U.S. patent application number 10/435521 was filed with the patent office on 2004-01-15 for canola oil having increased oleic acid and decreased linolenic acid content.
This patent application is currently assigned to Cargill, Incorporated, a Delaware corporation. Invention is credited to DeBonte, Lorin R., Hitz, William D..
Application Number | 20040010819 10/435521 |
Document ID | / |
Family ID | 24711418 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040010819 |
Kind Code |
A1 |
DeBonte, Lorin R. ; et
al. |
January 15, 2004 |
Canola oil having increased oleic acid and decreased linolenic acid
content
Abstract
An endogenous oil extracted from Brassica seeds is disclosed
that contains, after crushing and extraction, greater than 86%
oleic acid and less than 2.5% .alpha.-linolenic acid. The oil also
contains less than 7% linoleic acid. The Brassica seeds are
produced by plants that contain seed-specific inhibition of
microsomal oleate desaturase and microsomal linoleate desaturase
gene expression. Such inhibition can be created by cosuppression or
antisense technology. Such an oil has a very high oxidative
stability in the absence of added antioxidants.
Inventors: |
DeBonte, Lorin R.; (Fort
Collins, CO) ; Hitz, William D.; (Wilmington,
DE) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
3300 DAIN RAUSCHER PLAZA
60 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Cargill, Incorporated, a Delaware
corporation
|
Family ID: |
24711418 |
Appl. No.: |
10/435521 |
Filed: |
May 8, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10435521 |
May 8, 2003 |
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09966888 |
Sep 28, 2001 |
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6583303 |
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09966888 |
Sep 28, 2001 |
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09482287 |
Jan 13, 2000 |
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6441278 |
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09482287 |
Jan 13, 2000 |
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08907608 |
Aug 8, 1997 |
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6063947 |
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08907608 |
Aug 8, 1997 |
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08675650 |
Jul 3, 1996 |
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5850026 |
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Current U.S.
Class: |
800/281 ;
554/9 |
Current CPC
Class: |
C12N 15/8247 20130101;
C12N 9/0083 20130101; C11B 1/10 20130101; A01H 5/10 20130101; C12N
15/8218 20130101; A23D 9/00 20130101 |
Class at
Publication: |
800/281 ;
554/9 |
International
Class: |
A01H 001/00; C12N
015/82; C11B 001/00 |
Claims
What is claimed is:
1. An endogenous oil obtained from Brassica seeds, said oil having
an oleic acid content of greater than about 80%, an
.alpha.-linolenic acid content of less than about 2.5% and an
erucic acid content of less than about 2%, said oleic acid content,
linolenic acid content and erucic acid content determined after
hydrolysis of said oil.
2. The oil of claim 1, further comprising a linoleic acid content
of from about 1% to about 10%, said linoleic acid content
determined after hydrolysis of said oil.
3. The oil of claim 1, wherein said oleic acid content is from
about 84% to about 88% and said .alpha.-linolenic acid content is
from about 1% to about 2%.
4. The oil of claim 3, further comprising a linoleic acid content
of from about 3% to about 7%, said linoleic acid content determined
after hydrolysis of said oil.
5. The oil of claim 1, wherein said seeds are Brassica napus
seeds.
6. A Brassica plant containing at least one recombinant nucleic
construct, said at least one construct comprising: a) a first
seed-specific regulatory sequence fragment operably linked to a
wild-type microsomal delta-12 fatty acid desaturase coding sequence
fragment; and b) a second seed-specific regulatory sequence
fragment operably linked to a wild-type microsomal delta-15 fatty
acid desaturase coding sequence fragment, wherein said plant
produces seeds, yielding an oil having an oleic acid content of
about 86% or greater and an erucic acid content of less than about
2%, said oleic acid content and erucic acid content determined
after hydrolysis of said oil.
7. The plant of claim 6, wherein said plant contains first and
second recombinant nucleic acid constructs, said first construct
comprising said delta-12 desaturase coding sequence fragment and
said second recombinant nucleic acid construct comprising said
delta-15 desaturase coding sequence fragment.
8. The plant of claim 6, wherein said delta-12 desaturase coding
sequence fragment comprises a full-length Brassica delta-12
desaturase coding sequence.
9. The plant of claim 6, wherein said delta-15 desaturase coding
sequence fragment comprises a full-length Brassica delta-15
desaturase coding sequence.
10. A Brassica plant containing at least one recombinant nucleic
acid construct, said at least one construct comprising: a) a first
seed-specific regulatory sequence fragment operably linked to a
wild-type microsomal delta-12 fatty acid desaturase coding sequence
fragment; and b) a second seed-specific regulatory sequence
fragment operably linked to a wild-type microsomal delta-15 fatty
acid desaturase coding sequence fragment, wherein said plant
produces seeds yielding an oil having an oleic acid content of 80%
or greater, an .alpha.-linolenic acid content of about 2.5% or less
and an erucic acid content of less than about 2%, said oleic acid
content, linolenic acid content and erucic acid content determined
after hydrolysis of said oil.
11. The plant of claim 10, wherein said first and second regulatory
sequence fragments are linked in sense orientation to said delta-12
and delta-15 desaturase coding sequence fragments,
respectively.
12. The plant of claim 10, wherein said plant contains a first
recombinant nucleic acid construct comprising said delta-12
desaturase coding sequence fragment and a second recombinant
nucleic acid construct comprising said delta-15 desaturase coding
sequence fragment.
13. The plant-of claim 10, wherein said delta-12 desaturase coding
sequence fragment comprises a full-length Brassica delta-12
desaturase coding sequence.
14. The plant of claim 10, wherein said delta-15 desaturase coding
sequence fragment comprises a full-length Brassica delta-15
desaturase coding sequence.
15. The plant of claim 10, wherein said plant produces seeds
yielding an oil having an oleic acid content of about 84% to about
89%, an .alpha.-linolenic acid content of about 1% to about 2% and
an erucic acid content of less than about 2%, said oleic acid
content, linolenic acid content and erucic acid content determined
after hydrolysis of said oil.
16. The plant of claim 15, wherein said oleic acid content is from
about 86% to about 89% and said .alpha.-linolenic acid content is
from about 1% to about 1.7%.
17. A method of producing an endogenous oil from Brassica seeds,
comprising the steps of: a) creating at least one Brassica plant
having a seed-specific reduction in microsomal delta-12 fatty acid
desaturase gene expression and a seed-specific reduction in
microsomal delta-15 fatty acid desaturase gene expression; b)
crushing seeds produced from said plant; and c) extracting said oil
from said seeds, said oil having an oleic acid content of about 86%
or greater and an erucic acid content of less than about 2%, said
oleic acid content and erucic acid content determined after
hydrolysis of said oil.
18. The method of claim 17, wherein said seed-specific reduction in
delta-12 desaturase expression is created by cosuppression.
19. The method of claim 17, wherein said seed-specific reduction in
delta-12 desaturase expression is created by antisense
suppression.
20. The method of claim 17, wherein said seed-specific reduction in
delta-15 desaturase expression is created by cosuppression.
21. The method of claim 17, wherein said seed-specific reduction in
delta-15 desaturase expression is created by antisense
suppression.
