Strategies to enhance cottonseed oil contents and reshape fatty acid profile employing different breeding and genetic engineering approaches

lram Sharif, Jehanzeb Farooq, Shahid Munir Chohan, Sadaf Saleem, Riaz Ahmad Kainth, Abid Mahmood, Ghulam Sarwar

1 Cotton Research Station, Faisalabad 38000, Pakistan

2 Ayub Agricultural Research Institute, Faisalabad 38000, Pakistan

Abstract Cottonseed oil is the valuable byproduct extracted after seed cotton processing for lint. It confers a huge contribution to total vegetable oil production and ranked the 2nd to meet global edible oil requirements. Over centuries, breeders mainly focused to improve lint production and fiber quality. Now attention has been given to improve the cottonseed oil percentage,its functional and nutritional properties. However, these efforts are less than other major oilseed crops which left cottonseed oil market behind in term of consumer demand and kept cottonseed oil industry at vulnerable position. Considerable progress has been made to alter the relative percentage of fatty acid composition still intensified efforts have been required to meet the global oilseed demand. The objective of this review is to explore the cotton germplasm variation for seed oil carrying potential, its utilization in suitable breeding programs, seed oil biosynthetic pathways, major genes, and QTLs linked to quantity and quality enhancement of oil and deployment of modern genomic tools, viz., gene silencing and transgenic development to ameliorate its nutritional properties.

Keywords: biosynthesis, edible oil, gene silencing, oil quality, oleic acid, stearic acid

1. Introduction

Upland cotton is referred as “king of fibers” because of its immense importance in the social and economic affairs of more than 80 countries. In addition to fiber production,“edible oil” is also extracted from the whole cottonseed which is the 2nd major source of vegetable oil throughout the world (Ashokkumar and Ravikesavan 2013). Cottonseed oil is considered the world’s best edible oil for being free of cholesterol so termed as “heart oil”. Particular ratios of saturated and unsaturated fatty acids in cottonseed oil give it a peculiar taste and cooking quality (Agarwal et al. 2003).

Five major producers of cottonseed oil are China, the United States, India, the former Soviet Union, and Pakistan with 27, 12, 11, 10, and 9% production respectively that contribute 70% of global vegetable oil production (Song and Zhang 2007). Cottonseed oil ranks the 3rd in term of volume in the United States to fulfill the local fat demand and has become one of the main cottonseed oil exporters in the world market with 100 000 tons annual volume (Paterson 2009). In Pakistan, it meets 17.7% of edible oil requirement(Malik and Ahsan 2016).

During cotton ginning, fiber is removed from seed cotton and utilized in textile industry. The leftover seed is known as fuzzy cottonseed which may be used directly for feeding of cattle or processed further into different by-products:meal (45%), hull (26%), oil (16%), and linter (9%) while remaining 4% is lost during processing (Cherry and Leffler 1984). More than 10-15% of cotton grower’s income is expected to derive from these valuable byproducts. In last 15 years, global edible oil consumption has raised from 10.13 million tons in 2001-2002 to 20.08 million tons in 2014-2015. In Pakistan, the estimated demand for edible oil up to 2019-2030 will be 5.36 million tons out of which 1.98 million tons will be produced locally (Malik and Ahsan 2016). Therefore, it has become the global concern to enrich the oil potential of locally available cotton cultivars to meet the ever increasing edible oil demand.

Prospects are also increasing towards modification of cottonseed fatty acid profile to ameliorate its nutritional properties. Cottonseed oil rich in palmitic acid and other oxidative stable fatty acids (oleic and stearic acids) could cut off the need of partial hydrogenation of vegetable oils.It may also a potential replacement of palm oil import in Pakistan which is a significant contributor of palmitic acid for baking purpose and food processing (L’Abbé et al. 2009;Hayes and Pronczuk 2010).

In major oilseed crops, intensified efforts have been made to increase the nutritional value, quality, and oil percentage which left cottonseed oil far behind in term of consumer preference and kept cottonseed oil industry at vulnerable position (Paterson 2009). A little improvement in oil contents will be helpful to overcome the crises of edible oil in developing countries. Available literature regarding the inheritance studies of cottonseed oil contents and effective breeding methods is not enough for sizable improvement.This review addresses to organize literature on prevailing genetic diversity for utilization in cottonseed oil improvement and purposes future trends for production of designer oils having unique oil properties.

