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BREEDING FOR DESIGNER OIL SEED CROPS

R.Rameshkumar and S.P.Singh


Division of Genetics and Plant Breeding
National Botanical Research Institute, Lucknow.

1) INTRODUCTION:
Oil seed crops are important sources of energy for human consumption and also
provide raw material for a wide range of industrial products. Earlier animal products
such as lard, beef tallow, butter characterized by saturated fats were major sources of
fat supply. Since a number of nutritional and medical studies indicated strong
relationship between high levels of saturated fats and cholesterol, and incidence of
Coronary Heart Disease (CHD), there was a major shift in consumption from animal
fats to vegetable oils. The Institute of Shortenings and Edible Oils (ISEO) has reported
that there was a shift from two-thirds of the visible fat as animal fat to one-third in 1966
and to 95% of the visible fat as vegetable origin in 1992.
Since vegetable oils are predominantly composed of unsaturated fatty acids they
lack the characteristic stability, texture and flavour, imparted by saturated fats. So to
provide the same texture, flavour and stability, oils were partially hydrogenated, which
created a new issue: trans fatty acids. Elevated levels of trans fatty acids led to risk of
CHD. So genetic modification of oil seed crops through breeding and genetic
engineering, to produce oils with increased stability and flavour, without affecting their
characteristic nutritional value is considered as the best alternative.
The vegetable oils most commonly used in the trend towards “healthy” are
soybean, canola, sunflower, cottonseed, safflower, olive and peanut. About 54% of the
world oilseed production is soybean, 12% cotton seed, 11% rapeseed, 10% peanut, 9%
sunflower and 4% others. All these crops except rapeseed falls into oleic-linoleic group
(Swern, 1964). However in the development of canola from rapeseed , the erucic acid
was replaced by oleic placing canola within the oleic-linoleic group (Ronald, 1996).
Oleic and linoleic acids accounts for 70% of the world fatty acid supply (Khanna and
Singh, 1991) and known to lower blood cholesterol level preventing heart attacks, but
in terms of stability former dominates latter. Medium Chain (saturated) Fatty Acids
synthesized by wild genus Cuphea, that provides quick energy without raising
cholesterol level and α- linolenic acid produced by linseed, that imparts immunity in
human beings, are gaining importance in recent years. Also available are synthetically
structured fats, which contributes fewer calories and less fat.
The fats and oils industry has reacted to a rapidly changing nutritional
awareness of the effect of fats in the diet. Now consumers have moved from buying oils
“off the rack” to an industry which now offers “tailored oils” which have been coined
the term “designer fats and oils”.
The chapter covers in brief the chemistry, uses and biosynthesis of fats to
provide an understanding on the need for designing oil and basic principles involved in
it. Also various approaches for designing oil have been discussed with detailed
elaboration of crop wise breeding and genetic engineering approaches.

2) THE CHEMISTRY OF FATS:


Fats or lipids are esters of two molecules i.e. glycerol and fatty acids. Glycerol
is a sweet viscous colourless liquid. Structurally it has two primary and one secondary
alcohols. Depending upon the number of fatty acids attached to glycerol, mono- (1 fatty
acid), di- (2 fatty acids), and tri-glycerides (3 fatty acids) are formed. Most fat in our
bodies and in the food we eat is in the form of triglycerides (TGs). The word “fat” is
ordinarily used to refer to TGs that are solid or, more correctly semi solid at ordinary
temperatures, whereas the word “oil” is used for TGs that are liquid under the same
conditions.
2.1) Fatty acid:
Fatty acid is a hydrocarbon chain with a methyl group (CH3) at one end and
carboxyl group (COOH) at the other end. The three fatty acids in a TG may all be alike
or may be different. The composition of fatty acids in the fat or oil determines their
physical properties and nutritional qualities. So designing oil or fats involves
manipulation of fatty acids to achieve the desired quality suited for specific purpose.
2.2) Classification of Fatty Acids:
Generally fatty acids are classified into Saturated Fatty Acids (SFA) and
Unsaturated Fatty Acids (USFA) based on bonding between carbon atoms (Table: 1). In
SFA the carbons are connected to each other only by single bond. They are not a single
family of fats, but comprises three sub groups depending on chain length (Kabara,
2000):
a) Short Chain Fatty Acids (SCFA) – C2:0 to C6:0.
b) Medium Chain Fatty Acids (MCFA) – C8:0 to C12:0.
c) Long Chain Fatty Acids (LCFA) – C14:0 to C24:0.

Table: 1 Classification and Sources of Some Important Fatty Acids.


Common Abbreviation Fatty Major Sources
Name Acid
Famil
y
Saturated Fatty Acids
Caprylic C8:0 Cuphea
Capric C10:0 Cuphea
Lauric C12:0 Cuphea, Coconut, Oilpalm, Californian Bay
Myristic C14:0 Cuphea, Coconut
Palmitic C16:0 Milk, Animal fat, Cuphea, Oil palm
Stearic C18:0 Cuphea, Milk, Animal fat
Arachidic C20:0 Ground nut
Behenic C22:0 Ground nut
Unsaturated Fatty Acids
Oleic C18:1 n-9 * Sunflower, Safflower, Sesame, Olive, Oil
Linoleic C18:2 n-6
palm, Corn, Groundnut, Cottonseed.
α-Linolenic C18:3 n-3 Linseed, Marine oils
γ-Linolenic C18:3 n-6 Borage, Evening Primrose, Black Currant
Erucic C22:1 n-9 Brassica
Arachidonic C20:4 n-6 Groundnut
* Oleic and linoleic acids share common sources.
USFA have double bonds between carbon atoms. They are called unsaturated
because they could hold more hydrogen atoms than they do. Based on number of
double bonds they are grouped in to two:
a) Monounsaturated Fatty Acid (MUFA): They have one double bond in
the form of two carbon atoms double bonded to each other and therefore
lack two hydrogen atoms.
b) Polyunsaturated Fatty Acid (PUFA): They have two or more pairs of
double bond and therefore lack four or more hydrogen atoms.
Fig: 1 - Omega Fatty Acids.

One system of naming USFA is to indicate the position of first double bond
counting from the methyl end. The terminal carbon atom is called omega carbon
atom. Fatty acids having their first double bond after carbon 3 (counting from and
including omega carbon) are called Omega 3 Fatty Acids (α-linolenic acid). Likewise
fatty acids with first double bond at sixth carbon atom are called Omega 6 Fatty
Acids (linoleic acid). These omega fatty acids (Fig: 1) are popularly called as
“immunonutrients”.

