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ALTERING FATTY ACID
COMPOSITION IN OIL SEED CROPS
Joe W. Burton,1 Jerry F. Miller,2 B. A. Vick,2 Rachael Scarth3
and C. Corley Holbrook4
1USDA-ARS, Soybean & Nitrogen Fixation Unit, Raleigh, North Carolina, USA2USDA-ARS, Northern Crop Science Laboratory, Fargo North Dakota, USA
3University of Manitoba, Faculty of Agricultural & Food Science, Manitoba, Canada4USDA-ARS, Crop Genetics and Breeding Unit, Tifton, Georgia 31793, USA
I. SoybeanA. IntroductionB. Unsaturated Fatty AcidsC. Saturated Fatty AcidsD. Genetic Engineering Soybean Oil with Novel Fatty Acids
II. SunflowerA. IntroductionB. Nusun Sunflower OilC. Reducing Saturated Fatty Acids in Sunflower OilD. Tocopherol Role in Nusun Sunflower Oil
III. BrassicaA. Intraspecific and Interspecific CrossesB. MutagenesisC. TransformationD. Production of Modified Oil Cultivars
IV. PeanutA. Introduction, Breeding Objectives, and RationaleB. Increasing Oleic AcidReferences
World consumption of vegetable oils increased steadily in the lastdecade, from 62.6 million metric tons (MMT) in 1993 to 87.8 MMT in 2000(Goblitz, 2002). This demand has been primarily due to increased use ofedible oils in food preparation. Yet, vegetable oils are being used in manyindustrial products including fuels. Part of this has resulted from alterationof the fatty acid composition of vegetables oils making them more versatilein their uses.
The four major oilseed crops are soybean (Glycine max L. Merr.), sunflower(Helianthus annuus L.), rapeseed (Brassica), and peanut (Arachis hypogaea L.).The seed oil of these has been genetically altered through standard plantbreeding methodology and molecular genetic engineering. The following
273
Advances in Agronomy, Volume 84Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved.
DOI 10.1016/S0065-2113(04)84006-9
is a review of recent developments in the genetic manipulation of thesecrop plants to change seed oil quality. q 2004 by Elsevier Inc.
I. SOYBEAN
A. INTRODUCTION
Oil from soybeans is in high demand and is the most plentiful seed oil
being produced. Each year, consumption nearly equals production. Even
though soybean oil is generally considered to have lower intrinsic quality
than other vegetable oils, production and use increased each year in the last
decade. Currently, soybean oil accounts for about 44% of edible vegetable oil
produced. About 1/3 of the oil produced is consumed in the US. Increased
demand for soybean meal is a major reason for the increase in soybean oil
production. Some will argue that soybean should be considered a protein-seed
rather than an oil-seed because the amount of protein meal produced is about
four times the oil produced. In this sense, soybean oil is a valuable by-
product of meal production, and continued demand for high quality soy-
protein meal insures the production of a large supply of soybean oil.
Because production and consumption of soybean oil is so large, there has been
a desire to improve the nutritional and functional quality of the oil through genetic
alteration of the soybean seed lipids. Most of this work has been done using
traditional plant breeding methods. More recently, molecular genetic engineering
has also been used. While there are no popular cultivars today with radically
different oil quality, considerable progress has been made over the past 20 years in
the development and release of improved germplasm with altered oil composition.
Breeding goals vary, but the following would be considered by most oil users to be
improvements: 30 g kg21 or less linolenic acid, 70 g kg21 or less total saturated
fatty acids, and 550–650 g kg– 1 oleic acid. There is also research to increase
saturates, stearic and/or palmitic acids. There is germplasm with both traits. If a
higher saturated soybean oil proves to be useful to the food industry, then this trait
might be added as a breeding objective. Transgenic research is being used to bring
fatty acids that are different from those normally found in soybean oil.
B. UNSATURATED FATTY ACIDS
1. Decreasing Linolenic Acid Concentration
Reduction of linolenic acid in soybean oil was proposed by Howell et al.
(1972) over 30 years ago. They cited evidence that linolenic acid is broken down
J. W. BURTON ET AL.274
by enzymes or spontaneous oxidation to form secondary products that give the oil
a bad odor and flavor. At that time, cooking oil was routinely hydrogenated in
processing to lower the concentration of linolenic acid. Thus, reduction of
hydrogenation costs in oil processing was the primary benefit associated with a
genetically altered soybean oil. Today, because of public concern about trans
fatty acids produced in the hydrogenation process, oil processors want a more
stable oil that requires less hydrogenation.
Results from initial breeding research suggested that concentrations of
unsaturated fatty acids in soybean oil were polygenic traits (Howell et al., 1972).
Therefore, C.A. Brim (USDA-ARS, NC State University) applied recurrent mass
and within half-sib family selection to a population that had two plant
introductions (PI90406 and PI92567) in the parentage. Both of these lines had
relatively higher oleic acid and lower linolenic acid than standard cultivars.
Because linolenic acid results from the consecutive desaturation of oleic acid and
linoleic acid, concentrations of oleic acid and linolenic acid are negatively
correlated. Brim selected for higher oleic acid concentration as a way to
indirectly decrease linolenic acid concentration. In this experiment, oleic acid
was increased from 227 to 416 g kg21 in eight cycles of selection, and linolenic
acid decreased from 87 to 59 g kg21 (Carver et al., 1986). This increase in oleic
acid had very little effect on the saturated fatty acids.
A line, N78-2245, derived from the fifth cycle of this experiment was
the source of genes for higher oleic acid in subsequent research. Work with
N78-2245 showed that the low-linolenic phenotype was quite sensitive to
environmental differences (Carver et al., 1983). To stabilize this trait, N78-2245
was mated with PI123,440, a plant introduction with low linolenic acid
concentration compared with other accessions in the soybean germplasm
collection, for maturity groups V to VIII. This mating produced progeny in the
F3 generation that ranged from 3.4 to 10.9% linolenic acid. Because the extremes
in this phenotypic distribution were transgressive segregates when compared
with either parent, a combination of at least two different gene loci, one from
N78-2245 and one from PI123,440, must have been responsible. Because N78-
2245 had a relatively high oleic acid concentration, it was thought that the gene or
genes from that parent were responsible for less desaturation of oleic acid
(Wilson and Burton, 1986). The gene contributed by PI123,440 was thought to
cause less desaturation of linoleic acid. Further work with these materials led to
the development and registration of the germplasm line, N85-2176, which has
44% oleic and 3.3% linolenic acid (Burton et al., 1989).
Linolenic germplasms have also been developed through mutagenesis. Wilcox
et al. (1984) treated seeds of the cultivar “Century” with the mutagen, ethyl
methanesulfonate (EMS). In screening 5000 M2 plants, one was found that had oil
with 34 g kg21 linolenic acid and in this case, oleic acid was not unusually high,
22 g kg21, compared to 191 g kg21 for Century. This line was designated C1640.
Subsequent studies of the inheritance of this trait revealed it to be controlled
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 275
by a single recessive gene (Wilcox and Cavins, 1985) and the gene symbol fan was
assigned to the mutant (Wilcox and Cavins, 1987). The heterozygote Fan/fan was
found to be intermediate to the two homozygotes (Table I). They also found that the
low-linolenic trait segregated among F2 seeds from a single F1 plant indicating
that it was under embryonic rather than maternal control.
Another low linolenic acid germplasm line, A5, was developed through
mutagenesis by Hammond and Fehr (1983). They treated seeds of a line FA9525,
with EMS. This line had lower linolenic acid and higher oleic acid than standard
cultivars. The line A5 (3.9% linolenic acid) was selected from the M4 generation.
A fourth source of low linolenic acid germplasm, PI361088B, was identified
by Rennie et al. (1988). Rennie and Tanner (1989, 1991) have shown that the
three sources, (PI123,440, A5, and PI316088B) carry genes that are either allelic
or identical to the fan allele in C1640 (Table I). Variation in phenotype among the
three suggests that background genotype is also influential in actual oil
composition. Other mutations at this locus have been reported. One was found
in Japan by X-ray irradiation of seeds of the cultivar Bay (Rahman et al., 1996b).
