6Transgenic Oils

Thomas A. McKeon

USDA-ARS Western Regional Research Center

Albany, California

1. INTRODUCTION

Most commodity seed oils consist of triacylglycerols containing varying per-

centages of palmitic, stearic, oleic, linoleic, and linolenic acids esterified to the

glycerol backbone. These oils and the fatty acids derived from them are used pri-

marily for food and feed; yet they have important industrial applications as well.

They provide precursors for soaps and other surfactants, derivatives used in produc-

tion of certain plastics and polyamides, applications in low volatile organic carbon

(VOC) coatings, cosmetics, as well as lubricant and grease compounds. The useful-

ness of an oil for food use lies in caloric value and the presence of essential fatty

acids, specifically, those fatty acids that are not produced by humans, linoleic acids,

and a-linolenic acids. In many cases, the usefulness of a seed oil to industry derivesfrom a high proportion of a specific fatty acid in the oil; for example, the high

linolenate content of linseed oil results in its good properties as a drying oil.

This article will briefly cover progress in developing new oils, examine the state

of the art, and describe industrial oilseed crops projected to be developed for

commercial use.

Oils containing high levels of oleic acid are considered to be beneficial for

human health consequences. Such oils are stable to oxidation compared with oils

Baileys Industrial Oil and Fat Products, Sixth Edition, Six Volume Set.Edited by Fereidoon Shahidi. Copyright # 2005 John Wiley & Sons, Inc.

155

containing polyunsaturated fatty acids (PUFAs) and contain low levels of saturated

fatty acids. Olive oil (70% oleate) is generally considered to have a beneficial com-

position for oleate content. It should be noted that continued research is adding

information for fatty acid requirements and benefits. Linoleic and a-linolenic acidshave been known for decades to be essential fatty acids. More recently, the need for

eicosapentaeneoic and docosahexaenoic acids as part of the diet, especially for the

developing fetus, and even g-linolenic, underlie the need for intake of oils and fatsfrom multiple sources that can supply what seems to be an expanding list of nutri-

titionally important fatty acids.

Although oils containing PUFAs can be converted to high monounsaturate con-

tent by partial hydrogenation, the process results in the production of trans-

fatty acids. There is a negative perception of trans-fatty acids, which are thought

to behave physiologically as saturated fatty acids. These acids are considered to

increase arterial plaque formation and may contribute to the development of type II

diabetes. Thus, a considerable research-and-development effort has been put into

designing food oils with a high content of oleic acid. However, for commercial

use, the market for food-grade oils is often driven by price, with quality traits pro-

viding premium value.

Most vegetable oil that is produced is consumed as food. These food oils also

have important industrial uses. For example, approximately 15% of soybean oil

is used for industrial products, including inks, plasticizers, coatings, and composite

materials. Other commodity oils are useful industrially because they contain un-

common fatty acids. Castor oil is 90% ricinoleate (12-hydroxy-octadec-9-enoate),

and the hydroxy group imparts dramatically different physical and chemical proper-

ties that make castor oil an important industrial feedstock. Rapeseed oil contains

up to 60% erucate (docosa-13-enoate), which is used in several lubricant appli-

cations. Tung oil contains up to 80% eleostearate (octadeca-9c,11t,13t-trienoate),

a conjugated fatty acid that makes tung oil a prized drying oil because it does

not yellow during the drying process. Palm-kernel oil and coconut oil both contain

high levels of the medium-chain saturated fatty acids laurate (C12) and myristate

(C14), which have excellent foaming properties for production of soaps and other

surfactants. Thus, several features of a vegetable oil can impart industrial chemical

value. Chemical functionality can alter physical properties or provide reactive sites

that allow useful derivatives to be made. Another industrially useful feature is the

presence of a highly enriched single component. Some oils also have unique uses as

a result of their composition; e.g., cocoa butter is unique in its melting character-

istics, which makes it an excellent component of cosmetics in addition to its food

uses. Consistent composition is also important for industry, and this is usually

closely tied to a high content of a desired component. The goal of developing oil-

seeds for industrial use is to introduce one or more of these desirable characteristics

into the oil of an agronomically suitable crop.

Seed oils also contain potentially useful fatty acids that have not been introduced

into commerce because the plant has not yet been adapted to large-scale planting.

Examples of such plants include Vernonia anthelmintica and Euphorbia lagascae,

156 TRANSGENIC OILS

which produce oils high in vernolate (octadeca-12,13-epoxy, 9-enoate); Cuphea sp.,

many of which produce oils containing medium-chain fatty acids from caprylate

(C8) to myristate at levels up to 95% of a single fatty acid; Lesquerella sp., which

contain up to 55% lesquerolate (eicosa-14-hydroxy-9-enoate) that can replace

ricinoleate in some applications. Each crop has been the target of New Crops

research, which is aimed at breeding out undesirable agronomic characteristics

and introducing desirable traits, such as higher yield or indehiscence. Although

considerable progress has been made in each crop, the problem encountered is

a Catch 22: It is hard to get farmers to grow the crop because there is not yet

a significant market for the product, and it is hard to develop a market for the crop,

because no reliable source exists.

Breeding programs have expanded the potential uses of vegetable oils over the

years. Canola, high oleic safflower, high-oleic sunflower, and low-saturate soybean

oil are all the result of extensive traditional breeding programs, based on crossing

available crop germplasm. The introduction of mutagenesis provided a new tool

to breeders, and it is most often useful in eliminating an undesirable trait. The intro-

duction of genetic engineering greatly expanded the ability of the breeder to

introduce desired traits, with genes from incompatible crops, microorganisms,

insects, animals, or any other organism being added to the breeders toolbox.

Moreover, genomic sequencing efforts have revealed the extent of synteny among

plants. Synteny refers to a correspondence in genomic arrangement, and this has

allowed identification of specific genes associated with agronomically useful traits,

e.g., dwarfing. By comparing genomes, useful traits across species and genera can

be identified and selected for directly rather than through extensive breeding

programs.

Crop genetic engineering holds great promise as a means for developing oilseed

crops with unique characteristics that add both commercial and nutritive value,

increase utilization, and benefit the environment. Currently, the four genetically

engineered (transgenic) crops that have been adopted are all oilseed crops: soy, corn,

cotton, and canola. They are a commercial success and account for 99% of trans-

genic crops planted worldwide. Over 70% of the soy, 50% of the corn, and 70% of

the cotton grown in the United States are genetically engineered. Canola is a rela-

tively small crop in the United States, but approximately 60% of the canola planted

in the United States is transgenic. Most canola grown in Canada, a leading pro-

ducer, is transgenic. An increasing number of countries have adopted the techno-

logy. The United States, Argentina, Canada, Brazil, China, and South Africa account

for 99% of the transgenic crops produced, with an additional 12 countries adopting

the technology (1). The growth in planting of transgenic crops is remarkable in that

it has all occurred in the last 8 years, from the time the first transgenic crops were

introduced in 1996. At this time, each crop has been modified for input traits,

reducing or eliminating the need for chemical applications by the introduction of

genes encoding herbicide tolerance (soy, canola), insect resistance (corn, cotton),

or both (cotton). Currently, cotton is the only crop with a significant share of the

crop carrying both (stacked) traits. The introduction of genes for input traits is

INTRODUCTION 157

required for maintenance of substantial equivalence in the crop. Table 1 displays

data for soybean oil, cottonseed oil, and corn oil from plants lacking or including a

gene for glyphosate tolerance, and it indicates that the introduced gene has had

essentially no effect on composition, especially given the variability that is observed

in varietal and climate-related differences in fatty acid composition.