22. A method of producing an endogenous oil from Brassica seeds,
comprising the steps of: a) creating at least one Brassica plant
having a seed-specific reduction in microsomal delta-12 fatty acid
desaturase gene expression and a seed-specific reduction in
microsomal delta-15 fatty acid desaturase gene expression; b)
crushing seeds produced from said plant; and c) extracting said oil
from said seeds, said oil having an oleic acid content of about 80%
or greater, an .alpha.-linolenic acid content of 2.5% or less and
an erucic acid content of less than about 2%, said oleic acid
content and erucic acid content determined after hydrolysis of said
oil.
23. The method of claim 22, wherein said seed-specific reduction in
delta-12 desaturase expression is created by cosuppression.
24. The method of claim 22, wherein said seed-specific reduction in
delta-12 desaturase expression is created by antisense
suppression.
25. The method-of claim 22, wherein said seed-specific reduction in
delta-15 desaturase expression is created by cosuppression.
26. The method of claim 22, wherein said seed-specific reduction in
delta-15 desaturase expression is created by antisense suppression.
Description
TECHNICAL FIELD
[0001] This invention relates to a Brassica canola oil having an
elevated oleic acid content and a decreased linolenic acid profile
in the seed oil. The invention also relates to methods by which
such an oil may be produced.
BACKGROUND OF THE INVENTION
[0002] Diets high in saturated fats increase low density
lipoproteins (LDL) which mediate the deposition of cholesterol on
blood vessels. High plasma levels of serum cholesterol are closely
correlated with atherosclerosis and coronary heart disease (Conner
et al., Coronary Heart Disease: Prevention, Complications, and
Treatment, pp. 43-64, 1985). By producing oilseed Brassica
varieties with reduced levels of individual and total saturated
fats in the seed oil, oil-based food products which contain less
saturated fats can be produced. Such products will benefit public
health by reducing the incidence of atherosclerosis and coronary
heart disease.
[0003] The dietary effects of monounsaturated fats have also been
shown to have dramatic effects on health. Oleic acid, the only
monounsaturated fat in most edible vegetable oils, lowers LDL as
effectively as linoleic acid, but does not affect high density
lipoproteins (HDL) levels (Mattson, F. H., J. Am. Diet. Assoc.,
89:387-391, 1989; Mensink et al., New England J. Med., 321:436-441,
1989). Oleic acid is at least as effective in lowering plasma
cholesterol as a diet low in fat and high in carbohydrates (Grundy,
S. M., New England J. Med., 314:745-748, 1986; Mensink et al., New
England J. Med., 321:436-441, 1989). In fact, a high oleic acid
diet is preferable to low fat, high carbohydrate diets for
diabetics (Garg et al., New England J. Med., 319:829-834, 1988).
Diets high in monounsaturated fats are also correlated with reduced
systolic blood pressure (Williams et al., J. Am. Med. Assoc.,
257:3251-3256, 1987). Epidemiological studies have demonstrated
that the "Mediterranean" diet, which is high in fat and
monounsaturates, is not associated with coronary heart disease.
[0004] Intensive breeding has produced Brassica plants whose seed
oil contains less than 2% erucic acid. The same varieties have also
been bred so that the defatted meal contains less than 30 .mu.mol
glucosinolates/gram. Brassica seeds, or oils extracted from
Brassica seeds, that contain less than 2% erucic acid (C.sub.22:1),
and produce a meal with less than 30 .mu.mol glucosinolates/gram
are referred to as canola seeds or canola oils. Plant lines
producing such seeds are also referred to as canola lines or
varieties.
[0005] Many breeding studies have been directed to alteration of
the fatty acid composition in seeds of Brassica varieties. For
example, Pleines and Freidt, Fat Sci. Technol., 90(5), 167-171
(1988) describe plant lines with reduced C.sub.18:3 levels
(2.5-5.8%) combined with high oleic content (73-79%). Roy and Tarr,
Z. Pflanzenzuchtg, 95(3), 201-209 (1985) teaches transfer of genes
through an interspecific cross from Brassica juncea into Brassica
napus resulting in a reconstituted line combining high linoleic
with low linolenic acid content. Roy and Tarr, Plant Breeding, 98,
89-96 (1987) discuss prospects for development of B. napus L.
having improved linolenic and linolenic acid content. Canvin, Can.
J. Botany, 43, 63-69 (1965) discusses the effect of temperature on
the fatty acid composition of oils from several seed crops
including rapeseed.
[0006] Mutations can be induced with extremely high doses of
radiation and/or chemical mutagens (Gaul, H. Radiation Botany
(1964) 4:155-232) High dose levels which exceed LD50, and typically
reach LD90, led to maximum achievable mutation rates. In mutation
breeding of Brassica varieties, high levels of chemical mutagens
alone or combined with radiation have induced a limited number of
fatty acid mutations (Rakow, G. Z. Pflanzenzuchtg (1973)
69:62-82)
[0007] Rakow and McGregor, J. Amer. Oil Chem. Soc., 50, 400-403
(October 1973) discuss problems associated with selecting mutants
affecting seed linoleic and linolenic acid levels. The low
.alpha.-linolenic acid mutation derived from the Rakow mutation
breeding program did not have direct commercial application because
of low seed yield. The first commercial cultivar using the low
.alpha.-linolenic acid mutation derived in 1973 was released in
1988 as the variety Stellar. (Scarth, R. et al., Can. J. Plant Sci.
(1988) 68:509-511). The .alpha.-linolenic acid content of Stellar
seeds was greater than 3% and the linoleic acid content was about
28%.
[0008] Chemical and/or radiation mutagenesis has been used in an
attempt to develop an endogenous canola oil having an oleic acid
content of greater than 79% and an .alpha.-linolenic acid content
of less than 5%. Wong, et al., EP 0 323 753 B1. However, the lowest
.alpha.-linolenic acid level achieved was about 2.7%. PCT
publication WO 91/05910 discloses mutagenesis of a starting
Brassica napus line in order to increase the oleic acid content in
the seed oil. However, the oleic acid content in canola oil
extracted from seeds of such mutant lines did not exceed 80%.
[0009] The quality of canola oil and its suitability for different
end uses is in large measure determined by the relative proportion
of the various fatty acids present in the seed triacylglycerides.
As an example, the oxidative stability of canola oil, especially at
high temperatures, decreases as the proportion of tri-unsaturated
acids increases. Oxidative stability decreases to a lesser extent
as the proportion of di-unsaturated acids increases. However, it
has not been possible to alter the fatty acid composition in
Brassica seeds beyond certain limits. Thus, an endogenous canola
oil having altered fatty acid compositions in seeds is not
available for certain specialty uses. Instead, such specialty oils
typically are prepared from canola oil by further processing, such
as hydrogenation and/or fractionation.
SUMMARY OF THE INVENTION
[0010] An endogenous oil obtained from Brassica seeds is disclosed.
The oil has an oleic acid content of greater than about 80%, an
.alpha.-linolenic acid content of less than about 2.5% and an
erucic acid content of less than about 2%, which contents are
determined after hydrolysis of the oil. Preferably the oleic acid
content is from about 84% to about 88% and the .alpha.-linolenic
acid content is from about 1% to about 2%. The oil may further have
a linoleic acid content of from about 1% to about 10%, also
determined after hydrolysis of the oil. The oil can be obtained
from Brassica napus seeds, for example.
