DK175657B1 - Process for producing an endogenous vegetable oil - Google Patents

Description

DK 175657 B1! t

Rape (ie Brassica napus and Brassica campestris) is grown as an increasingly important oilseed crop in many parts of the world. As a source of vegetable oil, rape is currently only surpassed by soybeans and palm trees, and together with sunflower, rape shares third place in terms of commercial importance. The oil 5 is used as both cooking oil and lettuce oil worldwide.

In its original form, rapeseed oil was found to have detrimental effects on human health due to the relatively high content of erucic acid, which is usually present in natural culture varieties at concentrations of 30 to 10 50% by weight based on the total fatty acid content. Plant science professionals have previously identified a germplasm source of low erucic acid rapeseed oil, after which they began to incorporate this characteristic into commercial varieties. Reference can be made in this connection to Chapter 6 entitled "The Development of Improved Rapeseed Cultivars" by 15 B.R. Stefansson from "High and Low Erucic Acid Rapeseed Olis" edited by John K.G. Kramer, Frank D. Sauer, and Wallace J. Pigden, Academic Press Canada (1983).

In Canada, plant science professionals have focused their efforts on providing so-called "double-low" varieties which had a low erucic acid content in the oil and a low content of glucosinolates in the solid residue remaining after extraction of the oil ( determined an erucic acid content of less than 2.0% by weight based on the total fatty acid content and a content of glucosinolates of less than 30 pmol per gram of the oil-free residue). These 25 higher quality rapeseed developed in Canada are known as canola. In contrast, European plant experts have worked on obtaining only "single-low" types, which had a low erucic acid content, but which did not aim to improve the quality of the solid residue, which had a glucosinolate content of about 100 pmol per liter. grams of the oil-free residue. The result of this significant change in the fatty acid composition of rapeseed oil was the creation of a completely new oil profile, which often contained about 62% by weight oleic acid based on the total fatty acid content. Since the total oil percentage in the seeds did not change significantly as the new varieties with low erucic acid content

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teams were developed, so it seemed that the erucic acid had simply been converted

nets for other fatty acids, primarily oleic acid. This oleic acid level I

tended to vary within a fairly narrow range of about

about 55 to 65% by weight based on the total fatty acid content. There in this I

5 context is referred to chapter 7 entitled The Introduction of Low Eru- I

H cic Acid Rapeseed Varieties Into Canadian Production "by J.K. Daun of the I

In the above mentioned Academic Press Canada (1983) publication. Weight percent- I

the other fatty acids also varied to some extent, but not sufficiently

to define the individual types for specific applications or

In 10 increased commercial value. You can also refer to "Prospects for the I

In Development of Rapeseed (B. napus L.) With Improved Linoleic and I

in Linolenic Acid Content ”by N.N. Roy and A.W. Tarr, Plant Breeding, Vol. 98, I

In pages 89-96 (1987). IN

I 15 Currently, canola oil is marketed by Procter & Gamble under I

In the brand Puritan. Such a vegetable oil is typically free of chocolate content

In cholesterol, and the fatty acids present in the oil are about 6% saturated

In fatty acids in the form of stearic and palmitic acid, about 22% by weight of linolenic acid

In acid containing 2 double bonds per molecule of 18 carbon atoms, I

In about 10% by weight of α-linolenic acid, which contains 3 double bonds per mo- I

18 carbon atoms, about 62% by weight oleic acid containing 1 L

In double bond per molecule of 18 carbon atoms, and less than 1% by weight are I

In cyanide, which also contains only 1 double bond per molecule of 22 car * I

In bone atoms. IN

I 1 25 I

Over the years, scientists have tried to improve the fatty acid profile of canola oil. IN

For example, in the oxidative stability of the vegetable oil is related to the number I

I of double bonds in its fatty acids. This means that molecules with several double I

In belt bindings is considered to be more unstable. The researchers have therefore tried to:

In order to reduce the content of α-linolenic acid in order to improve storage I

In the stability and oxidative stability, in particular under the influence of I

In heat. This has not been possible through the use of natural I

In occurring germplasm and the reported values of α-linolenic acid with I

Such germplasm has been above 6% by weight (for example over 6% by weight and up to about 12% by weight). As reported by Gerhard Robbelen in Chapter 10 entitled "Changes and Limitations of Breeding for Improved Polyenic Fatty Acids Content in Rapeseed" from "Biotechnology for 5 the Olis and Fats Industry", edited by Colin Ratledge, Peter Dawson and James Rattray, American Oil Chemists' Society (1984), in a mutagenesis experiment, was able to obtain lines of less than ca. 3.5% by weight of α-linolenic acid based on the total fatty acid content. The profiles of these lines indicate that almost the entire amount of α-linolenic fatty acid was directed to linoleic acid and that the level of oleic acid was only increased by 1 or 2%. Nevertheless, the oil seemed to have certain advantages over normal canola oil.

