Turkish Journal of Biology
Turk J Biol
(2016) 40: 826-836
© TÜBİTAK
doi:10.3906/biy-1509-16
http://journals.tubitak.gov.tr/biology/
Research Article
Inducing osmotic stress leads to better genetic transformation efficiency in cotton
(Gossypium hirsutum L.)
1
2,
3
3
Surendra BARPETE , Allah BAKHSH *, Emine ANAYOL , Sancar Fatih ÖZCAN ,
1
4
1
Muhammet Cağri OĞUZ , Ömer Cem KARAKOÇ , Sebahattin ÖZCAN
1
Department of Field Crops, Faculty of Agriculture, Ankara University, Ankara, Turkey
2
Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Niğde University, Niğde, Turkey
3
Central Research Institute for Field Crops, Ministry of Food, Agriculture, and Livestock, Ankara, Turkey
4
Yapraklı Vocational School, Çankırı Karatekin University, Çankırı, Turkey
Received: 04.09.2015
Accepted/Published Online: 24.11.2015
Final Version: 21.06.2016
Abstract: The present study investigated the effect of different salts on cotton shoot regeneration and transformation efficiency. Twoday-old germinating embryos of a local cotton cultivar (SG-125) were pretreated with 50 mM each of NaCl, CaCl2, and KCl for 60 min.
The embryo explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid
pTF101.1 that carried the insecticidal gene (cry1Ac) under control of wound-inducible promoter (AoPR1) and bilanafos acetyl reductase
(bar) gene for plant selection. The salt-pretreated embryos showed maximum response on regeneration MS medium containing 0.50
mg/L 6-benzylaminopurine (BAP) and 0.10 mg/L indole-3-butyric acid (IBA), also supplemented with 5 mg/L bialaphos for in vitro
screening of the transformed plantlets. The primary transformants were further screened by molecular techniques for integration
and expression of the introduced gene. Maximum transformation efficiency (1.10%) was noted on KCl-treated explants compared to
nontreated (control) explants. In conclusion, pretreatment of explants with 50 mM KCl for 60 min induced positive effects and triggered
shoot regeneration in primary cotton transformants.
Key words: Cotton, in vitro, osmotic treatment, regeneration, transformation
1. Introduction
Cotton (Gossypium hirsutum L.) is an important fiber and
oil crop with great economic value worldwide. Genetically
modified cotton is cultivated on more than 24 million
hectares worldwide (FAO, 2014) and makes a significant
contribution to the income of 16.5 million small, resourcepoor farmers in developing countries (James, 2014; Bakhsh
et al., 2015). The global adaptation rates of genetic modify
cotton have increased 70% compared with conventional
cotton (James, 2014).
Recent trends in the development of agricultural crops
are inclined towards development of traits for improved
fiber-quality and resistance against insect pests, herbicides,
insecticides, and pathogens using genetic transformation
techniques (Mishra et al., 2003; Ahmed et al., 2014;
Barampuram et al., 2014). Different cultivars of cotton
have been bred by hybridization as most regeneration
protocols are genotype specific, and researchers need to
develop genotype-independent protocols for cotton. It
is well established that cotton is a very difficult plant to
regenerate (recalcitrant) under in vitro conditions (Han et
*Correspondence: [email protected]
826
al., 2009; Barpete et al., 2013), and a major limitation for
the genetic manipulation of cotton is the lack of efficiency
of existing transformation techniques(s) in creating
subsequently reliable and efficient regeneration.
Although many shoot-regeneration and genetic
transformation protocols have been developed in cotton
culture, the regeneration and transformation frequency is
very low and highly variable among cultivars (Satyavathi
et al., 2002; Mishra et al., 2003; Ahmed et al., 2014). It has
been proved in various crops that osmotic stress affects cell
growth as well as physiological metabolism during culture
growth (Huang and Liu, 2002; Huang et al., 2012).