Developments affecting output traits, the product of interest in the case of this

article being oil, have not yet achieved commercial success. In addition to oilseed

crops derived from breeding and mutagenesis, this article will describe the two

transgenic oilseeds that have been commercially introduced, briefly describing the

biochemical basis of their development and the problems faced in their commercia-

lization. That discussion will be followed by a description of other oils that have

been proposed for development through transgenic technology. Finally, the article

will discuss issues related to acceptance of transgenic crops.

2. TECHNOLOGY FOR ALTERING FATTY ACID COMPOSITION

Several approaches lead to oilseed crops with altered fatty acid composition. The

most ancient is evolution, which is a long-term, seemingly random process.

Although it is not a practical means for purposefully altering fatty acid composi-

tion, especially in a brief time span, evolution has, in fact, yielded a broad range

of oilseeds with differing characteristic fatty acid compositions. In the same species

and genera, these differences usually consist of varying percentages of the same

fatty acids. In some plant families and genera, considerable variation exists as well

in the types of fatty acid made in seed oil. These differences provide the variants

needed for successfully breeding varieties with altered fatty acid composition.

Breeding programs have successfully used available germplasm to develop major

TABLE 1. Composition of Transgenic Oilseeds (%).

Crop 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 Xa

Cotton-ctrl 27.7 0.6 2.7 15.3 43.2 0.2 0.2 2.3

Cotton-GM 26.8 0.7 2.7 15.5 45.9 0.3 0.2 1.7

Corn-ctrl 9.9 1.9 27.4 58.7 1.1 0.4 0.3 0.2

Corn-GM 9.9 1.9 27.5 58.6 1.1 0.4 0.3 0.2

Soybean-ctrl 11.2 4.1 19.7 52.5 8.0 0.4 0.2 0.5

Soybean GM 11.2 4.1 19.7 52.3 8.2 0.4 0.2 0.5

Canola-ctrl 4.6 0.3 1.6 57.5 19.4 13.8 0.6 1.4 0.3

Canola-12:0 3.3 0.2 1.1 35.1 14.6 8.8 0.3 0.7 0.2 35.5

Xa: uncommon fatty acid content; for cotton, malvalic, sterculic, and dihydrosterculic; Canola-12:0, 31.3% 12:0

and 4.2% 14:0.

Cotton control is Coker 312; Cotton GM is glyphosate tolerant, selection 1445 (Monsanto), from (2); Corn

control is GA21 segregant lacking the gene for glyphosate tolerance; GM variety is GA21 segregant carrying

glyphosate tolerance gene, from (3); Soybean control is A5403, and Soybean GM is GTS 40-3-2, derived from

particle-bombardment of A5403 with genetic material containing the CP4 ESPS gene for glyphosate tolerance

(4); Canola, control (Westar) compared to canola seed with gene for acyl-ACP thioesterase from California

Bay laurel (5).

158 TRANSGENIC OILS

crops soy, corn, rapeseed (Canola), and sunflower that have a more desirable oil

content or fatty acid composition. Where evolution may not have provided suitable

germplasm, approaches also can be taken to alter fatty acid composition. Random

mutagenesis followed by screening and breeding has produced varieties with altered

fatty acid composition in oil (6). As the mutagenic approach is geared to eliminat-

ing genes, usually this approach has reduced levels of undesirable fatty acid compo-

nents or increased levels of a desired fatty acid. A recent innovation in this approach

is targeting induced local lesions in genomes (TILLING), which uses a

mutagenic approach but introduces high throughput screening of the M2 generation

(second-generation, mutated lines that have been self-pollinated) to identify specific

genes that have been altered or inactivated by mutagenic events (7). Plant selections

carrying these mutated genes can then be screened directly for desired characteris-

tics. The TILLING process thus moves most of the screening effort into the labo-

ratory, which considerably reduces the population that would otherwise have to be

grown in the field for later screening.

The ability to manipulate fatty acid composition in oilseeds by genetic engineering

has resulted from a combination of three approaches. Biochemical characterization

has identified most steps in fatty acid biosynthesis (8, 9). Genetic identification

and chemical characterization of fatty acid biosynthetic mutants in mutagenized

Arabidopsis thaliana has provided an extensive genetic map of fatty acid and lipid

biosynthetic steps during plant growth and development (10). Identification, char-

acterization, and cloning of enzyme activities in plants that produce nutritionally

useful fatty acids, such as g-linolenic acid, or uncommon, industrially useful fattyacids, such as vernolic acid (12,13-epoxy oleate), have provided the additional in-

formation needed to broaden the spectrum of fatty acids available from commodity

oilseeds (11). Hundreds of other fatty acids with unusual chemical functionalities

are produced by one or more oilseed plants. A considerable amount of research has

gone into elucidating the biosynthetic process by which such fatty acids are made,

and much enzymology underlying the introduction of unsaturation, conjugated

unsaturation, hydroxyl, acetylenic, and epoxy functionality is now understood. The

enzymes that carry out each reaction are interrelated, can be interconverted by

engineering appropriate amino acid residues, and to a limited extent, can have their

specificity for chain length and positional-selectivity altered in a predictable

manner (12). The specificity of the chemistry carried out on what is essentially a

straight hydrocarbon chain is unprecedented for traditional bench chemistry, and

in the future, it may represent the development of green chemistry carried out in

plants to produce desired chemical precursors.

3. CANOLA FROM TRADITIONAL BREEDING OF OILSEED CROPS

Rapeseed has long been a source of cooking oil and has important industrial uses

such as lubricants for high-temperature applications, especially those leading to

environmental release of the lubricant; antislip agents in plastics manufacturing;

fabric softeners; and additional oleochemical applications. However, the erucic acid

CANOLA FROM TRADITIONAL BREEDING OF OILSEED CROPS 159

component has been considered a potential health problem, and as a result, a

low-erucic acid rapeseed (LEAR) was desired to meet food, feed, and export needs.

An intensive breeding program was initiated in the 1950s by R.K. Downey of Agri-

culture Canada in Saskatoon (13) and B.R. Stefansson (14) of the University of

Manitoba to reduce the content of erucic acid and eliminate glucosinolates from

the seed, as these were feeding deterrents and impeded use of rapeseed meal for

animal feed. Each researcher identified lines of rapeseed with very low erucic acid,

based on crossing out Brassica napus with B. juncea. The low-erucic varieties

developed had less than 2% erucic, compared with 55% in rapeseed. The low-erucic,

low-glucosinolate double-low varieties derived from this research were renamed

Canola in 1979 by the Western Canadian Oilseed Crushers but are also known as

LEAR. Although Canola is a minor crop in the United States, it is the major oilseed

grown in Canada and Northern Europe. Although the oil is used primarily for food,

its high-oleate content (60%) makes it useful for industrial processes requiring an

oxidatively stable oil. Its high yield of oil (per hectare) has resulted in its use as a

major source of biodiesel in Europe. Other changes have been bred in Canola

selections; these include low linolenate and lower saturate content. Other high-

oleate oils have been obtained through traditional breeding in sunflower, safflower,

and corn, whereas peanut and olive oil, among others, have a naturally high oleate

content.

