[Advances in Agronomy] Advances in Agronomy Volume 84 Volume 84 || Altering Fatty Acid Composition...

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.


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


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.


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


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)


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)


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)


et al. (2000)


et al. (2000)

fap2-b/fap2-b fap4/fap4 fap5/fap5 fap7/fap7 402 Stoltzfus

et al. (2000)


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


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


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


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.


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


(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


Colby, KS

NuSun hybrids, 1996–2000 2416 452 636


Minot, ND

NuSun hybrids, 1996–2000 2475 470 571



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


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.


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.


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


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).


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


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


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


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


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


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


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.


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