[0011] Also disclosed herein is a Brassica plant containing at
least one recombinant nucleic construct. The construct(s) comprise
a first seed-specific regulatory sequence fragment operably linked
to a wild-type microsomal delta-12 fatty acid desaturase coding
sequence fragment and a second seed-specific regulatory sequence
fragment operably linked to a wild-type microsomal delta-15 fatty
acid desaturase coding sequence fragment. Such a plant produces
seeds that yield an oil having an oleic acid content of about 86%
or greater and an erucic acid content of less than about 2%, which
are determined after hydrolysis of the oil. In some embodiments,
the plant contains first and second recombinant nucleic acid
constructs, the first construct comprising the delta-12 desaturase
coding sequence fragment and the second recombinant nucleic acid
construct comprising the delta-15 desaturase coding sequence
fragment. The delta-12 or delta-15 desaturase coding sequence
fragments may comprise either a partial or a full-length Brassica
delta-12 or delta-15 desaturase coding sequence.
[0012] Another Brassica plant containing at least one recombinant
nucleic acid construct is disclosed herein. The construct(s)
comprises a first seed-specific regulatory sequence fragment
operably linked to a wild-type microsomal delta-12 fatty acid
desaturase coding sequence fragment and a second seed-specific
regulatory sequence fragment operably linked to a wild-type
microsomal delta-15 fatty acid desaturase coding sequence fragment.
The plant produces seeds yielding an oil having an oleic acid
content of 80% or greater, an .alpha.-linolenic acid content of
about 2.5% or less and an erucic acid content of less than about
2%, which contents are determined after hydrolysis of the oil. Such
a plant may have first and second regulatory sequence fragments
linked in sense orientation to the delta-12 and delta-15 desaturase
coding sequence fragments, respectively. Alternatively-the first
and second regulatory sequence fragments may be linked in antisense
orientation to the corresponding coding sequence fragments. The
delta-12 or delta-15 desaturase coding sequence fragments may
comprise a partial or full-length Brassica delta-12 or delta-15
desaturase coding sequence. The plant may produce seeds yielding an
oil having an oleic acid content of 80% or greater, an
.alpha.-linolenic acid content of about 2.5% or less and an erucic
acid content of less than about 2, which contents are determined
after hydrolysis of the oil.
[0013] A method of producing an endogenous oil from Brassica seeds
is disclosed herein. The method comprises the steps of: creating at
least one Brassica plant having a seed-specific reduction in
microsomal delta-12 fatty acid desaturase gene expression and a
seed-specific reduction in microsomal delta-15 fatty acid
desaturase gene expression; crushing seeds produced from the plant;
and extracting the oil from the seeds. The oil has an oleic acid
content of about 86% or greater and an erucic acid content of less
than about 2%, determined after hydrolysis of the oil. The
seed-specific reduction in delta-12 or delta-15 desaturase
expression may be created by cosuppression or antisense.
[0014] Another method of producing an endogenous oil from Brassica
seeds is disclosed herein. The method comprises the steps of:
creating at least one Brassica plant having a seed-specific
reduction in microsomal delta-12 fatty acid desaturase gene
expression and a seed-specific reduction in microsomal delta-15
fatty acid desaturase gene expression; crushing seeds produced from
the plant; and extracting the oil from the seeds. The oil has an
oleic acid content of about 80% or greater, an .alpha.-linolenic
acid content of 2.5% or less and an erucic acid content of less
than about 2%, determined after hydrolysis of the oil. The
seed-specific reduction in delta-12 or delta-15 desaturase
expression may be created by cosuppression or by antisense.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The term "fatty acid desaturase" refers to an enzyme which
catalyzes the breakage of a carbon-hydrogen bond and the
introduction of a carbon-carbon double bond into a fatty acid
molecule. The fatty acid may be free or esterified to another
molecule including, but not limited to, acyl-carrier protein,
coenzyme A, sterols and the glycerol moiety of glycerolipids. The
term "glycerolipid desaturases" refers to a subset of the fatty
acid desaturases that act on fatty acyl moieties esterified to a
glycerol backbone. "Delta-12 desaturase" refers to a fatty acid
desaturase that catalyzes the formation of a double bond between
carbon positions 6 and 7 (numbered from the methyl end), (i.e.,
those that correspond to carbon positions 12 and 13 (numbered from
the carbonyl carbon) of an 18 carbon-long fatty acyl chain.
"Delta-15 desaturase" refers to a fatty acid desaturase that
catalyzes the formation of a double bond between carbon positions 3
and 4 (numbered from the methyl end), (i.e., those that correspond
to carbon positions 15 and 16 (numbered from the carbonyl carbon)
of an 18 carbon-long fatty acyl chain. "Microsomal desaturase"
refers to the cytoplasmic location of the enzyme, while
"chloroplast desaturase" and "plastid desaturase" refer to the
plastid location of the enzyme. It should be noted that these fatty
acid desaturases have never been isolated and characterized as
proteins. Accordingly, the terms such as "delta-12 desaturase" and
"delta-15 desaturase" are used as a convenience to describe the
proteins encoded by nucleic acid fragments that have been isolated
based on the phenotypic effects caused by their disruption. They do
not imply any catalytic mechanism. For example, delta-12 desaturase
refers to the enzyme that catalyzes the formation of a double bond
between carbons 12 and 13 of an 18 carbon fatty acid irrespective
of whether it "counts" the carbons from the methyl, carboxyl end,
or the first double bond.
[0016] Microsomal delta-12 fatty acid desaturase (also known as
omega-6 fatty acid desaturase, cytoplasmic oleic desaturase or
oleate desaturase) is involved in the enzymatic conversion of oleic
acid to linoleic acid. A microsomal delta-12 desaturase has been
cloned and characterized using T-DNA tagging. Okuley, et al., Plant
Cell 6:147-158 (1994). The nucleotide sequences of higher plant
genes encoding microsomal delta-12 fatty acid desaturase are
described in Lightner et al., WO94/11516.
[0017] Microsomal delta-15 fatty acid desaturase (also known as
omega-3 fatty acid desaturase, cytoplasmic linoleic acid desaturase
or linoleate desaturase) is involved in the enzymatic conversion of
linoleic acid to .alpha.-linolenic acid. Sequences of higher plant
genes encoding microsomal and plastid delta-15 fatty acid
desaturases are disclosed in Yadav, N., et al., Plant Physiol.,
103:467-476 (1993), WO 93/11245 and Arondel, V. et al., Science,
258:1353-1355 (1992).
[0018] Brassica species have more than one gene for endogenous
microsomal delta-12 desaturase and more than one gene for
endogenous microsomal delta-15 desaturase. The genes for microsomal
delta-12 desaturase are designated Fad2 while the genes for
microsomal delta-15 desaturase are designated Fad3. In
amphidiploids, each gene is derived from one of the ancestral
genomes making up the species under consideration. The full-length
coding sequences for the wild-type Fad2 genes from Brassica napus
(termed the D form and the F form) are shown in SEQ ID NO:1 and SEQ
ID NQ:5, respectively. The full-length coding sequence for a
wild-type Fad3 gene is disclosed in WO 93/11245.
[0019] The inventors have discovered canola oils that have novel
fatty acid compositions, e.g., very high oleic acid levels and very
low .alpha.-linolenic acid levels. Such oils may be obtained by
crushing seeds of transgenic Brassica plants exhibiting a
seed-specific reduction in delta-12 desaturase and delta-15
desaturase activity; oil of the invention is extracted therefrom.
Expression of Fad2 and Fad3 in seeds is reduced such that the
resulting seed oil possesses very high levels of oleic acid and
very low levels of .alpha.-linolenic acid. The fatty acid
composition of the endogenous seed oil, as determined after
hydrolysis of fatty acid esters reflects the novel fatty acid
composition of such seeds.