2. Nutritional importance of cottonseed oil

Nutritional value and industrial utilization of cottonseed oil can be derived by having glance at fatty acids profile which carries different carbon chain lengths and degrees of unsaturation. It is reported that consumption of one tablespoon of cottonseed oil provides 120 calories, 3.5 g saturated fatty acid along with vitamin A, K, and antioxidants(Malik and Ahsan 2016). It provides essential amino acids like lipase, phytase, and lecithin. It has prolonged shelf life than other seed oils due to higher amount of natural antioxidants and alpha-tocopherols (35 mg 100 g-1) in it,which promotes vitamin E activity (Agarwal et al. 2003).It is a good source of phosphoros (1%). It contains the modest level of cyclopropenoid fatty acids (0.5-1%) which are regarded anti-nutritional (Dowd et al. 2010). Genetic modification of minor components in cottonseed oil which have nutritional importance like vitamin E, neuroactive N-acylethanolamines, and phytosterols could increase cottonseed value significantly (Paterson 2009). It is a premium vegetable oil, little improvement in its composition can resolve the issues that would facility in expansion of its market value.

3. Fatty acid profile of cottonseed oil

Cottonseed oil consists of 65-70% unsaturated fatty acids while saturated fatty acids are 26-35%. Among unsaturated fatty acids, a major proportion (55%) is contributed by linoleic acid followed by oleic acid (15%) and linolenic acid less than 1%. The saturated fatty acids carry palmitic acid (26%)and stearic acid (2%) (Hui 1996). In addition to major fatty acids, cottonseed oil also contains little amount of several fatty acids (0.1-1% each) like myristic, lignoceric, arachidic,cis-vaccenic, sterculic, malvalic, palmitoleic, behenic, and α-linolenic acids (Dowd et al. 2010).

The unsaturated fatty acids are beneficial for health but deep frying for longer period convert them into short chain hydroperoxide, aldehydes, and keto derivatives responsible for off type flavor (Liu et al. 2002). Presence of higher percentage of saturated fatty acid (palmitic acids) is responsible for cottonseed oil oxidative stability during frying which compensates the instability of unsaturated fatty acids(Lindsey et al. 1990). Therefore, partial hydrogenation is used to increases oil stability by converting polyunsaturated fatty acids into monounsaturated and statured fats by keeping the oil in liquid state (Liu et al. 2002). Partial hydrogenation has its own side effects particularly produces trans fatty acids which uplift the level of LDL-cholesterol and reduce the HDL-cholesterol in blood serum (Mozaffarian et al. 2006). Monounsaturated fatty acid (oleic acid) is comparatively stable towards oxidative decomposition at high temperature. High oleic acid containing oils offer improved cooking stability for deep frying and are relatively more resistant to oxidative deterioration (Liu et al. 2009). Therefore, it could be increased at the cost of polyunsaturated fatty acids for improving quality.

Saturated fatty acids do not cause health risk by themselves but production of trans fatty acids as byproduct during the process of vegetable oils hydrogenation have significant cholesterol-raising properties (Mozaffarian et al.2007; De Souza et al. 2015). Cottonseed oil having elevated levels of palmitic acid is undesirable due to associated health risks (Qian et al. 2016; Zong et al. 2016). On the other side,enhancement in palmitic acid contents is necessary for oxidative stability of oil to make margarine, shortening, and confectionery products (Liu et al. 2017). Among saturated fatty acids, stearic acid is classified as desired dietary component as compared to palmitic acid and myristic acid as it does not enhance LDL-cholesterol but may have a role in lowering it (Bonanome and Grundy 1988; Dougherty et al. 1995). Stearic acid increases melting point of oil and enhances solidity and plasticity required for margarine and shortening (Tarrago-Trani et al. 2006) and provides the chance to substitute the extensive usage of hydrogenated oils (Liu et al. 2002).

4. Uses of cottonseed oil

Fig. 1 Trend of Pakistan’s cottonseed oil market regarding production and consumption in 1 000 t of cottonseed oil. Source,Department of Agriculture, the United States.

Cottonseed oil is also known as “naturally hydrogenated”due to balanced amount of stearic, oleic, and palmitic acids which ensures stable frying without additional processing(Malik and Ahsan 2016). Its flavor is neutral and maintains the natural flavor of the items to be cooked and used mostly in making edible products (Qayyum et al. 2009). Utilization of vegetable oil as food could be divided as cooking,marinades, pastries, margarines, dressing, shortening,whipped toppings, salads, icings, baked goods, and oriental dishes. By esterification of vegetable oils to sucrose,substitute of non-digestible fats is formulated. Beyond its consumption in food area, small volume of cottonseed oil is used in cosmetic products, specialty soaps, detergents,as lubricants, in pharmaceutical industry, for protective coating, fabric dispersants, in rubber manufacturing, inks,plastics, during the process of leather manufacturing and resins (Wakelyn and Chaudhry 2010). Cottonseed oil was also tested as biodiesel for single-cylinder engine performance for various diesel parameters including electrical efficiency, oil heating value, refractive index, acid value, iodine number, and saponification value. Based on its fuel properties, cottonseed oil proposed as the best possible green substitute for internal combustion engines with electrical generators (Bayindir 2010; Eevera and Pazhanichamy 2013).

