Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
  • A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
  • A01H1/104—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine involving modified lipid metabolism, e.g. seed oil composition
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/10—Seeds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
  • A01H6/202—Brassica napus [canola]
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
  • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils

Definitions

• Rape i.e. , Brassica napus and Brassica camoestris
• Rape is being grown as an increasingly important oilseed crop in many parts of the world.
• As a source 5 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.
• rapeseed oil was found to have deleterious effects on human health due to its relatively high level of erucic acid which commonly is present in native cultivars in concentrations of 30 to 50 percent by weight based upon the total fatty acid 5 content.
• canola oil is being marketed by Procter & Gamble under the Puritan 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 which contains three double bonds per molecule of 18 carbon atoms, approximately 62 percent by weight oleic acid which contains a single double bond per molecule of 18 carbon atoms, and less than one percent by weight erucic acid which contains a single double bond per molecule of 22 carbon atoms.
• the oxidative stability of the vegetable oil is related to the number of double bonds in its fatty acids. That is molecules with several double bonds are recognized to be more unstable.
• 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 germplasm have been greater than 6 percent by weight (e.g.. greater than 6 up to approximately 12 percent by weight) .
• 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 (1) an unusually high oleic acid content of at least 79 percent by weight based upon the total fatty acid content, (2) an erucic acid content of no more than 2.0 percent by weight based on the total fatty acid content, and (3) a glucosinolate content in the solid component of less than 100 micromoles per gram.
• a substantially uniform stand of rape plants 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 (1) an unusually high oleic acid content of at least 79 percent by weight based upon the total fatty acid content, (2) an erucic acid content of no more than 2.0 percent by weight based on the total fatty acid content, and (3) 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 (1) an unusually high oleic acid content of at least 79 percent by weight based upon the total fatty acid content, (2) 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.
• a method of enhancing the oleic acid content of rapeseeds comprises:
• step (b) regenerating said cells to produce a rape plant and to form rapeseed in at least one generation subsequent to that of step (a) ,
• step (c) selecting a rapeseed produced in step (b) which has an oleic acid content of at least 79 percent by weight based upon the total fatty acid content,
• step (d) producing a rape plant on the basis of said selection of step (c) , and
• step (e) self-pollinating the rape plant of step (d) for a sufficient number of generations to achieve substantial genetic homogeneity and to form rapeseeds which contain at least 79 percent oleic acid by weight based upon the total fatty acid content.
• rapeseed plants whether Brassica napus or Brassica campestris. have formed rapeseeds which possess an oleic acid content of well under 79 percent by weight based upon the total fatty acid content.
• 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 analyzed for fatty acid content by gas liquid chromatography using a .capillary column which allows separation on the basis of the degree of unsaturation and chain length.
• plant cells may be derived from Brassica napus or Brassica campestris plants.
• the Brassica napus plants may be of either the summer or winter types.
• the plant cells derived from a rapeseed plant which 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 a rape plant is produced on the basis of this selection which is self-pollinated for a sufficient number of generations (e.g..).
• the plant cells which are subjected to mutagenesis also commonly are from plants which form rapeseeds having an alpha- linolenic content of greater than 5.0 percent by weight (e.g.. greater than 3.5 percent by weight), and selection concurrently is made for a reduced alpha- linolenic acid content.
• the mutagenesis preferably is carried out by subjecting the plant cells (e.g..
• 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 plant.
• 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 (e.g. , a rapeseed) in a dosage of approximately 60 to 200 Krad. , and most preferably in a dosage of approximately 60 to 90 Krad. It should be understood that even when operating at radiation dosages within the ranges specified, some plant cells (e.g.. 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, ethylnitrosourea, etc. , and by the use of physical means such as x-ray, etc.
• mutagenesis treatment will result in a wide variety of genetic changes within the rape plants which are produced. Many of these changes will be deleterious to the viability of the resulting plant over an extended period of time. Some changes also will produce viable plants which possess deficient agronomic characteristics. Such off-types may be simply discarded. However, if desired plants which have undergone mutation with 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.
• rape plants are regenerated from the treated cells using known techniques. For instance, the resulting rapeseeds may be planted in accordance with conventional rape growing procedures and following self-pollination rapeseed is formed thereon. Alternatively, doubled haploid plantlets 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 subsequent generation is harvested and is subjected to analysis for oleic acid content.
• Brassica napus and Brassica campestris are dicotyledons
• the analysis for oleic acid can be carried out on a halfseed, and the remaining halfseed can be retained for possible future germination 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.
• oleic content of at least 79 percent by weight (preferably at least 80 percent by weight)
• the oleic acid content of such selection preferably will be 79 to 90 percent by weight (e.g.. 80 to 85 percent by weight) .
• the other halfseed i.e.. cotyledon
• the other halfseed which will be genetically 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 stabilization 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 rapeseed grower, or as a parent in the production of hybrid rapeseed with the high oleic acid 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 (e.g.. less than 0.05 percent 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, 2- hydroxy-3-butenyl glucosinolate, and 2-hydroxy-4- pentenyl gluconsinolate.
• the gluconsinolate determination preferably is made on the air-dry-oil- free 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.