4. HIGH-OLEIC SUNFLOWER FROM MUTAGENESISOF OILSEED CROPS

When available germplasm with desired characteristics is limited, mutagenesis can

help to provide additional germplasm. Chemical and radiation mutagenesis have

been used in breeding programs to obtain suitable germplasm for generating novel

traits. Sunflower has been mutagenized with and screened for fatty acid composi-

tion (15). The normal composition of sunflower oil is high in linoleate (6075%),

but some mutagenized lines were found that had higher oleate (>80%). In back-crosses, the high oleate trait remained stable, which indicates a nonrevertible muta-

tion had resulted in the high-oleate phenotype. Later research demonstrated that

the mutation is in the oleate desaturase gene (16), a single copy gene in sunflower.

Biochemically, this process would block conversion of oleate to linoleate and allow

oleate to accumulate in the seed (Figure 1). Mutagenesis has altered fatty acid

composition of oils derived from other crops, including soybean, cotton, flax, and

canola (15).

5. APPLICATIONS OF HIGH-OLEATE OILS

High-oleate oils are highly desirable for food use. They are stable to oxidation and

therefore good for frying and can be stored without spoilage for a longer time than

oils with high polyunsaturate content. Oleate is the prevalent fatty acid in the

160 TRANSGENIC OILS

Mediterranean diet based on olive oil and popularly thought to be the best fat

to consume for long-range health benefits. The oxidative stability of high-oleate oils

also meets industrial needs (17). Such oils are useful in cosmetic applications

as they are established to be safe for consumption. They are useful as sources of

Acetyl-ACP2 ACP

Acetyl-CoA

CO2

Malonyl-CoA

ACP 3Malonyl-ACP

Acetyl-ACP+

Malonyl-ACP

5

4

Butyryl-ACP

Malonyl-ACP 5

Caproyl-ACP

5

5

5

5

5

6

7

Capryloyl-ACP

Capryl-ACP

Lauroyl-ACP

Malonyl-ACP

Malonyl-ACP

Malonyl-ACP

Malonyl-ACP

Malonyl-ACP

Malonyl-ACP

B

B

Myristoyl-ACP

Palmitoyl-ACP

Stearoyl-ACP

Oleoyl-ACPOleoyl-CoA

10

11

Oleoyl-PC

Linoleoyl-PC Palmltate

Stearate

Oleate9 8

1. AcetylCoACarboxylase2. AcetylCoA ACPTransacylase3. MalonylCoA ACPTransacylase4. Condensing Enzymes,KAS III5. Condensing Enzymes,KAS I6. Condensing Enzymes,KAS II7. StearoylACPDesaturase8. AcylACP Thioesterase9. Fatty AcylCoASynthetase10. LysophosphatidicAcid AcylCoATransacylase11. Oleoyl Desaturase

1

Figure 1. Fatty acid biosynthetic pathway.

APPLICATIONS OF HIGH-OLEATE OILS 161

oleate, as they can reduce or eliminate the need for purification from other fatty acid

components, which adds significant expense to the cost of obtaining pure oleate. As

they remain liquid at room temperature and below, and have high-oxidative stabilty,

they are useful in applications such as hydraulic fluids and oil-based insulators.

Although problems are associated with using seed oil in these latter applications,

the oil has the benefit of being biodegradeable and nontoxic in case of a spill (17).

6. ALTERED POLYUNSATURATE CONTENTTHROUGH MUTAGENESIS

Linseed oil from the flax plant (Linum usitatissimum L.) has a high content of

a-linolenic acid, an essential fatty acid for the human diet. Although present inmany seed oils at levels from near 0% to 15%, it makes up 55% of the fatty acid

content of linseed oil. It is this high content of the oxygen-sensitive linolenate that

imparts excellent drying oil qualities to linseed oil, which makes it useful for

production of coatings and compound materials such as linoleum. As a source of

o-3 fatty acid, it provides an essential fatty acid for the human diet and thus canplay an important role in many physiological processes. However, linseed oil is

highly susceptible to air oxidation, turning rancid and developing objectionable

odors on exposure to air. Because the meal from the flaxseed has been found to con-

tain valuable nutritive components, such as the lignans, it is desirable to have both

linseed oil and flax meal that are more stable to air oxidation. To this end, a research

group led by Allan Green developed low linolenate strains of flax by mutagenesis of

flaxseed followed by screening for low linolenate and high oil content (18). The

resulting isolates are high in linoleic acid and less susceptible to rancidity than

linseed oil. The normal composition of linseed oil is approximately 13% linoleic

and 49% linolenic, whereas the oil derived from these plants, designated Linola

or solin, is up to 70% linoleic and 2% or less linolenic (19).

7. IMPROVED OIL COMPOSITION OF A TRANSGENIC SOYBEAN

Soybean oil linolenic acid content normally ranges from 5% to 12%, with most in

the 8% range, which makes the oil susceptible to oxidation and spoilage. As a

result, soy oil is often partially hydrogenated to stabilize the oil by reducing the

linolenate content. However, the hydrogenation process introduces trans-fatty

acids, which are considered undesirable dietary components. It has been shown

that reducing the content of linolenic acid in soy oil would significantly stabilize the

oil, which makes it useful for frying and other high-temperature cooking operations

without the need for hydrogenation (20). A recent introduction, the Vistive soybean,

has been bred to contain less than 2% a-linolenic acid, thereby producing an oil thatdoes not need to be hydrogenated for food use. Interestingly, the low linolenate trait

was introduced by traditional breeding into a soybean line genetically engineered

162 TRANSGENIC OILS

to carry the gene for glyphosate resistance. It is thus a hybrid of traditional and

transgenic technology. Vistive will be commercialized in 2005 (21).

8. IMPROVED INDUSTRIAL USE FROM GENETICALLYENGINEERING OILSEED CROPS

8.1. Laurate Canola

The first commercial oilseed genetically modified for industrial use is laurate

canola, altered to produce lauric acid (22). Laurate oils produce soaps and other

surfactants because of the excellent foaming properties of the medium-chain fatty

acid, and no temperate climate crop produces laurate. Because the price of laurate

oils, derived from coconut and palm kernel, is subject to considerable fluctuation,

it was thought that development of a stable, temperate climate crop that could

produce laurate would provide a valuable renewable resource to meet a significant

domestic need. Moreover, the higher temperature melting properties of laurate oils

make them useful in baking and confections where a melting temperature similar to

butter or cocoa is desired.