[0020] The fatty acid composition of oils disclosed herein is
determined by techniques known to the skilled artisan, e.g.,
hydrolysis of esterified fatty acids (triacylglycerides and the
like) in a bulk seed sample followed by gas-liquid chromatography
(GLC) analysis of fatty acid methyl esters.
[0021] In one embodiment, an oil of the invention has an oleic acid
content of about 80% or greater, as well as a surprisingly low
.alpha.-linolenic acid content of about 2.5% or less. The oleic
acid content is preferably from about 84% to about 89%, more
preferably from about 86% to about 89%. The .alpha.-linolenic acid
preferably is from about 1% to less than about 2.5%, more
preferably from about 1% to about 2%.
[0022] The linoleic acid content of an oil of this embodiment
typically is less than about 10%, preferably less than about 7%,
more preferably from about 2% to about 6%.
[0023] Canola seed is crushed by techniques known in the art. The
seed typically is tempered by spraying the seed with water to raise
the moisture to, for example, 8.5%. The tempered seed is flaked
using smooth roller with, for example, a gap setting of 0.23 to
0.27 mm. Heat may be applied to the flakes to deactivate enzymes,
facilitate further cell rupturing, coalesce the oil droplets and
agglomerate protein particles in order to ease the extraction
process.
[0024] Typically, oil is removed from the heated canola flakes by a
screw press to press out a major fraction of the oil from the
flakes. The resulting press cake contains some residual oil.
[0025] Crude oil produced from the pressing operation typically is
passed through a settling tank with a slotted wire drainage top to
remove the solids expressed out with the oil in the screw pressing
operation. The clarified oil can be passed through a plate and
frame filter to remove the remaining fine solid particles.
[0026] Canola press cake produced from the screw pressing operation
can be extracted with commercial n-Hexane. The canola oil recovered
from the extraction process is combined with the clarified oil from
the screw pressing operation, resulting in a blended crude oil.
[0027] Free fatty acids and gums typically are removed from the
crude oil by heating in a batch refining tank to which food grade
phosphoric acid has been added. The acid serves to convert the
non-hydratable phosphatides to a hydratable form, and to chelate
minor metals that are present in the crude oil. The phosphatides
and the metal salts are removed from the oil along with the
soapstock. The oil-acid mixture is treated with sodium hydroxide
solution to neutralize the free fatty acids and the phosphoric acid
in the acid-oil mixture. The neutralized free fatty acids,
phosphatides, etc. (soapstock) are drained off from the neutralized
oil. A water wash may be done to further reduce the soap content of
the oil. The oil may be bleached and deodorized before use, if
desired, by techniques known in the art.
[0028] A transgenic plant disclosed herein contains at least one
recombinant nucleic acid construct. The construct or constructs
comprise an oleate desaturase coding sequence fragment and a
linoleate desaturase coding sequence fragment, both of which are
expressed preferentially in developing seeds. Seed-specific
expression of the recombinant desaturases results in a
seed-specific reduction in native desaturase gene expression. The
seed-specific defect in delta-12 and delta-15 desaturase gene
expression alters the fatty acid composition in mature seeds
produced on the plant, so that the oil obtained from such seeds has
the novel fatty acid compositions disclosed herein.
[0029] Typically, the oleate and linoleate desaturase sequence
fragments are present on separate constructs and are introduced
into the non-transgenic parent on separate plasmids. The desaturase
fragments may be isolated or derived from, e.g., Brassica spp.,
soybean (Glycine max), sunflower and Arabidopsis. Preferred host or
recipient organisms for introduction of a nucleic acid construct
are oil-producing species, such as Brassica napus, B. rapa and B.
juncea.
[0030] A transgenic plant disclosed herein preferably is homozygous
for the transgene containing construct. Such a plant may be used as
a parent to develop plant lines or may itself be a member of a
plant line, i.e., be one of a group of plants that display little
or no genetic variation between individuals for the novel oil
composition trait. Such lines may be created by several generations
of self-pollination and selection, or vegetative propagation from a
single parent using tissue or cell culture techniques. Other means
of breeding plant lines from a parent plant are known in the
art.
[0031] Progeny of a transgenic plant are included within the scope
of the invention, provided that such progeny exhibit the novel seed
oil characteristics disclosed herein. Progeny of an instant plant
include, for example, seeds formed on F.sub.1, F.sub.2, F.sub.3,
and subsequent generation plants, or seeds formed on BC.sub.1,
BC.sub.2, BC.sub.3 and subsequent generation plants.
[0032] A seed-specific reduction in Fad2 and Fad3 gene expression
may be achieved by techniques including, but not limited to,
antisense and cosuppression. These phenomena significantly reduce
expression of the gene product by the native genes (wild-type or
mutated). The reduction in gene expression can be inferred from the
decreased level of reaction product and the increased level of
substrate in seeds (e.g., decreased 18:2 and increased 18:1),
compared to the corresponding levels in plant tissues expressing
the native genes.
[0033] The preparation of antisense and cosuppression constructs
for inhibition of fatty acid desaturases may utilize fragments
containing the transcribed sequence for the Fad2 and Fad3 fatty
acid desaturase genes in canola. These genes have been cloned and
sequenced as discussed hereinabove.
[0034] Antisense RNA has been used to inhibit plant target genes in
a tissue-specific manner. van der Krol et al., Biotechniques
6:958-976 (1988). Antisense inhibition has been shown using the
entire cDNA sequence as well as a partial cDNA sequence. Sheehy et
al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); Cannon et al.,
Plant Mol. Biol. 15:39-47 (1990). There is also evidence that 3'
non-coding sequence fragment and 5' coding sequence fragments,
containing as few as 41 base-pairs of a 1.87 kb cDNA, can play
important roles in antisense inhibition. (Ch'ng et al., Proc. Natl.
Acad. Sci. USA 86:10006-10010 (1989); Cannon et al., supra.
[0035] The phenomenon of cosuppression has also been used to
inhibit plant target genes in a tissue-specific manner.
Cosuppression of an endogenous gene using a full-length cDNA
sequence as well as a partial cDNA sequence (730 bp of a 1770 bp
cDNA) are known. Napoli et al., The Plant Cell 2:279-289 (1990);
van der Krol et al., The Plant Cell 2:291-299 (1990); Smith et al.,
Mol. Gen. Genetics 224:477-481 (1990).
[0036] Nucleic acid fragments comprising a partial or a full-length
delta-12 or delta-15 fatty acid desaturase coding sequence are
operably linked to at least one suitable regulatory sequence in
antisense orientation (for antisense constructs) or in sense
orientation (for cosuppression constructs). Molecular biology
techniques for preparing such chimeric genes are known in the art.
The chimeric gene is introduced into a Brassica plant and
transgenic progeny displaying a fatty acid composition disclosed
herein due to antisense or cosuppression are identified. Transgenic
plants that produce a seed oil having a fatty acid composition
disclosed herein are selected for use in the invention.
Experimental procedures to develop and identify cosuppressed plants
involve breeding techniques and fatty acid analytical techniques
known in the art.
[0037] One may use a partial cDNA sequence for cosuppression as
well as for antisense inhibition. For example, cosuppression of
delta-12 desaturase and delta-15 desaturase in Brassica napus may
be achieved by expressing, in the sense orientation, the entire or
partial seed delta-12 desaturase cDNA found in pCF2-165D. See WO
04/11516.