This mutant in line M-5 has 51 g kg21 linolenic acid. The other mutation was
developed in Canada by EMS mutagenesis of C1640. This mutant allele in
Table I
Sources of Soybean Germplasm with Reduced Concentration of Linolenic Acid in Seed Lipids,
Their Phenotype, and Alleles Associated with the Germplasm
Genotype
Linolenic acid
Germplasm Locus 1 Locus 2 Locus 3 (g kg21) Reference
Centurya Fan/Fan 72 Wilcox and Cavins (1985)
Fan/fan 52 Wilcox and Cavins (1985)
C1640 fan/fan 32 Wilcox and Cavins (1985)
PI123,440 fan/fan 49 Wilson and Burton (1986)
PI361,088B fan/fan 38 Rennie et al. (1988)
A5 fan/fan 51 Fehr et al. (1992)
A23 fan2/fan2 56 Fehr et al. (1992)
A16 fan/fan fan2/fan2 22 Fehr et al. (1992)
RG10 fan-b/fan-b ,25 Stojsin et al. (1998)
M5 fan/fan Fanx/Fanx 51 Rahman et al. (1998)
fan/fan fanxa/fanxa 24 Rahman et al. (1998)
M-24 Fan/Fan fanxa/fanxa 61 Rahman et al. (1998)
Bayb Fan/Fan Fanx/Fanx 92 Rahman et al. (1998)
A29 fan/fan fan2/fan2 fan3/fan3 13 Ross et al. (2000)
aCentury is a cultivar which was mutagenized to produce C1640 fan/fan genotype. It is presumably
wild-type at the second locus.bBay is a cultivar which was mutagenized to produce M5 and M-24.
J. W. BURTON ET AL.276
soybean line RG10 further lowers linolenic acid to ,25 g kg21. This allele has
been designated fan-b (Stojsin et al., 1998).
A second locus, fan2 was discovered by Fehr et al. (1992) in the line
A23, which had been selected for its high palmitic acid content. When both
recessive loci are combined in the homozygous condition in the line A16
( fan/fan fan2/fan2), linolenic acid is reduced to 2.2% (Fehr et al., 1992).
Combining recessive alleles at a third locus, fan3, with fan/fan and fan2/fan2
reduced the linolenic acid to 13 g kg21 (Ross et al., 2000). In Japan, Rahman
et al. (1998) have also discovered a gene locus independent of fan that
reduces linolenic acid. This gene was also mutagenized by irradiating seeds
of cultivar Bay. This gene that they designated, fanxa, in combination with
fan produces 2.4% linolenic acid. Because soybean is an ancient diploidized
tetraploid, there are many duplicate gene loci. Thus, it is likely that fan2 and
fanxa are mutations at the same locus.
Most of the linolenic acid in soybean oil is synthesized by a mircosomal
omega-3 desaturase that catalyzes desaturation of 18:2-PC (linolenic acid
esterified to phosphotidylcholine) to 18:3-PC. A single gene, FAD3, has been
cloned that encodes an omega-3 desaturase. Using Southern blot analysis, Byrum
et al. (1997) has shown that the cDNA encoding the omega-3 desaturase from the
A5 mutant, has a DNA fragment missing. This suggests that the low-linolenic
phenotype in A5 is at least partially due to a deletion in the FAD3 gene. Thus, fan
is probably a FAD3 gene. The other locus, fan2, showed no DNA polymorphism
between the mutant and wild-type. However, because soybean genomes possess
more than one isoform of the FAD3 gene, it is possible that the fan2 locus
corresponds to a second copy of the omega-3 desaturase gene that was
mutagenized in A23. Blocking the FAD3 gene expression, via gene silencing
molecular techniques, which should suppress the activity of all FAD3 isoforms,
soybean lines with ,1.5% linolenic acid were produced (Kinney, 1995).
2. Increasing Linolenic Acid Concentration
Oils with high levels of polyunsaturates would have application in the
manufacture of lubricants and drying oil. Also, linolenic acid is an omega-3 fatty
acid, which is essential in mammalian diets. So there is some interest in a high
linolenic acid oil from a nutritional standpoint. It would seem, however, that the
instability of such an oil would require that the high-linolenic soybean be
consumed as a vegetable or as tofu or some other soyfood.
Two sources of increased linolenic have been reported. One was developed by
Takagi et al. (1989) through mutagenesis using X-ray irradiation of seeds of the
cultivar Bay. This mutant B739, has 130 g kg21 linolenic compared with Bay at
90 g kg21. Some plant introductions in the wild soybean collection have elevated
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 277
levels of linolenic acid (Pantalone et al., 1997). One of these, PI424031, has
150 g kg21 linolenic. Crosses between G. max and G. soja yielded a line with
13 g kg21 linolenic.
3. Increasing Oleic Concentration
As previously mentioned, oleic acid was increased in a recurrent selection
experiment (Carver et al., 1986). Continued recombination and selection of these
higher oleic materials have resulted in a . 550 g kg21 oleic phenotype (Wilson
et al., 2003). The inheritance of this oleic phenotype is being determined, and it is
being incorporated into higher yielding materials.
Two higher oleic mutants have been created in Japan through irradiation of the
cultivar Bay (Rahman et al., 1996a). These mutants M23 and M11 have 530 and
390 g kg21 oleic acid, respectively. Segregation analysis of F1, F2, and
backcrosses between the two mutants and between the two mutants and Bay,
shows that a single locus is involved. They have designated the allele in Bay as
Ol, the allele in M23 as ol, and the allele in M11 as ola. The gene Ol is partially
dominant to ol, and ola is completely dominant to ol. They found no maternal or
cytoplasmic effects.
Desaturation of oleic to linoleic acid is catalyzed by microsomal omega-6
desaturases. There are at least two of these, FAD2-1 and FAD2-2. FAD2-1 is
expressed only in the seeds and the other, FAD2-2, is expressed constitutively in
all tissues (Heppard et al., 1996). Silencing the FAD2-1 gene in somatic embryos
and subsequent regeneration into plants has resulted in transgenic soybean lines
with very high .800 g kg21 oleic acid content (Kinney, 1995).
These single gene manipulations are successful in dramatically changing oleic
acid. But previous research suggests that the end phenotype in a field situation
may be under a more complex genetic control. For instance, it has been shown
that oleic acid can be influenced by the maternal genotype. Brim et al. (1968) and
Erickson et al. (1988a) have demonstrated this effect with reciprocal crosses
between low and mid-oleic lines. This effect was further demonstrated using
reciprocal grafts between mid-oleic line N78-2245 and the cultivar Essex (Carver
et al., 1987). When “Essex” as scion was grafted to a N78-2245 stock and then
defoliated after pods had set, the fatty acid concentrations of Essex seeds became
more similar to those of N78-2245 seeds. For instance, oleic acid of Essex seeds
changed from 17.8 g kg21 as an autograft to 38.3 g kg21 as a defoliated scion
N78-2245. Likewise, the oleic acid of N78-2245 decreased from 50.1 g kg21 as
an autograft to 27.6 g kg21 as defoliated scion. Because this effect was greatest
when the scion was defoliated, it was concluded the maternal influence on fatty
acid composition is expressed via translocated factors that probably originate in
leaf tissue.
J. W. BURTON ET AL.278
Several experiments have shown that temperature affects unsaturated fatty
acid synthesis. An experiment by Howell and Collins (1957) showed shifts in
linolenic acid of 4.3 percentage points in day temperatures that range from 21 to
298C. Heppard et al. (1996) looked at the affect of temperature on transcription
levels of both FAD2-1 and FAD2-2 genes. They studied plants of the soy cultivar
Rye at temperatures from 32/28 to18/128C. In both leaf and seed tissues, linoleic
and linolenic acid decreased as temperature increased, however transcript levels
of FAD2-1 and FAD2-2 were relatively constant in developing soybean seeds at
the different growth temperatures. Thus, temperature must be acting at the
translational or post-translational levels, perhaps by altering desaturase enzyme
activity or stability.
C. SATURATED FATTY ACIDS
1. Reducing Saturated Fatty Acids Concentrations
Medical studies have shown diets high in saturated acyl components, palmitic
and stearic fatty acids may contribute to increased blood serum cholesterol levels.
High blood cholesterol increases the risk of coronary heart disease. Accordingly,
substantial markets have emerged for low saturate oils, with FDA labeling
regulations requiring that a “low-saturated” vegetable oil must contain
,70 g kg21 total saturates. Although soybean oil is relatively low in total
saturates (120–140 g kg21), a 50% reduction in saturated fat is needed to
enhance the utility of soybean oil in this new market. When one considers the
large amount of soybean oil annually consumed in the US (<12 million pounds),
a 50 g kg21 reduction in the saturated fats of soybean oil would have a significant
impact on saturated fat consumption of our population without dietary change. Of
the two saturated fatty acids in soybean oil, palmitic and stearic, palmitic is of the
greatest concentration and has received the most attention. In fact from a health
standpoint, palmitic has been identified as the more problematic of the two.
Studies have shown that a minimum of two loci control the low palmitic acid trait
in soybean. The additive allele, fap1, was obtained as a mutation following EMS
treatment of seeds of the cultivar “Century” (Erickson et al., 1988b). Inheritance
studies showed a 12 g kg21 decrease in palmitic acid with each additional copy of
fap1. Fehr et al. (1991) developed a mutant line, A22, containing the fap3 allele,
which segregates independently of fap1. Palmitic acid concentration in A22 is
approximately 70 g kg21 (Stoltzfus et al., 2000). Another low palmitic genotype,
N78-2077-12, (Burton et al., 1994) was selected from the 5th cycle of recurrent
selection for increased oleic acid (Table II).