3) Nutritional and Non-nutritional Functions of Fatty Acids:


About 90% of world’s oil production is used for edible purposes and remaining
is used for industrial purposes. In industries it is used in manufacture of pharmaceutical
products, soaps and detergents, paints, coatings and resins, chemicals, technical
products and biofuels. The oil is commonly used in human consumption for cooking,
baking and frying purposes, as well as in products such as margarine and salad oils.
3.1) Caloric and related functions:
Fats are important source of energy in the diets, furnishing about 9 calories of
energy per gram, as compared with about 4 calories each furnished by proteins and
carbohydrates. They also have the highest caloric density of any foodstuff, since they
are consumed in a relatively water-free condition whereas proteins and carbohydrates
often occur with large quantities of water. The daily recommended intake of dietary fat
is 15-35% of total calories required (Anonymous, 1994).
3.2) Non caloric functions:
1) They act as carriers for important fat soluble vitamins A, D, E and K.
2) They are needed for the conversion of carotene to Vitamin A, for mineral
absorption and host of other processes.
3) They are important constituents of cells and cell membranes and serve as
precursors to certain harmones and acts as metabolic regulators of vital processes.
4) Humans can’t synthesize two of the fatty acids essential for health- linoleic
acid (Omega 6 fatty acid) and α- linolenic acid (omega 3 fatty acid) which are to be
obtained from external source. The dietary lipids are the only source to mitigate the
essential fatty acids deficiency. Omega 3 and omega 6 fatty acids help lower
cholesterol and blood TGs and prevent clot in arteries, which may result in strokes,
heart attacks and thrombones. It can also protect body against high blood pressure,
inflammation, water retention, sticky platelets and lowered immune function. They
are also especially important for normal fetal and infant growth and development, in
particular for brain development and visual activity (Anonymous, 1994).
Since linoleic acid and α- linolenic acid compete for the same enzymes and
have different biological roles, the balance between them in the diet is of considerable
importance. The recommended intake in the diet is, the ratio of linoleic and α-
linolenic acid should be between 5:1 and 10:1. (Anonymous, 1994)
5) Medium chain triglycerides (MCT) are a class of dietary lipids containing
fatty acids ranging from C6 to C12. They are metabolized differently than Long Chain
triglcerides (LCT). LCTs are hydrolyzed, then re-esterified to triglycerides, and then
imported into chylomicrons, which enter the lymphatic system. MCTs bypass the
lymphatic system. They are hydrolyzed to MCFA, which are transported via the
portal vein directly to liver, where they are oxidized for energy and are not likely to
be stored in adipose tissue (Ronald, 1996). MCTs provide quick energy similar to
glucose but with twice the caloric value (8.3 calories/ gram). They can benefit weight
conscious individuals because they supply energy while not contributing to fatty acid
deposits. Also research has indicated that heart disease, breast cancer and colon
cancer are reduced when MCTs are used as primary dietary lipid source (Babayan,
1981; Bach and Babayan, 1982; Babayan, 1987).
6) They have impact on the functioning of the cardiovascular system. A wealth
of nutritional and medical studies indicated strong relationship between dietary fats,
cholesterol and incidence of CHD. Increased serum concentration of low-density
lipoprotein (LDL) cholesterol is a major risk factor for cardio vascular disease while
the concentration of serum high-density lipoprotein (HDL) cholesterol is inversely
related to incidence of CHD. Also high serum TGs concentration increases the risk of
coronary heart disease (Kratz et al. 2002)
The SFAs – lauric, myristic and palmitic acids and trans-unsaturated fatty acids
are cholesterol raising fatty acids. They increase LDL/HDL ratio leading to incidence
of CHD. Another major SFA, stearic acid produces very little change in serum
cholesterol concentration and is apparently neutral in its effect on cholesterol.
The MUFA (oleic acid) and PUFA (linoleic and α- linolenic acid) reduce the
LDL/HDL ratio in serum, eliminating the risk of CHD. The amount of SFA is
positively and amount of MUFA and PUFA are inversely associated with the risk of
cardio vascular diseases. So SFA should make up less than 10% of calories, PUFA less
than 10% of calories and MUFA 10-15% of calories in our diet (Ronald, 1996).
3.3) Non-Nutritional Functions of Edible Fats and oils:
Major use of edible oil is in cooking where it is an efficient heat transfer
medium and imparts flavour and palatability to foods. All cooking oils are vegetable
products. Large quantities of fats are used in the production of baked goods. They are
also used in products such as margarines and shortenings. The non-nutritional uses of
edible fats and oils are influenced by various physical properties as follows:
3.3.1) Shelf Life:
The double bonds in the USFA are more prone to oxidation and
polymerization, which leads to rancidity and results in spoilage of edible fats and oil.
(Reimenschneider, 1955). Oxidation of linoleate and linolenate is approximately 10
and 25 times higher, respectively than that of oleic acid (Carlson, 1995; Horrobin,
1995; Lands, 1997). In contrast due to lack of double bonds the SFA have more
oxidative stability and thus increased shelf- life.
3.3.2) Melting Point:
The double bonds are rigid and introduce a kink in the molecule. This
prevents the fatty acids from packing close together and as a result unsaturated fatty
acids have a lower melting point than do saturated fats. Also melting point is
influenced by chain length, longer chain length tends to promote a higher melting
point than shorter ones. Generally plant fats tend to be unsaturated, therefore liquid
and animal fats tend to be saturated, therefore solid at room temperature.
3.3.3) Crystal Formation:
Types of crystals formed by fats are important in maragarine, which
needs crystalline structure to maintain semi-solid consistency, and in shortenings,
which needs creamier structure. The length of fatty acids and their position on the
glycerol backbone determine the type of crystals formed.
3.3.4) Stability while cooking:
Due to high temperature while cooking and moisture present in foods,
the fatty acids undergo hydrolysis which results in a poor quality oil with a reduced
smoke point, darkened colour and altered flavour. Oils rich in polyunsaturates have
lower stability and can’t be reused. In homes where oils are normally used for much
shorter periods of time and discarded after being used once or twice, stability
problems play a lesser role. It is more problem in catering operations where heating
is intermittent and oils are used for long period.

