Journal of Integrative Agriculture 2019, 18(10): 2205–2218

Available online at www.sciencedirect.com

ScienceDirect

REVIEW

Strategies to enhance cottonseed oil contents and reshape fatty

acid profile employing different breeding and genetic engineering
approaches

Iram Sharif1, Jehanzeb Farooq1, Shahid Munir Chohan1, Sadaf Saleem1, Riaz Ahmad Kainth1, Abid
Mahmood2, Ghulam Sarwar1

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

“edible oil” is also extracted from the whole cottonseed

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,
Received 12 March, 2018 Accepted 2 November, 2018
Correspondence Jehanzeb Farooq, E-mail: jehanzeb1763@
hotmail.com
© 2019 CAAS. Published by Elsevier Ltd. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
doi: 10.1016/S2095-3119(18)62139-2
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
2206 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218
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
 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218 2207

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
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

700 700
Production
Consumption
600 600

500 500
Production (×1 000 t)

Production (×1 000 t)

400 400

300 300

200 200

100 100

0 0
1968
1970
1972
1974
1976
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
2098
2000
2002
2004
2006
2008
2010
2012
2014
2016
18
19

Market year

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.
2208 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218

oil recovery is less as compares to 20 total seed cotton Min oil (%) Max oil (%)
production depicting the huge gap between total production 30

and oil recovery (Fig.  2). Under such circumstances,

25
an attempt has been made for quick access to available
information for successful hybridization.

Oil percentage (%)

20

5.1. Oil percentage diversity in wild cotton species 15

10
Oil percentage in 20 wild species of Gossypium ranged from
11.22 to 24.82% (Fig. 3). Oil contents were the highest in 5
G. lobatum (24.82%) followed by G. harknessi (24.22%)
while two species G. stocksii and G. somalense showed 0

um

um

e
the minimum percentage (11.22%). Delinted seed weight

ns
u
re

ce

ut

de
irs
o

ba
of these genotypes ranged from 43.33 to 54.54 mg/seed.

ba
rb

.h
er
.a

ar
G
.h

.b
G
Collectively seed oil contents and average seed weight were

G
the highest in G. arboreum and its races. Cultivated species

5.2. Oil percentage diversity in cultivated cotton Fig. 3 Oil percentage comparison in 20 wild species of
species Gossypium.

Glance at prevailing genetic diversity of cotton depicts a

while G. arboreum and G. herbaceum (desi cotton) are
wide range of oil content percentage in various genotypes
classified as diploid cotton. Oil percentage is comparatively
growing in different geographical regions. Out of 50 species
low in desi cotton as compare to tetraploid species. One of
of cotton only four are cultivated named: G. hirsutum and
the possible reasons of less oil percentage in desi cotton
G. barbadance (new world cotton) lies in tetraploid group
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)
35 000
studied nine cultivars of G. arboreum with oil content range
Cotton production (low bales)

30 000 varied from 14.4–18.7%.

25 000 Dani (1988) observed that seed oil contents were higher
in F1 ranging from 21.20 to 26.30% than parents having
20 000
range of 21.48–24.16% which shows that heritability for oil
15 000 content is higher. Bolek et al. (2016) studied 124 genotypes
10 000 of upland cotton for measurement of cottonseed oil, protein
5 000
content, and other quality determining parameters. In these
lines, oil contents ranged from 23.11 to 37.70% with an
0
average value of 31.0%. Genotypes with the maximum crude
1 400 oil were Delcerro, Acala 172, Paymaster 2379, Cirpan 60,
1 200 Fibermax 958, and Maydos Yerlisi having 33.89, 33.49,
Cottonseed oil (×1 000 t)

35.15, 37.7, 32.28, and 32.42% oil percentages, respectively.

1 000
In another study, Konuşkan (2017) investigated physic-
800 chemical properties of three cottonseed oil genotypes
600 naming Cukurova 1518, BA 119, and PAUM 15 with 17.8,
400
17.2, and 19.6% oil contents, respectively. PANUM15 was
found the most suitable one due to its highest oil contents
200
and improved quality for edible oil consumption. Singh
0 (1988) studied 162 entries of G. arboreum varnadan by using
India China USA Pakistan Brazil
Nuclear Magnetic Resonance (NMR). In this experiment,
the oil percentage range varied from 14 to 25.8%.
Fig. 2 Comparison of cotton and cottonseed oil productions
in five major cotton producing countries. Source, Cotton Oil content percentage in six Pakistani cotton varieties
Production by Agriculture Department, the United States. named CIM-534, CIM-496, AA-802, Z-33, Desi, and N-121
 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218 2209