A critical step in biosynthesis of palmitic acid in soybean seeds is release
of free palmitic acid from 16:0-ACP in the plasmid, followed by its transport
into the cytoplasm where it becomes esterified to CoA. The enzyme involved
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 279
is the 16:0-ACP thioesterose, which is encoded by the Fat B gene. Southern
blot analysis has shown that N78-2077-12 has a deletion in one isoform of
the Fat B gene (Wilson et al., 2001). Segregation analysis has shown the
gene, fapnc, to be allelic to fap3. However, Southern analysis has shown that
unlike fapnc, fap3 is not a Fat B deletion (Wilson et al., 2001). Nevertheless,
either fap3 or fapnc in combination with fap1 reduces palmitic to 4% or less
(Fehr et al., 1991; Wilcox et al., 1994).
Another interesting part of this picture is that even though the biosynthetic
pathways for fatty acids and triacylglycerol are well understood, heritable genetic
variation for modifiers that affect reduced saturated fatty acid content can be found.
Rebetzke et al. (1998) developed lines from a single cross that were homozygous
for normal and reduced palmitic acid alleles. Palmitic acid ranged 19 g kg21 in the
first and 15 g kg21 in the second. Heritabilities of this variation were 83 and 86.
Results for stearic acid were similar with heritabilities of 85 and 89. Another set of
materials from a different cross yielded similar results (Rebetzke et al., 1998).
Thus attempts to move the reduced palmitic trait into cultivated soybean should be
made emphasizing selection for alleles of major and minor effect.
2. Increasing Saturated Fatty Acids Concentrations
In the US, concerns about heart disease risk by consumers prompted food
industries to sharply decrease their use of tropical oil due to their high-palmitic
acid content. Because saturated fats (solid fats) are needed for many food products,
cottonseed oil has been substituted for tropical oil. Soybean oil is also
hydrogenated to increase the saturated fatty acids. It has been suggested that
soybean oil with high stearic acid might be used instead as a replacement for
tropical oils and decrease the need for hydrogenation. Stearic acid does not pose
the same health risk for coronary heart disease that palmitic acid does and would
Table II
Sources of Soybean Germplasm with Reduced Concentrations of Palmitic Acid in Seed Lipids,
Their Phenotype, and Alleles Associated with the Germplasm
Genotype
Germplasm Locus 1 Locus 2 Palmitic acid (g kg21) Reference
Century Fap1/Fap1 Fap3/Fap3 111 Erickson et al. (1988b)
C1726 fap1/fap1 Fap3/Fap3 84 Erickson et al. (1988b)
N79-2077-12 Fap1/Fap1 fapnc/fapnc 60 Burton et al. (1994)
C1943 fap1/fap1 fapnc/fapnc 40 Burton et al. (1998)
N94-2575 fap1/fap1 fapnc/fapnc 39 Burton et al. (1998)
A8 fap1/fap1 Fap3/fap3 44 Fehr et al. (1991)
J. W. BURTON ET AL.280
thus have a definite advantage if it had the processing qualities needed by the
food industry.
There are three alleles at a single locus that cause an increase in stearic acid
concentration, fas, fasa and fasb (Graef et al., 1985). All three were developed by
chemical mutagenesis. The fas and fasb alleles (Hartman et al., 1997), both
increase stearic acid about 4-fold, fasa causes a 7-fold increase (Table III). There
is evidence that another locus may be involved (Bubeck et al., 1989; Rahman
et al., 1997).
There are also mutants that cause increases in palmitic acid (Table IV; Erickson
et al., 1988b; Bubeck et al., 1989; Stoltzfus et al., 2000). These may be useful if it is
determined that the optimum saturated fatty acid content for solid products such as
margarine and shortening require some combination of increased palmitic and
stearic acid. One locus with two alleles, fap and fap2-b, cause increases of palmitic
acid from 120 to 180 and 210 g kg21, respectively. A second locus, fap4, causes
increases to 180 g kg21. Combining fap2-b and fap4 raises the concentration of
palmitic acid above 250 g kg21 (Schnebly et al., 1994). Three other loci, fap5,
fap6, and fap7 have been identified (Stoltzfus et al., 2000) which separately have
palmitic acid concentrations between 140 and 160 g kg21 (Table IV). These loci
can act additively to increase palmitic acid. A line homozygous for fap2-b, fap4,
fap5, and fap7 alleles had 402 g kg21 palmitic acid.
D. GENETIC ENGINEERING SOYBEAN OIL WITH
NOVEL FATTY ACIDS
Because the biosynthetic pathways for fatty acid and triacylglycerol synthesis
in oil seeds are well understood, and many of the genes cloned, they can be
manipulated using molecular genetic techniques. Given the large number of fatty
acids that exist in the oil of exotic oil seed species, A.J. Kinney of DuPont
Agricultural Products says “there should be no theoretical barriers to producing
exotic fatty acids in temperate oil seed crops” (Kinney, 1997). One exotic species
Table III
Sources of Soybean Germplasm with Increased Concentration of Stearic Acid in Seed Lipids,
Their Phenotypes, and Alleles Associated with the Germplasm
Genotype
Line Locus 1 Locus 2 Stearic acid (g kg21) Reference
FA8077 Fas/Fas ? 43 Graef et al. (1985)
A81-606085 fas/fas ? 187 Graef et al. (1985)
A6 fasa/fasa ? 304 Graef et al. (1985)
FA41545 fasb/fasb ? 155 Graef et al. (1985)
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 281
of interest “meadow foam” (Limnanthes douglasii) has a high content of C20 and
C22 fatty acids with D5 unsaturation. Eicosenoic acid (20:1 D5) accounts for 60%
of the total fatty acids. This oil is useful in cosmetics, surfactants and lubricants.
Using particle bombardment, Cahoon et al. (2000) transformed soybean somatic
embryos with cDNAs of a D5-desaturase gene and a FAE1 (fatty acid elongase)
homolog, with a strong seed specific promoter. The desaturase and elongase
genes were expressed in the somatic embryo, and in some embryos, eicosenoic
acid accumulated to amounts 18% of the total fatty acid.
In another example, Kinney reports transferring an epoxy fatty acid gene from
Vernonia to soybean and producing soybean oil containing 10% D12 epoxy fatty
acids. These oils are useful for PVC plasticizer application.
A third example involves the production of conjugated double bond fatty
acids, a class of fatty acids in which the double bonds are not separated by a
methylene group as is found with conventional fatty acids. These are the fatty
acids in tung oil and are the drying agents in paints and inks. Cahoon et al. (1999)
isolated a class of cDNAs which represent a divergent form of the D12-oleic
acid desaturase found in Momordica charantia and Impatiens balsamina tissues
that accumulate a-eleostearic (18:3 D9cis11trans13trans) and a-parinaric
(18:4 D9cis11trans13trans15cis) acids. Soybean somatic embryos transformed with
Table IV
Sources of Soybean Germplasm with Increased Concentration of Palmitic Acid in Seed Lipids,
Their Phenotypes, and Alleles Associated with the Germplasm
Genotype
Palmitic
Line Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 acid (g kg21) Reference
Century Fap2/Fap2 86 Erickson
et al. (1988b)
C1727 fap2/fap2 173 Erickson
et al. (1988b)
A21 fap2-b/fap2-b 200 Schnebly
et al. (1994)
A24 fap4/fap4 180 Schnebly
et al. (1994)
A19 fap2-b/fap2-b fap4/fap4 .280 Schnebly
et al. (1994)
A27 fap5/fap5 162 Stoltzfus
et al. (2000)
A25 fap6/fap6 159 Stoltzfus
et al. (2000)
A30 fap7/fap7 146 Stoltzfus
et al. (2000)
fap2-b/fap2-b fap4/fap4 fap5/fap5 fap7/fap7 402 Stoltzfus
et al. (2000)
J. W. BURTON ET AL.282
these cDNAs accumulated both a-eleostearic and parinaric acids to a combined
level of 17% of the total fatty acids.
While all of the above genetic alterations of soybean oil hold promise, the
percentage of the novel fatty acid produced within the soybean is much lower
than is observed in the native species in which these fatty acids are found, and
their yields need to be increased significantly before such products can become
commercially viable. Suboptimal substrate specificities of the cognate acyl-
transferases in the lipid biosynthetic pathways or lipase activities may be limiting
the accumulation of the novel fatty acids into soybean triacylglycerols.