4) SYNTHESIS OF FATTY ACIDS:


Fatty acid biosynthesis in plants is intracellularly compartmentalized and
regulated in a tissue and development specific manner. Denovo fatty acid
biosynthesis occurs predominantly in plastids. The free fatty acids produced in
plastids travel to endoplasmic reticulum to get synthesized to TGs. Acetyl Co-A
(two carbon compound), the precursor of fatty acid biosynthesis is converted to
malonyl Co-A (three carbon compound) catalyzed by acetyl-CoA carboxylase. The
acetyl Co-A and malonyl Co-A undergoes condensation catalyzed by condensing
enzyme, fatty acid synthase to form a five carbon compound. This compound
undergoes a cycle of reduction, dehydration and reduction reactions to form
saturated acyl groups having four carbons. This saturated acyl groups becomes the
substrate in another condensation with malonyl group and enters into second cycle
of reactions. With each passage through the cycle, the fatty acid chain is extended
by two carbon atoms. Since fatty acids are synthesized from fragments containing
two carbons, the number of carbon atoms in the chain is almost always an even
number.
When the growing acyl-chain reaches the length of C18:0 –ACP, stearyol-ACP
desaturase introduces a double bond converting C18:0 –ACP (saturated) to C18:1 –ACP
(unsaturated). The growing acyl groups are covalently attached via a thioester bond
to the acyl carrier protein (ACP), which is cleaved by acyl-ACP thioesterase to
release free fatty acids at the level of C16:0 or C18:0 or C18:1. In MCFAs producing
plants such as Cuphea TE cleaves the bonds in medium chain acyl-ACPs making
them free. The free fatty acids then cross the plastid envelope and be re-esterified to
Acyl-CoA moeties and eventually be utilized as substrate for the synthesis of
glycerol lipids including TGs.

5) Designing Oil:
Oils or fats can be designed by the following three methods:
1) Chemical and physical processes.
2) Breeding and genetic engineering.
3) Synthetically structured fats.
5.1) Chemical and Physical Processes (Hegenbart, 1991):
5.1.1) Blending of Oils:
Different oil types can be blended to achieve functional changes but the possible
modification is limited and using a single type of oil may be desirable in certain
products.
5.1.2) Hydrogenation:
This method is used to convert liquid oils to solid fats, which are used in
shortenings and margarines. It is a chemical process of adding hydrogen atoms to fatty
acid chains by reacting oil with hydrogen in the presence of a catalyst (nickel). This
results in saturation and conversion of double bonds from cis to trans configuration
(trans fatty acid). Both these effects straighten out the molecules so they can lie closer
together and become solid rather than liquid.
5.1.3) Fractionation:
This is the process by which oil may be separated into its different TG portions
based on their individual melting points. The oil is held at a predetermined temperature
and the solids present are filtered out or are separated in some way. The synthetic
MCTs are manufactured from caprylic and capric acids obtained by fractionating
coconut and palm-kernel oils.
5.1.4) Rearranging:
This process modifies oil properties by exchanging fatty acids on TG molecules.
The procedure is called interesterification if the fatty acids simply switch positions on
the molecule and transterification if the fatty acids actually change TGs. This is often
done in fats designed for confections or margarines. Interesterification also can be
directed in such a way as to produce a solid fat from oil without hydrogenation.
5.2) Breeding and genetic engineering:
The usage of oil depends on its fatty acid profile. The fatty acid profile in turn depends
upon the activity of fatty acid biosynthetic enzymes, which is ultimately decided by the
genes encoding these enzymes. So breeding for an desirable oil composition involves
concentration of these genes to enhance the level of require fatty acid in the cultivar.
Conventional breeding approaches utilize either the natural genetic variation or
generated variation (mutation) to develop cultivars possessing novel traits through
selection or hybridization. Molecular breeding approaches hasten the process of
conventional breeding and reduce the time needed to develop new varieties. Through
molecular breeding approaches, creation of novel genetic variation (somaclonal
variation), breaking sexual crossability barriers (embryo rescue, protoplast fusion),
rapid generation of homozygous lines (doubled haploids) and precise selection for
desirable traits (molecular markers) can be achieved. Thus classical and molecular
breeding approaches in combination offers a wide spectrum of methods for efficiently
designing oil with desired fatty acid composition. However further adjustments will not
be realized satisfactorily without the assistance of genetic engineering (Friedt and
Wilfried). Combination of these methods have led to drastic modification of fatty acid
profile of various oilseed crops (Table: 2) which has been discussed crop wise.

Table: 2 - Fatty acid profiles of traditional and modified oilseed crops.


Oil type/seed Origin/method 12:0 14:0 16:0 18:0 18:1 18:2 18:3 20:1 22:1 Others
variety n-9 n-6 n-3 n-9 n-9
Rapeseed
High-erucic Traditional - - 3 1 11 12 9 8 52 4
acid rapeseed
Double-low / Spontaneous - - 4 2 60 21 10 1 1 1
Canola mutant
Low-linolenic Mutagenesis - - 4 2 61 28 3 1 - 1
Canola
Laurate Genetic 37 4 3 1 33 12 7 - - 3
Canola engineering
High myristate Genetic - 18 23 2 34 15 4 - - 4
/palmitate engineering
High-oleic Mutagenesis / 4 1 84 5 3 1 - 2
Canola transgenic
Soybean
Conventional Traditional - - 11 4 23 54 8 - - -
Low Mutagenesis - - 10 5 23 60 2 - - -
linolenate
High palmitate Mutagenesis - - 17 3 17 55 8 - - -
High stearate Mutagenesis - - 8 28 20 35 7 - - 2
High oleate Genetic - - 7 4 85 1 2 - - 1
engineering
Sunflower
Conventional Traditional - - 6 4 18.5 71 0.5 - - -
High oleic Mutagenesis / - - 4 4 78.5 11.5 - - - -
transgenic
Linseed
Conventional Traditional - - 6.5 4.5 19.5 16.5 53.0 - - -
Low linolenic Mutagenesis - - 6.0 4 16 71.5 2.0 - - -