Eg. For example, the refining process required less hydrogenation than normal canola oil, and it showed better roasting stability.

15 Studies conducted in recent years have established the value of monounsaturated fatty acids as constituents of foods. This has led to "Mediterranean dishes" becoming popular, placing the emphasis on olive oil, which is a naturally occurring, rich source of oleic acid. With such a diet it is believed to be able to avoid the problem of arteriosclerosis, which occurs as a result of saturated fatty acids. However, even when following such a diet plan, olive oil is perceived to be less than ideal because it contains a certain amount of saturated fatty acids. Canola oil is potentially an excellent cooking oil, since about half of its saturated fatty acids are in the form of olive oil, and because its relatively high 25 alpha-linolenic acid, which is detrimental to storage stability and oxidative stability, can be advantageous from a nutritional standpoint. point of view. It is believed that α-linolenic acid is a precursor for the body's synthesis of a family of chemical compounds that can reduce the risk of cardiovascular disease.

It is recognized in the literature that the content of oleic acid in canola oil varies slightly depending on the environment, temperature and availability of moisture as the rapeseed is formed. As previously mentioned, the oleic acid content of available canola varieties is usually about 55 to 65% by weight; see e.g.

DK 175657 B1 I

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Table V on page 171 of Chapter 7 entitled The Introduction of Low Erucic Acid I

Rapeseed Varieties Into Canadian Production "by J.K. Daun, found in I

"High and Low Erucic Acid Rapeseed Olis", Academic Press Canada (1983). IN

H As reported in the same article, rapeseed varieties containing I

5 larger concentrations of erucic acid, contain even inferior amounts I

oleic acid. IN

Occasionally, higher oleic acid levels have been mentioned, but these have not

I have been made available to rapeseed growers. For example, it is mentioned on page I

In 10 23 in the presentation of the 7th International Rapeseed Congress, held in Poznan, I

I; Poland, May 11 to 14, 1987, that Mon has encountered a canola test with an I

I in oleic acid content of 79.0% by weight under given growth conditions while I

; under other growth conditions found an oleic acid content of 74% by weight. IN

This plant was claimed to have been produced by recurring selection I

In 15 using unidentified parent plants. However, there is speech I

'of an incomplete description which does not enable the reader to

You produce a rapeseed plant which produces rapeseed with such increased I

In oleic acid content. IN

I As stated in U.S. Patent Nos. 4,517,763, Nos. 4,658,084, and Nos. 4 I

In 658 085 as well as the publications mentioned in these patents, it is known

In bridging processes suitable for the production of rapeseed. IN

It is an object of the present invention to provide one in the art

In 25 late uniform collection of enhanced rapeseed yielding a vegetable oil I

with increased stability. IN

It is an object of the present invention to provide an I

process for preparing an endogenous vegetable oil as set forth in I

The preamble of claim 1 for increasing the oleic acid content of rapeseed and for I

thereby improving the stability of the vegetable oil produced from these. IN

Such a method is peculiar to the features set forth in the I

characteristic part of claim 1. I

in 5 DK 175657 B1

These and other objects and advantages associated with the invention will become apparent from the following description and claims.

5

The invention provides a substantially homogeneous collection of mature rape seeds capable of providing a vegetable oil of increased stability under heat exposure, which oil has (1) an unusually high oleic acid content of at least 79% by weight based on the total fatty acid content, 10 (2) a content of erucic acid not exceeding 2.0% by weight based on total fatty acid content and (3) a content of glucosinolates in the solid component of less than 100 μιτιοΙ per liter. gram.

A substantially uniform stock of rapeseed, obtained by self-pollination, is capable of forming rapeseed which provides a vegetable oil of increased stability when exposed to heat, where the rapeseed has an unusually high oleic acid content of at least 79% by weight based on the total fatty acid content, (2) a content of erucic acid not exceeding 2.0% by weight based on the total fatty acid content and (3) a content of glucosinolates in the solid component of less than 100 pmol per. gram.

From such rapeseed, an improved vegetable oil of increased stability can be prepared under heat exposure, the rapeseed having (1) an exceptionally high oleic acid content of at least 79% by weight based on the total 25 fatty acid content, (2) a content of erucic acid not exceeding 2.0% by weight based on the total fatty acid content and (3) an alpha-linolenic acid content of less than 5% by weight based on the total fatty acid content.

It has been found that a method for increasing the rapeseed content of oleic acid comprises that in DK 175657 B1

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(a) exposing at least one generation of cells derived from a rapeseed plant;

forming rapeseed with an oleic acid content of less than 79% by weight based on I

the total fatty acid content, for a technique selected from gamma irradiation, con

with a chemical mutagen and a combination of these techniques

5 views on inducing mutagenesis, I

(b) regenerating the cells to produce a rapeseed plant and to form I

rapeseed for at least one generation after generation from step (a), I

I 10

(c) selects rapeseed produced in step (b) having an oleic acid content.