Furthermore, the process of getting these materials from the lab to the farm to
the processor to the end user is complicated. Strong economic incentives are
needed before new oil products are developed and commercialized. Health
claims or labeling regulations represent additional complicating factors. For the
farmer, a new oil cultivar must have very good yielding ability or they will
probably be unwilling to produce it. Even so, reducing saturates, increasing oleic
acid and decreasing linolenic should make cooking oil that is superior in quality
and functionality to current standard soybean oil. If the demand by oil users for
this type of oil continues to increase, eventually there may be large scale
commercial production of soybeans with this type oil.
II. SUNFLOWER
A. INTRODUCTION
Traditional high-linoleic sunflower (Helianthus annuus L.) oil has been
viewed as a healthful vegetable oil with desirable flavor and is considered a
premium oil in world markets because of its high percentage of polyunsaturated
fatty acids. Its popularity in European and East Asian countries for salad oil,
cooking oil, or for margarine production was based on its oil composition and the
absence of cholesterol.
Selection of an oil for frying purposes depends on several criteria: (1)
oxidative stability, (2) product flavor, (3) product texture, (4) mouth feel, (5)
availability, (6) cost, (7) nutritional needs, and (8) consumer issues (Gupta,
1998). Most important of these criteria are oxidative stability and product flavor.
Unlike soybean and canola oils, sunflower oil has negligible linolenic acid, which
is highly susceptible to oxidation. Thus, when soybean and canola oils are used as
frying oils, they typically require partial hydrogenation to eliminate linolenic
acid. Hydrogenation leads to the formation of trans fatty acids, which are dietary
risk factors for cardiovascular disease. Increasing the oleic acid concentration of
sunflower oil increases its oxidative stability without the need for hydrogenation,
thus eliminating trans fatty acids and any related consumer issues regarding this
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 283
subject. However, a threshold level of linoleic acid must be maintained for
acceptable product flavor. A low saturated fat concentration would be beneficial
to consumers concerned with dietary requirements and cardiovascular disease.
A report by USDA-ARS scientists (Miller et al., 1987) creating a mid-oleic
level of oleic acid prompted E.D. Campbell of Archer Daniels Midland Co. in
Decatur, IL, and M.K. Gupta, formerly with the Frito Lay Co. in Plano, TX, to
recognize the potential of oleic-enhanced sunflower oil for the frying industry. In
meetings with the National Sunflower Association (Bismarck, ND), a task force
was formulated to investigate the creation of a mid-oleic sunflower oil, which
would be given the trademark NuSun. The composition of NuSun sunflower oil
was defined as 550–650 g kg– 1 oleic acid, 200–280 g kg21 linoleic acid, less
than 90 g kg21 saturated fatty acids, and 1 g kg21 linolenic acid. Comparisons of
NuSun to traditional sunflower oil and olive oil are made in Table V.
The objectives of the USDA-ARS Sunflower Research Unit were to
investigate the genetic inheritance of oleic acid concentration in sunflower oil
and to initiate hybrid trials testing the feasibility of NuSun hybrids in sunflower
production areas of the US. Also, studies to lower the saturated fatty acid level of
sunflower oil were initiated. The feasibility of changing the homologue of
tocopherol from a-tocopherol presently in sunflower oil to g-tocopherol is also a
breeding objective. With stronger antioxidant properties than a-, g-tocopherol in
combination with mid-oleic fatty acid levels could significantly increase the
oxidative stability of the oil.
B. NUSUN SUNFLOWER OIL
1. Inheritance
The development of a sunflower with a high oleic acid concentration was first
reported by Soldatov (1976). Seed of the variety VNIIMK 8931 was treated with
a 0.5% solution of dimethyl sulfate, a chemical mutagen. Seed was advanced to
the M3 generation and screened for differing levels of oleic acid. The individual
Table V
Fatty Acid Composition of NuSun Sunflower Oil, Traditional Sunflower Oil, and Olive Oil
Oil type
Fatty acid NuSun sunflower (g kg21) Traditional sunflower (g kg21) Olive (g kg21)
Oleic 630 230 770
Linoleic 270 720 80
Saturated 90 130 140
Linolenic 10 10 10
J. W. BURTON ET AL.284
seeds that were selected contained about 500 g kg21 oleic acid. By bulking the
superior plants with high-oleic concentration, the Pervenets variety was created
and released to producers in Russia by the VNIIMK research center (Pukhalsky
and Dvoryadkin, 1978). Seed of this cultivar grown in North Dakota averaged
699 g kg21 with individual plants having as high as 900 g kg21 of oil (Miller
et al., 1987). Oil from seed of 46% of the plants had .800 g kg21 and 21% had
,600 g kg21 oleic acid concentration. In 1978, Russian scientists reported that
they had created breeding lines with oleic concentrations as high as 895 g kg21
(Pukhalsky and Dvoryadkin, 1978).
Seed of the Pervenets variety (PI 483077) was planted in the 1982 spring
greenhouse of the USDA-ARS Sunflower Research Unit at Fargo, ND, and plants
were self-pollinated. Plants were harvested and the fatty acid composition of the
self-pollinated seed was determined by capillary gas chromatography. Ten
selections were made, each having an oleic acid concentration greater than
850 g kg21, and analysis of single seeds of these plants confirmed that they were
homozygous for oleic acid concentration. Four of these plants, designated
Pervenets 302, 304, 305 and 306, were utilized to determine the inheritance of the
high oleic acid trait in sunflower.
Plants of the inbred line HA 89, a traditional high-linoleic sunflower line, were
hand-emasculated and pollinated with the Pervenets selections. At maturity, seed
of the parental self-pollinated plants and F1 crosses were harvested and fatty acid
composition was determined. The seed oil of the Pervenets selections averaged
846 g kg21 of oleic acid, whereas HA 89 averaged 110 g kg21 (Table VI). The
average oleic acid concentration of the F1 crosses was 520 g kg21 of oil when the
high-oleic Pervenets selections were used as the female parent. This cross was
Table VI
Average Oleic Acid Concentration in Seed Oil of Parents and
Reciprocal F1 Crosses Grown in the Field at Fargo, ND
Parent or cross
Oleic acid concentration
(g kg21)
High-oleic Pervenets selections 846
Range 790–894
HA 89 110
Range 102–181
F1 seed, high-oleic/HA 89 520
Range 427–634
F1 seed, HA 89/High-oleic 390
Range 370–491
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 285
the first mid-oleic or NuSun sunflower hybrid created (Miller et al., 1987). When
the high-oleic Pervenets 306 was used as male parent in the cross, the F1 seed
averaged 390 g kg21, indicating maternal influence but not complete maternal
inheritance.
The F1 plants of the cross between Pervenets 306 and HA 89 were self-
pollinated and F2 seed were grown in the field nursery at Fargo, ND. Analysis of
F2 seed showed a trimodal distribution for oleic acid concentrations (Table VII).
An intermediate class was clearly evident, ranging in oleic concentration from
480 to 720 g kg21. The high-oleic class ranged from 820 to 920 g kg21, whereas
the low-oleic class was similar to HA 89 and ranged from 110 to 180 g kg21. The
number of seeds in the intermediate class was too large to support a single,
dominant gene theory. A Chi-square analysis for goodness of fit of segregation
3:9:4 high:intermediate:low was proposed and accepted for the four crosses, for
the pooled families, and the heterogeneity analysis (Table VII). The study
confirms the presence of a single, dominant gene, designated Ol. This gene
produced seed with oleic composition levels of 500–700 g kg21. A second gene,
designated Ml, appears to modify the oleic concentration upward. When in the
recessive form mlml and combined with the gene Ol, oleic levels in seed were
820 g kg21 or higher. Therefore, the theoretical genotype Ol_Ml_ would produce
oleic acid concentrations desirable for NuSun. F3 progeny tests were conducted
on intermediate F2 seeds. These F2 seeds produced very few high-oleic F3
segregants, as expected with the two-gene model proposed. Since this study,
Fernandez-Martinez et al. (1989) proposed a three-gene model, further
supporting the modifier gene aspect of mid-oleic sunflower oil.
Table VII
Distribution of Seed in Oleic Classes and Chi-Square Analysis for Goodness of Fit of Segregation
Observed in F2 Seeds (Derived on F1 Plants) Involving High- and Low-Oleic Acid Parents
No. of seeds in oleic classes
Pedigree High Intermediate Low Pa
F2 population (3:9:4 ratio tested)
HA 89/Pervenets Sel 302 6 15 9 0.50–0.80
HA 89/Pervenets Sel 304 18 42 30 0.20–0.50
HA 89/Pervenets Sel 305 11 26 13 0.50–0.80
HA 89/Pervenets Sel 306 13 31 13 0.50–0.80
Pooled Chi-square 48 114 65 0.05–0.20
Heterogeneity Chi-square 0.50–0.80
aProbability of a larger x2 value due to chance.