Many solutions are accumulated during osmotic stress
on explants while treating or adding in a culture such as
alkaline solution (Daud et al., 2012), soluble sugars (Jimenez,
2005), free amino acids (Huang and Liu, 2002), or abscisic
acid (Lee and Huang, 2014). To date, there are few reports
on the signaling and metabolic pathways of carbohydrates
during cell culture (Huang and Liu, 2002; Huang et al.,
2006). In addition to serving as an energy source, sucrose
acts as a hypotonic and hypertonic osmotic agent in tissue
BARPETE et al. / Turk J Biol
culture medium, depending on concentration. Sucrose
uptake from the medium is hydrolyzed into glucose and
fructose for subsequent metabolism and transportation of
growth particles. Therefore, hyper/hypotonic solution can
fill or flow without bursting the cell wall through passive
transport, which uses no energy and flows through the
pores in cell membranes (Lee and Huang, 2014).
Furthermore, osmotic stress has been highly related
to shoot-direct organogenesis, but the underlying
molecular mechanism is still unclear (Huang and Liu,
2002). Although the influence of osmotic stress on plant
regeneration is partially clear from the literature (Huang
et al., 2006; Lee and Huang, 2014), no studies on cotton
have been reported. The present study was conducted to
investigate possible influence of osmotic stress on cotton
explants (embryos) before bacterial infection and its
effects on shoot regeneration and genetic transformation.
2. Materials and methods
2.1. Plasmid construction
In our previous studies, we excised promoter (woundinducible AoPR1) and cry1Ac gene fragments from different
source plasmids [pAoPR1-GUS-INT (Özcan et al., 1993)
and pKUC (Cheng et al., 1998), respectively] using specific
restriction enzymes and cloned in an intermediate vector
(pJIT61) containing CaMV terminator. The fragment
(AoPR1-cry1Ac-CamV Terminator) was further cloned
in pTF101.1 binary vector. The resultant binary plasmid
was named pAoPR1AcBAR.101 (Figure 1). The vector
pTF101.1 contains bar gene (used as plant-selectable
marker), which confers resistance against nonselective
bialaphos herbicide. The standard molecular approaches
of PCR and plasmid restriction analysis were followed to
confirm the construct (data not provided).
2.2. Seed sterilization and explant treatment
The seeds of Gossypium hirsutum L. ‘SG-125’ were
delinted using commercial sulfuric acid (95% H2SO4)
at a concentration of 100 mL/kg and washed with tap
water to remove traces of acid. The surface sterilization
was performed using 25% hydrogen peroxide (H2O2) and
0.01% Tween-20 for 20 min. This was followed by 3 × 5
min rinsing with sterile distilled water. The sterilized seeds
were blotted on tissue papers and germinated at 30 °C
under 16 h light (25 µmol m–2 s–1) photoperiod conditions
in a growth chamber. After 2 days of seed germination, the
mature embryos were excised under aseptic conditions,
and each lot of 1000 embryos was subjected to 50 mM
each of NaCl, CaCl2, and KCl for 60 min with subsequent
co-culture using Agrobacterium tumefaciens (OD0.6). The
control explants were not subjected to salt treatments and
were directly used in the transformation process. These
concentrations were optimized during earlier experiments
in the laboratory. Thereafter, salt-treated (1000 embryo
explants per treatment) and control explants were cultured
on MS regeneration medium (Murashige and Skoog,
1962) supplemented with 3% (w/v) sucrose and solidified
with 0.3% (w/v) Gelrite. Moreover, each MS regeneration
medium in the study contained 2 g/L activated charcoal.
2.3. Cotton transformation
The pretreated and control embryos were mixed with
Agrobacterium tumefacines suspension (LBA4404)
harboring plasmid pTF101AoPR1AcBar that contained
cry1Ac, under the control of wound-inducible promoter
(AoPR1), and co-cultivation was carried out on MS
medium supplemented with 0.1 mg/L kinetin at 28 ± 2 °C
for 3 days. After co-cultivation, explants were subcultured
on regeneration selection medium, i.e. MS medium with
0.50, 1.0, and 2.0 mg/L 6-benzylamino purine (BAP) with
a constant concentration of 0.10 mg/L indole-3-butyric
acid (IBA), also supplemented with 5 mg/L bialaphos for
in vitro screening of the transformed plantlets. Augmentin
(Amoxillin and clavulanic acid) was also added into MS
medium at rate of 500 mg/L to suppress bacterial overgrowth.