5.2.1) Rapeseed and Mustard


Rapeseed and mustard are important brassica oil crops, belonging to family
Crucifereae and accounting for 13.9% of world’s vegetable oil production (Mc Calla
and Carter, 1991) supplying more than 12.5% of global edible oil (Röbbelen, 1987).
Three most important species that accounts for 95% of world’s rapeseed and mustard
oil production are B. napus, B. campestris and B. juncea (Khanna and Singh, 1991). B.
campestris was previously the most dominant species but later replaced by B. napus
due to its higher yield potential and improved oil and meal quality. B. juncea (Indian or
Brown Mustard) and B. campestris var sarson (Sarson) are the major sources of edible
oil in India.
The brassica seed oil is characterized by presence of LCFA: eicosenoic and
erucic acids. The oil was extensively used for edible purpose until the myocardial
damage in rats due to erucic acid was demonstrated. The feeding experiments on
animals during 1950’s revealed that reducing erucic acid to less than 2% could
significantly enhance nutritional value of oil. This led to the development of low erucic
acid variety now known as canola. In contrast high erucic acid cultivars are regaining
interest in industrial contexts. Also brassica is bred for low linolenic and high oleic
types to increase their nutritive value and shelf life.
Extensive studies on nature and number of genes controlling erucic acid
synthesis has been made by several workers. In B. campestris high erucic acid content
is controlled by a single (Dorrel and Downey, 1964) partially dominant gene (Davik
and Heneen, 1996). Harvey and Downey (1964) reported that erucic acid inheritance in
B. napus is controlled by four alleles (digenic) acting in additive manner. Krzymanski
and Downey (1969) reported a fifth allele and designated five alleles as e, Ea, Eb,Ec and
Ed contributing <1%, 10%, 15%, 30% and 3.5% erucic acid respectively. The e, Eb, and
Ec alleles were identified for B. campestris and e, Ea and Ed alleles were identified for
B. napus. Ea and Ed are truly allelic and present on B. oleraceae genome. In B. juncea
two erucic acid loci, one each from B. campestris and B. nigra genomes has been
reported (Krik and Oram, 1981) and the four alleles acting in a additive manner were
designated as E0, E1, E2 and E3, each contributing 0%, 12%, 20% and 20% erucic acid
respectively.
B. juncea (AABB) and B. napus (AACC) are digenomic natural amphidiploids
evolved by crossing among diploid species with AA, BB and CC genomes,
incorporated by B. campestris, B. nigra and B. oleraceae respectivelty. In an effort to
assign erucic acid genes to individual genomes Liu and Guan (1997) studied the
interspecific progenies between B. juncea and B. napus which revealed that A and B
genomes each carry a pair of genes controlling seed erucic acid content. Similarly
studies on fatty acid profile of resynthesized B.napus from their progenitors B.
campestris and B. oleraceae indicated equal contribution of AA and CC genomes
(Rahman, 2002).
A linkage map of Restriction Fragment Length Polymorphism (RFLP) was
constructed for B.napus and genes controlling erucic acid synthesis were mapped
(Teutonic and Osborn, 1994). Ecke et al (1995) mapped erucic acid genes to two
linkage groups on the RFLP map and also mapped three Quantitative Trait Loci (QTL)
for seed oil content on three different linkage groups. Two of the QTLs for oil content
showed a close association in location of the two erucic acid genes, indicating a direct
effect of the erucic acid genes on the oil content.
Genetic studies using “Stellar” a low linolenic acid cultivar demonstrated that
two major genes L1 and L2 control low linolenic acid content. A desaturase gene fad3
is linked to L1 (Jourdren et al. 1996) and a second gene is linked to the second locus L2
(Barret et al. 1999). Molecular markers such as RAPD (Rajcan et al.1999) and SCAR
(Ho et al. 1999) for linolenic acid are available.
Plant breeding and genetic engineering approaches have resulted in drastic
modification of fatty acid composition of brassica seed oil. Brassica genotypes and
cultivars with high/low saturated fatty acid oil, low linolenic oil (<3.5%), mid-oleic oil
(65-75%), high oleic oil (>75%), zero or low erucic acid oil (<2%) and high erucic acid
oil (60-80%) are available. Designing of brassica oil began with the identification of
low erucic acid genotypes in brassica germplasms by plant breeders. These genotypes
were used in hybridization programmes either to transfer low erucic acid trait to
existing brassica cultivars or to develop new cultivars with low or zero erucic acid
content. Now several cultivars with zero erucic acid (Hanna, Kizakinonatane,
Asukanonatane, etc.,) and low erucic acid (Cyclone, Ericka, Sunrise, etc.,) are
available. Conversely high erucic acid cultivars (Millenni UM 03, Millenni UM 02,
Neptune, Castor, Sterling, Venus, etc.,) are also available.
The low linolenic acid trait was produced by seed mutagenesis of the B. napus
cultivar Oro, which led to isolation of a mutation line M11 with an altered C18:2/
C18:3 ratio (Rakow, 1993). Stellar a B. napus cultivar with low linolenic acid (3%) was
developed in a backcrossing programme of M11 with cultivar Regent (Scarth et al.
1988).
Doubled haploids (DH) obtained through microspore culture have been
extensively used for fatty acid modification in brassica. Microspore derived embryoids
of B. napus at cotyledonary stage contains large amount of storage lipids, equal or
similair in fatty acid composition to those found in seeds of homozygous donor plants.
At this stage, one cotyledon can be dissected for determination of fatty acid
composition and remaining part of the embryo can be cultured to give a regenerated
plant. The correlation between cotyledons and the seeds produced by regenerated plants
for erucic acid content (Albrecht, et al. 1995) and oleic acid content (Möllers et al.
2000) was found to be significant. So selection for desired fatty acid composition can
be made at cotyledonary stage itself. Cegielska et al. (1999) developed 9 DH lines with
high erucic acid content ranging from 56.8 – 60.4%. Comparison of doubled haploid
breeding with conventional breeding revealed that best conventionally produced line
gave an erucic acid yield of only 10.7 dt/ha in comparison with 11.9 dt/ha of the first
generation DH line (Listl, 1992). DH B. rapa lines with reduced saturated fat levels
have been developed through microspore mutagenesis and this variation can be
introduced in to B. napus, to produce low saturate germplasm for further cultivar
development (Scarth and McVetty).
Protoplast fusion of zero erucic acid cultivar Hanna with Lesquerella fendleri
followed by crossing with High Erucic Acid Rape line (Schröder et al. 1999) and
protoplast fusion of B. oleraceae var botrytis and B. rapa var oleifera to resynthesize B.
napus (Heath and Earle, 1995) have resulted in high erucic acid types.
Oleate that is transported from ACP track (chloroplast) to the CoA track
(endoplasmic reticulum) by catalytic action of β-Ketoacyl-ACP Synthases (KAS) is at
the branch point in the erucic acid metabolism. Either oleate can be elongated to erucic
acid by fatty acid elongase or enter the phospholipid by lysophosphatidyl ethanolamine
acyltransferase (LPAAT) which acylates the sn-2 hydroxyl group of lysophosphatidyl
ethanolamine (LPE) to form phosphatidylethanolamine (PE), a precursor of
triacylglycerol. The oleate at sn-2 position of PE is then desaturated to linoleate and
linolenate by desaturase (Fig: 2). Manipulating the levels of the enzymes KAS and
LPAAT can control the accumulation of erucic acid in seed lipids.
Over expression of LPAAT in transgenic plants reduce the availability of oleate
for the formation of erucic acid and there by redirecting the pathway towards PUFA
synthesis. Alternatively erucic acid content can be increased by reducing the levels of
LPAAT through antisense expression and there by increasing the availability of oleate
for elongation reaction (Rajashekaran). Genes encoding LPAAT has been isolated from
yeast (SLC1-1), brassica and groundnut systems.

