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2. REVIEW OF LITERATURE
2.1 Sesame
2.1.1 Morphology
Sesame is an erect herbaceous annual crop that grows to a height of 0.4 m to 2 m (Fig.
1). The plants are often highly branched, but some varieties are relatively unbranched. Stem is
square with grooves. Leaves are hairy on both sides with variable shape (ovate to lanceolate)
and size, and may be opposite or alternate (Fig. 2). Bell-shaped, pale purple to white flowers
begin to develop at leaf axils within 6-8 weeks after planting. A single flower is produced at
each leaf axil starting from the lower axils and the plant continues blooming until the uppermost
flowers on the stem are open (Day, 2001). Multiple flowering is common in varieties with
opposite leaves (Oplinger et al., 1990). Sesame is predominantly self-pollinated, although
cross pollination by insects is common (Pathirana, 1994). The fruit is an oblong capsule, 1 to 3
inches long, containing 50 to 100 or more seeds. The seeds are oval and may be white, yellow
red, brown or black. The seeds mature 4 to 6 weeks after fertilization. Sesame grows
indeterminately, producing new leaves, flowers and capsules at the same time as long as the
weather conditions permit. The growth cycle is completed within 70 to 180 days depending on
the variety and growth conditions
2.1.2 Taxonomy and cytogenetics
The genus Sesamum consists of many species and the most cultivated is Sesamum
indicum L. (Ashri, 1998). According to Kobayashi et al. (1990), 36 species have been identified
of which 24 species are recorded from Africa, five in Asia and seven in both Africa and Asia.
There are three cytogenetic groups of which 2n = 26 consist of the cultivated S. indicum along
with S. alatum, S. capense, S. schenckii, S. malabaricum; 2n = 32 consist of S. prostratum, S.
laciniatum, S. angolense, S.angustifolium; while S. radiatum, S. occidentale, S. schinzianum
belong to 2n = 64. Mainly due to the difference in chromosomal numbers across the three
cytotaxonomic groups, there is limited cross compatibility among the species. Therefore, it has
been difficult to transfer desirable characteristics such as tolerance to drought, pests and
pathogens, from wild relatives into cultivated sesame (Carlsson et al., 2008).
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2.1.3 Origin and distribution
Sesame is one of the oldest cultivated crops known to humans. Archeological remains of sesame
dating back to 5,500 BC have been found in the Harappa valley in the Indian subcontinent
(Bedigian and Harlan, 1986). The origin of the crop has been a major subject of discussion, with
proposals for an African or Indian domestication. Based on various lines of evidence including
cytogenetics, biochemical composition, nuclear DNA marker comparisons and cultural history to
name a few, Bedigian (2003, 2004) has concluded that this species was first domesticated on the
Indian subcontinent. From there, it spread to Africa, the Mediterranean, and the Far East, and
into the Americas following trade routes. Today sesame, as an oilseed, is widely grown in China,
Japan, Korea, Turkey, India, USA, South America and parts of Africa.
2.1.4 Genetic diversity in sesame
According to studies on morphological variation, sesame shows extensive variation (Bedigian
and Harlan, 1986; Baydar et al., 1999; Bisht et al., 1998; Xiurong et al., 2000). For example,
diversity of an Indian sesame collection was determined for 100 accessions representing different
agro-ecological zones for morphological and agronomic characters. The accessions were
classified into seven clusters to create a core collection of sesame (Bisht et al., 1998). A sesame
germplasm collection in China was also established via morphological grouping (Xiurong et al.,
2000). Another morphological study was performed by Baydar in 2005. In this study, to improve
the ideal sesame plant type, classic breeding techniques and examination of generations were
applied based on eight features. Consequently, researchers showed that branching type is related
with high yield and that plants with low yield contain high oil content. A similar study was
performed by Sharmila et al. (2007). They found additive, dominant, and epistatic gene
interactions for seven quantitative traits via generation mean analysis in different sesame plants.
In Turkey, Uzun and Cagiran (2006) compared determinate and indeterminate types for
agronomic traits and they showed that determinate mutant types have some disadvantages and
they need further development. Parameshwarappa et al. (2010) evaluated 64 sesame genotypes
for yield and yield attributing characters to study the genetic diversity existing among them by
using Mahalanobis D2 statistics. Analysis of variance revealed significant difference among
genotypes for all the nine characters studied and 64 genotypes were clustered into 9 groups.
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2.1.5 Economic importance of sesame
Sesame is appropriately called the queen of oilseeds, as it is used worldwide in different
forms and for different purposes. Sesame is mainly cultivated for its seed, oil and protein.
Composition and uses of sesame seed, oil, seed cake and seed meal are dealt with in details here:
2.1.5.1 Sesame seed: Sesame seeds are small in size (1,000 seeds weight 2.0 – 3.5 gm)
having color of seed varies from white, grey, brown, violet and black. White and black varieties
are commonly cultivated in India while brown seed varieties are relatively less grown (Chadha,
1976).
Composition: Sesame seed contain 25% protein, 50% oil, 20% sugar, 6% fibre and various
minerals. They are rich in calcium (about 0.3%) and phosphorous (about 0.5%) content. Sesame
seeds contains slightly low amount of lysine but is rich in other amino acids like methionine,
cystine, arginine and leucine (Moazzami et al., 2006). The main constituents of seed are water
(5.8%), protein (8%), fat (49%), crude fibre (3.2%) and carbohydrate (18%) (Nayar and Mehra,
1970). Both white and black sesame have a higher phenolic content in their hull fraction as
compared to their whole seed counterpart, because endosperms contain very low amount of
phenolics compared to hulls. Antioxidant content of total of whole sesame seeds & hulls are as
follows – Black sesame hull > whole black sesame > White sesame hull > whole white sesame.
It is apparent that there exits a correlation between total phenolic contents of different sesame
fraction and their respective total antioxidant activity (Shahidi et. al., 2006).
Uses: Sesame seeds are used in the preparation of number of food products such as
tahini, halva, pinni, bakery and sweets confection. Whole seeds are found in many salads &
baked snacks. Seeds are sometimes added to breads including bagels & tops of hamburger buns.