5. Genetic variability for cottonseed oil contents

Cotton is mainly grown for fiber purpose, therefore, the scientist did not give much attention toward the improvement of quality and quantity of oil contents even though huge potential lies for the improvement of this trait. Despite the fact that cottonseed oil has major contribution toward edible oil requirements, its production is still lagging behind the consumption in Pakistan (Fig. 1). Genetic potential for oil contents is highly enough for sizable improvement in different geographic regions. At global level, cottonseed oil recovery is less as compares to 20 total seed cotton production depicting the huge gap between total production and oil recovery (Fig. 2). Under such circumstances,an attempt has been made for quick access to available information for successful hybridization.

5.1. Oil percentage diversity in wild cotton species

Oil percentage in 20 wild species of Gossypium ranged from 11.22 to 24.82% (Fig. 3). Oil contents were the highest in G. lobatum (24.82%) followed by G. harknessi (24.22%)while two species G. stocksii and G. somalense showed the minimum percentage (11.22%). Delinted seed weight of these genotypes ranged from 43.33 to 54.54 mg/seed.Collectively seed oil contents and average seed weight were the highest in G. arboreum and its races.

5.2. Oil percentage diversity in cultivated cotton species

Fig. 2 Comparison of cotton and cottonseed oil productions in five major cotton producing countries. Source, Cotton Production by Agriculture Department, the United States.

Fig. 3 Oil percentage comparison in 20 wild species of Gossypium.

Glance at prevailing genetic diversity of cotton depicts a wide range of oil content percentage in various genotypes growing in different geographical regions. Out of 50 species of cotton only four are cultivated named: G. hirsutum and G. barbadance (new world cotton) lies in tetraploid group while G. arboreum and G. herbaceum (desi cotton) are classified as diploid cotton. Oil percentage is comparatively low in desi cotton as compare to tetraploid species. One of the possible reasons of less oil percentage in desi cotton may be its smaller seed siize (Gopalakrishnan 2007;Pahlavani et al. 2009). Genetic potential of four cultivated cotton species is illustrated in Fig. 4. Sharma et al. (2009)studied nine cultivars of G. arboreum with oil content range varied from 14.4-18.7%.

Dani (1988) observed that seed oil contents were higher in F1ranging from 21.20 to 26.30% than parents having range of 21.48-24.16% which shows that heritability for oil content is higher. Bolek et al. (2016) studied 124 genotypes of upland cotton for measurement of cottonseed oil, protein content, and other quality determining parameters. In these lines, oil contents ranged from 23.11 to 37.70% with an average value of 31.0%. Genotypes with the maximum crude oil were Delcerro, Acala 172, Paymaster 2379, Cirpan 60,Fibermax 958, and Maydos Yerlisi having 33.89, 33.49,35.15, 37.7, 32.28, and 32.42% oil percentages, respectively.In another study, Konu?kan (2017) investigated physicchemical properties of three cottonseed oil genotypes naming Cukurova 1518, BA 119, and PAUM 15 with 17.8,17.2, and 19.6% oil contents, respectively. PANUM15 was found the most suitable one due to its highest oil contents and improved quality for edible oil consumption. Singh(1988) studied 162 entries of G. arboreum varnadan by using Nuclear Magnetic Resonance (NMR). In this experiment,the oil percentage range varied from 14 to 25.8%.

Fig. 4 Relative distribution of oil contents in cultivated species of Gossypium.

Oil content percentage in six Pakistani cotton varieties named CIM-534, CIM-496, AA-802, Z-33, Desi, and N-121 was found to be 15.15, 18.35, 16.02, 17.63, 15.06, and 16.63%, respectively. Fatty acid profile analysis depicted that unsaturated fatty acids were the maximum (73.17)in variety CIM-534 while CIM-496 carried the highest(627.8 mg kg-1) tocopherols contents. Genotypes having high tocopherols content would be more stable against oxidation damage and ultimately would be safe for prolonged storage and processing of cottonseed oil (Kouser et al.2015). Yield parameters of eight cotton genotypes were investigated for oil contents. Oil percentage ranged from 27.52 to 30.15% while the maximum percentage (30.15%)was reported in genotype SLH-279 followed by CIM-506(29.10%) while the minimum oil contents (27.52%) were found in cultivar CIM-499. Broad sense heritability and selection response were high (0.87 and 1.28%) for the oil contents that reveals its potential for improvement (Khan et al. 2010).