In holdings of at least 79% by weight based on the total fatty acid content, I

I (d) produces a rapeseed plant based on the selection in steps (c) and I

I 15

(e) self-pollinating the rapeseed plant from step (d) of an additional I

sufficient number of generations to achieve substantial genetic homogeneity I and to form rapeseed seeds containing at least 79% oleic acid based on the weight of the total fatty acid content.

I 20

The rapeseed plants that have been available so far, whether in I: whether Brassica napus or Brassica campestris, have produced rapeseed with an oleic acid content substantially below 79% by weight based on the total fat-I acid content. For the purposes of the present invention, the oleic acid content of a given collection of rapeseed is determined by a standard procedure in which the oil is removed from the rapeseed by crushing them and then extracted as one methyl ester after reaction with methanol and sodium hydroxide. Then, the resulting ester for its fatty acid content is analyzed by gas chromatography I using a capillary column which allows a separation on the basis of the degree of unsaturation as well as the chain length. This I analysis procedure is described in an article by J.K. Daun et al., J. Amer. Oil I Chem. Soc. 60: 1751-1754 (1983). The higher quality canola varieties of rapeseed available for commercial planting generally have an oleic acid content of not more than 65% by weight based on the total fatty acid content. As a result, there has so far been a need for improved canola varieties which exhibit a significantly higher oleic acid content.

In accordance with the concepts of the present invention, plant cells that can undergo regeneration (e.g., seeds, microspores, seed plants or vegetative parts) are preferably selected from any of the canola varieties known to have sufficiently good agronomic characteristics. Such plant cells can be derived from Brassica napus or Brassica 10 campestris plants. The brassica napus plants can be either summer or winter type. The plant cells derived from a rapeseed plant which produces rapeseed with an oleic acid content of less than 79% by weight based on the total fatty acid content, are then subjected to mutagenesis for at least one generation, after which a rapeseed plant is regenerated from the cells to form a rapeseed plant. and rapeseed for at least one subsequent generation. Rapeseed is selected with an oleic acid content of at least 79% by weight based on the total fatty acid content, after which a rapeseed plant is produced based on this selection, which is self-pollinated for a sufficient number of generations (e.g. 2 to 8 additional generations) to obtain a substantial genetic 20 homogeneity and to form rapeseed seeds containing at least 79% by weight oleic acid based on the total weight of the fatty acids present. The plant cells that are subject to mutagenesis usually also originate from plants that form rapeseed with a content of α-linolenic acid of more than 5.0% by weight (for example over 3.5% by weight), and a selection is made in parallel. for a reduced α-linolenic acid content.

The mutagenesis is preferably carried out by subjecting the plant cells (e.g., rapeseed) to a technique selected from gamma irradiation, contact with a chemical mutagen, and a combination of these techniques, which is of a duration sufficient to induce it. desired increase of oleic acid content (and preferably also therein desired reduction of α-linolenic acid content) via a genetic modification, but insufficient to destroy cell viability and their ability to be re-expressed.

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In generated for a plant. A rapeseed seed preferably contains a moisture content

amount of about 5 to 6% by weight at the time of such mutagenesis. IN

Preferably, the mutagenesis is carried out by gamma irradiation, for example I

In the kind that can be obtained from one. Cesium 137 source. Preferably, I is supplied

In the gamma irradiation of the plant cells (e.g. rapeseed) at a dose of I

In about 60 to 200 Krad, especially in a dosage of about 60 to 90 Krad. The ten

It should be understood that even when operating with radiation doses within the I

In designated areas, some plant cells (for example, rapeseed) will lose their living

In skill, after which they are discarded. The desired mutagenesis can also be I

I 10 is made by chemical means, such as by contact with ethyl methyl I

In sulphonate, ethyl nitrosourine or the like, as well as physical I

In agents such as X-rays or the like. IN

It is clear that the mutagenesis treatment will result in a wide variety of genes

In 15 neat changes to the rapeseed plants that are produced. Many of these inheritances

Inferences will be detrimental to the viability of the resulting plant I

For an extended period of time. Furthermore, some of the changes will lead to I

viable plants which, however, have deficient agronomic character- I

istika. Such less fortunate types can simply be discarded. If you wish- I

In 20 this happens, however, you can save the plants that have undergone mutation I

I with respect to oleic acid production coupled with undesirable agronomi- I

In distinguishing characteristics. These plants can be used for propagation or as source material

In rial from which to derive plants having the desired traits I

In coupled with satisfactory agronomic characteristics. IN

I 25 I

After mutagenesis, the rapeseed plants from the treated cells are regenerated at I

Using techniques known per se. Eg. can the resulting rapeseed I

You are planted in accordance with conventional rapeseed procedures,

In that after self-pollination, rapeseed is formed on the plants. Alternatively, you can

In 30 people, haploid seedlings doubled. The planting of the treatment I

In dyed canola seeds is preferably carried out in a greenhouse where the pollination is controlled

You are carefully read and monitored. The additional rape seeds formed as a result of I

In such a self-pollination in the present or a subsequent generation,

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a 9 DK 175657 B1 | collected and subjected to analysis for the oleic acid content. Since Brassica napus and Brassica campestris are two-seeded plants, the analysis for the oleic acid content can be performed on the half seed, while the other half seed can be saved for possible later germination if the oleic acid content is found to be favorable due to the mutagenesis. The rapeseed can be carefully separated into two half seeds using known techniques.