[0038] Seed-specific expression of native Fad2 and Fad3 genes can
also be inhibited by non-coding regions of an introduced copy of
the gene. See, e.g., Brusslan, J. A. et al. (1993) Plant Cell
5:667-677; Matzke, M. A. et al., Plant Molecular Biology
16:821-830). One skilled in the art can readily isolate genomic DNA
containing sequences that flank desaturase coding sequences and use
the non-coding regions for antisense or cosuppression
inhibition.
[0039] Regulatory sequences typically do not themselves code for a
gene product. Instead, regulatory sequences affect the expression
level of the mutant coding sequence. Examples of regulatory
sequences are known in the art and include, without limitation,
promoters of genes expressed during embryogenesis, e.g., a napin
promoter, a phaseolin promoter, a oleosin promoter and a cruciferin
promoter. Native regulatory sequences, including the native
promoters, of delta-12 and delta-15 fatty acid desaturase genes can
be readily isolated by those skilled in the art and used in
constructs of the invention. Other examples of suitable regulatory
sequences include enhancers or enhancer-like elements, introns and
3' non-coding regions such as poly A sequences. Further examples of
suitable regulatory sequences for the proper expression of mutant
or wild-type delta-12 or mutant delta-15 coding sequences are known
in the art.
[0040] In preferred embodiments, regulatory sequences are
seed-specific, i.e., the chimeric desaturase gene product is
preferentially expressed in developing seeds and expressed at low
levels or not at all in the remaining tissues of the plant.
Seed-specific regulatory sequences preferably stimulate-or induce
expression of the recombinant desaturase coding sequence fragment
at a time that coincides with or slightly precedes expression of
the native desaturase gene. Murphy et al., J. Plant Physiol.
135:63-69 (1989).
[0041] Transgenic plants for use in the invention are created by
transforming plant cells of Brassica species. Such techniques
include, without limitation, Agrobacterium-mediated transformation,
electroporation and particle gun transformation. Illustrative
examples of transformation techniques are described in U.S. Pat.
No. 5,204,253, (particle gun) and U.S. Pat. No. 5,188,958
(Agrobacterium), incorporated herein by reference. Transformation
methods utilizing the Ti and Ri plasmids of Agrobacterium spp.
typically use binary type vectors. Walkerpeach, C. et al., in Plant
Molecular Biology Manual, S. Gelvin and R. Schilperoort, eds.,
Kluwer Dordrecht, C1:1-19 (1994). If cell or tissue cultures are
used as the recipient tissue for transformation, plants can be
regenerated from transformed cultures by techniques known to those
skilled in the art.
[0042] One or more recombinant nucleic acid constructs, suitable
for antisense or cosuppression of native Fad2 and Fad3 genes are
introduced, and at least one transgenic Brassica plant is obtained.
Seeds produced by the transgenic plant(s) are grown and either
selfed or outcrossed to obtain plants homozygous for the
recombinant construct. Seeds are analyzed as discussed above in
order to identify those homozygotes having native fatty acid
desaturase activities inhibited by the mechanisms discussed above.
Homozygotes may be entered into a breeding program, e.g., to
increase seed, to introgress the novel oil composition trait into
other lines or species, or for further selection of other desirable
traits (disease resistance, yield and the like).
[0043] Fatty acid composition is followed during the breeding
program by analysis of a bulked seed sample or of a single
half-seed. Half-seed analysis useful because the viability of the
embryo is maintained and thus those seeds having a desired fatty
acid profile may be advanced to the next generation. However,
half-seed analysis is also known to be an inaccurate representation
of the genotype of the seed being analyzed. Bulk seed analysis
typically yields a more accurate representation of the fatty acid
profile in seeds of a given genotype.
[0044] Procedures for analysis of fatty acid composition are known
in the art. These procedures can be used to identify individuals to
be retained in a breeding program; the procedures can also be used
to determine the product specifications of commercial or pilot
plant oils.
[0045] The relative content of each fatty acid in canola seeds can
be determined either by direct trans-esterification of individual
seeds in methanolic H.sub.2SO4 (2.5%) or by hexane extraction of
bulk seed samples followed by trans-esterification of an aliquot in
1% sodium methoxide in methanol. Fatty acid methyl esters can be
extracted from the methanolic solutions into hexane after the
addition of an equal volume of water.
[0046] For example, a seed sample from each transformant in a
breeding program is crushed with a mortar and pestle and extracted
4 times with 8 mL hexane at about 50.degree. C. The extracts from
each sample are reduced in volume and two aliquots are taken for
esterification. Separation of the fatty acid methyl esters can be
carried out by gas-liquid chromatography using an Omegawax 320
column (Supelco Inc., 0.32 mm ID.times.30 M) run isothermally at
220.degree. and cycled to 260.degree. between each injection.
[0047] Alternatively, seed samples from a breeding program are
ground and extracted in methanol/KOH, extracted with iso-octane,
and fatty acids separated by gas chromatography.
[0048] A method to produce an oil of the invention comprises the
creation of at least one Brassica plant having a seed-specific
reduction in Fad2 and Fad3 gene expression, as discussed above.
Seeds produced by such a plant, or its progeny, are crushed and the
oil is extracted from the crushed seeds. Such lines produce seeds
yielding an oil of the invention, e.g., an oil having from about
80% to about 88% oleic acid, from about 1% to about 2%
.alpha.-linolenic acid and less than about 2% erucic acid.
[0049] Alternatively, such a plant can be created by crossing two
parent plants, one exhibiting a reduction in Fad2 gene expression
and the other exhibiting a reduction in Fad3 gene expression.
Progeny of the cross are outcrossed or selfed in order to obtain
progeny seeds homozygous for both traits.
[0050] Transgenic plants having a substantial reduction in Fad2 and
Fad3 gene expression in seeds have novel fatty acid profiles in oil
extracted from such seeds, compared to known canola plants, e.g.,
the reduction in both desaturase activities results in a novel
combination of high oleic and lower .alpha.-linolenic acid in seed
oils. By combining seed-specific inhibition of microsomal delta-12
desaturase with seed-specific inhibition of microsomal delta-15
desaturase, one obtains very low levels of seed .alpha.-linolenic
acid, without adversely affecting agronomic properties.
[0051] It is noteworthy that Fad2 and Fad3 cosuppression constructs
provide a novel means for producing canola oil having 86% oleic
acid or greater. A method of producing a canola oil having greater
than 86% oleic acid comprises the creation of a transgenic Brassica
plant containing at least one recombinant nucleic acid construct,
which construct(s) comprises an oleate desaturase coding sequence
expressed preferentially in developing seeds and a linoleate
desaturase coding sequence expressed preferentially in developing
seeds. A proportion of the plants that are homozygous for the
transgenes have seed-specific cosuppression of the native linoleate
desaturase. Seeds produced by such transgenic cosuppressed plants
are crushed and the oil is extracted therefrom. The oil has about
86% or greater oleic acid and less than about 2% erucic acid. The
oleic acid content can be as high as 89%.
[0052] Transgenic plants exhibiting cosuppression of Fad2 and Fad3
produce seeds having a very high oleic acid content. This result
was unexpected because it was not known if one could obtain plants
in which inhibition of Fad2 and Fad3 via cosuppression was
sufficient to achieve an oleic acid level of 86% or greater in
seeds. Indeed, it was not known if two cosuppressed genes in fatty
acid metabolism could be introduced in canola without the first
cosuppression gene interfering with the second cosuppression gene,
or without adversely affecting other agronomic traits.
