie Stove rcno t osdrd which is cited to establish the Publication 41ateil Of another document of particular relevance;- the claimed invention citation or ether special reason (as specified) cannot be Considered to involve an Inventive ase when the document referring to aon oral disclosur~ use, eitmition or document is combined with one or more other such doCu- Other means mania, such combination being obvious to a person slled "P4 document published prior to the international filing date buft Iin*eat later than Ithe prior ity date claimed "S"document member of the same patent f8aiY IV. CERTIFICATION Date of the Actual Completionm of the International Search I Dale of Mailing of this Itniationt Search Report MAY 1989 1JL18 International SearChingi Authority Signs lure of Authrftd Officer ISA/US DAVID T.FOX ~1 International Application No. Ineratonl ppictin o. PCT/US89/00835 7- Ill. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED~ FROM THE SECOND SHEET) Category Citation al Document, with indication, where appropriate. of tihe relevant passages IRelevant to Claim No Volume 1r,0, issued October 1973, (Champaign, Illinois, USA), RAKOW, "Opportunities arnd problems in modification of levels of rapeseed C18 unsaturated fatty acids," pages 400-403, see pages 400-401 in particular., Y INDI'AN JOURNAL OP AGRICULTURAL SCIENCE, 1-27 Volume 48, issued July 1978, (New Delhi, India), Rahman, "Relationships between major fatty acids of oleiferous specieslof Brassica,"1 pages 401-406, see pages 402k11 404 and 405 in particular. >P untPCTM~2tO gem IMi) t1~w.1142) International Application No, PCT/US89/00835- FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET Y ZEITSCHRIFT FUR PFLAN-ZENZUCHTUNG, Volume 75, 1,2,5-9, issued June 1975, (Berline West Germany), 12-16', RGBBELEN, "Genetical and7--physiological
14. 19-27 on mutants for polyip~oic fatty iirapeseed," pages 93-105, see pages 93, 94, 96 and 100 in particular. y ZEITSCHRIFT FUR PFLANZENZUCHTUNG, Volume 95, 1-27 issued September 1985, (Berlin, West Germany), (IROY, "IXLIN- an interspecific-,ource for hich linoleic and low linolenic aLA. content in" rapeseed". pages 201-209, see pages 201,202, an 0 npatclr OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLEI This interralional search report has not cen established in respect of certain claims under Article 17(2) for the following reasons: Claim numbers *because they .elate to subject matter il not required to be searchied by this Authority, namnely. Claim numbers .because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out 12. specifically.* Claim numbers because tey are dependent claims not drafted in accordance with the second and third sentences of PCT Rule 6.4(a). VI.Q: OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authority found mulltiple inventions in this international application as follows: At all required additional search tees were timely paid by the applicant, this International search report covers all searchable claims of the International applicatfdn. As only some of the rentrired addritional search fees were tindy paid by the applicant, this Internationai search report covers only thoe" claims of tho international applicatio:V 'Iiich fees were paid, as eifically claims: No required additiona'k search ?es were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned I~ the claims: itis covered by claim numbers: 4. 5 As all searchable claims could be searched without effort jusifying ant additional fee. the Internationral Searching Authority did not invite payment ot any additional fee. Rernarl on Protest 'the additional &Z-arch foes were accompanied by appicant*s protest, No protest accompanied the payment of additional stitch fees Feiti peZSAM.2tsitati*S (Z 1147)