Twenty two genotypes were investigated along with three checks in three different environments and results proved that environment had significant effects on total variation in oil percentage. Percentage of mean oil content ranged from 20.14 to 25.51% among lines while 21.33 to 22.04%range was observed between checks. Highest oil contents(24.51%) were observed in line CNPA2011-5 followed by CNPA2011-5 and CNPA2011-14 with value of 24.51 and 23.81%, respectively. High genotype (G)×enviroment(E) interaction heritability was still high due to genotypic component with high genetic gain which shows selection could improve the oil content percentage (Carvalho et al.2017).

6. Association of seed oil contents with other parameters

During breeding for simultaneous improvement of cotton seed yield, fiber quality, and oil contents, breeders should be well aware of the direction and magnitude of genetic correlation between these economic importance traits which may assist in selection of breeding strategies. The association of oil seed contents was found highly positive with seed weight (Pahlavani et al. 2008), bolls/plant, cotton seed yield, lint index, staple length, and fiber fineness(Qayyum et al. 2010) while positive with linoleic acid(Gubanova 1989), seed index (Taneja et al. 1993), boll weight (Azhar and Ahmad 2000), fiber maturity (Turner et al.1976), okra type leaf, long fruiting branches (Liu et al. 1994),seed vigor (Snider et al. 2014), fuzzless seeds (Bellaloui et al. 2015), and size of seed (Pahlavani et al. 2009).

Many researchers reported contradictory results, e.g.,negative correlation of oil contents with lint index (Singh 1986), protein percent (Taneja 1998), and seed cotton(Ashokkumar and Ravikesavan 2010). Further studies found that oil seed contents are also linked negatively with palmitic, stearic, and oleic acids (Gubanova 1989), harvest dates (Taneja et al. 1993) and percentage of immature seeds(Turner et al. 1976). The results revealed that selection of high oil containing lines will assist in identification of genotypes with economically important morphological traits.The correlation of different traits with seed oil contents is described in Table 1. In different studies, it was reported that yield, nutritional value, and chemical composition seed oils are not only mainly affected by genetic constitution but also due to variable agroclimatic condition and geographic regions (Figueiredo et al. 2008). Further studies found that oil seed contents are also linked negatively with palmitic, stearic, and oleic acids (Gubanova 1989), harvest dates (Taneja et al. 1993) and percentage of immature seeds (Turner et al. 1976). It is also notable that palmitic acid contents in cottonseed oil reported to have positive correlation with oleic acid but not with stearic acid and thushigh palmitic oils would expense some predominant loss of stearic acid (Liu et al. 2017).

Table 1 Correlation of cottonseed oil contents with other traits

7. Cottonseed oil biosynthesis

In oilseeds, short chain saturated and monounsaturated fatty acids are produced in plastids and later then they moved toward to cytosol for further modifications and production of polyunsaturated fatty acids. Chiefly oleic acid (18:1), minor proportion of stearic acid, and palmitic acid are released in cytosol (Ohlrogge and Browse 1995). Fatty acid synthases(FAS) and β-ketoacyl ACP synthase (KAS) are complex proteins enzyme families having central role in metabolic pathways of fatty acid biosynthesis. Both the enzymes catalyze the addition of C2 units to the newly developing fatty acyl chain through Claisen condensation (Ohlrogge and Jaworski 1997). KAS??? initiates the fatty acid de novo biosynthesis by catalyzing the condensation conversion of C2-CoA to C4 in plastids, further events be catalyzed by KAS? activity through conversion of C4- to C14-ACP followed by production of palmitoyl-ACP which is the key branching point determinant of amount of palmitic, stearic, and oleic acids in final seed oil (Cahoon and Shanklin 2000).Palmitoyl-ACP could act as substrate for two pathways;Conversion of palmitoyl-ACP into stearoyl-ACP through catalytic activity of KAS??, and subsequent desaturation into oleyol ACP by Delta9 stearoyl-ACP desaturase(SAD) leads to accumulation of stearic acid in seed oil.Alternatively palmitoyl-ACP could be directly transformed into free palmitic acid by the activity of palmitoyl-ACP thioesterase (FatB). Competing action of KAS?? and FatB for accomplishment of palmitoyl-ACP precursor determines the different fatty acid contents in final seed oil profile (Martz et al. 2006; Pidkowich et al. 2007; Sun et al. 2014; Aslan et al. 2015).

Conventionally fatty acid biosynthesis in plastids terminates into oleic acid synthesis which is then provided to cytosol, where alteration of 18:1 occurs via two pathways.Carbon units may be added to elongate 18:1-CoA chain into 20:1-CoA to 22:1-CoA esters by the action of a fatty acid elongase, FAE1 (Kunst et al. 1992). Major pathway employs entry of oleic acid into the membrane bound lipid phosphatidylcholine (PC) apparatus followed by endoplasmic reticulum localized fatty acid desaturation by oleate desaturase (FAD2) and then linoleate desaturase(FAD3) to synthesize linoleic acid (18:2) and α-linolenic acid (18:3) respectively (McConn et al. 1993; Okuley et al.1994). Polyunsaturated fatty acids (PUFA) synthesized here through acyl editing then took part in triacylglycerols(TAG) assembly (Shanklin and Cahoon 1998; Zhang et al.2009; Bates and Browse 2012). The information about the key regulatory enzymes and the exact mechanisms involved in fatty acids acyl editing is lacking yet. However,in Arabidopsis thaliana, two genes LPCAT1 and LPCAT2 have been proposed to regulate the PUFA synthesis on PC(Bates et al. 2012).