Oil percentage in wild species (%)

30
25
20
15
10
5
0
m

rip m

um

tru ds

. k avid i
ii
um

um

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om sii

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en

i
. g ne

ca
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al

. c san

an

rid

at

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oc
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. b hyl

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ca

tia

us
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m
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al

gi
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.a

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ar
ai
fri

no

bo

iti

.t

sc

.t
.l

os
G

.r
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ap

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.d
G
.t
.a

ar

G
.s

.l
tz

.s
G
G

G
G

G
G

lo
G

G
G
G

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

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 thus
2210 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218

Table 1 Correlation of cottonseed oil contents with other traits

Sr. no. Character Species1) Associated trait Correlation Reference
1 Oil contents Asiatic cotton Seed cotton yield Positive Singh (1986)
2 Oil contents Asiatic cotton Lint index Negative Singh (1986)
3 Oil contents G. barbadense Linoleic acid Positive Gubanova (1989)
4 Oil contents G. barbadense Oleic acids, palmictic, stearic Negative Gubanova (1989)
5 Oil contents G. hirsutum Seed protein Negative Xiang et al. (1984)
6 Oil contents G. hirsutum Seed index Positive Taneja et al. (1993)
7 Oil contents G. hirsutum Harvest dates Negative Taneja et al. (1993)
8 Oil contents G. hirsutum Protein percent Negative Taneja et al. (1998)
9 Oil contents G. hirsutum Boll weight Positive Azhar and Ahmad (2000)
10 Oil contents G. arboreum Seed cotton yield Negative Rashmi et al. (2004)
11 Oil contents G. hirsutum Seed weight Positive Pahlavani et al. (2008)
12 Oil contents G. hirsutum Seed cotton yield Negative Ashokkumar and Ravikesavan
(2010)
13 Oil contents G. hirsutum Maturity of fiber Positive Turner et al. (1976)
14 Oil contents G. hirsutum Percentage of immature seeds Negative Turner et al. (1976)
15 Oil contents G. barbadense Development of okra type leaves Positive Liu et al. (1994)
and extended fruit bearing
branches
16 Oil contents G. hirsutum Seed vigor Positive Snider et al. (2014)
17 Oil contents G. hirsutum No. bolls/plant, boll wight, and highly positive Qayyum et al. (2010)
seed cotton yield
18 Oil contents G. hirsutum Lint index, staple length and fiber highly positive Qayyum et al. (2010)
fineness
19 Oil contents G. hirsutum Fuzzless seeds Positive Bellaloui et al. (2015)
20 Oil contents G. hirsutum Seed size Positive Pahlavani et al. (2009)
1)
G., Gossypium.

high palmitic oils would expense some predominant loss of
stearic acid (Liu et al. 2017).
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). KASǀǀǀ initiates the fatty acid de novo
biosynthesis by catalyzing the condensation conversion of
C2-CoA to C4 in plastids, further events be catalyzed by KASǀ
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 KASǀǀ, 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 KASǀǀ 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
 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218
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 F1 and F2 populations, 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
F2 suggest 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 BC2 populations. 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
2211
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.
2212 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218
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 engineering Genetic 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 silencing Different 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
 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218 2213

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 efficacy

Table 2 Genetic manipulation for improvement of cottonseed oil quality

Change in fatty acid contents in
Fatty acid Gene Expression Technology seed oil (%) Reference
Conventional seed Trangenic
Palmitic acid GhKAS2 Down-regulation Gene silencing 26 65 Liu et al. (2017)

GhSAD1 Down-regulation Gene silencing 26 15 Liu et al. (2002)

GhFAD2 Down-regulation Gene silencing 26 15 Liu et al. (2002)
Oleic acid BnFAD2 Transgene expression Transgenic 15 30 Chapman et al. (2008)
development
GhFAD2 Down-regulation Endogenous 15 40 Chapman et al. (2001)
expression
GhFAD2 Down-regulation Gene silencing 15 77 Liu et al. (2002)
Linoleic acid GhFAD2 Up-regulation Endogenous 55 80 Liu et al. (1999)
expression
Stearic acid GhSAD1 Down-regulation Gene silencing 2 40 Liu et al. (2002)
2214 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218
of 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).
Identification of QTLs/genes Oil 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). Stearoyl-
acyl 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 selection Cottonseed 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 CO2 and 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
 Iram Sharif et al. Journal of Integrative Agriculture 2019, 18(10): 2205–2218
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.
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