• the resulting rapeseeds also are selected which have an alpha- linolenic acid content less than 5 percent by weight based upon the total fatty acid content (e.g.. preferably no more than 3.5 percent by weight based upon the total fatty acid content) .
• 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 (e.g. 6 to 7 percent by weight) .
• the desired traits described herein e.g. , unusually high oleic acid content
• the desired traits described herein can be readily transferred into other plants within the same Brassica napus or Brassica campestris species by conventional plant breeding techniques involving cross- pollination and selection of the progeny. It has been demonstrated that the characteristics are highly heritable, can be 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 napus and campestris species using the same conventional plant breeding techniques involving pollen transfer and selection. The transfer of other traits, such as low erucic acid content, between the napus and campestris species by standard plant breeding techniques is already well documented in the technical literature.
• campestris variety 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) .
• desired traits described herein e.g. f unusually high oleic acid content
• campestris variety such as
• Tobin, Horizon, or Colt and carry out an interspecific cross with an appropriate plant of the napus breeding lines discussed hereafter (e.g.. FA677-39, Topas H6-90 and FA677M5-132) .
• other napus breeding 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.
• members of the F ⁇ generation are self-pollinated to produce F2 seed. Selection for the desired traits (e.g...
• the rapeseeds possessing the specified combination of characteristics are multiplied to form a substantially uniform assemblage of such seeds (e.g.. 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.
• the vegetable oil is present in a quantity convenient for commercial or domestic use (e.g.. a quantity of at least one liter) .
• mutagenesis has been found to be capable of increasing the oleic acid content to such high levels in rape is considered to be complex and incapable of simple explanation.
• 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.
• Example I Seeds of the Regent variety of Brassica napus 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 north central region of the United States, the western prairie area of Canada, and other areas where summer rape is 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 (i.e.. 2.0 grams) of the mature seeds of the starting material prior to subjection to gamma radiation 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: Number of Number of Carbon Atoms Double Bonds Weight
• 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.
• 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 5.5 percent by weight following air drying.
• Seeds of the Regent variety i.e.. approximately 10 grams
• a Gammacell 1000 gamma irradiation apparatus manufactured by Atomic Energy of Canada, Ltd. where they were subjected 90 Krad. of irradiation produced by a Cesium 137 source at a 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 ⁇ 3 ⁇ C, 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.
• 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 50 seed samples from each plant to be crushed and analyzed by gas liquid chromatography. When these representative 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 acid content (i.e.. 80 percent) . Sixty-five seeds from this plant were planted to grow the M5 generation.
• cotyledon analysis 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:
• Linoleic 18 2 4.4
• 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.
• the average oleic acid content of all of the samples analyzed was 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 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 M5 generation designated FA 677-39 have been deposited under the Budapest Treaty in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., 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 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 laws.
• Seeds (i.e. f the M2 generation) were next harvested from 153 plants (i.e.. the Ml 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 higher based upon the total fatty acid content. Three of these selections were found to contain an oleic acid content 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 approximately 25 ⁇ 3°C. and a night temperature of approximately 18 ⁇ C, plants were formed, and seeds were formed as the result of self-pollination (i.e.. 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 seed 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 non-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 30 micromoles per gram.
• the plants resulting from the germination of the seeds exhibited a substantially uniform phenotype.
• Comparable rapeseeds of the M3 generation designated FA677M5-132 have been deposited under the Budapest Treaty in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., 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 rights granted under the authority of any government in accordance with its patent or breeder's rights laws.
• Topas variety of Brassica napus 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 rape is adapted. The Topas variety was registered in 1987 by Svaldof AB of Sweden. Planting seed for this variety is available from Bonis & Company Ltd. of Lindsay, Ontario, Canada. This variety typically exhibits an oleic acid content of approximately 65 percent by weight based upon the total fatty acid content and an alpha-linolenic acid content of approximately 8 percent by weight based upon the total fatty acid content.
• Ten thousand seeds of the Topas variety were subjected to mutagenesis while employing a chemical mutagen. More specifically, seed lots were formed consisting of 1,000 seeds each and were treated with ethylnitrosourea as previously described. The resulting seeds were planted in flats present in a greenhouse containing a soilless greenhouse growing media. 500 seeds were planted in each flat and can be termed Ml seeds. Seeds (i.e.. the M2 generation) were formed as the result of self-pollination on 111 surviving fertile plants (i.e.. the Ml plants) .
• M2 seeds when subjected to cotyledon analysis exhibited on oleic acid content of 82.07 percent by weight and an alpha-linolenic 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 C, a plant was formed, and seeds were formed as the result of self-pollination (i.e.. 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 3.55 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 30 micromoles per gram.
• the plants resulting 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.
• Topas H6-90-99 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:
• Seeds produced from H6-90-99 will continue to exhibit a glucosinolate content in the solid component of less than 30 micromoles per gram.

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• 1989
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