2.5.1.2 Oil: Sesame oil is mechanically extracted by applying pressure in a mechanical
expeller and is tolerant to minimal heating. The oil yield is dependent upon the growing
condition and seed variety. It is highly polyunsaturated and is semi-drying in nature. Various
lignans in sesame have been isolated from sesame oil.
Composition: Sesame oil contains linoleic acid (48%), oleic acid (38%), palmitic acid (9%)
and stearic acid (5%) with lesser amount of linolenic acid (Nayar and Mehra, 1970).Many
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secondary metabolites such as phenolic acid, tocopherols, sterols and flavonoids have been
isolated from the sesame seed oil. Various lignans have been isolated from sesame oil. The
potent antioxidant properties of sesame seed oil are attributed mainly to the presence of the
lignans such as sesamin and sesamolin.
Uses: Sesame oil is used as cooking oil. It is used in salad, for marinating meat and
vegetables, used in paints, soaps, perfumes, insecticides (Bedigian and Harlan, 1986), lighting
and as a lubricant. Refined sesame oil is mainly used in pharmaceuticals and cosmetic products.
It is known to be used for massaging and health treatment of the body in the ancient Indian
ayurvedic system, with the type of massage called abhyanga and shirodhara. The oil is said to be
laxative and to promote menstruation. It is used in the preparation of Iodinol and Brominol,
which are employed for external.
2.5.1.3 Cake: After the extraction of oil from sesame seeds, cake is obtained. It usually
contains large quantity of fibres & oxalic acids.
Uses: It is used as livestock feed. It is often blended with flours for baking. S. radiatum and
S. indicum are also employed as green manure (Nayar and Mehra, 1970).
2.5.1.4 Meal: Defatted seed meals are hydrophilic antioxidants (Shyu et al., 2004). The
defatted (oil free) Sesame meal contains nearly 50% protein and high methionine content. It is
also rich in calcium, phosphorous and vitamin E. The meal protein contains all the major amino
acids found in meat (Nayar and Mehra, 1970). Sesame meal contains water soluble lignans like
sesaminol, pinoresinol etc which shows antioxidant property. It is valued in feeds and human
food.
2.1.6 Status of production of sesame
India is the fourth largest oilseed producing country after USA, China and Brazil. Among the
large number of oilseeds grown here, sesame lies at the sixth position of production after
soyabean, cotton seed, groundnut, sunflower and mustard (National productive council, New
Delhi). Even though the crop is highly valuable nutritionally, medicinally and agriculturally, it
is losing out as an oilseed. In 2010, sesame was cultivated in an area of 1.84 million hectares (as
against 1.87 in 2009; FAO) with a production of 6.23 million tonnes (6.57 mt in 2009) and a
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world average productivity of 467 kg/ha (Fig 3). India had been the dominant producer till 2005
accounting for almost 25 per cent of the world output. However, it lost to China in 2006
followed by Myanmar from 2007 (Fig 4). This decreasing crop yield can be attributed to its
cultivation in un-irrigated areas, lack of varietal replacement through development of hybrids,
vagaries of nature and production losses due to pests and diseases (FAOSTAT data, 2011).
Dedicated and integrated efforts have to be made to bridge the ever increasing gap between the
potential achievable yield (about 1,000 kg/ha) and the average yield (467 kg /ha). For this
purpose, advantages of crop needs to be explored such as oil quality, antioxidants, drought
tolerance, low cost of production and improvement in soil water percolation. The dramatic
increase in demand for sesame oil places increasing pressure on plant breeders to continuously
improve seed and oil yields, the overall agronomical performance and the quality of the oil and
of the meal that remains after oil extraction.
2.1.7 Oil content and fatty acid composition of sesame seeds
Sesame has a relatively superior oil quantity as well as quality in comparison to many major oil
crops. The oil content ranges from 34.4 to 59.8% but is mostly about 50% of seed weight (Ashri,
1989, 1998). Azeez and Morakinyo (2011) reported that seed oil content was 53.23–55.12% in
cultivated while 53.35–58.56% in wild accessions. Values of up to 63.2% have been reported in
some varieties by Baydar et al. (1999) and Uzun et al. (2002). Both genetic and environmental
factors influence the oil content in sesame. Were et al. (2006) could establish correlation between
fatty acid and oil concentration in a three year study of sesame. They reported that oil content
correlated negatively with palmitic and linoleic acids, and positively with stearic and oleic acids.
Late maturing cultivars are reported to have higher oil content than early ones (Yermanos et al.,
1972). Uzun et al. (2002) observed that indeterminate cultivars accumulated more oil than
determinate ones. Variation also occurs between capsules at different positions on the same
plant, such that seeds from the basal capsules on the main stem contain more oil than those
located towards the apex and on side branches (Mosjidis and Yermanos, 1985; Muthuswamy and
Thangavelu, 1993). Black seeded cultivars often have lower oil content than brown or white
seeded ones, indicating a possible linkage between oil content and the pathway that contributes
to the seed coat colour. Kamal-Eldin and Appelqvist (1994) have attributed the low oil content in
black seeded sesame to a high amount of crude fibre in the seed coat. Black seed coat is usually
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thicker than lighter coloured ones. Philip, J.K. (2011) reported nutritional variation in black and
white sesame wherein protein, fat, zinc, copper, sodium, magnesium, glucose, sucrose, maltose,
Vit C, E and K being higher in white seeds.
The genus sesame has limited variability in the seed fatty acid proportions (Kamal-Eldin
et al., 1992). The seed fatty acid composition varies considerably among the different cultivars of
sesame worldwide (Yermanos et al., 1972; Brar, 1982; Baydar et al., 1999). The oil contains four
major fatty acids namely, palmitic, stearic, oleic and linoleic acids, along with small quantities of
vaccenic, linolenic, arachidic, behenic and eicosenoic acids (Weiss, 1983; Kamal Eldin et al.,
1992; Ashri, 1998; Were et al., 2006). Oleic and linoleic acids occur in nearly equal amounts,
constituting about 85% of the total fatty acids. Sesame treated with mutagens shows fatty acid
variation having saturated fatty acids higher than control besides lower concentration of
polyunsaturated fatty acids. As regards the oleic acid, the high yielding/branched mutant was
revealed the highest oleic acid content (Savant and Kothekar, 2011).