Subculturing was performed every 2 weeks. The elongated
and multiplied regenerated shoots were rooted on halfand full-strength MS medium supplemented with 0.10 and
0.50 mg/L IBA for rooting using 7–8 multiplied shoots per
treatment in three replications. Data on root induction (%),
mean number of roots, and mean root length per explant
were recorded after 3 weeks of culture.
Healthy plantlets from both control and pretreated
groups with well-developed roots and shoots were
selected for acclimatization. The plants were removed
and transferred to plastic pots containing peat moss or
peat moss + perlite (4:1). The acclimatized plants were
maintained under ambient daylight conditions at 19–23 °C
in a greenhouse. Transformation efficiency was calculated
by dividing total number of polymerase chain reactionpositive plants by the total number of explants used in the
experiments, and this was expressed as a percentage.
2.4. Molecular evaluation of putative transformants
The genomic DNA of putative transgenic plants was
extracted following the protocol described by Doyle (1991).
The standard molecular technique of PCR was carried out
to confirm gene presence in the plant genome. PCR was
carried out using the gene-specific primer to amplify the
internal fragment of cry1Ac (412 bp) and bar gene (310
bp) from putative transgenic plants. The amplification
reaction proceeded with gene-specific primers cry1Ac
(forward: 5’-ATGGACA ACCCAAACATC-3’ and reverse:
5’-TCATGTCGTTGAA TTGAATACG-3’) and bar gene
(forward: 5’-TGCACCATCGTCAACCACTA-3’ and
reverse: 5’-ACAGCGACCACGCTCTTGAA -3’). PCR
was performed at 94 °C for 4 min, 94 °C for 1 min, 52 °C
(for cry1Ac and 62 °C for bar gene) for 1 min, and 72 °C for
1 min followed by 34 cycles. The plasmid DNA was used
827
BARPETE et al. / Turk J Biol
Figure 1. Schematic representation of pTF101AoPR1AcBar plant expression vector containing cry1Ac gene under wound-inducible
(AoPR1) promoter. The plasmid pTF101.1 contains bar gene; therefore, bialaphos was used for plant selection at a concentration of
5 mg/L.
as positive control, whereas DNA from untransformed
plants was used as negative control. The amplified DNA
fragments were electrophoresed on 1.0% agarose gel and
visualized by ethidium bromide staining under ultraviolet
(UV) light. PCR was also performed with Phire Plant
Direct PCR kit (Thermo Scientific, cat. #F-130WH) for
this purpose by using plasmid DNA as positive control; a
small leaf disc from untransformed plants was the negative
control.
2.5. Bioassay test
To determine the efficacy of the introduced insecticidal
gene (cry1Ac) against targeted insect pests, cotton
828
primary transformants in T0 progeny were subjected to
leaf bioassays with second instar larvae of army worm
(Spodoptera exigua L.). Five fresh leaves from each plant
were taken and placed on wet filter paper in petri plates
accommodating one leaf per plate. Five 2nd instar larvae
prefasted for 4–6 h were released in each plate and allowed
to feed on the leaf. The data on insect mortality were
recorded up to 72 h. The entire study (experiment) was
repeated twice.
2.6. Statistical analyses
All experimental data were subjected to one-way analysis
of variance (ANOVA, SPSS for Windows v. 11., SPSS,
BARPETE et al. / Turk J Biol
USA), and the post hoc tests were performed using least
squares difference (LSD), Duncan’s, and Tukey’s b-tests.
Data given in percentages were subjected to arc sine (√X)
transformation (Snedecor and Cochran, 1967) before
statistical analysis.
3. Results
3.1. Seed sterilization and explant establishment
The cotton seeds surface-sterilized with hydrogen peroxide
(25% H2O2) for 20 min showed good results with no sign of
contamination in germinating embryos. The germinated
embryos were excised under aseptic conditions. The
optimization of bialaphos experiments with embryo
explants showed that 5 mg/L bialaphos concentration was
optimum for screening of primary putative transformants
that killed all nontransformed explants (Figure 2) under
culture conditions. The effect of plant-growth regulators
on cotton regeneration was obvious and significant
(Table 1) using the control group of explants (without salt
treatment). The regeneration selection medium containing
0.50 mg/L BAP, 0.10 mg/L IBA, and 5 mg/L bialaphos along
with 2 g/L activated charcoal resulted in maximum shoot
induction (93.30%), whereas minimum shoot induction
was observed using MS medium containing 2.0 mg/L
BAP and 0.10 mg/L IBA, respectively. The shoot induction
percentage ranged from 40.00% to 93.30%.