In the usual fatty acid biosynthetic pathway (Figure 1), the major product is

oleate with varying amounts of palmitate and stearate produced, depending in

part on the relative activities of the acyl-ACP (acyl carrier protein) thioesterases,

stearoyl-ACP desaturase, and keto-acyl-ACP synthase II (KAS II). Certain plants

that produce uncommon fatty acids have different enzyme(s) present that result

in alterations of this pathway. Researchers at Calgene (Davis, CA) identified an

enzyme in California Bay laurel seed, which produces oil containing approximately

60% laurate. The enzyme, a lauroyl-ACP thioesterase (FATB gene), specifically

releases laurate during the course of fatty acid biosynthesis, which prevents the fatty

acid chain from being further elongated and makes the laurate available for incorpo-

ration into the triacylglycerol fraction (22). By introducing the gene for this enzyme

into Canola, and driving the expression of the gene with a promoter for napin, a

seed storage protein that is highly expressed in Brassica sp., laurate-producing

cultivars averaging 40%, and some up to 60% (on a mole basis), laurate were

obtained (Table 1, reference 23). The triacylglycerols in the oil were acylated with

laurate in the sn-1 and sn-3 positions. Because coconut oil contains laurate in all

3 sn- positions, the coconut lysophosphatidic acid acyltransferase (LPAAT) was

purified and was shown to specifically incorporate laurate into the sn-2 position.

The gene for the enzyme was cloned, expressed in laurate canola, and resulted in

cultivars with a laurate content averaging over 50% (molar basis) (24). Despite the

overwhelming scientific success of this approach, and the development of what is

logically a valuable industrial crop, the commercialization of laurate canola has not

yet been successful. The crop has reduced yields compared with normal canola

(25), requires special handling to keep it separate from other canola, has added

cost to recoup the research and development of the crop, and includes a premium

paid to contracted growers. Given these cost items and the coinciding low price of

IMPROVED INDUSTRIAL USE FROM GENETICALLY ENGINEERING OILSEED CROPS 163

laurate oils from tropical sources, laurate canola could not achieve commercial

success as a replacement for palm-kernel and coconut oils.

8.2. High-Oleate Soybean

High-oleate soybean oil, which contains over 80% oleic acid, was developed and

commercialized by the DuPont Corporation (Delaware) (26). The presence of high

levels of linoleate in a food oil is undesirable, as the presence of two methylene-

interrupted double bonds in a fatty acid makes it more sensitive to oxidation than

those high in oleate, which reduces its applicability in certain long-term uses. As

linoleate is further desaturated to a-linolenate in soy, this make the oil even moresensitive to oxidation. Moreover, oleate is generally considered a more desirable

fatty acid for dietary intake. Linoleate is derived from the action of the oleoyl-

desaturase; thus, if the oleoyl desaturase activity could be suppressed in soybean,

the oil composition should be primarily oleate, which is ideal for food use and some

industrial uses mentioned above. Although antisense technology (in which a gene

is introduced to be transcribed in the reverse, or antisense, direction) often blocks

gene expression, the DuPont group used gene suppression, which results in

stable reduction of gene expression when the gene is inserted in the sense (same)

direction. The cultivars obtained produced oil containing up to 80% oleate, with

concomitant reduction in the amount of linoleate, some reduction in the amount

of linolenate, and little difference in levels of palmitate and stearate. However,

the expense of the seed, the availability of other oilseed crops that can also produce

high oleate, and the expense of identity preservation (IP) to keep the seeds separate

from normal soybean have inhibited commercial success for the high-oleic trans-

genic soybean as well (27).

9. FUTURE DIRECTIONS FOR TRANSGENIC OILSEEDS

Research efforts are geared to developing oils that meet changing food, feed, and

chemical feedstock needs. Hundreds of fatty acids are produced in plant sources,

and hundreds more are produced in organisms from microbes to mammals. Some

of these would be of great value if they were available in suitable amounts from a

crop source. The two general transgenic approaches used to develop such sources

are as follows:

Transgenic crops genetically modified to produce the desired fatty acid Transgenic modification of a source plant to make it agronomically suitable

Research groups are pursuing one or both courses to enhance the value and uses of

vegetable oil for food and to expand industrial crop production and develop renew-

able resources that can replace products derived from petroleum. Although none of

these have been commercialized yet, the following examples present anticipated

new oils.

164 TRANSGENIC OILS

10. POTENTIAL NEW OILS FOR FOOD, FEED,AND INDUSTRUIAL USE

10.1. New Polyunsaturated Fatty Acid Components

In addition to the essential fatty acids linoleate and a-linolenate, it is becomingclear that dietary intake of other polyunsaturated fatty acids has important benefits

for proper development and health. Eicosapentaenoic acid (20:55,8,11,14,17) (EPA)and docosahexaenoic acid (22:64,7,10,13,16,19) (DHA) are known to play an impor-tant role in fetal neurological development (28), and they are also associated with

reduction of chronic inflammatory diseases and improved psychological mood (29).

These o-3 fatty acids are derived from fish oils in the human diet, with algae andphytoplankton providing the original source of the fatty acids for fish. Both EPA

and DHA can be produced by humans, via successive elongation and desaturation

of a-linolenate. Linoleate is also subject to the same set of elongation reactions,which leads to production of arachidonic acid (20:45,8,11,14) (AA). EPA andDHA lead to formation of the o-3 eicosanoids, which tend to be anti-inflammatory,whereas AA leads to the formation of o-6 eicosanoids, which tend to promoteinflammation (29). The two types, thus, counterbalance each other. However, the

modern diet tends to be richer in linoleate, so there is considerable interest in

expanding the availability of EPA and DHA in the diet.

Numerous biosynthetic routes to EPA and DHA exist across the spectrum of

organisms that synthesize them (29). One research group (30) has combined genes

encoding enzymes from a marine microalgae (Isochrysis galbana), from Euglena

gracilis, and from the oleaginous fungus Mortierella alpina to introduce the bio-

synthetic steps for EPA biosynthesis into Arabidopsis thaliana. The resulting triple-

transformed plant produced 3% EPA in its leaf tissue and 6.6% arachidonic acid.

This successful engineering feat can be followed up to provide seed oil containing

AA, EPA, and DHA as a more concentrated product of these fatty acids (31).

An alternative route to EPA and DHA can come from elongation and further

desaturation of stearidonic acid (18:46,9,12,15). Certain plants, including black-berry, borage, and evening primrose, contain up to 25% of g-linolenic acid(18:36,9,12) in their seed oil, with considerably smaller amounts of stearidonicacid. The g-linolenate arises from the action of a 6-desaturase on linoleate. Smallamounts of o-3-desaturase present in these seeds account for the stearidonateproduced. When 6-desaturase from borage was introduced into soy, plants produ-cing up to 29% g-linolenate (precursor to arachidonate), with up to 4% stearidonatein oil, resulted (32). Further desaturation to stearidonate could be promoted with

high expression of an o-3-desaturase.