Fig: 2 - Biosynthesis of oleate and its desaturation .


Elongase
Acetate → → → 16:0-ACP → 18:0 -ACP → 18:1-ACP
↓ Thioesterase
Plastid stroma (ACP track) Oleic acid

Acyl-CoA synthetase
Oleoyl-CoA

LPE → PE-18:1 → PE-18:2 → 18:3


Acyltransferase δ -12 desaturase δ -15 desturase
Endoplasmic Reticulum
Adapted from Rajashekaran.

KAS and LPAAT are the key enzymes in erucic acid synthesis of which the
former is characterized well. Earlier to brassica the gene (FAE 1) encoding KAS has
been isolated in Arabdiopsis. From B. napus Barret et al (1998) isolated two sequences,
CE7 and CE8 homologus to Arabdiopsis FAE 1 gene and found tight linkage of one of
the FAE 1 genes to E1 locus. Fourmann et al. (1998) designed PCR primers
corresponding to FAE 1 gene and amplified two B. napus genes Bn-FAE 1 and Bn-
FAE 2 corresponding to its parental species B. rapa (B. campestris) and B. oleraceae
respectively. Also they reported co-segregation of these genes with E1 and E2 loci
respectively. These genes had 98%, 86%, 84% and 58% nucleotide similarity with FAE
1 genes from B. napus, B. juncea, Arabdiopsis thaliana and Simmondsia chinensis
respectively. The FAE 1 genes from low and high erucic acid lines differ at 13
positions at amino acid sequence level, mainly localized in the central part of protein
sequence (Das et al. 2002). This altered amino acid sequence in variant β-Ketoacyl-
ACP Synthase proteins due to mutated FAE 1 gene leading to lack of acyl-CoA
elongation activity is responsible for low level of erucic acid in brassica seed oil.
Since erucic acid alleles are additive in nature, theoretical expectation of erucic
acid yield through conventional breeding is only 66-67%. This is because erucic acid
occupies only sn-1 and sn-3 positions of the glycerol molecule (Appelqvist, 1971).
However through genetic engineering it might be possible to increase the levels of
erucic acid up to 80% by incorporating erucic acid in the sn-2 position also (Katavic et
al. 2000). Lassner et al. (1995) through transgenic experiments demonstrated that erucic
acid could be positioned in sn-2 indicating the feasibility of altering stereochemical
composition of brassica seed oils. Expression of Arabdiopsis FAE-1 gene and yeast
SLC-1 gene in high erucic acid cultivar Hero resulted in increased proportion of erucic
acid in transgenic Hero lines (Katavic et al. 2001; Taylor et al. 2002).
High oleic canola (> 86%) 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). Cargill’s Clear Valley 75 is a high oleic (75%) cultivar (Scarth and
McVetty). High laurate canola is the world’s first transgenic oilseed crop in commercial
production. The high laurate trait was the result of insertion of the acyl-ACP TE,
isolated from Umbellularia california (Californian bay).

5.2.2) Soybean:
Soybean (Glycine max) tops among all oilseed crops with 54% of world oilseed
production. Besides oil, soybean seeds contain high amount of protein (42-45%). Seed
oil of soybean consists largely of unsaturated fatty acids predominating in linoleic acid
(50-51%) followed by oleic (28-29%) and linolenic acids (6-7%). It also has significant
amount of palmitic acid (9-10%). The high content of linoleic acid makes soya oil
suitable for edible purposes. However, the oil gets oxidized readily leading to off type
flavour and poor keeping quality due to high amount of linolenic acid. So oil from
soybean cultivars with <1% linolenic acid, which will have improved oxidative
stability, reducing the formation of undesirable flavour compounds is considered
desirable. Soybean oil with elevated palmitate content may be useful for producing
solid fat (for baked products) at room temperature without hydrogenation. Also oils rich
in oleic acid that have improved flavour and nutritional value is preferred. So designing
of soya oil is primarily targeted towards low linolenic followed by high oleic and
palmitic types.
Genetics of three major fatty acids of soybean oil viz., palmitic, oleic and
linolenic have been studied well. The inheritance of oil content is influenced by
maternal effects (Brim et al. 1968) and governed by additive genes (Mc Kendry et al.
1985). Correlation among individual fatty acids and fatty acids with oil content, protein
content and seed size have been reported by several authors (Zhang, 1991; Liu et al.
1995; Nian et al. 1996; Maestri et al. 1998; Stolzfus et al. 2000; Kwon and Shin, 2002).
Oil and protein content are negatively correlated traits, however oleic acid is positively
correlated with crude protein content. Linoleic and linolenic acids are positively
correlated with each other and negatively associated with oleic acid. The association of
palmitic acid is positive with linoleic and linolenic acids and negative with oleic acid.
Seed size is positively correlated with oleic and stearic acids and negatively correlated
with linoleic and linolenic acids. So in breeding programmes selection for larger seeds
will increase oleic and decrease linolenic acid content in seed oil.
M11 and M23 (Rahman et al. 1994) are high oleic acid mutants of soybean
derived from cultivar Bay via treatement with X-rays. Oleic acid alleles of Bay, M11
and M23 are designated as Ol, ola, and ol respectively (Takagi and Rahman, 1996;
Rahman et al. 1996). The genotypes of Bay, M11 and M23 are OlOl, olaola, olol
respectively with average oleic acid content 27.8%, 30.8% and 48.6% of the total fatty
acids respectively. Ol allele for low oleic acid in Bay is partially dominant to the allele
ol in M23 and completely dominant to the allele ola in M11. Maternal effects influence
oleic acid inheritance and there is complete inverse relationship between oleic and
linoleic acid contents in both the mutants which indicates that the mutant alleles ol and
ola may also control the linoleic acid content by blocking the synthesis of this acid at the
step of oleic acid desaturation. The linolenic acid locus designated as fan, and fanxa
locus is responsible for low linolenic acid content (Rahman et al. 2001). Ol and fanxa
are independently inherited and combination of these two loci resulted in a germplasm
line DHL with high oleic and low linolenic acid content.
Two cDNA sequences FAD 2-1 and FAD 2-2 encoding microsomal -6 fatty
acid desaturase have been isolated and characterized in soybean (Heppard et al. 1996).
The FAD 2-1 gene is strongly expressed in developing seeds whereas the FAD 2-2
gene is constitutively expressed and the former plays major role in controlling
conversion of oleic to linoleic acid in storage lipids during seed development. So down
regulation of FAD 2-1 will elevate oleic acid content in seed oil. RFLP analysis of
Bay, M11 and M23 using microsomal -6 fatty acid desaturase cDNAs as probe
resulted in identification of a band (4.6 kb) present in Bay and M11, and absent in M23
(Kinoshita et al. 1998). The band fitted with the expected F2 ratio 1:2:1 and their
intensities were completely consistent with oleic acid content indicating that high oleic
acid in M23 is due to some nucleotide modification of the ol locus encoding for an
isoenzyme of microsomal -6 fatty acid desaturase.
Through mutation breeding low linolenate lines (A5, A6), low palmitate line
(A18), high palmitate line (A19) and high oleate lines (M11 and M23) are developed.
Soybean cultivars with high oleic acid (Bay) and low linoleic acid (Murayutaka) have
been bred. Also genetically engineered high oleic acid soybean cultivars are available.
High oleic soybean oil showed greater oxidative stability than other high oleic oils,
including sunflower, canola and corn (Ronald, 1996).