[0053] Marker-assisted breeding techniques may be used to identify
and follow a desired fatty acid composition during the breeding
process. Such markers may include RFLP, RAPD, or PCR markers, for
example. Marker-assisted breeding techniques may be used in
addition to, or as an alternative to, other sorts of identification
techniques. An example of marker-assisted breeding is the use of
PCR primers that specifically amplify the junction between a
promoter fragment and the coding sequence of a Fad2 gene.
[0054] While the invention is susceptible to various modifications
and alternative forms, certain specific embodiments thereof are
described in the general methods and examples set forth below. For
example the invention may be applied to all Brassica species,
including B. rapa, B. juncea, and B. hirta, to produce
substantially similar results. It should be understood, however,
that these examples are not intended to limit the invention to the
particular forms disclosed. Instead, the disclosure is to cover all
modifications, equivalents and alternatives falling within the
scope of the invention.
EXAMPLE 1
Constructs For Cosuppression of Delta-12 Fatty Acid Desaturase and
Delta-15 Fatty Acid Desaturase
[0055] The wild-type Brassica cDNA coding sequence for the delta-12
desaturase D form was cloned as described in WO 94/11516, which is
incorporated herein by reference. Briefly, rapeseed cDNAs encoding
cytoplasmic oleate (18:1) desaturase were obtained by screening a
cDNA library made from developing rapeseed using a heterologous
probe derived from an Arabidopsis cDNA fragment encoding the same
enzyme. (Okuley et al 1994). The full-length coding sequence of
Fad2 is found as SEQ ID NO:1. Rapeseed cDNAs encoding the
cytoplasmic linoleate (18:2) desaturase (Fad3) were obtained as
described in WO 93/11245, incorporated herein by reference. See
also (Yadav et. al 1993). Seed specific expression of these cDNAs
in transgenic rapeseed was driven by one of four different seed
storage protein promoters, napin, oleosin and cruciferin promoters
from B. napus and a phaseolin promoter from Phaseolus vulgaris.
[0056] Detailed procedures for manipulation of DNA fragments by
restriction endonuclease digestion, size separation by agarose gel
electrophoresis, isolation of DNA fragments from agarose gels,
ligation of DNA fragments, modification of cut ends of DNA
fragments and transformation of E. coli cells with plasmids have
been described. Sambrook et al., (Molecular Cloning, A Laboratory
Manual, 2nd ed (1989) Cold Spring Harbor Laboratory Press); Ausubel
et al., Current Protocols in Molecular Biology (1989) John Wiley
& Sons). Plant molecular biology procedures are described in
Plant Molecular Biology Manual, Gelvin S. and Schilperoort, R. eds.
Kluwer, Dordrecht (1994).
[0057] The plasmid pZS212 was used to construct binary vectors for
these experiments. pZS212 contains a chimeric CaMV35S/NPT gene for
use in selecting kanamycin resistant transformed plant cells, the
left and right border of an Agrobacterium Ti plasmid T-DNA, the E.
coli lacZ .alpha.-complementing segment with unique restriction
endonuclease sites for EcoRI, KpnI, BamHI and SalI, the bacterial
replication origin from the Pseudomonas plasmid pVS1 and a
bacterial Tn5 NPT gene for selection of transformed Agrobacterium.
See WO 94/11516, p. 100.
[0058] The first construct was prepared by inserting a full-length
mutant Brassica Fad2 D gene coding sequence fragment in sense
orientation between the phaseolin promoter and phaseolin 3' poly A
region of plasmid pCW108. The full-length coding sequence of the
mutant gene is found in SEQ ID NO:3.
[0059] The pCW108 vector contains the bean phaseolin promoter and
3' untranslated region and was derived from the commercially
available pUC18 plasmid (Gibco-BRL) via plasmids AS3 and pCW104.
Plasmid AS3 contains 495 base pairs of the Phaseolus vulgaris
phaseolin promoter starting with 5'-TGGTCTTTTGGT-3' followed by the
entire 1175 base pairs of the 3' untranslated region of the same
gene. Sequence descriptions of the 7S seed storage protein promoter
are found in Doyle et al., J. Biol. Chem. 261:9228-9238 (1986) and
Slightom et al., Proc. Natl. Acad. Sci. USA, 80:1897-1901 (1983).
Further sequence description may be found in WO 91/13993. The
fragment was cloned into the Hind III site of pUC18. The additional
cloning sites of the pUC18 multiple cloning region (Eco RI, Sph I,
Pst I and Sal I) were removed by digesting with Eco RI and Sal I,
filling in the ends with Klenow and religating to yield the plasmid
pCW104. A new multiple cloning site was created between the 495 bp
of the 5' phaseolin and the 1175 bp of the 3' phaseolin by
inserting a dimer of complementary synthetic oligonucleotides to
create the plasmid pCW108. See WO 94/11516. This plasmid contains
unique Nco I, Sma I, Kpn I and Xba I sites directly behind the
phaseolin promoter.
[0060] The phaseolin promoter:mutantFad2:phaseolin poly A construct
in pCW108 was excised and cloned between the SalI/EcoRI sites of
pZS212. The resulting plasmid was designated pIMC201.
[0061] A second plasmid was constructed by inserting the
full-length wild type Brassica Fad2 D gene coding sequence into the
NotI site of plasmid pIMC401, which contains a 2.2 kb napin
expression cassette. See, e.g., WO94/11516, page 102. The
5'-napin:Fad2:napin poly A-3' construct was inserted into the SalI
site of pZS212 and the resulting 17.2 Kb plasmid was termed
pIMC127. Napin promoter sequences are also disclosed in U.S. Pat.
No. 5,420,034.
[0062] A third plasmid, pIMC135, was constructed in a manner
similar to that described above for pIMC127. Plasmid pIMC135
contains a 5' cruciferin promoter fragment operably linked in sense
orientation to the full-length wild-type Brassica Fad2 D gene
coding sequence, followed by a cruciferin 3' poly A fragment. The
5'-cruciferin:Fad2 D:cruciferin polyA cassette was inserted into
pZS212; the resulting plasmid was termed pIMC135. Suitable
cruciferin regulatory sequences are disclosed in Rodin, J. et al.,
J. Biol. Chem. 265:2720 (1990); Ryan, A. et al., Nucl. Acids Res.
17:3584 11989) and Simon, A. et al., Plant Mol. Biol. 5:191 (1985).
Suitable sequences are also disclosed in the Genbank computer
database, e.g., Accession No. M93103.
[0063] A fourth plasmid, pIMC133 was constructed in a manner
similar to that described above. Plasmid pIMC133 contains a 5'
oleosin promoter fragment operably linked in sense orientation to
the full-length Brassica Fad2 D gene coding sequence, followed by a
3' oleosin poly A fragment. See, e.g., WO 93/20216, incorporated
herein by reference.
[0064] A napin-Fad3 construct was made by first isolating a
delta-15 desaturase coding sequence fragment from pBNSF3-f2. The
fragment contained the full-length coding sequence of the
desaturase, disclosed as SEQ ID NO: 6 in WO 93/11245, incorporated
herein by reference. The 1.2 kb fragment was fitted with linkers
and ligated into pIMC401. The 5'napin:Fad3:3'napin cassette was
inserted into the Sal I site of pZS212; the resulting plasmid was
designated pIMC110.