10.2. Oils Containing Hydroxy Fatty Acids for Industrial Use

Castor (Ricinus communis) is a model industrial crop. The seed is up to 60% oil,

which is composed of 90% ricinoleic acid (12-hydroxy oleate), a fatty acid that

produces literally hundreds of products, which include lithium grease, low VOC

POTENTIAL NEW OILS FOR FOOD, FEED, AND INDUSTRUIAL USE 165

coatings, plasticizers, Nylon 11, and cosmetics, among others. The laxative effect

of the oil proscribes use of castor as a food crop, and it seems to be a monotypic

genus. Thus, many concerns expressed for genetic modification of food crops do not

apply to castor. However, the presence of a potent allergen and the toxic protein

ricin in the seed complicate utilization of the seed meal remaining after oil extrac-

tion and prevent widespread cultivation of castor as a crop (33). Initial research

efforts were aimed at producing a castor oil substitute in an alternative crop.

The gene for the enzyme that made ricinoleate, the oleoyl-12-hydroxylase, was

cloned (34) and expressed in plants including Arabidopsis and canola (35). How-

ever, these transgenic plants never made oil containing more than 20% hydroxy

fatty acid. It became apparent that in addition to the oleoyl hydroxylase, other

enzymes involved in the biosynthetic pathway for high ricinoleate oil may also

have evolved with the pathway, and developed substrate specificities not present

in alternative crop plants. This result seem to be the case for the diacylglycerol

acyltransferase (DGAT), the terminal step in triacylglycerol biosynthesis. The

enzyme from castor displays preference for substrates containing ricinoleate

when compared with a DGAT from Arabidopsis (36). The biochemical elucidation

of castor oil biosynthesis should eventually provide the molecular tools necessary

to engineer synthesis of a high-ricinoleate oil in an agronomically suitable crop

(37, 38).

As the toxin and allergen are both proteins and have previously been identified

and cloned, it is possible to use transgenic technology to block their expression.

This approach is being pursued, and has resulted in the development of a transfor-

mation system for castor, a plant that had proven to be recalcitrant to transformation

and regeneration of intact plants (39).

10.3. Oils Containing Novel Monounsaturated Fatty Acids

The fatty acid petroselenic acid (octadeca-cis-6-enoate) is produced in Umbellifereae

plants such as coriander, with levels approaching 90% in the oil. Unlike oils with

high oleate, oils high in petroselenate are solid at room temperature and are a

precursor of adipic acid for Nylon 6,6. Although first postulated as arising from

a simple variant of the stearoyl-ACP desaturase that produces oleate, its production

in plants is more complicated. The biosynthesis of the fatty acid occurs by desatura-

tion of palmitoyl-ACP at the C-4 position, followed by elongation to petroselenate

(40). Although biochemically analogous to stearoyl desaturation, the protein

factors, such as ACP and ferredoxin, involved in the reaction appear specific for

the petroselenate pathway. It is now thought that, in general, when a plant produces

an unusual fatty acid, it may require an entire complement of additional genes to

effectively produce the fatty acid (41, 42).

Numerous fatty acids have considerable commercial potential if they can be

produced in suitable crops at a high level (42). Some of these are included in

Table 2. With perhaps the exception of medium-chain fatty acids, there is not yet

a major success in producing commercially useful levels of any uncommon fatty

acid in a transgenic crop plant.

166 TRANSGENIC OILS

11. ISSUES RELATED TO TRANSGENIC OILSEEDS

One inhibiting factor in commercial development of transgenic oilseeds with novel

traits is public acceptance. The primary principle upon which approval has been

based is known as substantial equivalence, which means that aside from any

introduced changes, the composition of the plant or seed remains essentially

unchanged. However, the concept of unintended consequences expands the

scope of substantial equivalence, which establishes criteria that must be examined

and met. Satisfying the concern for unintended consequences broadened the con-

cept of substantial equivalence to include transcripts, the proteome, metabolome,

and even genome sameness (43). In the approval process for a transgenic plant,

these issues become a key part of the risk assessment both for food crops (44)

and for industrial crops (45).

Most transgenic oilseeds with altered fatty acid composition remain research

subjects, with commercial introduction limited to two crops, neither of which have

yet achieved success in the marketplace. The expected benefits from transgenic

crops with altered fatty acid composition include improved stability properties;

enhanced nutritive value; expanded use of renewable resources to replace petroleum

derived materials; replacement of chemical processes, such as epoxidation of fatty

acid double bonds; and gradual expansion of agriculture as a chemical industry, a

concept long ago known as chemurgy. It is possible to predict some issues that

TABLE 2. Industrially Useful Fatty Acids for Transgenic Plant Production.

Fatty Acid (type) Source Use

Eleostearic (conjugated)

Octadeca-9c,11t,13t-trienoic

Tung, Bitter Melon Drying oil

Erucic (very long chain)

Docosa -13c-enoic

Rapeseed, Crambe Lubricants,

Anti-slip agent

g-LinolenicOctadeca-6c,9c,12c-trienoic

Borage, Blackberry Nutraceutical

Medium chain (saturated)

6 to 14 carbons

Cuphea, Coconut,

Bay Laurel

Detergents

Oleic

Octadeca-9c-enoic

Many Hydraulic oil,

Oleochemicals

Petroselenic (isomer)

Octadeca-6c-enoic

Coriander Nylon 6,6

Ricinoleic (hydroxylated)

Octadeca-9c,12-OH-enoic

Castor Lubricants,

Polymers

Vernolic (epoxy)

Octadeca-9c,12,13-O-enoic

Vernonia, Euphorbia lagascae Coatings, plasticizer

Very long chain

polyunsaturated VLCPUFA

Algae nutraceutical

ISSUES RELATED TO TRANSGENIC OILSEEDS 167

will arise from commercialization and widespread planting of these crops. It is

noteworthy that the major commercially successful transgenic crops are all oil-

seeds, and some understanding of issues and effects of transgenic oilseeds can be

drawn from these crops.

The major transgenic crops grown in the United States and elsewhere are

soybean, cotton, maize, and canola. Considerable effort has gone into and continues

in optimizing these crops for food use. Industrial applications serve as a supplemen-

tal market for vegetable oils. The key to converting oilseeds to enhance food value

or expand their use as an industrial feedstock lies in predictable alterations of bio-

synthetic pathways that will lead to production of the desired product. In the case

of these four commercial crops, they have been engineered for the input traits,

herbicide tolerance and insect resistance. Studies presenting long-range predictions

on profitability of transgenic crops done in the 1980s, before any crops were near

commercialization, indicated that sales of seeds for transgenic crops would be the

major source of profit. Thus, several manufacturers of agricultural chemicals

acquired seed companies and developed research programs that addressed financial

and environmental concerns. Some seeds developed under these programs relied on

application of a product from the company, thus providing a secure market for the

seed and the agrochemical. At the same time, the transgenic crops required consid-

erably less pesticide or herbicide applied, which provided benefit to the farmer via

lower capital outlay, an increase in yield, and considerably lower amounts of agro-

chemicals applied to crops and released into the environment. The secure market for

herbicides also provided an economic incentive for carrying out the mandated regis-

tration of agrochemicals for each crop.