J. W. BURTON ET AL.286
2. Methods of Producing NuSun Sunflower Hybrids
Based on the inheritance studies, there are four basic methods of producing
NuSun sunflower hybrids with a mid-oleic level of oleic acid. At present, most
seed industry companies are utilizing the first two strategies, with a few
companies applying the third strategy.
(a) High £ Low Crosses or Low £ High Crosses: Crossing a high-oleic
female parent (OlOlmlml) with a low-oleic male parent (traditional linoleic)
(ololMlMl) produces hybrid seed with the genotype OlolMlml. Planting of seed
with this genotype has produced sunflower seeds which generally average 520–
620 g kg21 oleic acid. The crossing of a low-oleic female parent (ololMlMl) with
a high-oleic male parent (OlOlmlml) also produces hybrid seed with the genotype
OlolMlml. However, planting of seed of this cross has typically produced a
sunflower crop that averages 500–560 g kg21. This result is due to the maternal
influence factor, or the influence of the genotype of the maternal parent on the
resulting oleic concentration of seed produced on that plant (Miller et al., 1987).
It appears that the high-oleic female parent continues to influence the oleic
concentration of seed produced on that plant.
(b) Modified Single-Crosses [(High £ Low Female) £ High Male]: Crossing
a high-oleic female parent (OlOlmlml) with a related low-oleic, female parent
(ololMlMl) produces a cytoplasmic male-sterile hybrid plant of mid-oleic
phenotype (OlolMlml). This male-sterile hybrid could then be crossed with a
high-oleic male (OlOlmlml), producing a mixture of male-fertile genotypes with
varying oleic acid concentrations in the producer’s field. This cross appears to
produce a sunflower crop that averages 600–680 g kg21 oleic acid content, much
higher than the strict single-cross hybrid.
(c) Mid-oleic £ Mid-oleic Crosses: The crossing of a true-breeding mid-oleic
female parent (OlOlMlMl) with a true-breeding mid-oleic male parent
(OlOlMlMl) produces a hybrid with the mid-oleic trait because the genotype of
the hybrid is also OlOlMlMl. Seed of this plant should be more stable than
high £ low or low £ high crosses. However, the breeding effort appears much
more involved and it is difficult to create these genotypes. The obstacle may lie in
the number of modifier genes having an effect on the oleic concentration. Hybrids
must be tested extensively to confirm that specific modifier genes are the same in
both genotypes, ensuring that the correct mid-oleic level will be produced by the
hybrid. An industry breeder must have two programs, both male and female, for
the selection of specific mid-oleic genotypes.
(d) High-oleic £ High-oleic Crosses with Differing Numbers of Modifier
Genes: The crossing of a high-oleic female parent having 800–820 g kg– 1 oleic
acid and possessing a modifier gene with a slight effect on oleic concentration
(OlOlml1ml1Ml2Ml2ml3ml3) with a high-oleic male parent possessing a
different modifier gene, also with a slight effect on oleic concentration
(OlOlml1ml1ml2ml2Ml3Ml3), would produce a genetically complex hybrid
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 287
(OlOlml1ml1Ml2ml2Ml3ml3). Planting of seed with this genotype has produced a
sunflower crop which generally averages 700–780 g kg21 oleic acid. This result
is due primarily to the small effect of each of the different modifier genes on the
Ol gene, lowering the oleic concentration from a potentially high level. Several
hybrids of this type were produced by the sunflower seed industry and USDA-
ARS when the female line HA 341 was used as a female parent in experimental
hybrids.
3. Progress of Industry
Acceptance of NuSun hybrids by industry and producers has been excellent,
and steady progress has been made in 2002 and 2003. According to the National
Sunflower Association, 2003 hybrid sunflower production was approximately
60–70% converted to NuSun sunflower hybrids. This is an outstanding feat when
considering that the idea was just proposed to companies during the year 1995.
The company of Proctor & Gamble, Cincinnati, OH, completely converted their
original Pringles Potato Crisp frying line to NuSun sunflower oil. North Central
regional companies Old Dutch and Barrel O’Fun also use NuSun sunflower oil in
producing potato chips (National Sunflower Association, Bismarck, ND, www.
sunflowernsa.com).
Producer acceptance has been enhanced by the performance of the seed
industry’s NuSun hybrids (Table VIII). Trials of NuSun sunflower hybrids have
Table VIII
Yield, Oil Content, and Oleic Acid Concentration of NuSun Sunflower Hybrids and Traditional
Check Hybrids Grown at Five Locations in the US from 1996 to 2000
Location and year grown Yield (kg ha21) Oil (g kg21) Oleic acid (g kg21)
Casselton, ND
NuSun hybrids, 1996–2000 2457 463 596
Traditional checks, 1996–2000 2366 467 225
Brookings-Gettysburg, SD
NuSun hybrids, 1996–2000 2146 464 612
Traditional checks, 1996–2000 2049 472 279
Colby, KS
NuSun hybrids, 1996–2000 2416 452 636
Traditional checks, 1996–2000 2428 459 277
Minot, ND
NuSun hybrids, 1996–2000 2475 470 571
Traditional checks, 1996–2000 2584 484 158
J. W. BURTON ET AL.288
been grown since 1996 at three locations in the US: Casselton, ND, Brookings-
Gettysburg, SD, and Colby, KS. A trial at Minot, ND, was later added to test
NuSun hybrids in a more northern location requiring earlier maturing hybrids.
The maturation of these hybrids at Minot in a cooler environment puts downward
pressure on oleic concentration. As expected, the oleic concentration of NuSun
hybrids was higher at the Kansas and South Dakota locations due to higher
growing degree day effects on oil composition. However, the difference between
oleic composition in hybrids grown in southern and northern locations was
smaller than expected, indicating that NuSun oleic values are more stable than
high-oleic hybrids. NuSun hybrid yield performance was nearly the same as the
traditional linoleic check hybrid performance, enhancing the acceptance of
NuSun hybrids by producers. However, the oil content of NuSun hybrids has
been lower, indicating that industry needs more effort to improve present hybrids
in this characteristic.
4. Identity Preservation
Identity preservation of NuSun hybrid seed versus traditional linoleic hybrid
seed has been a major challenge to the sunflower industry. A National Sunflower
Association committee was organized specifically to address this problem. The
country elevator was targeted as the point at which identity should be made, with
the elevator binning the two types separately. In 2000, nearly all country
elevators were equipped with a small oil press to extract approximately 5 ml of
oil from random samples taken throughout a truck load. The oil was then placed
on a hand-held refractometer. The refractometer will not provide an exact
numerical oleic concentration number but it will clearly show if the seed has
produced a traditional linoleic concentration or NuSun sunflower oil. The North
Dakota Grain Inspection Service provides sample bottles of NuSun oil with the
oil used to calibrate the hand-held refractometer. The 2001 minimum oleic
concentration level acceptable for NuSun processors was 550 g kg21.
C. REDUCING SATURATED FATTY ACIDS IN SUNFLOWER OIL
Reducing the saturated fatty acid concentration of NuSun sunflower oil would
benefit the sunflower industry through increased consumer preference for a low
saturated sunflower product. This factor is very important to industries using
NuSun oil for frying as consumers become more aware of labeling requirements
on food products.
In a project designed to reduce saturated fatty acids in sunflower oil, two
USDA maintainer inbred lines, HA 821 and HA 382, and two USDA pollen
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 289
fertility restorer inbred lines, RHA 274 and RHA 801, were selected for
mutagenesis treatment (Miller and Vick, 1999). Two chemical mutagens (NMU
and EMS) were evaluated at rates of 1 and 2 g kg21. Rates of 4, 6, and 8 g kg21 of
both mutagens were found to be lethal for sunflower seed. A total of 56,000 seeds
were treated, or 14,000 seeds per inbred line and 3500 per mutagen treatment
(two levels of two mutagens). The fatty acid concentration of approximately 6800
M5 lines was analyzed by gas chromatography.
Sixteen of the 6800 M5 lines analyzed showed significant deviation in fatty
acid concentration from their respective parental genotypes. Five lines were
lower in palmitic acid concentration, seven lines were lower in stearic acid
concentration, two lines had over 800 g kg21 oleic acid, and one line was
classified as a mid-oleic genotype (525 g kg21). Only one line had an elevated
level of a saturated fatty acid, with the palmitic acid concentration reaching
132 g kg21, compared with 59 g kg21 in the original HA 382 genotype. Also
found was a mutant line of RHA 274 having a high linoleic acid concentration of
786 g kg21, in comparison with 646 g kg21 for the parental genotype. No
significant reductions were found in the arachidic (20:0), behenic (22:0), or
lignoceric (24:0) saturated fatty acids.