8. Traditional and molecular breeding approaches for improvement of cottonseed oil quality and quantity

8.1. Traditional breeding approaches

Previously, improvement in fiber yield and quality were the major objectives of cotton breeders and efforts for meliorism of cottonseed oil and composition were neglected. Diversified application of cottonseed oil as food, feed, and biofuel along with competition created by other non-conventional oilseed crops has enhanced awareness for uplifting cottonseed oil percentage and quality. Now breeders have intensified their efforts regarding improvement in cottonseed oil and its composition without sacrificing lint yield and quality(Paterson 2009).

To address the problem in term of oil percentage along with quality improvement, different breeding strategies have been used with varying level of success (Cherry et al. 1981).In different crops, oil contents were elevated via accumulation of favorable genes in single line. Researchers used different breeding strategies for improvement of oil contents in cotton like pedigree selection, mass selection, backcross, and mutation breeding. However, pedigree breeding was the most effective one with 2-3% increase in oil content percentage(Singh and Narayanan 1991). Plant to row selection method was found effectively as compared to bulk selection for oil and lint improvement (Dani 1989). Maternal effects are involved significantly for determination of oil percentage in cotton (Dani and Kohel 1989) as seed and embryo size depend on maternal parent and both of these characters are linked positively with oilseed contents. For exploring the gene action in F1and F2populations, genetic components of variation were estimated and revealed that in F1, non additive gene action is prominent, however, additive gene action becomes more important in F2. High heritability estimates in F2suggest the implementation of recurrent selection for the improvement of oil contents in cottonseed. In another study,genetic architecture of cotton genotypes was investigated by using parents, F1, F2, BC1, and BC2populations. The results suggested that oil contents can be improved by 1-2 cycles of recurrent selection followed by pedigree method(Shanthi et al. 1999). In past, breeders mainly focused on the following aspects reported by Agarwal et al. (2003): (1)Reducing the hull and linters and increasing the kernel size; (2) enhancing oil contents and protein percentage; (3)uplifting of polyunsaturated fatty acids profile; (4) reducing Gossypol contents; and (5) decreasing the cyclopropene free fatty acids.

Conventional breeding approaches have been used for development of high oleic contents carrying genotypes in sunflower with oleate percentage 80-90%, soybean(83%), safflower (75%), rapeseed (75%), and olive (75%)(Murphy 2014). Efforts have been made for conversion of industrial linseed oil into edible oil (Green 1986). Application of mutation breeding for cottonseed oil improvement was followed by the utilization of naturally occurring mutation in other oil seed crops. Exploitation of naturally occurring mutations to improve cotton oil with high monounsaturated/polyunsaturated ratios may be preferable over genetic engineering tools due to amassed concerns of public against GMOs. In rapeseed naturally occurring mutations with reduced erucic acid have been selected by using mass screening (Downey and Craig 1964). Canola type was developed by accumulating such type of spontaneous mutation for development of cultivars with very low erucic acid (LEA)/low glucosinolate. In rapeseed, mutants for altered fatty acid profile were separated for enhancing the nutritional value of the rapeseed oil. The mutants having LEA and high oleic were among of them (Gupta 2015).However, the frequency of beneficial mutation is very low and occurs mostly in recessive form, therefore, masked by dominant genes. The frequency of effective mutation has been increased with the help of automated mutagenesis known as TILLING for manipulation of fatty acid profile(Xu 2010; Murphy 2011). Ethyl methanesulfonate (EMS)was used to develop mutant lines having less percentage of linolenic acid (Green and Marshall 1984). In addition to increase the oil percentage, chemical mutagenesis has also been exploited for development of reduced fuzzy lint on seed surface with increased seed oil percentage, which also increased oil extraction efficiency (Lowery et al. 2007). The pyramiding of these two favorable mutations may elevate the oil percentage up by 20% (Paterson 2009). Recently a naturally occurring mutant of fatty acid desaturase-2(FAD2-1D) gene has been identified in accession GB-713 of Gossypium barbadense mutant (FAD2), a potential candidate line for high-oleate trait with less linoleic acid in Gossypium hirsutum, however, identification of markers associated with this mutant locus and their deployment in breeding programs remains to be elucidated (Shockey et al.2017). Furthermore, due to narrow genetic base for fatty acid composition, traditional breeding approaches along with induced mutagenesis are not enough for significant improvement of cottonseed oil quality. Despite of many years’ efforts regarding cottonseed oil quality improvement,conventional breeding strategies exerted modest impact toward its quality enrichment.