Although the benefits of transgenic crops to consumers are somewhat abstract, as

the level of agrochemical residues on crops is already very low, the benefits to farm-

ers include higher profitability as a result of reduced chemical input and reduced

toxic exposure. For example, farmers in some nations experienced a 75% reduction

in exposure to the toxic effects of agrochemicals when growing transgenic cotton

(46). Reductions in chemical exposure are clearly beneficial to the farmer, farm

workers, and wildlife.

Although oilseed crops may be engineered for industrial use, areas of concern

relate to the food supply. The introduction of allergens in the form of new proteins

is a concern, highlighted by the Starlink episode (47). As yet, no human case of an

allergic reaction related to Starlink has been identified. However, any food crop

modified to produce a toxic, noxious, or bioactive compound can present a potential

health hazard. These hazards would include oilseeds expressing ricinoleate, which

is a laxative; vernolate, which might be an irritant; and other fatty acids with

unwanted physiological effects. Such crops and components of the crop must be

isolated from the food supply by using a sound IP system.

Any process that causes comingling or cross contamination of food and non-food

crops is a concern. Cross-pollination with food crops is a particular concern, and

several strategies for preventing it have been described (48). For example, crops

producing oils for use in industry and containing a non-food fatty acid should

not comingle or cross with related food crops. Such crops would have to be grown

168 TRANSGENIC OILS

in limited areas and surrounded by a buffer crop to block cross-pollination (or

low-probability revertants if male-sterile) (48, 49), a particular concern for canola,

which must be buffered from HEAR when grown. An additional concern for

nonsterile crops is seed drop during harvesting, which can result in germination

and growth of the crop among the crop planted in the next growing season.

Recently, some corn seed grown to produce a pharmaceutical protein in one grow-

ing season was left in a field and germinated among a crop of soybeans. As a result,

the soybeans had to be recovered and destroyed. The end result of this incident was

increased regulatory oversight of non-food and industrial transgenic crops to pre-

vent such incidents in the future.

Industrial oilseed crops are beneficial because they are renewable resources,

yield biodegradeable products, and are environmentally benign. To the extent they

supplant products derived from petroleum, they are clearly green alternatives

to synthetic chemicals and inherently benefit human health by reducing exposure to

atmospheric and aqueous emissions from petroleum processing plants. As much

controversy about genetic modification is concerned with the safety of food derived

from genetically modified plants, genetic modification of industrial crops should

be relatively free of controversy. This situation is not the case, for several reasons.

Many byproducts of industrial crop processing enter the food supply. For example,

after oilseeds, such as industrial rapeseed, have been processed to remove the oil,

the remaining meal is protein rich and processed for use as animal feed. In some

cases, the meal may produce foods for human consumption; for example, flax meal

from linseed oil processing provides nutritional benefit in the form of lignans and

omega-3 oil residue (50). Additionally, many crops have dual uses. Soybean is pri-

marily a food crop, but soybean oil and soybean protein are also used for non-food

applications, such as inks, coatings, and adhesives. The Starlink maize incident has

made it clear that approving a food crop for strictly non-human use (animal feed) is

not sufficient to prevent it from entering the human food supply. No genuinely

harmful consequences of the Starlink corn to human health were found, only to

positive perception of the transgenic crop industry (47). The case described above

involving comingling of transgenic corn carrying a therapeutic protein underlines

the need for a sound (IP) system and appropriate quarantine (both space and time),

if food crops are to produce potentially noxious products, and if food crops are to be

planted at a later date in the same field. In the case of Starlink, with animal feed

being an inherently cheaper end-use than human food, no economic motivation

existed to maintain it separately from other maize. In the case of the transgenic

corn, the product would be much higher value than any food use but extremely dif-

ficult to prevent some seed from remaining in the harvested field.

For transgenic crop approval in the United States, the action and approval

of three Federal agencies is required. The Food and Drug Administration evalu-

ates the crop for direct and indirect food use, the Environmental Protection Agency

registers the crop for potential environmental effects, and the U.S. Department of

Agriculture, Animal and Plant Health Inspection Service (USDA-APHIS) evaluates

genetically modified crops for their potential to disrupt the growth habit of domestic

plants. Canada and Australia have similar oversight, and it is likely that this

ISSUES RELATED TO TRANSGENIC OILSEEDS 169

multipartite regulatory process has engendered consumer confidence in the safety

of transgenic crops (44).

IP is the means by which a crop is maintained separately during storage, ship-

ping, and processing, and it has been in practice for several specialty crops that

have added value, for example, low-saturated soybean. Insofar as comingling a

transgenic industrial crop with other crops could result in health problems, it is

essential to segregate transgenic crops from other crops, in the field and postharvest.

The concept of IP and crop isolation is also important for market perception.

Currently, those persons opposed to eating food from transgenic crops require IP

of the nontransgenic crops. The estimated cost of IP is in the range of 510% of

the crops value (45). If a given transgenic crop delivers higher value to the end

user, at least an economic incentive exists to keep it from contaminating a lower

value crop.

11.1. Risk Assessment

The genetic engineering of oilseed crops to make them better suited for food and

industrial purposes necessitates an evaluation of their potential for hazard. The

primary consideration is currently whether the modification to the crop alters it

unpredictably; i.e., does the introduced gene maintain the crop as substantially

equivalent, or are unintended consequences associated with the introduced gene?

In every case, the designed crop, by definition, will produce the desired product.

Based on existing toxicological data, it is likely that either the chemical product

or related compounds will provide the means to develop a toxicological profile

and determine any potential harm arising from production. A procedure based on

close comparison of a nontransgenic control with the transgenic has been described

(51). A novel product in the plant will require an independent toxicological assess-

ment. The crop residue remaining after extraction of the product will also need to

be evaluated for the remaining product, as well as changes in the crop residue that

result from the alterations required to make the product. Changes in metabolism

brought about to enhance production of a single product can be predicted based

on knowledge of biosynthetic pathways affected by the alteration, but a broad-based

approach is required to identify unintended changes (51). Regulatory agencies

require a demonstration of substantial equivalence depending on the intended

uses of the product or the crop residue remaining (47). Finally, migration of the

transgene(s) into other crops must be evaluated from the standpoint of potential

for harm and the likelihood that it will actually occur. Knowledge of agronomic

habit allows assessment of the latter, and toxicological analysis provides the needed

information for the former. Any potentially toxic or noxious product can be

restricted to sterile strains. Although pollen release from transgenic oilseeds,

such as the Brassica sp., is a common scenario for concern, a more significant

problem may arise from comingling as a result of seed drop during harvest. It

is inevitable that crops that are not controlled to prevent gene release will

ultimately not be permitted (52). Transgenic technology holds out great promise

for expanding the use of renewable resources in production of industrial products.

170 TRANSGENIC OILS

Accordingly, it is essential that such a benefit be implemented in a way to prevent

any harmful effects.