Cultivars with exceptionally high (> 60%) oleic or linoleic acid are rare (Baydar et al.,
1999). Uzun et al. (2002) found differences in stearic, oleic and linoleic acids between
determinate and indeterminate cultivars. Determinate cultivars generally have higher stearic and
oleic acids, and lower linoleic acid compared to indeterminate ones. Capsule position on the
plant also affects the relative quantities of the fatty acids; palmitic, stearic and oleic acids tend to
increase up the stem while linoleic acid decreases (Brar, 1977). The fatty acid composition is
strongly influenced by environmental factors. Linoleic acid content has been reported to increase
under cool growing conditions (Uzun et al., 2002).
2.1.8 Dietary and health benefits of sesame oil
The fatty acid composition is a major determinant of edible oil quality. Oils having high
polyunsaturated fatty acids (PUFAs) content, in combination with low quantities of saturated
fatty acids are commercially and nutritionally desirable. Saturated fatty acids are associated with
high risk of heart disease whereas PUFAs are known to be beneficial for human health. Sesame
oil has a low level of saturated fatty acids (< 15%) and approximately equal quantities of monoand polyunsaturated fatty acids. The oil is nutritionally valuable as a source of linoleic acid and
linolenic acid which are essential to humans.
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Despite having a high content of linoleic acid, sesame oil is unusually stable to oxidation
compared to other vegetable oils with a similar fatty acid composition. This feature is attributed
to antioxidant activities of sesamol and sesaminol together with tocopherols present in the oil
(Kamal-Eldin and Appelqvist, 1994). A combination of the high stability and a nutritionally
acceptable fatty acid composition contributes significantly to the excellent oil quality, making a
high- value edible oil.
Recent studies have shown that sesame oil is beneficial in lowering cholesterol levels and
hypertension (Sankar et al., 2004; Frank et al., 2004), and reducing the incidence of certain
cancers (Hibasami et al., 2000; Miyahara et al., 2001). These health enhancing effects of sesame
oil are explained by the low level of saturated fatty acids and high levels of PUFA. Moreover,
the Sesamin is known to enhance the availability and functioning of vitamin E (tocopherol). An
elevated concentration of tocopherol in the blood is associated with reduced risk of heart disease
and some cancers e.g. of the upper gut. Thus, sesame oil could be beneficial for enhancing health
by improving the vitamin E levels in the body (Frank et al., 2004).
2.2. Biosynthesis of fatty acids in oil seeds
The fatty acid biosynthesis pathway is a primary metabolic pathway, because it is found in every
cell of the plant and is essential to growth. lnhibitors of fatty acid biosynthesis are lethal to cells,
and no mutations that block fatty acid synthesis have been isolated (Ohlroggeav and Browseb,
1995).
In higher plants, PUFAs are synthesized through both prokaryotic (chloroplast) and
eukaryotic (ER) pathways (Fig5) (Roughan et al., 1980; Browse et al., 1986). As an initial step
for 18:3 fatty acid synthesis, first double bond is introduced into stearic acid (18:0) by a soluble
stearoyl acyl carrier protein deasturase found in chloroplasts (Iba et al., 1993). In the second step,
two distinct mechanisms are responsible for further desaturation of 18:1 to 18:3 via 18:2. One
occurs in plasids and other in microsomes. In normal conditions like in Arabidopsis thaliana the
chloroplast ω-6 and ω-3 fatty acid desaturases encoded by fad 6 and fad 7 loci are involved in the
desaturation of 18:1 and 18:2, respectively. While microsomal ω-6 and ω-3 fatty acid desaturases
encoded by the fad 2 and fad 3 loci are responsible for the desaturation of 18:1 and 18:2 fatty
acids respectively and production of α- linolenic acid (Browse and Sommerville, 1991).
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2.2.1 Modification of fatty acid composition in plant storage oils: problems, progress and
prospects
Vegetable oils may sometimes lack the properties best suited for their intended use. For instance,
they could have undesirable nutritional attributes such as high proportion of saturated fatty acids
in comparison to the more acceptable unsaturated forms, or have melting behaviour that
contributes to poor quality of spreads. Such deficient oils would need to be modified to attain the
desired properties. Modification of lipid properties is conventionally carried out by chemical
processes namely, partial hydrogenation, fractionation or inter esterification (Bhattacharya et al.,
2000; Timms, 2005). These processing methods, however, are expensive and sometimes yield
undesirable products in the edible oils. For example, during the hydrogenation of highly
unsaturated oils for making margarine and shortenings trans fatty acids, known to confer health
risks in humans, are formed (Mozaffarian, 2005).
Development of crop varieties producing oils with quality appropriate for specific market
needs presents a better alternative to chemical modification of vegetable oils and a means to
circumvent the short comings associated with the technology. One way to achieve this is by
domesticating wild plants that accumulate oil with desirable characteristics. However, the long
time scale (of over 20 years) needed to adapt them to cultivation and the requirement for remodelling of agricultural machinery and processing equipment present a major limitation to
development of novel oil crops. In a variety of cultivated oil crops, the fatty acid composition has
been modified by means of conventional breeding methods to meet various consumer demands.
Using sexual hybridization as well as induced mutagenesis, new varieties of oil crops have been
generated which have diverse composition of fatty acids. Examples include the breeding and
establishment of LEAR (low erucic acid rapeseed) for edible oil, and the development of higholeic varieties of soybean, sunflower, brassica oilseeds and peanut (Burton et al., 2004). Though
successful, conventional breeding relies on the naturally occurring variation within a species or
genus and is therefore limited to cross compatible taxa. Some of the variations the breeders have
used is due to spontaneous random mutations affecting fatty acid synthesis e.g. in LEAR
although they are very rare. Induced mutagenesis has helped to create additional diversity in seed
fatty acid composition, as was done when developing high linoleic acid linseed (Linola) from a
high linolenic acid variety (Green, 1986). However, induced mutagenesis is disadvantageous as
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it lacks precision, generating many plants with defects and for this reason entails extensive
screening of lines to eliminate the bulk of abnormal ones.