5.2.3) Sunflower:
Sunflower oil is valued as premium oil in world market because of its high
content of linoleic acid (67%) associated with low linolenic acid content (0.5%).
However sunflower oil with high oleic acid is preferred due to its oxidative stability.
The development of sunflower with high oleic acid content was reported by
Soladatov (1976). A single, partially domonint gene designated as Ol, controls the high
oleic acid trait in sunflower. Miller et al. (1987) confirmed this, who further reported a
second gene, ml. The presence of recessive gene ml in homozygous condition along
with gene Ol, results in high oleic acid content. Pervenets is a high oleic acid cultivar
developed through mutation breeding (Miller and Vick, 1984).
The relative proportions of oleic and linoleic acids in sunflower oil are under
both genetic and environmental control. Several investigators have reported that there is
inverse correlation between prevailing temperature during growth period and linoleic
acid content; and the opposite is true for oleic acid (Kinman and Earle, 1964; Canvin,
1965; Kawanabe, 1979; Downes and Tonnet, 1982). A variety under constant 10◦C
produced about 80% linoleic acid while at 26.5◦C the linoleic acid content was dropped
to nearly 25%, and this was accompanied by simultaneous rise in oleic acid content
(Canvin, 1965). Temperature stable high oleic acid strain (86%) was developed by
treatment of cultivar Peredovik with chemical mutagens (Prudy, 1985; 1986).
However the sunflower industry had difficulty in maintaining very high oleic
acid level and elected to commercialize, a mid oleic acid (60%) oil composition
(Downey). ARS, USA in co-operation with private industries have released new class
of sunflower called “NuSun” having mid oleic composition (Johnson, 1998).

5.2.4) Linseed:
Linseed or Flax (Linum usitatissimum) has traditionally been utilized as a source
of industrial oil, for use in the production of paints, varnishes, inks and linoleum. The
high level of linolenic acid in the oil (45-65%) imparts rapid drying property in such
products. Since the demand for industrial quality linseed oil is declining due to
synthetic substitutes and markets for vegetable oil is expanding, efforts are on to
develop edible quality linseed oil. The presence of high linolenic acid in linseed oil
causes rancidity and renders it unsuitable for edible purpose. So to convert linseed in to
premium edible oil linolenic acid would have to be reduced considerably to a maximum
of 3%.
In contrary high linolenic acid in linseed has valued it as a nutritionally
desirable crop. Linseed is the richest plant source of (alpha) linolenic acid, an omega 3
fatty acid and also contains average content (18-20%) of linoleic acid (omega 6 fatty
acid). Encapsulated linseed oil is available commercially. Edible linseed oil with
desirable nutritional and keeping quality can be obtained by developing cultivars with
fatty acid profile fitting the recommended linoleic: α-linolenic acid ratio (5:1 to 10:1)
balanced with oxidative resistant oleic acid.
Extensive surveys of Linum usitatissimum germplasm collection revealed that
variety mean for linolenic acid content varied only between 45% to 65% (Zimmerman
and Klosterman, 1959; Green and Marshal, 1981) indicating mutation breeding to be a
promising approach rather than hybridization and selection to reduce linolenic acid
content (Green and Marshal, 1984). Two mutant lines M 1589 and M 1722 were
developed following EMS mutagenesis of cultivar Glenelg. The linolenic acid content
of these mutant lines constituted approximately 29% compared with 43% in Glenelg
(Green and Marshal, 1984). Further crossing these two mutant lines led to development
of a mutant genotype having less than 2% linolenic acid (Green, 1986a). The virtual
elimination of linolenic acid (<2%) from the seed lipids is accompanied by an
equivalent increase in the content of linoleic acid (>46%), the proportions of other fatty
acids remaining unchanged. These changes indicated that the mutation block the final
desaturation of linoleic to linolenic acid (Green and Marshal, 1984; Green, 1986a).
Studies on linolenic acid inheritance in mutant lines M 1589 and M1722
revealed that both lines are homozygous for a single gene mutation that reduce linolenic
acid content and these mutations are in different unlinked genes, exhibiting additive
(co-dominant) gene action (Green, 1986b). The mutant locus in M 1589 and M 1722
are designated as Ln1 and Ln2 respectively. Varieties producing edible linseed oil
called “Linola” (Dribnenki and Green, 1995; Dribnenki et al. 1996; Dribnenki et al.
1999) are now commercially grown and consumed in Canada, United States, Australia
and several European countries (Table: 3).