11.2. Allergenicity

It is expected that beyond the different product in an engineered transgenic oil-

seed, the crop will retain substantial equivalence to the nontransgenic crop. The

genes for given characteristics have been cloned and sequenced; perhaps quantities

of the protein have been isolated after being expressed in bacteria or yeast. In many

instances, the activity of the native protein has only been demonstrated by the

change brought about in planta. Therefore, its potential for becoming a problem

allergen remains unknown. In the case of oils, where the primary product is free

of the transgenic crop protein, the allergenicity of the protein is not a food health

issue. However, because most oilseed meals are used as food or animal feed, then

protein allergenicity clearly becomes a consideration. In plants that have undergone

metabolic engineering, the introduced gene(s) is (are) often overexpressed to

redirect the flow of metabolites to the desired product, which leads to a relatively

high level in the plant (23). Altered timing for expression may also be implemented.

Promoter technology is still in a relatively primitive state. In oilseeds, it is common

to use promoters that drive the synthesis of storage proteins and to restrict expres-

sion of the introduced gene to the seed (23). As storage protein promoters are

geared to produce a high level of protein, high levels of the expressed protein

can accumulate, and as storage proteins are expressed late into seed development,

proteins produced to alter metabolism of the immature seed may persist to a high

degree in the harvested seed, whereas they would not normally be present. Methods

for demonstrating potential allergenicity exist, for example, model pepsin digestion

reactions (47, 51) and a decision-tree for assessing allergenicity have been

described, with linear epitope analysis and partial sequence identity to allergens

as indicators (51). Animal models for allergenicity have also been proposed to sup-

plement the decision-tree. In cases where the protein is not available in isolation,

theoretical models to predict allergenicity from the protein sequence are essential to

ensure the safety of associated byproducts, such as seed meal.

11.3. Pollen Transfer

Ecological concern exists about transmission of pollen from some types of plant,

such as the Brassicas and tree crops, either into weedy relatives or into crops grown

at some distance. This problem is not limited to transgenic plants. Canola, a rape-

seed cultivar bred to produce low glucosinolates and low erucic acid, must be

planted in isolation from industrial rapeseed, as each crop will result in seed with

altered composition from the ideal: that is, excessive erucic acid in the canola

and less erucic acid in the industrial rapeseed. Because the products of industrial

crops are not intended for consumption, and may even be noxious, risk manage-

ment and containment, including the prevention of intercrop cross-pollination, is

ISSUES RELATED TO TRANSGENIC OILSEEDS 171

essential. The approaches described (53) can address the problem of out-crossing

from transgenic crops.

11.4. Economics

A major argument for promoting transgenic technology has been the need to pro-

vide more food. Some industrial applications use surplus products, for example,

soybean oil, and provide a buffer against surplus crops, to prevent a decline in

farm profitability. However, if industrial transgenic farming expands, it is not clear

to what extent agriculture may be diverted away from food production, which will

result in increased food costs, if industrial crops are grown in higher volume and

have a higher value than food crops.

If the success of transgenic industrial oilseeds is to be measured on the basis of

their commercial success (see (54) for an economic analysis of genetically modified

industrial crop profitability), then the success of such crops can affect the prosperity

of the industries they replace, such as chemical manufacturing. Although the over-

all benefit will be great, as renewable resources replace potentially limited resour-

ces, industries and workers may be displaced.

Several developing nations produce specialty crops that meet current industrial

needs, such as castor oil, rubber, and lauric acid, all of which are products from

Southeast Asia. If these products were to be replaced by transgenic crops grown

in temperate regions, economic displacement of the less wealthy countries could

occur. However, because transgenic products may also be produced more cheaply

in these countries, they should ultimately benefit from the same new technologies.

Where new uses, such as biodiesel fuel and fuel additives produced from castor oil

and laurate (45), will greatly expand demand, economic disruptions may be offset.

Many countries already have research programs in transgenic crop technology. The

impact of transgenic technologies in industrial agriculture on the world economy

remains to be seen.

Transgenic technology remains a powerful tool for developing a broad range of

useful food and industrial oils. To date, attempts to use this technology have been

limited to crop input traits, but in the long term, novel crops with altered output

traits will fill important niches in the food supply and will help to shift the petro-

leum economy to renewable resources.

REFERENCES

1. C. James. Global Status of Commercialized Transgenic Crops: 2003 Executive Summary

International Service for the Acquisition of Agri-Biotech Applications. (2004). Available:

http://www.isaaa.org.

2. D. L. Nida, S. Patzer, P. Harvey, R. Stipanovic, R. Wood and R. L. Fuchs, J. Agric. Food

Chem., 44, 19671974 (1996).

3. R. S. Sidhu, B. G. Hammond, R. L. Fuchs, J. N. Mutz, L. R. Holden, B. George, and

T. Olson, J. Agric. Food Chem., 48, 23052312 (2000).

172 TRANSGENIC OILS

4. S. R. Padgette, N. B. Taylor, D. L. Nida, M. R. Bailey, J. MacDonald, L. R. Holden, and

R. L. Fuchs, J. Nutr., 126, 702716 (1995).

5. W. E. Neff, T. L. Mounts, and W. M. Rinsch, Lebensrm. Wiss. u.-Technol., 30, 793799

(1997).

6. P. F. Knowles, in G. Robbelen, R. K. Downey and A. Ashri, eds., Oil Crops of the World,

McGraw-Hill, New York, 1985, pp. 260282.

7. S. Henikoff and L. Comai, Annu Rev. Plant Biol., 54, 375401 (2003).

8. J. L. Harwood, Biochim. Biophys. Acta, 1301, 756 (1996).

9. T. Voelker and A. J. Kinney, Annu. Rev. Plant Physiol. Mol. Biol., 52, 3353361 (2001).

10. J. Browse and C. Somerville, Annu. Plant Physiol. Plant Mol. Biol., 42, 467506

(1991).

11. J. J. Thelen and J. B. Ohlrogge, Metabol. Eng., 4, 1221 (2002).

12. J. Jaworski and E. B. Cahoon, Curr. Opin. Plant Biol., 6, 178184 (2003).

13. R. K. Downey, Can. J. Plant Sci., 44, 499504 (1964).

14. B. R. Stefansson, F. W. Hougen, and R. K. Downey, Can. J. Plant Sci., 41, 218219 (1961).

15. L. Velasco and J. M. Fernandez-Martinez, J. Crop Prod., 5, 309344 (2002).

16. S. Lacombe and A. Berville, Mol. Breeding, 8, 129137 (2001).

17. S. Erhan and S. Asadauskas, Ind. Crops Prod., 11, 277282 (2000).

18. A. G. Green, Can. J. Plant Sci., 66, 499503 (1986).

19. A. Green, Aus. New Crops Newsl. 3(1). (1995). Available: http://www.newcrops.uq.edu.

au/newslett/ncnl3-92.htm.

20. T. L. Mounts, K. Warner, G. R. List, W. E. Neff and R. F. Wilson, JAOCS, 71, 495499

(1994).

21. Monsanto Press Release. (2004). Available: http://www.monsanto.com/monsanto/layout/

media/04/0901-04.asp.

22. T. A. Voelker, A. C. Worrell, L. Anderson, J. Bleibaum, C. Fan, D. J. Hawkins, S. E. Radke,

and H. M. Davies, Science, 257, 7274 (1992).

23. T. A. Voelker, T. R. Hayes, A. M. Cranmer, J. C. Turner, and H. M. Davies, Plant J., 9,

229241 (1996).