Current research effort is directed towards creating plant oils having diverse fatty acid
composition by genetic engineering of the established oil crops. This approach is superior to
those previously used owing to its precision and applicability across taxa. By using molecular
techniques, it is possible to modify specifically the seed oil quality while keeping the rest of the
genetic background of the plant constant. Using techniques such as antisense repression, cosuppression and inverted repeat silencing, transgenic oil crops having novel fatty acid profiles
have been generated (Cartea et al., 1998; Stoutjesdijk et al., 2002). Examples include highstearate rape (Knutzon et al., 1992), high-laurate rape (Voelker et al., 1992), and high-oleate
cotton (Liu et al., 2002) among others.
The major edible oils contain predominantly unsaturated 18 carbon fatty acids and
palmitic acid. Key targets for genetic modification of these oils both for edible and industrial
uses have been identified (Murphy, 1999). One goal for modification of these oils for edible use
is to increase the amount of palmitic and stearic acids in order to minimise the need of
hydrogenation in the production of dietary fats. Other is to increase the amount of
polyunsaturated fatty acids for human health benefits.
2.2.2 Work done in sad (Stearoyl acyl carrier protein desaturase) locus
Stearoyl ACP desaturase is a soluble enzyme localizing in plastid, which catalyzes conversion of
stearoyl-ACP to oleoyl-ACP by primary introduction of a cis-double bond between carbon
positions 9 and 10. Since S-ACP-DES are the only plant enzymes which catalyze conversion of
18:0 to 18:1 in plants, their activity primarily regulates the ratios of saturated to monounsaturated
FAs (Kachroo et al., 2007). cDNAs encoding a stearoyl-ACP desaturase were isolated from
several dicotyledonous species: cucumber and caster (Shanklin and Somerville, 1991), potato
(Taylor et al., 1992), spinach (Nishida et al., 1992). In oilseeds like Brassica napus (Slocombe et
al., 1992), safflower (Thompson et al., 1991),Brassica rapa (Knutzon et al., 1992), jojoba (Sato
et al., 1992) and Linseed (Singh et al., 1994) cDNA of this gene were isolated. ESTs were
developed of sad gene in Arachis hypogea by Florin and group in 2011. Two different sad genes
in soybean were worked upon by Byfield et al., 2006. They were designated as A and B and
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there comparison gave two unique amino acid variations. Introduction of an antisense gene to
stearoyl-ACP desaturase resulted in a reduced ratio of unsaturated fatty acids to saturated fatty
acids in transgenic Brassica and Nicotiana plants (Cahoon et al., 1992; Knutzon et al., 1992),
implicating this enzyme as a key enzyme in determining the balance between saturated and
unsaturated fatty acids in higher plants. Therefore, genetic manipulation of stearoyl-ACP
desaturase expression may control the amount of unsaturated fatty acids.
2.2.3 Work done in fad2 (Fatty acid desaturase2) locus
The fad2 gene encodes the ER 18:l desaturase that controls the vast majority of polyunsaturated
lipid synthesis in plant cells. It is responsible for more than 90% of the polyunsaturated fatty acid
synthesis in non photosynthetic tissues, including the developing seeds of oil crops. Fad 2 was
cloned in 1994 by Okuley et al. from mutants of Arabidopsis thaliana. Mutants of Arabidopsis at
the fatty acid desaturarion 2 (fad2) locus are deficient in activity of the endoplasmic reticulum
desaturase (Mlquel and Browse, 1992). Mutant alleles at fad2 (or constitutive antisense
expression of fad2 sequences) in soybean and in canola considerably decrease the amount of
polyunsaturated fatty acids in vegetative tissues as well as in the seed oil. The expression of an
antisense fad2 gene under the control of a seed-specific promoter could alleviate the problem of
low-temperature sensitivity in the vegetative state but does not preclude the alteration of
membrane fatty acid composition in the seed, since the fad2 gene product is responsible for
desaturation of both membrane and storage lipids (Browse and Somerville, 1991). cDNA were
isolated of fad 2 from Arabidopsis thaliana (Okuley et al., 1994) and Olea europaea (Banilas et
al., 2005). Study in Arachis hypogea fad2 gene revealed that mutation in fad2A and reduced
expression of fad2b gave high oleic acid (Jung et al., 2000). Success has been found in producing
transgenic oil crop in case of soybean by expressing 2 copies of fad2 gene by Du Pont. Soybean
lines G94-1, G94-19 and G168 were developed using biolistic transformation and now they are
considered to be the best example of success in metabolite engineering.
2.2.4 Work done in o3fad (Omega3 fatty acid desaturase) locus
In 1992, workers have cloned o3fad from mutant of Arabidopsis thaliana (Arondel et al., 1992).
In Linseed, Vriental et al., (2005) identified two genes, Lu o3fadA and Lu o3fadB that encode
microsomal desaturases capable of desaturating linoleic acid. The deduced proteins encoded by
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these genes shared 95.4% identity. Low linolenic acid (18:3) is desirable in soybean oil to reduce
hydrogenation and trans-fat accumulation. Three independent recessive genes affecting omega-3
fatty acid desaturase enzyme activity are responsible for the lower 18:3 content in soybeans.