8.2. Molecular approaches

It is reported that about 30 enzyme dependent reactions are responsible for conversion of acetyl-CoA (produced during photosynthesis) into seed oil. This complex network of enzymatic reaction makes the oil improvement difficult only via conventional breeding (Paterson 2009).Conventional and non-conventional breeding approaches make cottonseed oil improvement feasible in term of oil contents, fatty acids composition, and modification of other nutritionally important components.

Genetic engineeringGenetic engineering is a quick and direct breeding tool for alteration of fatty acids profile. This approach can be used in development of oil with desired fatty acid profile. Genetically modified plants have been available in different oilseed crops for desired alteration in fatty acids profile depending upon consumer and industrial demand (Voelker et al. 1996). Transgenic cotton plants were developed to enhance the oleic acid level via Agrobacterium transformation by inserting previously designed binary vector for suppression of endogenous enzyme fatty acid desaturase 2 (FAD2) by sub-cloning of fad2 mutant allele present in rapeseed. The FAD2 enzyme is characterized for conversion of oleic acid into linoleic acid, suppressing the functionality of this enzyme could lead to increased percentage of oleic acid in cottonseed oil. These authors successfully enhanced oleic acid contents from 21 to 30%in primary transformants while 47% content in the progeny which shows three time increase in oleic acid percentage than standard cottonseed oil (Chapman et al. 2001).

Work on engineering cotton plants through key genes for the production of novel fatty acids is trending (Singh et al. 2005). These unusual fatty acids have prospective to bring revolution in the industries by replacing the petroleum derived industrial feed (Cahoon and Kinney 2005). Use of cottonseed oil as an alternative candidate for fuel oil is increasing day by day. More than 300 different types of unusual fatty acid exist worldwide (Snapp and Lu 2013),oilseed crops are being metabolically engineered using conventional and traditional approaches for the development of non-native fatty acids intending for industrial utilization(Jaworski and Cahoon 2003; Singh et al. 2005; Scarth and Tang 2006; Cahoon et al. 2007; Napier 2007). Another unusual fatty acid, vernolic acid, is an epoxy fatty acid having multiple industrial uses and acts as raw material for the production of resins, glues, plastics, and polymers(Kleiman et al. 1965; Kuksis and Pruzanski 2017). Fatty acids having less or more than 16 and 18 carbon atoms along with double bonds on a typical sites of unsaturation and unique functional groups (like hydroxyl, epoxy, and acetylenic residues) characterized as unusual fatty acids(Abebe et al. 2017; Patel Alpa and Bhalerao 2017).

Oilseeds such as Ricinus communis, Bernardia pulchella,members of Apiaceae and Araliaceae families contain approximately up to 90% of species specific unusual fatty acids (Cahoon and Kinney 2005). Vernolic acid production in transgenic cottonseed oil has been attained by overexpression of a fatty acid desaturase (FAD2) coped with transgenic expression of C. palaestina derived Δ12-epoxygenase gene (Cpal2). Co-expression of Cpal2 and a modified copy of FAD2 in cotton seeds conditionally having more amount of linoleic acid substrate resulted into vernolic acid accumulation up to 17% in oil which is the highest level of any non-native fatty acid reported so far (Zhou et al. 2006). In future, cottonseed oil has a wide scope as an alternate of petroleum products due to diminishing non-renewable fuel resources. These biofuel resources are attractive for the countries lacking the liquid fossil fuels (Eevera and Pazhanichamy 2013). Although significant improvements have been made in major oilseed crops via traditional breeding practices by employing natural or induced variations, howerver, in cotton, due to polyploidy nature of cotton genome and lack of remarkable genetic variability, improvement of oil quality is difficult via conventional breeding (Liu et al. 2002). On the other hand,advancement in molecular biology, increased understanding of biochemical process and cloning of different genes used in oil biosynthesis pathway could be used efficiently for targeted alteration of cottonseed oil in term of improvement of its functional properties and nutritional value.