EXAMPLE 2
Creation of Transgenic Cosuppressed Plants
[0065] The plasmids pIMC201, pIMC127, pIMC135, pIMC133 and pIMC110
were introduced into Agrobacterium strain LBA4404/pAL4404 by a
freeze-thaw method. The plasmids were introduced into Brassica
napus cultivar Westar by the method of Agrobacterium-mediated
transformation as described in WO94/11516, incorporated herein by
reference. Transgenic progeny plants containing pIMC201 were
designated as the WS201 series. Plants transformed with pIMC127
were designated as the WS687 series. Plants transformed with
pIMC135 were designated as the WS691 series. Plants transformed
with pIMC133 were designated as the WS692 series. Plants
transformed with pIMC110 were designated as the WS663 series.
[0066] Unless indicated otherwise, fatty acid percentages described
herein are percent by weight of the oil in the indicated seeds as
determined after extraction and hydrolysis.
[0067] From about 50 to 350 transformed plants (T1 generation) were
produced for each cDNA and promoter combination. T1 plants were
selfed to obtain T2 seed. T2 samples in which cosuppression events
occurred were identified from the fatty acid profile and from the
presence of the transgene by molecular analysis. The transformed
plants were screened for phenotype by analysis of the relative
fatty acid contents of bulk seed from the first transformed
generation by GC separation of fatty acid methyl esters.
[0068] T2 seed was sown in 4-inch pots containing Pro-Mix soil. The
plants, along with Westar controls, were grown at 25.+-.3.degree.
C./18.+-.3.degree. C., 14/10 hr day/night conditions in the
greenhouse. At flowering, the terminal raceme was self-pollinated
by bagging. At maturity, seed was individually harvested from each
plant, labelled, and stored to ensure that the source of the seed
was known.
[0069] Fatty acid profiles were determined as described in WO
91/05910. For chemical analysis, 10-seed bulk samples were hand
ground with a glass rod in a 15-mL polypropylene tube and extracted
in 1.2 mL 0.25 N KOH in 1:1 ether/methanol. The sample was vortexed
for 30 sec. and heated for 60 sec. in a 60.degree. C. water bath.
Four mL of saturated NaCl and 2.4 mL of iso-octane were added, and
the mixture was vortexed again. After phase separation, 600 .mu.L
of the upper organic phase were pipetted into individual vials and
stored under nitrogen at -5.degree. C. One .mu.L samples were
injected into a Supelco SP-2330 fused silica capillary column (0.25
mm ID, 30 M length, 0.20 .mu.m df).
[0070] The gas chromatograph was set at 180.degree. C. for 5.5
minutes, then programmed for a 2.degree. C./minute increase to
212.degree. C., and held at this temperature for 1.5 minutes. Total
run time was 23 minutes. Chromatography settings were: Column head
pressure--15 psi, Column flow (He)--0.7 mL/min., Auxiliary and
Column flow--33 mL/min., Hydrogen flow--33 mL/min., Air flow--400
mL/min., Injector temperature--250.degree- . C., Detector
temperature--300.degree. C., Split vent--1/15.
[0071] Table 1 shows the content of the seven major fatty acids in
mature seeds from transgenic cosuppressed plants homozygous for the
napin:Fad3 construct or the napin:Fad2 construct (T4 or later
generation). Over expression phenotypes and cosuppression
phenotypes were observed for both chimeric genes (oleate desaturase
and linoleate desaturase); data for plants exhibiting the
cosuppression phenotype are shown in the Table.
[0072] As shown in Table 1, the homozygous Fad2-cosuppressed seed
had a .alpha.-linolenic acid content of about 2.9%, which was less
than half that of the Westar control; the oleic acid content
increased to about 84.1%. The homozygous Fad3-cosuppressed seed had
an .alpha.-linolenic acid of about 1.2%; the oleic acid and
linoleic acid contents in Fad3-cosuppressed plants increased
slightly compared to Westar. The results demonstrate that
inhibiting gene expression of either enzyme by cosuppression
resulted in a change in fatty acid composition of the seed oil.
1TABLE 1 Fatty Acid Profiles in Oil From Cosuppression Canola Seed
FATTY ACID (% OF TOTAL FATTY ACIDS TRANSGENE CONSTRUCTION 16:0 18:0
18:1 18:2 18:3 20:0 20:1 22:0 24:0 non-transformed Westar 3.9 1.8
67.0 19.0 7.5 0.6 0.8 0.6 0.1 napin: Fad2 (co-suppression) 4.3 1.4
84.1 5.2 2.9 0.6 0.9 0.5 0.2 napin: Fad3 (co-suppression) 3.8 1.5
68.5 22.1 1.2 0.6 1.1 0.4 0.1
[0073]
2TABLE 2 Fatty Acid Profiles in Oil From Cosuppression Canola Seeds
Construct (Prommoter/ Fatty Acid Composition Line # coding
sequence) 16:0 18:0 18:1 18:2 18:3 663-40 napin/Fad3 3.9 1.4 71.2
20.1 1.2 687-193 napin/Fad2 4.0 1.5 82.8 5.9 3.7 691-215
cruciferin/Fad2 3.3 1.3 86.5 3.0 3.7 692-090-3 oleosin/Fad2 3.4 1.3
86.5 2.6 3.9 692-105-11 oleosin/Fad2 3.4 1.3 86.2 2.7 4.2 201-389
A23 phaseolin/MFad2 4.2 2.7 84.6 4.7 3.7
[0074]
3TABLE 3 Range of Fatty Acid Profiles for Fad2 and Fad3
Cosuppression Lines Tested in the Field Line Min/ Fatty Acid
Composition No. Vector Max C16:0 C18:0 C18:1 C18:2 C18:3 663-
pIMC110 Min 3.5 2.3 73.5 16.3 0.8 40 Max 4.7 2.2 64.0 24.2 1.5 687-
pIMC127 Min 3.4 3.1 93.3 3.8 2.3 193 Max 3.4 2.1 85.5 3.2 2.5 692-
pIMCl33 Min 3.7 2.7 84.6 2.B 2.4 105 Max 3.3 2.3 86.3 2.1 2.7 691-
pIMC135 Min 3.2 2.4 84.6 3.0 2.5 215 Max 3.0 2.0 86.3 2.6 2.5
[0075] Table 2 shows the fatty acid profile in T4 or later
homozygous seeds produced by six individual plants having various
promoter-desaturase gene combinations. The seeds were obtained from
greenhouse-grown plants. The results indicate that the oleic acid
content ranged from about 82.8% to about 86.5% among the lines
carrying the Fad2 constructs. The phaseolin:mutated Fad2 construct
was as successful as the wild-type Fad2 constructs in achieving
seed-specific Fad2 cosuppression.
[0076] The napin:Fad3 cosuppressed plant line had an unusually low
.alpha.-linolenic acid content of 1.2%. However, the oleic acid
content was only 71.2% and the linoleic acid content was similar to
that of the non-transformed control Westar in Table 1.
[0077] Homozygous seeds from four of the lines in Table 2 were
planted in a field nursery in Colorado and self-pollinated. Seed
samples from several plants of each line were collected and
separately analyzed for fatty acid composition. The results for the
663-40 plant having the minimum and the 663-40 plant having the
maximum linolenic acid content observed in the field are shown in
Table 3. The results for the 687-193, 692-105 and 691-215 plants
having the minimum and maximum oleic acid content in the field are
also shown in Table 3.