Using a candidate gene approach perfect markers for three microsomal omega-3 desaturase
genes have been characterized and can readily be used in for marker assisted selection in
breeding for low 18:3 by Pham et al. (2010). Yadav et al. (1993) reported the isolation of the
Arabidopsis microsomal 0-3 fatty acid desaturase gene by T-DNA tagging and the subsequent
use of its cognate cDNA to manipulate the levels of polyunsaturated fatty acids in transgenic
plant tissues. Several genes encoding o3fad have been isolated from Arabidopsis, soybean
(Glycine max), rapeseed (Brassica napus), tobacco (Nicotiana tabacum), tung (Aleurites fordii),
flax (Linum usitatissimum), perilla (Perilla frutescens), and rice (Oryza sativa) (Yadav et al.,
1993; Hamada et al., 1994; Kodama et al., 1997; Chung et al., 1998; Bilyeu et al., 2003; Dyer et
al., 2004; Vrinten et al., 2005). Arabidopsis harbors only a single copy of o3fad, which is
constitutively expressed (Beisson et al., 2003). In contrast, other plants, including soybean,
perilla, and flax, express additional o3fad gene(s) that are tightly regulated during seed
development (Chung et al., 1998; Bilyeu et al., 2003; Vrinten et al., 2005). The linolenic acid
level in Arabidopsis seeds is significantly reduced by a mutation of o3fad gene (James and
Dooner, 1990). Furthermore, a soybean mutant line A5, with low seed linolenic acid, shows a
deletion of the seed specific o3fad gene Gm o3fadA (Bilyeu et al., 2003). Two other seedspecific o3fad genes, Lu o3fadA and Lu o3fadB, control the amount of linolenic acid in flax seed
(Vrinten et al., 2005). Until now, the genes for both plastid and microsome-derived o-3
desaturases have been cloned from some plant species including Arabidopsis thaliana, soybean,
rapeseed, castor, mungbean, meadowfoam, tobacco, sesame, perilla, rice, wheat and maize, and
their molecular properties characterized (Arondel et al., 1992; Iba et al., 1993; Yadav et al.,
1993; Hamada et al., 1994; van de Loo and Somerville, 1994; Watahiki and Yamamoto, 1994;
Bhella and MacKenzie, 1995; Shoji, 1995; Lee et al., 1996; Kodama et al., 1997; Horiguchi et
al., 1998; Berberich et al., 1998).
2.2.5 Prospects and challenges to modify seed oil composition in sesame
A detailed knowledge of the metabolic pathways involved in the biosynthesis of fatty acids is a
prerequisite for genetic engineering of the seed fatty acid composition. Although the pathway for
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sesame is not documented, the fatty acid profile suggests synthesis via the known route common
to most major oil crops. Various genes encoding enzymes involved in fatty acid synthesis have
been isolated from the species and characterized. Yukawa et al. (1996) isolated two copies of the
9 stearoyl-ACP desaturase expressed in seeds. Recently, a gene encoding a microsomal 12
oleoyl-PC desaturase was simultaneously cloned by two research groups and characterized in
sesame (Jin et al. 2001). A detailed study of sesame fad2 promoter has been carried out by Kim
et al. (2008) reporting the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA
under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant.
Elsewhere, a 15 linoleoyl-PC desaturase cDNA has been isolated (GenBank Accession
E12718) although reports on their characterization are lacking. Shoji K. (1995) submitted the
sequence of omega-3 fatty acid desaturase mRNA, nuclear gene encoding chloroplast protein in
NCBI and the sequence was patented JP 1997065882-A 1 on 11-MAR-1997.The expression
pattern of the cloned genes is well- understood. Additional genes that would be useful in
modifying the fatty acid composition in sesame oil are the fatty acid desaturase 3. But to the best
of my knowledge, no fatty acid desaturase 3 from sesame have been characterized to date.
Table 1 shows a summary of target enzymatic steps that could be modulated by genetic
engineering to alter the seed fatty acid profile leading to accumulation of novel oils in sesame.
Using the various modification strategies (last column in Table 1), it is possible to specifically
vary the proportions of different fatty acids i.e. saturated or monounsaturated to polyunsaturated
acids and consequently create new oils that would be suited for other uses besides the common
use of sesame oil for frying and salad dressing. Considering that conventional sesame oil is
beneficial to human health, it seems appropriate that further improvement of quality should focus
on producing oils with new dietary, cosmetic, pharmaceutical and nutraceutical uses that would
embrace the known advantageous properties of the oil.
An important requirement for genetic modification of oil composition is the availability
of a strongly expressed seed-specific promoter. Besides, the promoter should display correct
temporal expression of the introduced genes since the synthesis of various storage products is
developmentally regulated. In sesame, fatty acid synthesis begins early (9 days after fertilization)
during seed development (Chung et al., 1995), and therefore, a late expressing promoter would
be unsuitable. Promoters to the seed expressed 9 and 12-desaturase genes have been cloned and
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their expression pattern characterized (Yukuwa et al., 1996; Jung et al., 2004). These promoters
are strong and turned on at the onset of lipid synthesis, making them ideal candidates for future
use in the engineering of sesame oil composition.
2.3 Biotechnological approaches for sesame
2.3.1 Tissue culture
The first report on tissue culture in sesame was that of Lee et al. (1985) on shoot tip culture
followed by George et al. (1987) using different explants. Effects of explants and hormone
combinations on callus induction was studied by Kim et al. (1987) in order to obtain herbicide
tolerant lines of sesame using in vitro selection. However, successful plant regeneration from a
herbicide tolerant callus was achieved by Chae et al. (1987). The effect of growth regulators on
organ cultures (Kim and Byeon, 1991) and their combination with cold pretreatment in anther
culture (Lee et al., 1988) of sesame revealed genotypic effects. In sesame, micropropagation is
achieved from shoot tip (Rao and Vaidyanath, 1997a), nodal explants (Gangopadhyay et al.,
1998) and leaf (Sharma and Pareek, 1998) cultures. Somatic embryos have been obtained from
zygotic embryos (Ram et al., 1990) and seedling-derived callus cultures (Mary and Jayabalan,
1997; Xu et al., 1997) with low plant conversion frequencies. Indirect adventitious shoot
regeneration from hypocotyl and/or cotyledon explants has also been reported but at low
frequencies (Rao and Vaidyanath, 1997b; Takin and Turgut, 1997; Younghee, 2001). Baskaran
and Jayabalan (2006) reported standardization of a reproducible micropropagation protocol in
cultivated varieties of sesame. Influence of macronutrients, plant growth hormones and genotype
on adventitious shoot regeneration from cotyledon explants was reported in sesame by Were et
al. (2006). High-frequency plant regeneration through direct adventitious shoot formation from
de-embryonated cotyledon segments of sesame was achieved by Seo et al. (2007).
