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

Name

Abbreviation Fatty

Acid

Family

Major Sources

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

palm, Corn, Groundnut, Cottonseed.Linoleic C18:2 n-6

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 variety

Origin/method 12:0 14:0 16:0 18:0 18:1n-9

18:2n-6

18:3n-3

20:1n-9

22:1n-9

Others

Rapeseed

High-erucic acid rapeseed

Traditional - - 3 1 11 12 9 8 52 4

Double-low / Canola

Spontaneous mutant

- - 4 2 60 21 10 1 1 1

Low-linolenic Canola

Mutagenesis - - 4 2 61 28 3 1 - 1

Laurate Canola

Genetic engineering

37 4 3 1 33 12 7 - - 3

High myristate /palmitate

Genetic engineering

- 18 23 2 34 15 4 - - 4

High-oleic Canola

Mutagenesis / transgenic

    4 1 84 5 3 1 - 2

Soybean

Conventional Traditional - - 11 4 23 54 8 - - -

Low linolenate

Mutagenesis - - 10 5 23 60 2 - - -

High palmitate Mutagenesis - - 17 3 17 55 8 - - -

High stearate Mutagenesis - - 8 28 20 35 7 - - 2

High oleate Genetic engineering

- - 7 4 85 1 2 - - 1

Sunflower

Conventional Traditional - - 6 4 18.5 71 0.5 - - -

High oleic Mutagenesis / transgenic

- - 4 4 78.5 11.5 - - - -

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, E a, 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 . ElongaseAcetate ® ® ® 16:0-ACP ® 18:0 -ACP ® 18:1-ACP ¯ ThioesterasePlastid stroma (ACP track) Oleic acid

Acyl-CoA synthetaseOleoyl-CoA

 LPE ® PE-18:1 ® PE-18:2 ® 18:3 Acyltransferase d-12 desaturase d-15 desturaseEndoplasmic 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 LinolaPalmiticStearic

Total Saturates

4 – 92 – 46 - 13

6410

OleicTotal Monounsaturates

14 – 3914 - 39

1616

Linoleicα-linolenic

Total Polyunsaturates

7 – 1935 – 6642 - 85

72274

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

Palmitic

C18:0

Stearic

C18:1

Oleic

C18:2

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 OthersCoconut 8 7 48 18 19Oil Palm

(Kernel Oil)3.5 4 50 14 28.5

C. procumbens 1.7 91.3 1.3 3.1 2.6C. lanceolata - 87.5 2.1 1.4 9

C. viscosissima 9.1 75.5 3.0 1.3 11.1C. koehneana 0.2 95.3 1.0 0.3 3.2

C. wrightii - 29.4 53.9 5.1 11.6C. 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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