When a mature semi-seed is found to have an oleic acid content of at least 79% by weight (preferably at least 80% by weight), it is selected and stored.

The oleic acid content of such a selection will preferably be between 79 and 90% by weight (e.g. 80 to 85% by weight).

The second semi-seed (ie, seedling), which will be genetically the same as the semi-seed selected for semi-seed analysis, can then be germinated to form a rapeseed plant which is subjected to self-pollination. Such propagation of the semi-seed is preferably also carried out in a greenhouse where the pollination is carefully controlled and monitored. The resulting rapeseed are harvested, propagated and self-pollinated for a sufficient number of generations to achieve significant genetic homogeneity. The genetic stabilization of the rapeseed plant material makes it possible to create plants that have a reasonably predictable genotype and which can be used as breeding or source material for use in the production of other improved rapeseed varieties, as a fully developed variety for use in rapeseed cultivation or as a parent plant. for use in the production of hybrid rapeseed, in which the high 25 oleic acid content is transferred to the offspring.

The resulting rapeseed are also selected in such a way that they have an eracic acid and glucosinolate content similar to canola. More specifically, the acetic acid content is at most 2.0% by weight based on the total fatty acid content and preferably below 0.1% by weight (e.g. below 0.05% by weight) based on the total fatty acid content. The content of glucosinolates in the solid component is less than 100 pmol per ml. grams (preferably below 30 pmol per gram). The glucosinolate content may be derived from 3-butenylglucosinolate, 4-pentenylglucosinolate,

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2-hydroxy-3-butenylglucosinolate, 2-hydroxy-4-pentenylglucosinolate or I

mixtures thereof. The glucosinolate content is preferably determined

on the air-dried, oil-free solid and it is measured by the gas chromatographic I

method developed by the Canadian Grain Commission. The prescribed I

5 erucic acid and glucosinolate levels are usually made possible by selecting I

starting materials which already contain particularly desirable levels of I

these components. In a preferred embodiment, the vegetable I

oil is thought to be used for frying, the resulting rapeseed are selected on a I

such that they have an α-linolenic acid content of less than 5% by weight based I

10 on the total fatty acid content (i.e., preferably not more than 3.5% by weight based on the I

total fatty acid content). In another preferred embodiment, it contains I

vegetable oil not exceeding 7% by weight of saturated fatty acids in the form of stearic acid and I

palmitic acid, calculated on the total fatty acid content (e.g. 6 to 7 l

% By weight). IN

I 15 I

I Once the desired characteristics described above (Example I

In pelvic an exceptionally high oleic acid content), have been established, they can easily

transferred to other plants within the same Brassica napus or Brassica I

campestris species using conventional plant breeding techniques, I

20 including cross-pollination and selection of the offspring formed. It has become you

shown that these characteristics are highly heritable, that they can be transmitted from I

parents of offspring and that they can be found in separated offspring in subsequent I

generations after the crossing. When the desired character traits are first you

established, they can also be transferred between napus and carripestris species at I

25 using the same conventional plant breeding techniques, including pole I

len transmission and selection. The transmission of other characteristics, such as an I

low erucic acid content, between napus and campestris species at stan- I

dard techniques in plant cultivation are already well documented in the I

technical literature. For example, refer to "Brassica Crops and Wild Allies I

30 Biology and Breeding ”edited by S. Tsunada, K. Hinata, and Comez Campo, I

Japan Scientific Press, Tokyo (1980). As an example of the transmission of the I

the desired character traits described above (for example, an unusually high I

oleic acid content) from napus to campestris one can select a commercial I

----—---- DK 175657 B1 11 available campestris variety, such as Topin, Horizon or Colt, and carry out an interspecific crossing with a suitable plant from the napus breeding lines described below (e.g. FA677- 39, Topas H6-90 and FA677M5-132).

Alternatively, other napus breeding lines can be reliably and independently developed by following the mutagenesis techniques described below. The Topin variety is available from Agriculture Canada, Saskatoon, and other distributors. The Horizon and Colt varieties are available from Bonis & Company Ltd.,

Lindsay, Ontario, Canada. After the interspecific crossing, the members of the Fi generation self-pollinate to produce F2 seeds. A selection 10 for the desired character traits (e.g., an unusually high oleic acid content) is then made on single F2 seeds, which are then crossed with the campestris parent through the number of generations required to obtain a euploid (n = 10) campestris. line exhibiting the desired properties (e.g., an unusually high oleic acid content).