Chattopadhyaya et al. (2010) established an efficient protocol for shoot regeneration from
sesame internodes using the transverse thin cell layer (tTCL) culture method. Abdellatef et al.
(2010) evaluated the in vitro regeneration capacity of sesame cultivars exposed to culture media
containing ethylene inhibitors such as cobalt chloride and silver nitrate, and found growth
promoting effects due to reduction in ethylene concentration followed by inhibition of ethylene
action.
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Review of literature
2.3.2 Genetic Transformation
The yield potential of sesame is very low when compared with major oil seed crops due to early
senescence and extreme susceptibility to biotic and abiotic stress factors including
photosensitivity (Rao et al., 2002). Wild species of sesame possess genes for resistance to biotic
and abiotic stresses (Joshi, 1961; Weiss, 1971; Brar and Ahuja, 1979; Kolte, 1985). However,
introgression of useful genes from wild species into cultivars via conventional breeding has not
been successful due to post-fertilization barriers. The only option left for improvement of sesame
is to transfer genes from other sources through genetic transformation techniques. The main
obstacle to genetic transformation is the recalcitrant nature of sesame to in vitro regeneration due
to overproduction of secondary metabolites (Baskaran and Jayabalan, 2006). There are very few
reports on shoot regeneration, with low frequencies in a few genotypes from cotyledon and/or
hypocotyl explants (Rao and Vaidyanath, 1997a; Taskin and Turgut, 1997; Younghee, 2001;
Were et al., 2006; Seo et al., 2007). Hairy root cultures using Agrobacterium rhizogenes have
been successfully established (Ogasawara et al., 1993; Jin et al., 2005). Although sesame has
been shown to be susceptible to Agrobacterium tumefaciens, no transformed plants were
recovered until last decade (Taskin et al., 1999).
For the first time, Yadav et al. (2010) reported conditions for establishing an A. tumefaciensmediated transformation protocol for generation of fertile transgenic sesame plants. This was
achieved through the development of an efficient method of plant regeneration through direct
multiple shoot organogenesis from cotyledonary explants, and the establishment of an optimal
selection system. Using hypocotyl and cotyledon explants from sesame seedlings, Chun et al.
(2009) established hairy root cultures and a peroxidase gene were cloned from the hairy roots.
The frequency of sesame hairy root formation was higher from hypocotyl compared to
cotyledonary explants. It was also observed that the peroxidase gene differentially expressed in
different tissues of sesame plants.
2.3.3 Domestication associated genes
In last few decades, there is an increase in studies on crop domestication with the development
of many informative molecular techniques (Zeder et al., 2006; Burke et al., 2007; Purugganan
and Fuller, 2009). Genome wide molecular markers have been used to investigate domestication
23
Review of literature
events successfully (Heun et al., 1997; Badr et al., 2000; Matsuoka et al., 2002; Morrell and
Clegg, 2007; Purugganan and Fuller, 2009). However, there is an increasing trend to study
evolutionary history of domestication through the selected loci (Sang, 2009; Gross and Olsen,
2010; Blackmann et al., 2011) and hence are successful in unraveling many domesticationassociated traits like Rht B1 and Rht D1 for plant stature and yield in wheat (Peng et al., 1999);
BoCAL for inflorescence morphology in cauliflower (Purugganan et al., 2000); fw2.2 for fruit
size in tomato (Frary et al., 2000); Waxy for biochemistry of kernel in rice (Olsen and
Purugganan, 2002) and tb1 for apical dominance in maize (Clark et al., 2004).
Domestication-associated genes offer an approach to reconstruct a crop’s domestication
history using associated trait with phylogeny and phylogeographic resolution. In sesame, various
decisive evidences have been provided to support that domesticated sesame arose from a
progenitor (Sesamum malabaricum) on the Indian subcontinent (Bedigian, 2003). However, no
domestication- associated genes have yet been identified in sesame. Moreover, variety of traits
getting evolved during domestication makes it more unclear as to what extent a single
domestication gene can be used to infer the domestication history of whole crop genome.
Through our study we aimed for the knowledge of domestication history of Indian sesame
germplasm using domestication- associated genes. Understanding the domestication genetics will
greatly facilitate the efforts of plant breeders, through discovery and utilization of rare but
potentially important alleles present in the genetic resources.
2.3.4 Molecular Marker
DNA markers provide a powerful tool for genetic evaluation and marker-assisted breeding of
crops and especially for cultivar identification. Genetic variability in sesame has also been
studied by molecular markers. Isozymes, RAPD, ISSR, AFLP, SSR, SRAP and EST SSR have
been used as molecular markers to date. The first molecular approach used to examine sesame
genetic diversity was performed by Isshiki and Umezaki (1997). They used isozymes for
determination of genetic variation in 68 accession of cultivated sesame (12 from Japan, 15 from
Korea, and 41 from Thailand). As a result, only one enzyme system isocitrate dehydrogenase
(IDH) showed variation. Bhat et al. (1999) evaluated genetic diversity of exotic sesame and
Indian germplasm via RAPD markers. They found a high level of genetic diversity but showed
24
Review of literature
that Indian germplasm has more genetic variation than exotics. A similar study with RAPD was
performed by Ercan et al. (2004) in Turkey and they showed important variation among
populations. To determine genetic variation in sesame populations, another study was done using
ISSR markers (Kim et al., 2002). They determined genetic diversity among 75 accessions of
Korean and exotic sesame. The accessions clustered into seven groups and showed that different
geographical origins are not completely distinct. Recent studies about sesame genetic diversity
were performed using the AFLP marker system. In 2006, Laurentin and Karlovsky (2006)
performed AFLP analysis to examine genetic relationships and diversity in sesame germplasm.