Two M5 lines, HA 821 LS-1 and RHA 274 LS-2, had lower stearic acid
concentration (41 and 20 g kg21), and one M5 line, RHA 274 LP-1, had lower
palmitic acid concentration (47 g kg21) than their respective parental lines.
Segregation ratios of F2 and testcross progeny indicated that the low stearic acid
concentration in HA 821 LS-1 was controlled by one gene, designated fas1, with
additive gene action. The low stearic acid concentration in RHA 274 LS-2 was
controlled by two genes with additive gene action. The first gene was designated
fas2, and the second gene was temporarily designated fasx. The allele fap1 was
identified in RHA 274 LP-1 to control palmitic acid concentration with additive
gene action.
Incorporation of the fas1, fas2, fasx, and fap1 genes into NuSun hybrids will
require extensive testing in early generation breeding material for development
of parental inbred lines. Utilizing half-seed analysis of segregating seeds derived
from one sunflower head will be feasible. For example, a genotype homozygous
for the fas1 fas1 fas2 fas2 fap1 fap1 alleles should be easily discerned from a
genotype with heterozygous alleles at any locus. The key to selection is testing
sufficient numbers of seeds to find the desired genotype. Marker assisted
selection would be a new tool to identify genotypes with the three alleles.
The combination of the alleles providing low stearic acid concentration with
alleles providing low palmitic acid concentration could substantially reduce the
saturated fatty acid concentration of NuSun seed oil. On the basis of results of this
study, a sunflower hybrid could be produced with a total saturated fatty acid
concentration of less than 80 g kg21, including the 20:0, 22:0, and 24:0 saturated
fatty acids.
J. W. BURTON ET AL.290
D. TOCOPHEROL ROLE IN NUSUN SUNFLOWER OIL
NuSun oil stability during use and storage can be enhanced by increasing the
natural antioxidants contained in the oil. Tocopherols (Vitamin E) are the most
powerful of the natural fat-soluble antioxidants (Demurin, 1993). They exist in
four homologues: alpha, beta, gamma, and delta. Each homologue differs in
antioxidant activity. Cultivated sunflower contains a high concentration of alpha
tocopherol (97% of total tocopherol), which has the lowest antioxidant property
of the four homologues.
1. Inheritance of Tocopherol in Sunflower
A mutation breeding program was initiated by the VNIIMK (Krasnodar,
Russia) research group working on sunflower oil quality. They found a line, LG-
15, with an oil tocopherol composition of 50% beta homologue and 50% alpha
homologue. This tocopherol composition was determined to be controlled by a
recessive gene, tph1 (Popov et al., 1988; Demurin, 1993) (Table IX). Another
mutant line, LG-17, was found to contain 95% gamma tocopherol and 5% alpha
tocopherol. The gene controlling the high gamma tocopherol composition was
also recessive and named tph2. The genes tph1 and tph2 were non-allelic and
unlinked, and neither was linked with the Ol gene controlling high oleic acid
concentration in the cultivar Pervenets.
The epistatic action of the tph2 gene on the tph1 gene results in approximately
84% of total tocopherol being the gamma homologue, 8% alpha tocopherol, and
8% delta tocopherol (Table IX). Expressivity of the tph1 and tph2 genes depends
both on seed maturity and temperature during maturation. An increase in
temperature from 20 to 308C during seed maturation resulted in an increase of the
Table IX
The Content of Tocopherols in the Seeds of Sunflower Lines
Tocopherol homologue (% of total tocopherol)
Line Genotype Alpha Beta Gamma Delta
VK373 Tph1, Tph1, Tph2, Tph2 97 ^ 1 0 3 ^ 1 0
LG15 tph1, tph1, Tph2, Tph2 50 ^ 5 50 ^ 5 0 0
LG17 Tph1, Tph1, tph2, tph2 5 ^ 2 0 95 ^ 2 0
LG24 tph1, tph1, tph2, tph2 8 ^ 2 0 84 ^ 5 8 ^ 4
Demurin, 1993.
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 291
alpha homologue of tocopherol. Combining both genes with the high-oleic gene
increased the stability and shelf life of sunflower oil dramatically.
2. Altering Tocopherol Homologues in NuSun Sunflower
Sunflower breeders in the US are currently incorporating the tph2 gene into
parental lines of NuSun sunflower hybrids. Since the gene control is recessive,
the tph2 gene must be incorporated into both the female and male parents of all
NuSun hybrids. Molecular marker technology will be beneficial in identifying
genotypes which have the tph2 gene in early generation selection.
III. BRASSICA
The development of the new technologies for trait modification has opened the
possibility of engineering a wide range of new oil profiles in Brassica. The new
Brassica oil qualities provide the opportunity to assess the effect of fatty acid
composition on the nutritional or functional properties of the oil. Cultivars with
modified oil profiles are being developed by Brassica breeding institutions
worldwide to compete with other modified oilseeds for a share in the world
vegetable oil market.
The categories of modified oils are as follows: low erucic acid oil, high and
super high erucic acid oil, reduced saturate and elevated saturate (saturates
defined as the sum of palmitic acid C16:0 þ stearic acid C18:0 þ arachidic acid
C20:0 þ behenic acid C22:0), low-linolenic oil (,35 g kg21 C18:3), mid-oleic
oil (between 650 and 750 g kg21 C18:1) and high-oleic oil (over 750 g kg21
C18:1). These categories are not mutually exclusive as a change in one fatty acid
is accompanied by changes in other fatty acids and in other minor components.
However, these categories do represent separate germplasm developments that
may then be combined to produce new oil profiles. The modified oil profiles have
been grouped in this review according to the method used to produce the new
fatty acid composition: intraspecific crosses and interspecific crosses, mutagen-
esis and transformation.
A. INTRASPECIFIC AND INTERSPECIFIC CROSSES
1. Low Erucic Acid Oil
Brassica napus and Brassica rapa. The term canola oil is used in this review to
describe the oil profile of the B. napus and B. rapa cultivars which produce oils
J. W. BURTON ET AL.292
with very low levels of erucic acid (less than 10 g kg21 C22:1), equivalent to the
terms low erucic acid rape or rapeseed (LEAR) and colza. The typical canola oil
profile can be represented as 70 g kg21 saturated fat, 610 g kg21 C18:1,
210 g kg21 linoleic acid C18:2 and 110 g kg21 C18:3. This fatty acid composition
represents the original modified oil profile in both B. napus and B. rapa.
The reduction in erucic acid was achieved through crossing and selection,
after the discovery of the variation in an accession of forage rapeseed Liho
(Stefansson et al., 1961). The LEAR profile was released first in the B. napus
cultivar Oro and was subsequently transferred through interspecific crossing into
B. rapa with the first LEAR cultivar Span.
Other Brassica and related species. The isolation of a B. juncea selection with
low levels of erucic acid (Kirk and Oram, 1981) has led to the development of
mustard germplasm with canola quality oil after crossing and selection. The low
erucic acid trait has also been established in B. carinata and in Sinapis alba at two
breeding organizations: AAFC Saskatoon and the University of Idaho, USA.
2. High Erucic Acid Oil
B. napus cultivars with very high levels of erucic acid C22:1 provide a
valuable source of C22:1 and its derivatives for industrial applications including
high temperature lubricants, surfactants, plasticisers, surface coatings and
solvents (McVetty and Scarth, 2002). There are also pharmaceutical and food
applications. The economics of extracting C22:1 from the oil for these
applications improves significantly when the erucic acid levels are increased
over 500 g kg21.
The level of C22:1 in unselected B. napus germplasm is between 350 and
400 g kg21. Selection following intraspecific crossing with B. napus germplasm
containing elevated C22:1 produced the high erucic acid (HEAR) cultivar strain
Hero (Scarth et al., 1991). The recently released HEAR cultivars Castor and
Millenium 01 have oil profiles with 550 g kg21 C22:1. (McVetty et al., 1998a,b).
3. Super High Erucic Acid Oil
Market applications for erucic acid would increase significantly with further
increases in the level of erucic acid. However, no Brassica germplasm has been
identified with erucic acid levels exceeding 660 g kg21 (McVetty and Scarth,
2002). The substrate specificity of the Brassica acyltransferase LPAAT
preferentially inserts C18:1 in the middle position (sn-2) of the glycerol
molecule, excluding the longer chain fatty acids including C22:1 and limiting the
maximum expression of erucic acid content to 660 g kg21 (Luhs et al., 1999).
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 293
Accessions of B. oleracea and B. rapa have been identified which esterify
erucic acid in the sn-2 position (Taylor et al., 1995; Taylor, personal
communication). This germplasm was used to resynthesize B. napus by
interspecific crossing followed by chromosome doubling. However, the erucic
acid levels expressed in the resynthesized B. napus did not exceed 660 g kg21
(McVetty, personal communication) A similar approach using interspecific
crosses to resynthesis high eruric B. napus was reported by Luhs and Friedt (1995).