In accordance with the present invention, the rapeseed seeds possessing the specified combination of characteristics are multiplied to form a substantially uniform collection of such seeds (e.g., a sack of such seeds) which can be used to produce a substantially 20 uniform stock of such rapeseed plants. The number of rapeseed contained in such a collection amounts to at least 250 seeds and the resulting substantially uniform stock of rapeseed plants amounts to at least 250 plants.

The improved vegetable oil according to the invention can be formed by simple extraction, which takes place directly from the rape seed seeds, for example by crushing and subsequent extraction according to known techniques. For example, Refer to Chapter 8 entitled "Rapeseed Crushing and Extraction" by DHC Beach, found in "High and Low Erucic Acid 30 Rapeseed Oils", Academic Press Canada (1983). In a preferred embodiment, the vegetable oil is present in a quantity convenient for commercial or household use (ie at least 1 liter).

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The theory by which the mutagenesis has been shown to be capable of increasing I

the oleic acid content to such high levels in rapeseed is assumed to be for I

complicated for simple explanation. Eg. the mutation may have a deleterious effect

5 has an effect on the formation of one or more enzymes which will normally contribute to I

the dehydrogenation of the fatty acids as the seeds mature. IN

The invention is further illustrated by the following specific examples. The ten

however, it is to be understood that the invention is not limited to the specific details

H 10 you, as shown in the examples. IN

EXAMPLE I

Seeds of the Regent variety of Brasslca napus were selected as the starting material

15 rials. This canola variety is of the summer type and is suitable for production I

of vegetable oil when grown in the north central region of the United States, it i

In western prairie areas of Canada and other places where summer rape has adapted

You see. The Regent variety was first introduced in 1977 by University I

You in Manitoba. Seeds for planting the Regent variety can be obtained from the Department of I

In 20 Plant Sciences at the University of Manitoba. A representative sample (i.e., I

2.0 g) of the mature seeds of the starting material before being exposed to I

gamma irradiation (as described below), contained the following fat I

acids in the indicated approximate concentrations based on the total weight of I

In the fatty acids present (determined by gas chromatography as previously described)

I 25 written): I

DK 175657 B1 i! 13 i j

Number of double-

Number of C-atom bonds per

Fatty acid per molecule molecule Weight% 5 Palmitic acid 16 0 4.8

Palmitoleic Acid 16 1 0.1

Stearic acid 18 0 1.6

Oleic Acid 18 1 65.4

Linoleic acid 18 2 19.3 10 α-Linolenic acid 18 3 6.9 j

Arachidic acid 20 0 0.6 i i

Eicosenoic acid 20 1 1.0 j

Behenic acid 22 0 0.3 in Erucic acid 22 1 not detectable

The content of glucosinolates in the solid component was 13.44 pmol per ml. grams as determined by the gas chromatographic method developed by the Canadian 20 Grain Commission.

Prior to the gamma irradiation, the seeds of the Regent variety of canola were stored under conditions such that viability could be maintained.

Specifically, the seeds were stored in a cold storage room with a temperature of about 10 ° C and a relative humidity of 40%. The seeds had a moisture content of about 5.5% by weight after air drying.

Seeds of the Regent variety (more specifically about 10 g) were then placed in a gamma irradiator of the brand Gammacell 1000 manufactured by 30 Atomic Energy of Canada, Ltd. where they were exposed to irradiation of the order of 90 Krad produced by a Cesium 137 source in an amount of 26.61 Krad per hour to induce mutagenesis. These seeds can be referred to as M1 seeds.

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Following the gamma irradiation, the M1 seeds were transplanted into a greenhouse in Georgetown, I

Ontario, Canada. The greenhouse had a daytime temperature of about 25 + 3 ° C

and a night temperature of about 18 ° C. About 40% of the gamma I

5 irradiated seeds produced fertile rapeseed plants which by self pollination I

gave M2 seeds. These M2 seeds were then propagated in open field in the same I

area for producing plants which, after pollination, yielded M3 seeds. IN

Representative M3 seeds produced on the M2 plants were then soaked in I

10 g of water, after which a seedling from each seed was carefully taken for analysis of I

In the fatty acid composition using the gas described previously

In chromatographic technique. Such semi-seed analysis was carried out in above I

In accordance with the procedure described in "Methods for Breeding for oil I

In Quality in Rape ”by R.K. Downey and B.L. Harvey reported in Canadian I

In 15 Journal of Plant Science, Vol. 43, pp. 271-275 (1963). From a total of 4490 germs

In leaf analyzes of M3 plants, 37 seedlings were found to contain an increased I

In the amount of oleic acid, more specifically between 70.2 and 76% based on wall I