Table: 3 – Fatty acid profile of linola in comparison with flax.


Fatty Acid Profile Flax Linola
Palmitic 4–9 6
Stearic 2–4 4
Total Saturates 6 - 13 10
Oleic 14 – 39 16
Total Monounsaturates 14 - 39 16
Linoleic 7 – 19 72
α-linolenic 35 – 66 2
Total Polyunsaturates 42 - 85 74
5.2.5) Safflower:
Like sunflower, safflower (Carathamus tinctorius) oil is predominant in linoleic
acid (70-80%) with low linolenic acid content. It is the first oil seed crop in which
individual gene control of oleic and linoleic acid was demonstrated due to efforts of
Furehally (reported by Knowles, 1989). The three genes that control production of
oleic, linoleic and stearic acids designated as olol, lili and stst respectively are major
recessive genes at different loci (Table: 4).
High linoleic/ low oleic acid allele is dominant over high oleic/ low linoleic acid
allele indicating single allele control of oleic to linoleic acid conversion. Increase in
stearic acid is accompanied by decrease in oleic or linoleic acid or both. An interesting
fact in this crop is that high or low oleic acid lines are stable to temperature changes but
the intermediate ones are affected indicating that different alleles respond differently to
temperature variations.
Safflower is one of the best examples of variability for fatty acid composition of
seed oil. Variants with high stearic (4-11%), high oleic (>80%) and high linoleic acid
concentration (>85%) have been identified and are currently available as varieties
(Muralidharan et al. 2002).

Table: 4 - Genetics of fatty acid content of different oil types in safflower.


Oil Type Genotype Fatty Acid Content in Safflower Oil (% range)
C16:0 C18:0 C18:1 C18:2
Palmitic Stearic Oleic Linoleic
Very High Linoleic OlOl lili StSt 3-5 1-2 5-7 87-88
High Linoleic OlOl LiLi StSt 6-8 2-3 16-20 71-75
High Oleic olol LiLi StSt 5-6 1-2 75-80 14-18
Intermediate Oleic ol’ol’ LiLi StSt 5-6 1-2 41-53 39-52
High Stearic OlOl LiLi stst 5-6 4-11 13-15 69-72
Adapted from Muralidharan et al. 2002.

5.2.6) Sesame:
The seed oil of sesame (Sesamum indicum) contains mainly four fatty acids viz.,
palmitic, stearic, oleic and linoleic acids. Sesame oil is unique in containing several
lignan antioxidants as well as some tocopherols. The lignans, sesamin and sesamolin
and their derivatives, sesamol and sesaminol, and their related compounds prevent the
oxidation of sesame oil contributing to the characteristic stability and extended shelf
life.
Oleic and linoleic acids constitute around 85% of oil composition and their
inheritance is controlled by single gene (Brar and Ahuja, 1979). The high oleic acid
allele (Ol) is dominant over low oleic acid allele (ol). Variation for fatty acid content
was induced by gamma rays (Lee et al., 1985) and by sodium azide (Kang, 1994).
These efforts resulted in release of Seodun in 1997 in South Korea, which has high
oleic acid content.

5.2.7) Groundnut:
Groundnut (Arachis hypogea) oil is composed of 80% unsaturated fatty acids
and unique in containing LCFAs, arachidic and behenic acids. These LCFAs are useful
in emulsification and stabilization of products like peanut butter, but their implications
in heart disease is a matter of concern (Kritchevsky et al. 1971). So far not much efforts
for fatty acid modification has been done in this crop.
The Virginia botanical types generally have higher oleic acid content and lower
linoleic acid content than Spanish or Valencia types. Crosses among these types
indicated that sufficient variability for fatty acid could be generated by recombination
of genes. A favourable feature for breeding is that fatty acid composition is determined
by growing embryo and few additive genes are involved in determination of oleic and
linoleic acid ratio (Khan et al. 1974).

5.2.8) Cuphea:
The genus Cuphea belongs to family Lythraceae (Graham, 1988; Singh, 2001)
and is largest in the family having more than 260 species of which C. lanceolata, C.
viscosissima and C. procumbens are promising ones. Cuphea seed oil is diverse in fatty
acid composition ranging from C8 – C18, predominating in MCFA (C8 – C12) (Earle et
al. 1960; Miller et al. 1964). There is no known genus in plant kingdom with such a
diverse fatty acid composition (Anonymous, 1985; Graham, 1989; Knapp, 1993a). So
the researchers can almost tailor-select the oil composition as per need of the society.
Caprylic and capric acids, which have potential nutritional applications, are
either derived from petrochemicals (non-renewable and dwindling source) or by
fractionating coconut and palm-kernel oil (costly source). The use of capric and
caprylic acids in human diet is severely restricted due to limited availability (Knapp,
1993a). Lauric acid that has industrial importance is currently derived from coconut and
palm kernel oil. Coconut and oil palm are perennial sources and also have relatively
less lauric acid content (50%) than Cuphea (60%) (Anonymous, 1985; Singh et al.
1998). The fatty acid composition of Cuphea in comparison with coconut and oil palm
is presented in table: 5.
The lauric acid content of Cuphea makes them potential substitute for coconut
and oil palm and the caprylic and capric acid contents suggest that they could
eventually supplement or replace petrochemicals. However Cuphea is yet to be
commercialized due to hindrance of wild characters such as seed shattering and seed
dormancy (Hirsinger and Röbbelen, 1980; Anonymous, 1985; Knapp, 1993a; Pandey et
al. 2000).

Table: 5 – Fatty acid composition of Cuphea in comparison with other sources.