3.2. Salt-treatment effect
The cotton embryos were treated with 50 mM each
of NaCl, CaCl2, and KCl to induce osmotic stress;
furthermore, nontreated (control) and treated explants
were subjected to A. tumefaciens-mediated transformation.
The salt-treated embryos were cultured on MS medium
supplemented with 0.50 mg/L BAP plus 0.10 mg/L IBA
and 2 g/L activated charcoal (Figures 3a and 3b). The
analysis of variance showed significant effects (P ≤ 0.01)
of pretreatments causing variations in plant height, leaf
area, and fresh biomass among regenerated plants (Table
2). Maximum plant height (10.26 cm), leaf area (4.62 cm),
and fresh biomass (0.313 g) were noted on KCl-pretreated
embryo explants. However, shoot morphology and growth
were affected variably by the type of salt used during
pretreatment after culture on the regeneration medium.
Leaf shape morphology was also affected by pretreatment
of salts. However, maximum embryo survival was noted
on untreated (control) embryos. CaCl2-treated embryos
had the maximum number (6.30) of leaves per explant.
Explants treated with NaCl showed an appreciable
percentage of embryo survival, but leaves per plant, leaf
area, and fresh biomass were considerably reduced.
3.3. Rooting and acclimatization
The regenerated shoots (pretreated as well as control) were
rooted on 1 × and 1/2 × MS medium supplemented with
Figure 2. Optimization of concentrations of bialaphos using germinating embryos as
explant on MS regeneration medium. Data were recorded after 1 week. Concentration
was optimized to 5 mg/L of bialaphos.
829
BARPETE et al. / Turk J Biol
Table 1. Effect of PGRs on shoot regeneration from mature embryonic explants in cotton (Gossypiun hirsutum L. ‘SG-125’).
Plant growth regulator (mg/L)
BAP
IBA
Callus **
induction (%)
Shoot **
induction (%)
Primary #shoot
per plant
Secondary
shoot per plant
0.50
0.10
0.00
93.30 a
1.00
8.00 a
1.00
0.10
0.00
64.60 b
1.00
6.00 b
2.00
0.10
10.00
40.00 c
1.00
3.00 c
Note: ** = values shown in a column followed by different small letters are significantly different using LSD test at 0.01 level of
significance; # = nonsignificant.
various concentrations of IBA and showed significant
differences among treatments (P ≤ 0.01) in root induction,
number of roots per plant, and root length in medium.
The regenerated shoots cultured on half-strength medium
supplemented with 0.50 mg/L IBA resulted in maximum
root induction (85.70%) with 10.60 roots per plant and
6.80 cm of root length (Table 3; Figure 3c). The root length
per plant ranged from 1.20 to 6.80 cm.
The rooted plants from both experimental groups
were hardened in pots containing either peat moss or peat
moss + perlite (4:1). The pots containing peat moss–perlite
showed 100% acclimatization on the substrate. The peatmoss–grown plants had stronger roots that were about 1.5–
2.5 mm in thickness at their origin. The acclimatized plants
exhibited healthy growth and development, including
better plant height than peat-moss–acclimatized plants.
Figure 3. In vitro regeneration of cotton. (a) Cotton seed embryo in co-cultivation medium, (b) embryo-raised seedlings in
selection MS medium containing 5 mg/L bialaphos, (c) in vitro root regeneration from embryo-raised seedlings, (d) prehardening
in growth chamber, and (e) putative transgenic cotton plant in glasshouse.
830
BARPETE et al. / Turk J Biol
Table 2. Effect of osmotic stress (50 mM each) salt treatments on in vitro regeneration of cotton (Gossypium hirsutum L. ‘SG-125’).