In ten of the total fatty acid content. The content of α-linolenic acid was between 5.4 and 1

In 13.1% based on the weight of the total fatty acid content. IN

I 20 I

One chose a M3 semi-seed, designated as FA677, which was found to contain I

In the following fatty acids at the indicated concentrations (based on the total I

By weight of the fatty acids present):

15 DK 175657 B1

Number of double-

Number of C-atom bonds per

Fatty acid per molecule molecule Weight% 5 Palmitic acid 16 0 4.0

Palmitoleic acid 16 1 not visibly

Stearic acid 18 0 1.4

Oleic acid 18 1 70.9 10 Linoleic acid 18 2 10.7 α-linolenic acid 18 3 11.0

Arachidic Acid 20 0.5

Eicosenoic acid 20 1 1.2

Behenic Acid 22 0 0.3 15 Erucic Acid 22 1 not visibly

All M3 semi-seeds, including FA677, were transplanted into greenhouses and allowed to undergo self-pollination and to form the M4 generation. Each of these plants produced enough seeds to randomly select 50 seed samples from each plant for crushing and subsequent analysis by gas chromatography. When these representative 50 seed samples from the M4 generation were analyzed, it was found that the oleic acid content was between 25 63 and 80% by weight based on the total fatty acid content and that the linolenic acid content was between 3.2 and 7.7 % by weight based on the total fatty acid content.

The individual plant, designated FA677, was found to have the highest oleic acid content (namely 80% by weight). 65 seeds were planted from this plant for cultivation of the M5 generation. For reference purposes, 50 of 30 of these 65 seeds were additionally subjected to a seedling analysis which revealed oleic acid content of between 74.0 and 85.0% by weight based on the total fatty acid content. The profile of the best plant that can be deduced from the seedling analysis (85.0% oleic acid) is *

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shown in the following table:

Number of double I

Number of C-atom bonds per IN

In 5 fatty acids per molecule molecule Weight% I

Palmitic Acid 16 0 3.2 I

Palmitoleic Acid 16 1 0.2 I

In Stearic Acid 18 0 2.4 I

I 10 Oleic Acid 18 1 65.0 I

In Linoleic Acid 18 2 4.4 I

α-linolenic acid 18 3 2.7 I

Arachidic acid 20 0 1.0 I

Eicosenoic acid 20 1 1.2 I

15 Behenic Acid 22 0 not on- I

Surely you

Erucic acid 22 1 not on- I

Surely you

j ''

i

20 I

I I The above identified specific FA677 plant which produced an I

In oleic acid content of 85% by weight based on total fatty acid content was lost in I

In the subsequent generation as a result of breaking the class in the greenhouse. When you

I; however, a sample of 50 seeds from a sister plant in M5-I was analyzed

In the 25th generation, designated FA677-39, after crushing, the following were observed

In fatty acids at the indicated concentrations based on the total weight of I

In: the fatty acids. The plants that arose from germinating the seeds showed one in it

Essentially uniform phenotype. IN

I 30 I

DK 175657 B1 17

Number of double-

Number of C-atom bonds per

Fatty acid per molecule molecule Weight% 5 Myristic acid 14 0 0.1

Palmitic acid 16 0 4.1

Palmitoleic Acid 16 1 0.3

Stearic acid 18 0 1.6

Oleic acid 18 1 79.2 10 Linoleic acid 18 2 7.1 α-linolenic acid 18 3 4.6

Arachidic Acid 20 0.7

Eicosenoic acid 20 1 1.5

Behenoic acid 22 0 0.5 15 Erucic acid 22 1 0.06

Lignoceric acid 24 0 0.3

The content of glucosinolates in the solid component was 10.94 mmol per ml. gram.

The plants produced from these seeds are seed resistant after * self-pollination and exhibit a substantially uniform phenotype. The average average oleic acid content of all analyzed samples (65 samples, 50 seeds per sample) was 77.1% by weight based on total fatty acid content, significantly indicating a stable high oleic acid level in all descendants of the plant designated FA677.

Further selections using the FA677-39 breeding line may result in the identification of plants exhibiting even higher oleic acid content. These plants can be preserved and multiplied using conventional techniques.

