2. REVIEW OF LITERATURE
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).
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.
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:
220.127.116.11 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.
18.104.22.168 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 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.
22.214.171.124 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).
126.96.36.199 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 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 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 mono- and polyunsaturated fatty acids. The oil is nutritionally valuable as a source of linoleic acid and linolenic acid which are essential to humans.
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).
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 high-oleic 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 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, co-suppression 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 high-stearate 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 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 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 seed-specific 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 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 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.
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. tumefaciens-mediated 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 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 domestication- associated 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 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 tag-simple 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 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 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.
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 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.
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. 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.
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.
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.