Treatment
Embryo*
survived (%)
Plant height*
(cm)
Number of
leaf/plant
Leaf area*
(cm)
Fresh
biomass*(g)
Dry #
biomass (g)
Control
93.33 a
5.66 b
5.00 ab
1.91 b
0.167 b
0.023
NaCl
90.83 ab
6.93 b
4.33 b
3.48 a
0.199 b
0.025
CaCl2
82.50 b
6.30 b
6.33 a
3.65 a
0.200 b
0.026
KCl
87.50 ab
10.26 a
5.66 ab
4.62 a
0.313 a
0.035
Note: * = values shown in a column followed by different small letters are significantly different using Duncan’s test at 0.01 level of
significance; # = nonsignificant.
Table 3. Root induction in in vitro grown explants derived from embryonic shoot explants in Gossypium hirsutum L.
‘SG-125.’
MS medium
strength
IBA mg/L
Root*
induction (%)
Number* of
roots per plant
Root*
lengths (cm)
1 × strength
0.10
10.66 c
1.60 a
1.20 d
1 × strength
0.50
13.40 c
2.53 c
1.90 c
1/2 × strength
0.10
66.60 b
4.40 b
3.20 b
1/2 × strength
0.50
85.70 a
10.60 a
6.80 a
Note: * = values shown in a column followed by different small letters are significantly different using Duncan’s test at
0.01 level of significance.
Healthy shoot growth had a positive interrelationship
with better development of roots and good growth. The
well-acclimatized plants (Figure 3d) were shifted to a
greenhouse and subjected to various molecular analyses to
confirm integration and gene expression (Figure 3e).
3.4. Transformation efficiency and molecular evaluation
Variable transformation efficiency was noted when
embryos were treated with KCl, NaCl, or CaCl2 salts
compared to control (nontreated) embryos in the present
study. A maximum transformation of 1.10% was noted
on the primary transformants that resulted from KCl
treatment, while transformation efficiency in control
remained at 0.40% (Table 4). However, embryos treated
with NaCl and CaCl2 exhibited a transformation efficiency
of 0.70% each. The PCR results of primary transformants
showed amplification of the required bands of 412 bp of
the cry1Ac gene and 310 bp of bar. No amplification was
observed in nontransformed plants (control) (Figure 4).
3.5. Bioassay test
The efficacy of the insecticidal gene in primary
transformants was evaluated using leaf bioassays against
Spodoptera exigua larvae. The results showed that cry1Ac
gene expression was sufficient to confer full protection
against the targeted insect (Figure 5). The levels of cry
protein increased with the passage of time and conferred
mortality of insect larvae up to 72 h (Table 5). However,
the number of dead larvae was variable. The plants with
a lower toxicity level of targeted larvae (>40%) were not
selected for further studies. No significant mortality was
observed in larvae fed on nontransgenic cotton leaves.
4. Discussion
As osmotic stress has been highly related to direct shoot
organogenesis (Huang and Liu, 2002), and most of the
genetic transformation studies in cotton emphasize direct
shoot organogenesis using embryos as explants (Rao et al.,
2011; Bajwa et al., 2013; Bakhsh et al., 2015), we designed
the present study to determine transformation efficiency
in cotton by subjecting cotton embryos to salt stress.
The surface sterilization of cotton seed with hydrogen
peroxide (25%) resulted in zero contamination as well as
good germination in MS medium. H2O2 is an effective
sterilizing agent for surface sterilization of cotton seeds
and reportedly decontaminates seed from bacteria
and fungi. Barampuram et al. (2014) and Barpete et
al. (2015a) reported that H2O2 is an efficient sterilizing
agent for cotton. Two-day-old germinated embryos were
osmotically stressed using 50 mM NaCl, CaCl2, and KCl
following germination.
The salt-stressed embryos along with control were
subjected to Agrobacterium-mediated transformation
831
BARPETE et al. / Turk J Biol
Table 4. Genetic transformation frequency (%) in in vitro regenerated cotton plant.
Treatment
Total number of
embryo infected with
bacteria and cultured
Surviving explant Surviving explant Surviving plant Total number
in regeneration
in rooting
in hardening
of PCR-positive
medium
medium
media
plants
Transformation
frequency (%)
NaCl
1000
159
108
81
7
0.70
CaCl2
1000
124
95
73
7
0.70
KCl
1000
103
86
64
11
1.10
Control
(untreated)
1000
133
96
77
4
0.40
A
B
Figure 4. Molecular evaluation of primary cotton transformants (T0 progeny).