They used 32 sesame accessions from the Venezuelan germplasm collection which represents
five diversity centers. Consequently, they tried eight primer combinations and recorded 457
AFLP marker that were 93% polymorphic. They found high genetic variability which was
independent of geographical origin. Also in 2007, Ali et al. used AFLP for determining the
genetic diversity of 96 sesame accessions collected from different parts of the world and found
low (35%) genetic diversity. Except for genetic diversity studies, there is only one molecular
genetic study related with trait mapping in sesame. Uzun et al. (2003) identified a molecular
marker linked to the closed capsule mutant trait via the AFLP method. The closed capsule
mutant (induced via gamma ray irradiation) prevents shattering, a major problem for sesame
production. Scientists used 72 primer combinations and one closely linked AFLP marker was
found. This marker will be used for breeding to modify undesired side effects of the closed
capsule
mutation
by
marker
assisted
selection.
SRAP
(sequence-related
amplified
polymorphism) was used for the analysis of 67 sesame (Sesamum indicum L.) cultivars widely
used in Chinese sesame major production areas from 1950 to 2007 (Zhang et al., 2010). A total
of 561 bands were amplified using 21 SRAP random primer pairs, with 265 of them
polymorphic, resulting in a polymorphism ratio of 47.2%. From the study it was confirmed that
the genetic basis of Chinese sesame main cultivars is relatively narrow. Expressed sequence tagsimple sequence repeat (EST-SSR) markers were developed using publicly available sesame
EST data (Wei et al., 2008). A total of 1785 non redundant EST sets were assembled among the
3328 identified sesame ESTs. The primers were used successfully to amplify sesame, cotton,
soyabean and sunflower accessions and thereby were considered valuable for genetic analysis,
linkage mapping, and transferability study among oil plants. More recently, phenotypic, ISSR,
and SSR markers were employed to determine the genetic diversity and relationships among 20
25
Review of literature
commercially cultivated sesame genotypes representing different geographical regions of India
(Kumar and Sharma, 2011). A narrow range of genetic dissimilarity (0.01–0.12) with mixed
clustering was observed in them. Gebremichael and Parzies (2011) used ten Simple Sequence
Repeats (SSRs) markers to study patterns of genetic variation within and among 50 sesame
populations representing the existing Ethiopian collections. They reported that the genetic
divergence between populations was smaller than genetic divergence within. Also, both
landraces and cultivars were line mixtures and segregants of past outcrossing results.
Single nucleotide polymorphisms (SNPs) are very important for the genetic association
studies of complex traits. In oilseeds, SNP has been worked upon extensively in last decade.
Certain groups preferred whole genome scan over candidate gene approach. In soybean (Zhu et
al., 2003; Van et al., 2004; Schmutz et al., 2010) whole genome scan was carried out to find SNP
in both coding and non coding regions. On the other hand Jeong and Saghai Maroof (2004) and
Shi et al. (2008) worked on candidate genes related to resistance in soybean.
Similarly, in Brassica ,Trick et al. (2009) and Park et al. (2010) reported SNP present in
whole genome while Li et al. (2009) reported SNP markers in candidate genes controlling
flowering time and leaf morphological traits. In Arachis, Lopez et al. (2000) and Barkeley et al.
(2011) worked on finding SNP in candidate genes delta 12 fad and in fad2A respectively while
Alves et al. (2008) did the same in resistance genes. Bertioli et al. (2009) found SNP in whole
genome of Arachis. As of now, there is no work reported on SNP in sesame
2.3.5 SNP genotyping
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms
(SNPs) between members of a species. Several methods are employed to study SNP genotyping.
The increasing need for large-scale genotyping applications of single nucleotide polymorphisms
(SNPs) in model and nonmodel organisms requires the development of low-cost technologies
accessible to minimally equipped laboratories. The method presented here allows efficient
discrimination of SNPs by allele-specific PCR in a single reaction with standard PCR conditions.
A common reverse primer and two forward allele-specific primers with different tails amplify
two allele-specific PCR products of different lengths, which are further separated by agarose gel
electrophoresis. PCR specificity is improved by the introduction of a destabilizing mismatch
26
Review of literature
within the 3′ end of the allele-specific primers. This is a simple and inexpensive method for SNP
detection that does not require PCR optimization (Gaudet et al., 2008). Alelle specific PCR has
been reported to be used in fad2 gene for oleic acid increase in Arachis (Chen et al., 2010). SNP
genotyping has probably not been worked upon yet in sesame.
2.3.6 Mapping
A molecular marker is a site of heterozygosity for some type of silent DNA variation not
associated with any measurable phenotypic variation. Such a “DNA locus,” when heterozygous,
can be used in mapping analysis just as a conventional heterozygous allele pair can be used.
Because molecular markers can be easily detected and are so numerous in a genome, when they
are mapped by linkage analysis, they fill the voids between genes of known phenotype (Griffiths
et al. 2000). There has been a lot of work done in map construction in oilseeds. SNP has been
invariably targeted in mapping oilseed populations, as Song et al. (2004); Choi et al. (2007) and
Hyten et al. (2010) could map SNP in soyabean. Similarly, Gupta et al. (2004); Suwabe et al.
(2006); Schwarzacher (2008) and Trick et al. (2009) were successful in making QTL and linkage
maps of Brassica.
Fatty acid desaturases have been mapped in several crops successfully in last few years.
In maize, an allelic variant of the gene fad2 was successfully associated with increased oleic acid
levels (Belo et al., 2008). Same was reported previously in Brassica (Schierholt et al. 2000)
where high oleic acid mutation was mapped in Brassica napus. There are other reports of
mapping in Brassica like QTL analysis of seed oil and erucic acid which was done by Qiu et al.
(2006). This was followed by mapping QTLs controlling fatty acid composition in Brassica
napus by Long et al. (2011).
Much work has not been done on genetic mapping of sesame. Wei et al. (2009) first
reported sesame genetic linkage map based on F2 segregating population of an intraspecific
cross between two cultivars. Using three types of PCR-based markers, 284 polymorphic loci
including 10 EST-SSR marker, 30 AFLP marker and 244 RSAMPL marker, respectively, were
screened. Subsequently, a total of 220 molecular markers were mapped in 30 linkage groups
covering a genetic length of 936.72 cM, and the average distance between markers was 4.93 cM.
In this map, the linkage groups contained from 2 to 33 loci each and ranged in distance from 6.44
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Review of literature
cM to 74.52 cM. The genetic linkage map would help in tagging traits of breeding interest and
further aid in the sesame molecular breeding. There is no history of mapping SNP in sesame.