The increased oleic acid content enables the rapeseed to produce a vegetable oil which exhibits increased stability upon exposure to heat. The

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H means that the resulting oil can be reliably used for frying I

of food over a prolonged period of time without any adverse results

tater, compared to the canola oil of the prior art. It re- I

induced α-linolenic acid content in the resulting vegetable oil gives end-I

5 further increases the oxidative stability of the oil. IN

Comparable rapeseed of the M5 generation designated FA677-39 have been I

deposited under the Budapest Treaty with the American Type Culture I

I Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, 31st

: December 10, 1987. These deposited seeds have been given the deposit number 40409 I

and will in time become available to the public. IN

However, the availability of these seeds should not be considered an op

claim to carry out the present invention in violation of the rights;

15 granted by the authorities of any State under the Patent Law or Law I

for the rights of growers. IN

EXAMPLE II I

Twelve M5 generation selections were discussed (discussed in Conn. I

In the case of Example I), which had the highest oleic acid content, an additional I

In is mutagenesis using a chemical mutagen. More specifically, I

two portions of composite seeds were formed from these selections, each of which I

You had 1000 seeds and were treated with ethyl nitrosourine. This I

In 25 ethyl nitrosourea was dissolved in dimethyl sulfoxide at a concentration of 8 mM. IN

25 ml of di-I was added during the preparation of the ethylnitrosourine solution

In methyl sulfoxide to 1 g of ethyl nitrosourea, then the resulting. IN

In solution was buffered to a pH of 5.5 using 5 mM morpholino-I

sulfonic acid. Each portion of seeds was placed in a large petri dish and added

30 ml of the resulting solution were added 30 ml. While the seeds were in contact with I

In the ethylnitrosourse solution, they were incubated in the dark at 20 ° C for 18 hours, after which they were purified 3 times with distilled water. Finally, the 19 DK 175657 B1 was planted in trays in a greenhouse, using a soilless growth medium for use in greenhouses. 500 seeds were planted in each tray.

* About 30% of the seeds treated with the ethyl nitroso urea solution grew into plants which were transferred to herbal pots. These plants were grown in a greenhouse, and it was found that about 25% of these plants exhibited sufficient fertility to undergo self-pollination and to form seeds (ie, the M2 generation after mutagenesis using a chemical mutagen).

10

Then, seeds (i.e., the M2 generation) were harvested from 153 plants (i.e., the M1 plants). From each of the plants, 10 seeds were individually analyzed by the previously described semi-seed analysis. A total of 276 seedlings were selected which had an oleic acid content of 77% by weight or more on the basis of the total fatty acid content. It was found that three of these selections had an oleic acid content of 84% by weight based on the total fatty acid content.

The remaining seedlings from the 276 selections were transplanted into a greenhouse in Georgetown, Ontario, Canada, which had a day temperature of about 25 20 + 3 ° C and a night temperature of about 18 ° C. After plant formation, seeds were formed as a result of self-pollination (i.e., the M3 generation after mutagenesis using a chemical mutagen). A selection designated FA677M5-132 in the M3 generation, using two randomized assays of each 50 seeds, was found to have an oleic acid content of 81.9 wt% 25 based on the total fatty acid content, an α-linolenic acid content of 4.03 % by weight based on the total acid content, an undetectable erucic acid content,

V

saturated fatty acid content of 6.59% by weight (in the form of stearic acid and palmitic acid) based on the total fatty acid content as well as a content of glucosinolates in the solid component of less than 30 pmol per gram. The plants that emerged as a result of germination of the seeds exhibited a substantially uniform phenotype.

Further selections from the breeding line FA677M5-132 will result in the identification of plants exhibiting even higher oleic acid content. These plants can be stored and propagated using conventional techniques. When

DK 175657 B1 I

I I

one for example. analyzes some seedlings from this breeding line, you have been able to

observe oleic acid content exceeding 85% by weight based on total fatty acid I

In content. IN

5 Comparable rapeseed from the M3 generation designated FA677M5-132 are I

been deposited in accordance with the Budapest Treaty with the I

In the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Mary- I

country 20852, USA, December 13, 1988. This sample has been deposited

ring number 40523 and will be made available to the public in due course. IN

10 I

However, the availability of these seeds should not be considered as an option

I for carrying out the present invention in violation of the rights granted by I

the authorities of any State under its patent or cultivation law- I

In secrecy law. IN

I 15 I

In Example III

Seeds of the Topas variety of Brassica napus were selected as the starting material

I ale. This canola variety is of the summer type and is well suited for production

In 20 ing of vegetable oil when grown in the United States, Canada, Sweden and others

In areas where summer rape has adapted. The topaz variety was recorded in I

1987 by Svaldof AB, Sweden. Seeds of this variety for planting are available from I

Bonis & Company Ltd., Lindsay, Ontario, Canada. This variety exhibits you

typically an oleic acid content of about 65% by weight based on the total I

25 fatty acid content and a content of α-linolenic acid of about 8% by weight, as well

This is based on total fatty acid content. IN

10,000 seeds of the Topas variety were subjected to mutagenesis using I

I of a chemical mutagen. Specifically, portions were formed, each serving

I 30 consisted of 1000 seeds, which were treated with ethyl nitrosourse as previously I

In described. The resulting seeds were planted in flat trays in a greenhouse, with I