Gene silencingDifferent strategies have been used for downregulation of endogenous factors like antisense,ribozyme, co-suppression, and RNAi via introduction of gene homologous for silencing of targeted gene. Quality of cottonseed oil contents has been improved through modification of fatty acid profile by mediating HP gene silencing technique for downregulation of ghSAD-1 and ghFAD2-1 encoding stearoyl-acyl-carrier protein Δ9-desaturase and oleoyl-phosphatidyl choline ω6-desaturase.The targeted hairpin constructs were transformed in G. hirsutum. Gene silencing of ghSAD-1 enhanced the stearic acid percentage from 2-3% up to 40% while downregulation of ghFAD2-1 gene uplifted oleic acid contents from 15 to 77%. Besides this, palmitic acid percentage was lowered significantly. The successful silencing of both of two genes may assist in future for development of cultivars having different percentages of oleic, linoleic, stearic, and palmitic acids which can be utilized directly without processing in deep frying and margarine (Liu et al. 2002). Such type of materials may fulfill the promise of manufacturing cottonseed oil food stuff with improved nutritional value for the consumers.

RNAi technology was utilized for downregulation of phosphoenolpyruvate carboxylase 1 gene which dramatically increased oil contents up to 16.7% without any drastic effect on phenotype. Transcriptome analysis provided the evidence that downregulation of GhPEPC1 led to elevated expression of genes linked to triacylglycerol biosynthesis which played roles in biosynthesis of lipids.These findings suggest the utilization of RNAi technique for development of high oil contents carrying genotypes(Xu et al. 2016).

In another study, percentage of palmitic acid was enhanced from 25 to 51% by using RNAi mediating downregulation of KASII which enhanced C16 fatty acids up to 65%. This type of oil has high melting and boiling points and could be used for making confectionary products (Liu et al. 2017). Previous reports suggested that downregulation of stearoyl-ACP desaturase in rapeseed may enhance stearic acid percentage from 1.8 to 39.8%by weight due to decline in oleic acid contents (Knutzon et al. 1992).

Higher uptake of saturated fatty acids leads to increase cholesterol level especially low-density lipoprotein (LDL)which enhances the chances of cardiovascular disease(Baum et al. 2012). Many researchers have tried to improve cottonseed oil quality by altering the gene expression(Table 2). Liu et al. (2017) used RNA interference technique to suppress the expression of GhFAD2-1 and GhFATB genes encoding oleated esaturase and palmitoyl-acyl carrier protein thioesterase respectively and regulate the saturated and unsaturated fatty acids proportion. RNAi reduced palmitic acid and linoleic acid and improved oleic acid contents, however, affected the seed vigor and germination adversely.

Phytotoxins in plants especially gossypols in cotton have vital role in conferring resistance against pathogens but their presence in oil make it unfit for edible use as well as for animal feed. Gossypol in free form has more toxic effects than bound form and is highly undesirable in oil.Scientists attempted to develop glandless cotton but it became susceptible for insects attack and commercially failed. Recently, glandless genotypes have been developed by using RNAi technique. The RNAi disturbed the gossypol contents with the helo if δ-cadinene synthase genes during development of biosynthesis pathways (Sunilkumar et al.2006). Expression of two genes GhCPS1 and GhCPS2(cyclopropane synthase homologues) associated with the production of cyclic fatty acids (CFA) in stems, roots,and seed. Expression level of both two genes is similar in seeds which suggest the targeted silencing of these genes to reduce the accumulation of undesirable cyclopropenoid fatty acids (Yu et al. 2011).

Lysophosphatidic acid acyltransferase (LPAAT), a multigene family in higher plants regulate “Kennedy pathway”, which catalyze phosphatidic acids synthesis and their incorporation for the production of phospholipids, which are one of the key ingredients in biological membranes(Okazaki et al. 2006; Arroyo-Caro et al. 2013). LPAAT genes have also been found to involve in seed oil, protein contents, and fiber quality related attributes in G. hirsutum and G. barbadense, however, natural sequence variations exist among LPAAT genes in both species. Reinforced overexpression of LPAAT genes specifically Gh13LPAAT5 within G. hirsutum through biotechnology can enhance the production of fatty acids in cottonseed oil (Wang et al.2017). Genes encoding major enzymes responsible for seed oil biosynthesis have been cloned (Liu et al. 2002). Seed based modifications of endogenous expression of napin,lectin, phaseolin, and α-globulin genes have been deployed in cotton fatty acid profile engineering (Chapman et al. 2001;Zhou et al. 2006). The success in gene silencing assists in developing cotton lines with different percentages of oleic,linoleic, stearic, and palmitic acids which provoke the way toward synthesis of designer oil. The comparative study of haipin and antisense constructs revealed higher efficacyof hpRNA-mediated silencing to produce more silenced individuals as compared to antisense technique without effecting seed germination and plant growth. This could be the remarkable contribution in crop plants particularly cotton which carries lesser transformation efficiency to build larger transgenic population (Liu et al. 2002).