Moreover, association mapping of fatty acid desaturase genes have probably never been targeted
in sesame anywhere in the world.
2.3.7 Marker assisted selection
Uzun et al. (2003) were the first investigators to identify a molecular marker linked to an
agronomically important trait in sesame. They identified an AFLP marker linked to the closed
capsule mutant trait in sesame using a bulked segregant analysis (BSA) approach to segregating
progenies of a cross between the closed capsule mutant line ‘cc3’, and the Turkish variety
‘Muganli-57’. They tested a total of 72 primer combinations to screen for linkage to the trait, but
only one closely linked AFLP marker was identified. The linkage was confirmed by analyzing
the AFLP profile from single plants. They suggested that this marker had the potential to
accelerate breeding programmes aimed at modifying unwanted side-effects of the closed capsule
mutation through marker-assisted selection.
The first report on molecular tagging of the dt gene which regulates determinate growth habit
in sesame came from Uzun and Cagirgan (2009). The development of determinate cultivars has
become a high priority objective in sesame breeding programmes. They investigated RAPD and
inter simple sequence repeat (ISSR) techniques for the development of molecular markers for
this induced mutant characteristic. Using the F2 segregating population and bulked segregant
approach, two ISSR marker loci originating from a (CT) 8AGC primer were detected. They
proposed that this marker would potentially be useful for assisting sesame breeding programmes
through marker assisted selection and to facilitate the integration of determinate growth habit
into new genetic backgrounds.
2.3.8 Genomics
In spite of extensive efforts to develop new sesame varieties by conventional and mutational
breeding, the lack of a non-shattering sesame variety is one of the major barriers to obtaining
high yield of sesame seeds (Yermanos et al., 1972; Ashri, 1987). In addition, after oil extraction,
the remaining meal, corresponding to 50% of seed dry weight, is wasted or used for poultry feed.
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Review of literature
Therefore, identification of novel genes involved in the biosynthesis of sesame-specific flavor or
lignans, and a better understanding of the metabolic pathways from photosynthates towards oil
which can be stored and used, are desirable as aids to improve the quality and quantity of oil in
sesame cultivars. Expressed Sequence Tags (ESTs) generated by large-scale single-pass cDNA
sequencing have proven valuable for the identification of novel genes in specific metabolic
pathways. In order to elucidate the metabolic pathways for lignans in developing sesame seeds
and to identify genes involved in the accumulation of storage products and in the biosynthesis of
antioxidant lignans, Suh et al. (2003) obtained 3,328 ESTs from a cDNA library of 5-25 days old
immature sesame seeds. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. They carried out a
comparative analysis between developing sesame and Arabidopsis seed ESTs for gene expression profiles during development of green and non-green seeds. Analyses of these two seed
EST sets helped to identify similar and different gene expression profiles during seed
development, and to identify a large number of sesame seed-specific genes. Seed-specific
expression of several candidate genes was confirmed by northern blot analysis. Suh et al. (2003)
identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin
and sesamolin, and suggested a possible metabolic pathway for the generation of cofactors
required for synthesis of storage lipid in non-green oilseeds. Moreover, 41,248 expressed
sequence tags (ESTs) were obtained from cDNA libraries from 5–30 days old immature seeds
(Ke et al., 2011). Also, sesame transcriptomes from five tissues were sequenced using Illumina
paired-end sequencing technology (Wei et al., 2011). Amongst the annotated unigenes, 10,805
and 27,588 unigenes were assigned to gene ontology categories and clusters of orthologous
groups, respectively. In total, 22,003 unigenes were mapped onto 119 pathways using the Kyoto
Encyclopedia of Genes and Genomes Pathway database (KEGG). Furthermore, 44,750 unigenes
showed homology to 15,460 Arabidopsis genes based on BLASTx analysis against The
Arabidopsis Information Resource (TAIR, Version 10) and revealed relatively high gene
coverage. In total, 7,702 unigenes were converted into SSR markers (EST-SSR). Dinucleotide
SSRs were the dominant repeat motif, followed by trinucleotide , tetranucleotide, hexanucleotide
and pentanucleotide SSRs.
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Review of literature
Justification of work:
One of the main goals of biotechnology as applied to Sesamum is to manipulate metabolism in
their seeds for the production of improved products such as vegetable oils. Though a number of
sesame cultivars have been developed by conventional breeding methods, there is still demand
for the development of sesame varieties producing economically and nutritionally more valuable
oils. From a long-term perspective the present work, through development of SNP markers (in
three important genes of fatty acid biosynthetic pathway sad, fad2 and o3fad), followed by their
association mapping to the fatty acid composition will help in marker assisted selection. Since,
there is no past report of such work done in sesame; this study will benefit molecular plant
breeders in future.
Also, domestication leads to reduction of genetic diversity in crops. Reduced diversity in
crop cultivars is growing concern because such crops lose wider adaptability and consistent
productivity and, may become susceptible to newly emerging diseases and insect pests. To
counter this concern, there have been increased efforts to widen genetic base of the crop by
including wild relatives and other exotic or indigenous germplasm in breeding programmes.
Therefore, the study included to know the domestication history of Indian sesame germplasm
using domestication- associated genes. For this study, sad, fad2 and o3fad DNA sequence
variations were evaluated within and among four populations of sesame genotypes: wild species,
landraces, introgressed and cultivars. Till date, no such work probably has been reported.
Moreover, there is insufficient variability in the fatty acid composition of sesame oil.
This study will identify cultivars with high beneficial fatty acid content and a composition
different from the rest that will later be developed further by genetic modification for the
production of novel oils. With the help of biotechnological tools this work will be highly useful
to molecular plant breeders for producing sesame cultivars with appropriate quality of oil.
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Review of literature
The present work was therefore undertaken with the following objectives:
1. Identify SNPs that control the synthesis of fatty acids in sesame.
2. Map the SNPs responsible for fatty acid synthesis and quality in sesame.
3. Develop simple SNP detection procedures which can be used in future for crop improvement
programmes.
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