SOURCES DISTRIBUTION (% OF TOTAL FATTY ACIDS)
C8:0 C10:0 C12:0 C14:0 Others
Coconut 8 7 48 18 19
Oil Palm 3.5 4 50 14 28.5
(Kernel Oil)
C. procumbens 1.7 91.3 1.3 3.1 2.6
C. lanceolata - 87.5 2.1 1.4 9
C. viscosissima 9.1 75.5 3.0 1.3 11.1
C. koehneana 0.2 95.3 1.0 0.3 3.2
C. wrightii - 29.4 53.9 5.1 11.6
C. laminuligera - 17.1 62.6 9.5 10.8

The seed oil of C. viscosissima, C. lanceolata and C. procumbens are caprylic


and capric acid rich. These species are nearly domesticated and could supply natural
MCTs with minimal processing. Lauric and myristic acids are minor constituents of
these species. So the spectrum of oils produced by these species has been increased by
several induced fatty acid mutants (Knapp and Tagliani, 1991; Knapp, 1993b and
Tagliani et al. 1995). Among these are several C. viscosissima mutant lines with
decreased capric acid – CPR-1, CPR-2, CPR-4, CPR-6, CPR-7 and CLM-1 (Knapp,
1993b; Tagliani et al. 1995). These lines are homozygous for mutations induced by
EMS and are near isogenic lines of the wild type C. viscosissima line PI-534911
(Knapp, 1993b; Tagliani et al. 1995). The inheritance pattern of these mutant lines was
studied by Knapp et al. (1997) and they reported that CPR-1 and CPR-2 are allelic
mutations affecting the cpr (capric acid) locus, CPR-5 is an allele of cpr locus or locus
tightly linked to cpr, and CPR-4 and CLM-1 are non allelic mutations. VS-320 an F3
line developed by crossing CPR-1 and CLM-1 produces oil with elevated levels of
medium and short chain TGs that may be a potential substitute for diesel fuel (Geller et
al. 1999).
Since wild characters have hindered commercialization of Cuphea, engineering
existing oilseed crops for MCFA synthesis has been considered as commercially viable
alternative. Knowledge of biosynthetic and catabolic enzymes regulating the
composition and levels of these unusual fatty acids is very much essential for
engineering seed oil biosynthesis. Cuphea has served as a model organism and key
enzymes, thioesterases (TE), β-Ketoacyl-ACP Synthases (KAS) and acyl transferases
are best characterized.
TE is the important enzyme that converts the composition of TGs from the
common LCTs to unusual MCTs. Based on sequence homology they are classified into
two families, Fat A and Fat B, that prefers C18:1-ACP and saturated acyl-ACPs as
substrates respectively. In C. lanceolata, a capric acid rich species Fat B gene family of
atleast four members was identified (Topfer et al. 1995). Two TE cDNAs, Cp Fat B1
and Cp Fat B2 have been isolated from C. paulistris that accumulates 64% myristate
and 20% caprylate (Dehesh et al. 1996a). Cp Fat B1 strongly prefers C8:0-ACP whereas
Cp Fat B2 strongly prefers C14:0-ACP and the latter is kinetically superior over former
resulting in predominance of C14:0 in C. paulistris seed oil (Dehesh, 2001). Similarly
two cDNAs from C. hookeriana (Ch Fat B1-3 and Ch Fat A) and C. wrightii (Cw Fat
B1 and Cw Fat B2) have been isolated (Dehesh et al. 1996b). Several transgenic
experiments in Arabdiopsis and canola revealed the discrepancy in fatty acid profile
and quantity of MCFAs between transgenic plants and native plants (Dehesh, 2001).
This indicated that apart from TEs, condensing enzymes (KAS) could also influence the
fatty acid chain length.
The first reported breakthrough in obtaining a condensing enzyme with altered
substrate specificity was the cloning of the C. wrightii KASA (Slabaugh et al. 1998).
The presence of encoded product of KASA, a 46 kD polypeptide, correlates strongly
with the synthesis of MCFAs in different species of Cuphea. Another KAS cDNA has
been cloned from C. hookeriana (Dehesh et al. 1998). Expression of this KAS in canola
together with Cp Fat B1 or Ch Fat B2 resulted in increase of the total levels of MCFAs
in transgenic pool upto 30% and also the proportions of C8:0 and C10:0 relative to the
transgenic plants expressing TE alone was altered dramatically (Dehesh, 2001). The
knowledge gained will certainly pave way for engineering MCFA biosynthesis in
traditional oil seed crops like canola increasing availability of MCFAs in the world oil
market.

5.3) Synthetically Structured Fats:


Synthetically structured fats are designed to look and act like fats, but they
contribute fewer calories and less fat (Ronald, 1996). Two approaches have been taken:
1) Attaching planned ratios of long-chain (LC) saturated fatty acids with very low
caloric density and shorter-chain (SC) fatty acids with slightly lower caloric density
than LC fatty acids (caprenin, salatrim) to glycerol back bone or 2) Attaching fatty
acids to a non-glycerol backbone in such a manner that the molecule is poorly absorbed
in the body (olestra). Since the first method results in a triglyceride found in nature the
regulatory route is simple whereas for second one it is more complex.
5.3.1) Glycerol Back Bone:
The first product commercialized under this grouping was Caprenin, a reduced-
calorie designer fat consisting of three fatty acids: capryllic, capric and behenic acid.
Behenic acid is only partially absorbed by the body, and the medium-chain fatty acids
have lower caloric densities than longer-chain fatty acids, resulting in a total caloric
density for caprenin of 5 kcal/gram. Caprenin was commercialized by Procter &
Gamble as a cocoa butter replacer and was launched in two products. Unfortunately, the
product has difficult tempering characteristics and appeared to increase the serum
cholesterol slightly, resulting in its withdrawal from the market.
Salatrim, is another family of restructured fats developed by Nabisco Foods.
Due to the lower caloric density of stearic acid and the short-chain fatty acids, salatrim
contributes a total of 5 kcal/gram. Safety studies revealed that the molecule has no
effect on serum cholesterol and on absorption of fat-soluble vitamins.
5.3.2) Sucrose Backbone:
Olestra is synthesized from sucrose and vegetable oil (cottonseed or soybean),
and it has physical properties comparable to conventional fats. The complexity of the
molecule inhibits the activity of digestive enzymes required to break it down.
Therefore, olestra passes through the body undigested, contributing no fat or calories to
foods. The product has been commercialized but it is unfortunate that it may cause
abdominal cramps and loose stools and inhibits the absorption of some vitamins and
other nutrients.

6) Conclusion:
With the advent of breeding and biotechnology approaches the native fatty acid
profile of major oilseed crops particularly brassica, soybean and sunflower have been
modified to desirable extent. All oils have a place, and individual points of identity, any
of which may be usable for certain functionalities. The fatty acids, which seem to be
undesirable today, may assume importance in the future. So designing oil is a continous
process, and it is a long-term commitment to work with consumers to develop oils with
certain fatty acid profile for specific purposes, and with certain nutritional properties.
So the nutritionist, breeders and biotechnologist, should have a long-term view and
integrated approach to provide oil with required quality at any point of time.

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