A growth-free medium was used in greenhouses for use in greenhouses. In each I

In the ground were planted 500 seeds, which can be termed M1 seeds. IN

B

21 DK 175657 B1

Seeds (ie the M2 generation) were formed as a result of self-pollination on 111 surviving fertile plants (ie the M1 plants). One of the M2 seeds * was subjected to seedling analysis which showed an oleic acid content of 82.07% by weight and an α-linolenic acid content of 5.12% by weight, both based on the total fatty acid content. The remaining M2 seedlings were transplanted in a greenhouse in Georgetown, Ontario, Canada, with a daytime temperature of about 25 + 3 ° C and a nighttime temperature of about 18 ° C. After formation of a plant, seeds (ie M3 generation) were formed as a result of self-pollination. This M3-10 generation was named Topas H6-90, and in two randomized analyzes of every 50 seeds, the M3 generation was found to have an oleic acid content of 81.17% by weight based on the total fatty acid content, an α-linolenic acid content of 3, 55% by weight, also based on the total fatty acid content, an undetectable erucic acid content, a content of saturated fatty acids of 6.17% by weight (in the form of 15 stearic acid and palmitic acid) based on the total fatty acid content and a glucosinolate content in the solid component of less than 30 pmol per gram. The plants that emerged from germination of the seeds exhibited a substantially uniform phenotype. Further selections from the breeding line Topas H6-90 (as indicated below) will result in the identification of plants with even higher oleic acid content. These plants can be stored and propagated using conventional techniques.

Comparable rapeseed of the M3 generation designated Topas H6-90 have been deposited in accordance with the Budapest Treaty of the 25 American Type Culture Collection, 12301 Parkiawn Drive, Rockville, Maryland 20852, USA, December 13, 1988. This deposit has been numbered 40524 and it will in time be made available to the public.

Upon further selection within the M3 generation of Topas H6-90, one could identify a selection termed Topas H6-90-99 which contained the following fatty acids in the indicated approximate concentrations based on the total fatty acid content (using the previous gas chromatographic technique described):

DK 175657 B1 I

I I

Number of double I

Number of C-atom bonds per IN

In fatty acid per molecule molecule Weight% I

I 5 Palmitic Acid 16 0 3.57 I

In Palmitoleic Acid 16 1 0.31 'I

In Stearic Acid 18 0 1.87 I

In Oleic Acid 18 1 85.84 I

I Linoisyre 18 2 3.54 I

I! 10 α-linolenic acid 18 3 2.62 I

Arachidic Acid 20 0. 0.49 I

Eicosenoic acid 20 1 1.29 I

In Behenic Acid 22 0 0.32 I

Erucic acid 22 1 not on- I

Certainly I

Lignoceric Acid 24 0 0.07 I

Seeds produced from H6-90-99 will continue to exhibit a glucosinolate-I

In 20 contents of the solid component of less than 30 μ moles per gram. IN

In the foregoing, the invention has been described by means of preferred embodiments

In embodiments, however, it should be understood that one can make variations and mo- / I

deficiencies within the scope of the invention. IN

Η I

Claims (11)

Process according to claim 1, characterized in that the oil is extracted from engineered seeds of Brassica napus plants Process according to claim 1 or 2, characterized in that the oil is extracted from rapeseed seeds having a moisture content of about 5-6% by weight and the seeds having been subjected to about 60-200, preferably 60-90 Krad 25 gamma radiation. % Process according to any one of the preceding claims, characterized in that the oil is extracted from rapeseed seeds, which have at least partially been subjected to a chemical mutagen. 30 Process according to claim 1, characterized in that the oil is extracted from seeds which further have an endogenous content of alpha linoleic acid which is less than 5% by weight based on the total content of fatty acids. DK 175657 B1 I I I Process according to claim 1, characterized in that the oil extracted from the engineered rapeseed seeds has an alpha linoleic acid content not greater than 3.5% by weight. . I I I Process according to any one of the preceding claims, characterized in that the oil extracted from the engineered rapeseed seeds has an oleic acid content I of at least 79% by weight based on the total fatty acids content, an erucic acid content of not more than 2 0% by weight based on the total content of I 10 fatty acids and a glucosinolate content in the solid material of less than 100 II micromoles per gram. IN Process according to claim 1, characterized in that the rapeseed I1 seeds are formed with substantial homogeneity on Brassica napus plants. I I 15 I Process according to claim 1, characterized in that the rapeseed seeds I I have a substantially high oleic acid content of I I at least 80% by weight based on the total fatty acid content. IN 10. A process according to claim 1, characterized in that the rapeseed seeds I1 have a substantially high oleic acid content of 80-190% by weight based on the total fatty acid content. IN 11. A process according to claim 1, characterized in that the rapeseed seeds I I 25, with substantial homogeneity, additionally have an alpha-linolenic acid content of I I less than 5% by weight based on the total fatty acid content. IN Process according to Claim 1, characterized in that the rapeseed I1 of substantial homogeneity additionally has an alpha-linolenic acid content of I130 not more than 3.5% by weight based on the total fatty acid content. IN