Table 2 Genetic manipulation for improvement of cottonseed oil quality

ldentification of QTLs/genesOil biosynthesis is the quantitative trait and controlled by many genes. Knowledge of QTLs associated with cottonseed oil quantity and quality may assist in its improvement. Despite of vast utility of cottonseed oil, genetic bases of oil and protein are not investigated thoroughly. Seventeen QTLs related to oil contents and 22 linked to protein contents were mapped on 12 chromosomes while three QTLs were identified for gossypol contents in backcross progeny of American cotton and Pima cotton by constructing genetic map of 392 SSR markers. It provided clear evidence that seed oil contents correlated negatively with protein contents as eight QTLs for oil and protein contents were localized in same region expressing additive effects opposite to each other.Among these, QTLs located on chromosomes 12 and 21 at the distance of 127 cM and 54.2-60.5 cM contributed 22-26% toward phenotypic variation. Therefore, they are known as major QTLs for oil contents (Yu et al. 2012). In another study, one significant QTL (qOP-D8-1) related to kernel oil contents was identified by using SSR markers(BNL2860_190 and NAU1369_400) (Song 2007). An IF2 population derived by intraspecific crossing of two hybrids was used to map the QTLs involved in synthesis of oil contents from embryo and female plant genome.Four QTLs named: qOC-18-1, qOC-18-2, qOC-LG-11,and qOC-22 were identified (Alfred et al. 2012). Stearoylacyl carrier protein desaturase (SADs) regulates the conversion of saturated fats into unsaturated fats and resultantly determines the fatty acid profile. Eighteen SADs genes were identified in TM-1 accession of G. hirsutum.Bioinformatic and phylogenetic analyses divided these genes into two different classes: GhSAD2 and GhSAD4.Expression of GhSAD2 and GhSAD4 was found 20-35 days after anthesis in developing ovule with varying degree of expression pattern in low and high yielding cotton genotypes.Association analysis revealed that GhSAD4-At expression regulates linoleic acid and oleic acid in cottonseed oil (Shang et al. 2017).

Marker-assisted selectionCottonseed oil being a polygenic trait and its expression is significantly influenced by the environmental factor, therefore, it becomes difficult to judge such traits just on the bases of morphological descriptor whose expression varies in different environments. Along with conventional breeding approaches, use of molecular genetics facilities the mapping of genes/QTLs involved in biosynthesis of oil contents and speeds up the selection process. Different molecular markers linked to QTLs could be used for direct selection that will not only improve selection efficiency but also save time and tedious working (Gopalakrishnan 2007). Linkage maps of cotton were constructed by using 228 SSR markers by utilizing 180 cotton genotypes. Fifteen SSR markers were mapped on 10 chromosomes (six from A genome and four from D genome) of upland cotton (Liu et al. 2015).A study investigated genetic diversity for oil contents and protein% in eight genotypes of cotton viz., CIM-70, H-449-3,FH-87, LRA-5166, NIAB-78, MNH-93, Lumain-1, and L.229-29-71 by using SSRs. The similarity index was the maximum (85.29%) between MNH-93 and LRA-5166 while two genotypes (CIM-70 and H-499) were founds dissimilar by showing the least similarity index (52.94%). Molecular markers for different traits may be used for increasing breeding efficiency by reducing backcross generations and also avoids tedious phenotypic selection (Qayyum et al.2009). Marker-assisted selection could speed up the varietal development program.

8.3. Foliar application of exogenous material

Foliar application of K increased oil contents significantly than non-treated cotton plants, this reveals the importance of potassium in boosting biochemical reaction. Potassium raises rate of photosynthesis in the leaves, accumulation of CO2and facilitates the movement of carbon (Sawan et al. 2006). In another study, application of Zn improved oil content per hectare as compared to untreated plants.Spraying of another micronutrient P also improves the yield contents by producing high-quality seeds. These findings ensure the significance of P as coenzyme involved in energy transfer process (Cakmak 2000).

9. Conclusion

To meet the global oil demand, it is necessary to improve the local varietal potential by utilizing high oil content carrying lines especially in major cotton growing regions.Recent trends focusing the improvement in fatty acid profile of cottonseed oil intended to fulfill the demand of consumers and industries. In tetraploid cotton, oil percentage varied in cultivars growing different agro ecological zones representing a huge a gap to be covered yet. The combination of appropriate conventional and biotechnological tools can be applied successfully to improve the cottonseed oil percentage, its nutritional value and for widespread industrial application. Conventional approaches could be used to improve the oil percentage to some extent by using natural variability and induced mutation. However, biotechnological tools have widened the breeder’s vision for attaining the desired percentage of fatty acid profile or improvement of nutritional value particularly by genetic engineering approach. Gene silencing produced fruitful results for endogenous suppression of targeted genes without affecting plant growth. Marker-assisted selection could be helpful in pyramiding of genes/QTLs linked with oil biosynthesis and quality improvement. Among agronomical practices foliar application of Zn, P, and K could increase oil percentage in cotton. The suitable combination of these approaches could revolutionize the cottonseed oil economy worldwide.