[0078] The results in Table 3 demonstrate that the fatty acid
profile in field-grown seeds of cosuppressed transgenic plants was
similar to that in the greenhouse-grown seeds (Table 2), indicating
that the cosuppression trait confers a stable fatty acid
composition on the oil. The results also indicate that an oil
having the combination of an oleic acid content of 86% or greater
and an .alpha.-linolenic acid content of 2.5% or less could not be
obtained from plants cosuppressed for either Fad2 or Fad3
alone.
EXAMPLE 3
Oil Content in Seeds of Plants Exhibiting Fad2 and Fad3
Cosuppression
[0079] Crosses were made between the napin:Fad3 cosuppressed line
663-40 and three Fad2 cosuppressed lines, 691-215, 692-090-3 and
692-105-11. F1 plants were selfed for 2 generations in the
greenhouse to obtain F3 generation seed that was homozygous for
both recombinant constructs.
4TABLE 4 Fatty Acid Profile in F3 Seeds of Lines Exhibiting Fad2
and Fad3 Cosuppression Line # Construct 16:0 18:0 18:1 18:2 18:3
663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2 691-215 cruciferin/ 3.9 1.3
86.5 3.0 3.7 Fad2 663- napin/Fad3 3.2 1.4 86.2 5.2 1.5 40X691-
& cruciferin/ 215 Fad2 663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2
692- oleosin/Fad2 3.4 1.3 86.5 2.6 3.9 090-3 662- napin/Fad3 3.4
1.5 85.5 5.0 1.7 40X692- & oleosin/ 0903-3 Fad2 663-40
napin/Fad3 3.9 1.4 71.2 20.1 1.2 692- oleosin/Fad2 3.4 1.3 86.2 2.7
4.2 105-11 663- napin/Fad3 3.4 1.4 86.8 4.6 1.4 40X692- &
oleosin/ 105-11 Fad2
[0080] The seed fatty acid profiles of the parent lines and a
representative F3 cosuppressed line are shown in Table 4. Plants
expressing both cosuppression constructs exhibited an oleic acid
level of about 86% or greater. Moreover, this high level of oleic
acid was present in combination with an unusually low level of
.alpha.-linolenic acid, less than 2.0%. However, the linoleic acid
content in the F3 seeds increased from about 2.6-3.0% to about
4.6-5.2%.
[0081] These results demonstrate that a canola oil can be extracted
from rapeseeds that contains greater than 80% oleic acid and less
than 2.5% .alpha.-linolenic acid. Results similar to those obtained
using cosuppression constructs are achieved when antisense
constructs are used.
[0082] The canola oil extracted from Fad2 and Fad3 cosuppressed F3
seed, or progeny thereof, is found to have superior oxidative
stability compared to the oil extracted from Westar seed. The
improved oxidative stability of such an oil is measured after
refining, bleaching and deodorizing, using the Accelerated Oxygen
Method (AOM), American Oil Chemists' Society Official Method Cd
12-57 for fat stability, Active Oxygen Method (revised 1989). The
improved oxidative stability is also demonstrated when using the
Oxidative Stability Index method. The improved oxidative stability
is measured in the absence of added antioxidants.
EXAMPLE 4
Oil Content in Seeds of Plants Having Fad3 Cosuppression and
Chemically-induced Fad2 Mutations
[0083] Q4275 is a doubly mutagenized B. napus line having defects
in the Fad2 gene. Q4275 was derived by chemical mutagenesis of B.
napus line IMC129, which carries a mutation in the Fad2 D gene; the
coding sequence of the mutated gene is shown in SEQ ID NO:3. Line
IMC129 was itself derived by chemical mutagenesis of the cultivar
Westar, as disclosed in WO 91/05910. Genetic segregation analysis
of crosses between Q4275 and other fatty acid mutant lines
indicated that Q4275 carried a mutation in the B. napus Fad2 F gene
in addition to the IMC129 Fad2 D gene mutation. Q4275 thus carries
chemically induced mutations in both Fad2 genes.
[0084] A cross was made between Q4275 and the napin:Fad3
cosuppressed line 663-40. F1 plants were selfed in the greenhouse
and F2 plants that were homozygous for the recombinant construct
and the Fad2 D and Fad2 F mutated genes were identified by fatty
acid profile analysis of the F3 generation seed. After selfing to
homozygosity, the fatty acid profiles in seeds of a representative
homozygous plant was analyzed and compared to the profile of the
parent plants, as shown in Table 5.
[0085] The results show that an oil having greater than 87% oleic
acid and less than 1.5% .alpha.-linolenic acid can be obtained from
a transgenic Brassica plant containing a seed-specific reduction in
Fad3 gene expression as well as chemically-induced mutations in
Fad2 genes.
5TABLE 5 Fatty Acid Profile of Fad3 Cosuppression, Fad2 Mutated
Seeds 16:0 18:0 18:1 18:2 18:3 663-40 3.9 1.4 71.2 20.1 1.2 Q4275
3.3 1.5 86.7 2.2 3.1 Q4275 .times. 663-40 3.2 1.6 87.6 4.2 1.3
[0086]
6TABLE 6 Range of Fatty Acid Profiles for Fad3 Cosuppression, Fad2
Mutated Lines Tested in the Field 16:0 18:0 18:1 18:2 18:3 663-40
Min 3.5 2.3 73.5 16.3 0.8 Max 4.7 2.2 64.0 24.2 1.5 Q4275 Min 3.2
3.3 85.0 1.8 2.0 Max 3.0 2.3 86.6 1.7 2.6 Q4275 .times. 663-40 Min
3.2 2.0 85.1 5.3 0.9 Max 3.2 2.9 84.0 6.0 1.5
[0087] Additional seed from the homozygous plant described above
was planted in the field and self-pollinated. Mature seeds from
several progeny plants were separately analyzed for their fatty
acid profile. The fatty acid profile for the progeny plant having
the minimum linolenic acid content and the plant having the maximum
linolenic acid content are shown in Table 6. The results show that
the homozygous plant having Fad2 mutations and Fad3 cosuppression
had a fatty acid profile in the field that was similar to that of
the greenhouse-grown seed (Table 5), indicating that the Fad3
cosuppression trait and the chemically-induced Fad2 mutants
conferred a stable fatty acid composition on seeds of this plant.
Thus, an oil of the invention can be obtained from either
field-grown seeds or greenhouse-grown seeds.
[0088] Because of the decreased .alpha.-linolenic acid content and
increased oleic acid content, an oil of the invention is useful in
food and industrial applications. Oils which are low in
.alpha.-linolenic acid have increased oxidative stability. The rate
of oxidation of lipid fatty acids increases with higher levels of
linolenic acid leading to off-flavors and off-odors in foods. The
present invention provides novel canola oils that are low in
.alpha.-linolenic acid.
[0089] To the extent not already indicated, it will be understood
by those of ordinary skill in the art that any one of the various
specific embodiments herein described and illustrated may be
further modified to incorporate features shown in other of the
specific embodiments.
[0090] The foregoing detailed description has been provided for a
better understanding of the invention only and no unnecessary
limitation should be understood therefrom as some modifications
will be apparent to those skilled in the art without deviating from
the spirit and scope of the appended claims.
Sequence CWU 1
1
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