4. Reduced Saturate Oil Quality
Canola oil contains a very low level (.71 g kg21) of total saturates, and only
40 g kg21 of the saturated fatty acids identified as contributing to elevated blood
cholesterol levels, i.e., lauric acid C12:0, C14:0 and C16:0. The labeling
regulations in the US and Canada allow oils with less than one gram of saturated
fat per 14 g of total fat or less than 70 g kg21 saturated fatty acid content in the oil
to be identified as low in saturated fatty acids. It is this trait that distinguishes
canola oil from the competing vegetable oils in the North American market such
as corn, soy, and peanut oil. Consumer preference for low saturate oils as a salad
oil and in cooking and frying applications has established a significant market
share for canola oil (Fitzpatrick and Scarth, 1998). Defense of this market share
requires at a minimum the maintenance of the 70 g kg21 limit on saturated fat.
Breeding institutions in Canada have focused on the reduction of saturates
initially below 70 g kg21, with the long-term objective of achieving reductions in
the sum of C16:0 þ C18:0 below 40 g kg21. Significant reductions in saturated
fatty acids are not available in current B. napus canola cultivars. B. napus
germplasm has been developed from interspecific crosses between B. rapa and B.
oleracea strains, with levels comparable to B. rapa (less than 60 g kg21 of the
total saturated fatty acids) (Raney et al., 1999a).
B. MUTAGENESIS
1. Low-Linolenic Oil Quality
Linolenic acid and linoleic acid are both essential fatty acids for human health.
Linolenic acid has a role in reducing plasma cholesterol levels (Eskin et al.,
1996). The ratio of C18:3/C18:2 in canola oil (1:2) is also regarded as
nutritionally favorable. However, vegetable oils high in C18:3 such as canola
have poor oxidative and flavor stability. The use of hydrogenation to reduce the
polyunsaturated fatty acids results in the formation of trans fatty acids.
Nutritionists are concerned with the trans isomers of cis-fatty acids raising the
serum low-density lipoprotein cholesterol (LDL-C) levels and reducing the serum
J. W. BURTON ET AL.294
high-density lipoprotein cholesterol (HDL-C). Elevated levels of LDL-C and
reduced levels of HDL-C are associated with enhanced risk of CVD. The current
recommendation from nutritionists is that the current levels of trans fatty acid in
the diet should not be increased (Fitzpatrick and Scarth, 1998). One solution is
the reduction of C18:3 to increase stability without the need for hydrogenation.
Low-linolenic oils with .35 g kg21 C18:3 have shown increased stability under
conditions of accelerated storage with no changes in overall odor intensity or
pleasantness. There were also significantly lower levels of free fatty acids during
frying with low-linolenic canola oil with better flavor quality of the French fry
product (Eskin et al., 1996). The reduction in C18:3 is typically accompanied by
an increase in C18:2 and C18:1.
Many breeding institutions worldwide have developed low-linolenic B. napus
cultivars. The low-linolenic profile is being developed in zero-erucic acid spring
turnip rape B. rapa at the University of Helsinki and Boreal Plant Breeding in
Finland. Current levels of linolenic acid are 70 g kg21 with a 1:4 ratio of
C18:3/C18:2, achieved through selection (Laakso et al., 1999). The most
reported method used to reduce C18:3 content has been mutagenesis applied to
imbibed seeds or to developing microspores. The low-linolenic trait was
produced by seed mutagenesis of the B. napus cultivar Oro, which led to the
isolation of a mutation line, M11 with an altered C18:3/C18:2 ratio (Raney et al.,
1999b). A program of backcrossing to the adapted cultivar Regent, combined
with selection, led to the release of the first low linolenic cultivar Stellar with
approximately 30 g kg21 C18:3 in the seed oil (Scarth et al., 1988).
2. Mid- and High-Oleic Oil Quality
Comparative studies of genetically modified oils for frying performance or life
of the oil, shelf-life, dietary benefits and sensory properties of end product
identified the optimum oil profile as 50–70 g kg21 saturate, 670–750 g kg21
C18:1, 150–220 g kg21 C18:2 and .30 g kg21 C18:3. The role of C18:2 in
enhanced sensory properties was noted as C18:1 levels over 750 g kg21 results in
a reduction in sensory properties (flavor and taste) and an increase in off-odors
(Warner and Mounts, 1993). Mid-oleic oils with increased C18:1 levels in
combination with reduced C18:2 and C18:3 provide stability without the
requirement for partial hydrogenation (Fitzpatrick and Scarth, 1998).
Enhanced C18:1 levels have been produced through mutagenesis, applied
both to seed and to microspore derived embryos. The University of Gottingen
Institute fur Pflanzenbau und Pflanzenzuchtung has produced high-oleic
quality in winter rapeseed developed from mutagenic lines from cv. Wotan
and other high-oleic lines. A study of the influence of the environment on
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 295
the high-oleic trait showed a high genetic heritability, but with a significant
environment and GXE component (Schierholt and Becker, 1999).
High-oleic oils (over 750 g kg21) are targeted for industrial end-use in the
oleate market or can be blended with conventional oils to lower oleic levels.
The competing oils in this market are the mid- and high-oleic sunflower oils. Seed
mutagenesis followed by crossing and selection resulted in Brassica genotypes
with .850 g kg21 oleic and reduced levels of linoleic and linolenic acid (Wong
et al., 1991). The oil profile with oleic acid levels over 800 g kg21 has been
protected through patent.
3. Reduced Saturate Oil Quality
Doubled haploid B. rapa lines with reduced saturate levels have been
developed from microspore mutagenesis conducted at the Plant Biotechnology
Institute in Saskatoon. This low saturate variation is being introduced into B.
napus, to produce low saturate germplasm for further cultivar development
(McVetty and Scarth, 2002). Raney and Rakow (2002) have also used
mutagenesis to produce B. napus germplasm with reduced saturate levels.
C. TRANSFORMATION
1. High Saturate Oil Quality Including Novel Fatty Acids
Oils with high levels of saturated fat have applications in the production of
solid fat products such as shortenings and margarines where unmodified liquid
oils cannot be used. Canola and soybean oils with high saturated fatty acid levels
can replace animal fats and offer the benefit of domestic production for temperate
countries, reducing dependence on tropical high saturate oils.
Transgenic modification using the Agrobacterium-mediated system has been
used to increase the total saturated fatty acid content of canola. High laurate
C12:0 oil canola was the world’s first transgenic oilseed crop in commercial
production. The high C12:0 trait was the result of the insertion of the acyl-ACP
thioesterase (TE) isolated from California Bay Laurel (Umbellularia californica).
The typical analytical value of Lauricale oil profile is 380 g kg21 C12:0,
40 g kg21 C14:0, 30 g kg21 C16:0, 310 g kg21 C18:1, 110 g kg21 C18:2,
70 g kg21 C18:3 and 60 g kg21 other fatty acids. The Lauricale product has
applications in the food industry in products such as confectionary, simulated
dairy products, icings and frostings.
Overexpression of the oleate preferring acyl-ACP TE from soybean (Glycine
max) increased the sum of C16:0 and C18:0 to approximately 200 g kg21 in
transgenic lines of the canola cultivar Westar (Hitz et al., 1995). Levels of C16:0
J. W. BURTON ET AL.296
have been increased to over 30 g kg21 by transformation with TE from elm
(Ulmus americanca) and nutmeg (Myristica fraganceae) (Voelker et al., 1992).
Transgenic lines with TE from Cuphea lanceolata have been characterized with
160 g kg21 C14:0 and 430 g kg21 (C14:0 þ C16:0) (Rudloff et al., 1999).
2. Mid- and High-Oleic Oil Quality
High-oleic canola (.860 g kg21 C18:1, ,70 g kg21 C18:2, ,25 g kg21
C18:3) has been produced using seed specific inhibition of microsomal oleate
desaturase and microsomal linoleate desaturase gene expression, either through
co-suppression or antisense technology. Co-suppression has been used in
combination with mutation treatments to produce modified fatty acid profiles
(Debonte and Hitz, 1996).
D. PRODUCTION OF MODIFIED OIL CULTIVARS
The effect of modifying oil quality on the other quality traits, including oil and
protein content, and on agronomic performance is a critical factor in the
successful production of the modified oil quality. In general, the performance of
the modified oil cultivars is not different from the non-transgenic parent cultivar
used in the original crosses, mutagenetic treatment or transformation event. The
challenge for the successful production of these cultivars is to achieve the
performance of the current conventional cultivars with the optimum expression
of the modified oil quality. The stability of the modified oil trait over different
production environments is another important factor.