24. D. S. Knutzon, T. R. Hayes, A. Wyrick, H. Xiong, H. M. Davies, and T. A. Voelker, Plant

Physiol., 120, 739746 (1999).

25. A. E. McCoy, M. Pippin, and R. D. MacNeill IV. Laurate Canola Variety Trial. (1998).

Available: http://www.griffin.peachnet.edu/swvt/1998/rp981rat.htm.

26. A. J. Kinney, in J. P. Williams, M. U. Khan and N. W. Lem, eds., Physiology, Biochemistry

and Molecular Biology of Plant Lipids, Kluwer Academic, Dordrecht, the Netherlands,

1997, pp. 298300.

27. R. Frerichs. (2003). Available: http://web.aces.uiuc.edu/value/factsheets/soy/fact-oleic-

soy.htm.

28. A. Rodriguez, D. Raederstorff, P. Sarda, C. Lauret, F. Mendy, and B. Descomps, Eur. J.

Clin. Nutr., 57, 727734 (2003).

29. O. V. Sayanova and J. A. Napier, Phytochemistry, 65, 147158 (2004).

30. B. Qi, T. Fraser, S. Mugford, G. Dobson, O. Sayanova, J. Butler, J. A. Napier, A. K.

Stobart, and C. M. Lazarus, Nature Biotech., 22, 739745 (2004).

31. A. G. Green, Nature Biotech., 22, 680682 (2004).

REFERENCES 173

32. S. Sato, A. Xing, X. Ye, B. Schweiger, A. Kinney, G. Graef, and T. Clemente, Crop Sci.,

44, 646652 (2004).

33. D. L. Auld, R. D. Rolfe, and T. A. McKeon,. J. New Seeds, 3, 6169 (2001).

34. F. J. van de Loo, P. Broun, S. Turner, and C. Somerville, Proc. Nat. Acad. Sci. (USA), 92,

67436747 (1995).

35. P. Broun and C. Somerville, Plant Physiol., 113, 933942 (1997).

36. X. He, C. Turner, G. Q. Chen, J. T. Lin, and McKeon, Lipids, 39, 311318 (2004).

37. T. A. McKeon and J. T. Lin, in T. M. Kuo and H. W. Gardner, eds., Lipid Biotechnology,

Marcel Dekker, New York, 2001, pp. 129139.

38. M. Smith, H. Moon, G. Chowrira, and L. Kunst, Planta, 217, 507516 (2003).

39. T. A. McKeon and G. Q. Chen, U. S. Patent 6,620,986 B1, 2003.

40. E. B. Cahoon and J. B. Ohlrogge, Plant Physiol., 104, 827837 (1994).

41. M. C. Suh, D. J. Schultz, and J. B. Ohlrogge, Planta, 215, 584595 (2002).

42. H. Drexler, P. Spiekermann, A. Meyer, F. Domergue, T. Zank, P. Sperling, A. Abbadi, and

E. Heinz, J. Plant Physiol., 160, 779802 (2003).

43. F. Cellini, A. Chesson, I. Colquhoun, A. Constable, H. V. Davies, K. H. Engel, A. M. R.

Gatehouse, S. Karenlampi, E. J. Kok, J.-J. Leguay, S. Lehesranta, H. P. J. M. Noteborn,

J. Pedersen, and M. Smith, Food Chem. Toxicol., 42, 10891125 (2004).

44. J. Thomson, Trends Food Sci. Technol., 14, 210228 (2003).

45. T. A. McKeon, Trends Food Sci. Technol., 14, 229241 (2003).

46. J. Huang, S. Rozelle, C. Pray, and Q. Wang, Science, 295, 674677 (2002).

47. H. A. Kuiper, G. A. Kleter, H. P. J. M. Noteborn, and E. J. Kok, Plant J., 27, 503528

(2001).

48. H. Daniell, S. J. Streatfield, and K. Wycoff, Trends Plant Sci., 6, 219226 (2001).

49. P. J. Dale, B. Clarke, and E. M. G. Fonte, Nature Biotech., 20, 567574 (2002).

50. B. D. Oomah, J. Sci. Food Agric., 81, 883888 (2001).

51. A. Konig, A. Cockburn, R. W. R. Crevel, E. Debruyne, R. Grafstroem, U. Hammerling,

I. Kimber, I. Knudsen, H. A. Kuiper, A. A. C. M. Peijnenburg, A. H. Penninks, M. Poulsen,

M. Schauzu, and J. M. Wal, Food Chem. Toxicol., 42, 10471088 (2004).

52. S. Smyth, G. G. Khachatourians, and P. W. Phillips, Nature Biotech., 20, 537541 (2002).

53. G. van den Eede, H. Aarts, H.-J. Buhk, G. Corthier, H. J. Flint, W. Hammes, B. Jacobsen,

T. Midtvedt, J. van der Vossen, A. von Wright, W. Wackernagel, and A. Wilcks, Food

Chem. Toxicol., 42, 11271156 (2004).

54. B. Hitz, Curr. Opin. Plant Biol., 2, 135138 (1999).

174 TRANSGENIC OILS

Front MatterTable of ContentsVolume 1. Edible Oil and Fat Products: Chemistry, Properties, and Health EffectsVolume 2. Edible Oil and Fat Products: Edible OilsVolume 3. Edible Oil and Fat Products: Specialty Oils and Oil Products3.1 Conjugated Linoleic Acid Oils3.2 Diacylglycerols3.3 Citrus Oils and Essences3.4 Gamma Linolenic Acid Oils3.5 Oils from Microorganisms3.6 Transgenic Oils3.6.1 Introduction3.6.2 Technology for Altering Fatty Acid Composition3.6.3 Canola from Traditional Breeding of Oilseed Crops3.6.4 High-oleic Sunflower from Mutagenesis of Oilseed Crops3.6.5 Applications of High-oleate Oils3.6.6 Altered Polyunsaturate Content through Mutagenesis3.6.7 Improved Oil Composition of a Transgenic Soybean3.6.8 Improved Industrial Use from Genetically Engineering Oilseed Crops3.6.9 Future Directions for Transgenic Oilseeds3.6.10 Potential New Oils for Food, Feed, and Industruial Use3.6.11 Issues Related to Transgenic OilseedsReferences

3.7 Tree Nut Oils3.8 Germ Oils from Different Sources3.9 Oils from Herbs, Spices, and Fruit Seeds3.10 Marine Mammal Oils3.11 Fish Oils3.12 Minor Components of Fats and Oils3.13 Lecithins3.14 Lipid Emulsions3.15 Dietary Fat Substitutes3.16 Structural Effects on Absorption, Metabolism, and Health Effects of Lipids3.17 Modification of Fats and Oils via Chemical and Enzymatic Methods3.18 Novel Separation Techniques for Isolation and Purification of Fatty Acids and Oil by-products

Volume 4. Edible Oil and Fat Products: Products and ApplicationsVolume 5. Edible Oil and Fat Products: Processing TechnologiesVolume 6. Industrial and Nonedible Products from Oils and FatsIndex