IV. PEANUT
A. INTRODUCTION, BREEDING OBJECTIVES, AND RATIONALE
In the US, peanut (Arachis hypogaea L.) is primarily used as a food crop,
however, peanut is one of the five most important oilseeds produced in the world
(Carley and Fletcher, 1995). About two thirds of the world’s peanut crop is utilized
for oil production (Savage and Keenan, 1994). The fatty acid composition of peanut
is an important quality attribute whether it is used as a food or a source for oil.
Two fatty acids, oleic (O) and linoleic (L), can account for up to 80% of the oil
content of peanut (Young and Waller, 1972). Oleic acid is an 18-carbon mono-
unsaturated (18:1) precursor to linoleic acid (18:2). Oleic acid is less reactive
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 297
with oxygen and therefore more stable. A commonly used index of the quality of
groundnut is the ratio of oleic to linoleic fatty acids (O/L ratio). A higher ratio of
oleic to linoleic acid in peanut oil and other peanut products is considered an
indicator of a more stable product.
Fatty acid composition of peanut oil is influenced by cultivar, degree of
maturity, and environmental conditions (Ahmed and Young, 1982). In the US,
approximately 70% of the peanuts are runners (small-seeded types of var.
hypogaea), 20% are virginias (large-seeded types of var. hypogaea), 10% are
spanish (var. vulgaris) and less than 1% are valencia (var. fastigiata) market
types, respectively (Knauft and Gorbet, 1989). Oil from seeds of different
botanical types of peanuts differ in their tendency to develop oxidative rancidity
or undesirable odors and flavors. Virginia botanical-type peanuts (var. hypogaea)
produce oil with slightly lower linoleic percentage and therefore tend to have
greater oil stability than spanish or valencia botanical types (Norden et al., 1982).
During seed maturation the proportion of oleic acid increases and the proportion
of linoleic acid decreases (Sanders, 1980a,b; Sanders et al., 1982). Peanuts
produced in the northern regions of the US peanut production areas often have
relatively low O/L ratios resulting in inferior oxidative stability (Worthington
et al., 1972; Brown et al., 1975). Holladay and Pearson (1974) observed that
higher temperatures during the last 4 weeks before harvest resulted in peanuts
with a higher proportion of oleic acid.
B. INCREASING OLEIC ACID
1. Breeding History
Historically, standard peanut cultivars have averaged 550 g kg21 oleic acid
and 250 g kg21 linoleic acid (Knauft et al., 1993). Norden et al. (1987) examined
the fatty acid composition of 494 genotypes and identified a breeding line (F435)
with 800 g kg21 oleic acid and 20 g kg21 linoleic acid. This was a major
deviation from previously known levels of fatty acid composition in peanut.
Moore and Knauft (1989) found that inheritance of the high-oleate trait was
controlled by duplicate recessive genes, ol1 and ol2, and therefore, easily
transferable to existing cultivars through backcrossing. F435 differed at both loci
from a virginia-type line but at only one locus from a runner line. Knauft et al.
(1993) found the trait to exhibit monogenic inheritance in crosses with 12
additional cultivars and breeding lines of the runner market-type. A cross with a
virginia market-type segregated in a 15:1 ratio typical of recessive digenic
inheritance. These results led the authors to conclude that one of the recessive
alleles occurs with high frequency in peanut breeding populations in the US and
that the other allele is rare. Isleib et al. (1996) examined five different cultivars of
virginia-type peanut cultivars and found that four were either Ol1Ol1ol2ol2 or
J. W. BURTON ET AL.298
ol1ol1Ol2Ol2 and one was Ol1Ol1Ol2Ol2. When only one gene transfer is required,
Isleib et al. (1998) were able to identify heterozygotes based on linoleate levels.
This will allow breeders to identify carriers of the recessive allele in successive
cycles of backcrossing without intervening generations of selfing and decrease
the time required to achieve the desired number of backcrosses.
Lopez et al. (2001) examined the inheritance of the high oleic acid in six
Spanish market-type peanut cultivars. Segregation patterns indicated that two
major genes were involved. However, the presence of low-intermediate O/L ratio
genotypes indicated that other genetic modifiers may be involved in the
expression of the O/L ratio in these genotypes. Isleib et al. (1998) also observed
an effect of other loci on fatty acid concentrations.
2. Progress
SunOleic 95R was the first peanut cultivar having the high-oleic trait
(Gorbet and Knauft, 1997). This was followed by the release of Flavorunner,
GK7 High Oleic, and SunOleic 97R (Gorbet and Knauft, 2000). Oil from high-
oleic peanut has a composition similar to olive oil with low levels of
polyunsaturates. In comparison to standard peanut oil, the high-oleic oil has a
much longer shelf-life before exhibiting rancidity (O’Keefe et al., 1993). This
increase in oil stability is achieved without chemical hydrogenation, which
introduces undesirable trans fatty acids. Rancidity is also delayed in whole
peanuts which are high-oleic (Mugendi et al., 1998) resulting in increased shelf
life for some peanut products.
Pattee and Knauft (1995) evaluated four high oleic acid breeding lines and
observed no change in roasted peanut attribute intensity in comparison to
Florunner. Pattee et al. (2001) observed that high-oleic cultivars and breeding
lines derived by backcrossing with Sunrunner had a high positive breeding value
for the roasted peanut attribute. It is not clear if this is a real genetic effect or an
artifact of the sensory evaluation since the protocol requires a storage period
during which some oxidation of linoleic acid in the Sunrunner seeds may occur
that may produce off-flavors
The first peanut cultivars containing high oleic acid were extremely
susceptible to tomato spotted wilt tospovirus (TSWV) (Culbreath et al., 1997)
and performed poorly in areas with high TSWV pressure. Moderate levels of
resistance to TSWV have been reported in the mid-oleic cultivars Florida MDR-
98 (Culbreath et al., 1997) and ViruGard (Culbreath et al., 2000). Recently, high-
oleic breeding lines have been produced with acceptable levels of resistance to
TSWV (Culbreath et al., 1999).
Several in vitro studies have indicated that fatty acid composition could either
directly or indirectly affect aflatoxin contamination (Fabbri et al., 1983; Passi
ALTERING FATTY ACID COMPOSITION IN OIL SEED CROPS 299
et al., 1984; Doehlert et al., 1993; Burrow et al., 1997). Holbrook et al. (2000)
evaluated the effect of altered fatty acid composition on preharvest aflatoxin
contamination in peanut, but observed no measurable effect of reduced linoleic
acid composition on preharvest aflatoxin contamination. They concluded that the
products of the lipoxygenase pathway that have been shown to affect aflatoxin
biosynthesis in vitro may not be present in sufficient quantities in developing
peanut seed. However, under conditions that simulated post-harvest conditions,
Xue et al. (2003) observed an increased ability of high-oleic lines to support
production of aflatoxin in comparison to normal-oleic lines. They urged special
care in handling and storage of high-oleic peanuts to prevent the growth of
Aspergillus spp.
Ray et al. (1993) determined that the high-oleic phenotype was due to
reduced activity of microsomal oleoyl-phosphatidylcholine (PC) desaturase.
Jung et al. (2000b) isolated two cDNA for this microsomal oleoyl-PC
desaturase, ahFAD2A and ahFAD2B. The amount of ahFAD2B transcript
was markedly reduced in plants with the high-oleic trait (Jung et al., 2000a),
and this decreased activity was consistent with the observed inheritance of the
high-oleic phenotype.
Comparison of the base sequences of the reading frames of the ahFAD2A and
ahFAD2B genes showed 11 base pair differences resulting in four differences in
amino acids (Jung et al., 2000a). The change of aspartate at position 150 to
asparagine was a change in a residue that is absolutely conserved among other
desaturases. Brunner et al. (2001) used site-specific mutagenesis to change the
aspartate in the high activity enzyme to asparagine and to change asparagine in
the lower activity enzyme to aspartate. Subsequent expression in yeast resulted in
nearly complete loss of activity of the previously more active desaturase and
restored activity to the previously less active desaturase indicating that this
mutation is the molecular basis of the high-oleic phenotype in peanut. Lopez et al.
(2000) observed a similar polymorphism in Spanish market-type lines with the
high-oleic trait.
It is not clear how widely accepted the high-oleic characteristic will be
in the US peanut industry. The trait should be valuable in the northernmost
production areas since undesirable fatty acid profiles have historically been a
problem in peanuts from these regions. This trait adds much less value to
peanuts produced in the southern production areas. The value of this trait is also
dependent on the end product of the peanuts. An extended shelf life would be of
great value for some peanut products whereas for other products, such as
peanut butter, stabilizers are already used to extend shelf life. Negotiations
are ongoing to determine the cost for using this trait and who will bear these
costs. The University of Florida has been granted three patents related to this
trait (Knauft et al., 1999, 2000a,b). These patents encompass peanut seed,
oil, and products.
J. W. BURTON ET AL.300
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