(A) PCR assay showed the amplification of required cry1Ac band (412 bp). Lane
1: negative control DNA from nontransformed plant, lane 2: DNA ladder 100 bp
(Thermo Scientific), lane 3: positive control (plasmid DNA), lane 4: empty well,
lanes 5–13–14: representative putative transgenic plants showing amplification of
cry1Ac, lane 14: DNA ladder 100 bp (Thermo Scientific). (B) PCR assay showed
the amplification of required bar fragment (310 bp). Lane 1: negative control DNA
from nontransformed plant, lane 2: DNA ladder 100 bp (Thermo Scientific), lane
3: positive control (plasmid DNA), lane 4: empty well, lanes 5–13: representative
putative transgenic plants showing amplification of bar gene, lane 14: DNA ladder
100 bp (Thermo Scientific).
following Gould et al. (1998), with some modifications.
The MS medium containing BAP and IBA had a
significant role in shoot induction in this study. Satyavathi
et al. (2002) and Mishra et al. (2003) emphasized in
vitro effects of plant growth regulators to shoot and root
differentiation in cotton plants. The salt-treated osmotic
832
stress exhibited variable positive effects on regeneration of
explants compared to the control. However, KCl-treated
explants regenerated efficiently and improved plant height,
leaf area, and fresh biomass in the culture condition.
Potassium (K) is an essential macronutrient for plants.
Current knowledge regarding biochemical and molecular
BARPETE et al. / Turk J Biol
Figure 5. Leaf biotoxicity assays conducted were on leaves of T0 primary transformants of cotton. The transgenic plants showed
appreciable level of resistance against Spodoptera exigua. (a, b) S. exigua larvae still alive and chewing leaf of nontransgenic
cotton plant, (c, d, and e) S. exigua larvae found dead when fed to transgenic leaf after 12, 24, and 72 h, respectively.
Table 5. Bioassay test for selection of Spodoptera resistance transgenic (T0) progeny at
different time regimes.
Transgenic (T0)
plant number
Insect died after (in h)
12 h*
24 h**
48 h**
72 h**
1
0.00 b
1.00 cde
3.00 abcd
4.33 a
2
0.66 b
3.33 a
4.66 a
5.00 a
3
0.33 b
0.66 de
1.33 dc
4.33 a
4
0.33 b
2.33 abcd
3.33 abc
4.66 a
5
0.33 b
2.66 abc
2.66 bcd
3.66 a
6
0.66 b
1.33 bcde
2.33 cd
3.33 a
7
2.00 a
3.33 a
4.00 abc
5.00 a
8
0.66 b
3.00 ab
4.33 ab
5.00 a
Control
0.00 b
0.00 e
0.00 e
0.00 b
Note: * and ** = values shown in a column followed by different small letters are
significantly different using Tukey’s test at 0.01 and 0.05 levels of significance, respectively.
833
BARPETE et al. / Turk J Biol
events that form the basis of the interaction between K
and primary metabolism is rudimentary. There is no lack
of mechanisms through which K could affect primary
metabolism. It is assumed that KCl treatment may have
affected transmembrane potentials and pH gradients,
long distance transport, ribosomal function, and changes
in enzyme activities in the treated plants, in agreement
with Marschner (1995). However, KCl treatment may also
enhance translocation of photosynthates and mobilize
stored materials, in agreement with Mengle (1980), and
has beneficial effects on ATP synthesis (Watanbae and
Yoshida, 1970) that improve transformation efficiency.
Although the maximum numbers of leaves per explant
were recorded on CaCl2-treated explants, it was not as
efficient in transformation as KCl. It is possible that
osmotic changes in the concentration gradient among
the salt water solutions may have positively affected
cotton regeneration. Molassiotis et al. (2006) and Lee and
Huang (2014) emphasized that osmotic stress regulates
endogenous levels of auxins interacting with cytokinin
and abscisic acid, positively affecting carbohydrate
metabolism to trigger shoot regeneration in rice. NaClregenerated embryos induced a reduction in number of
leaves per plant, leaf area, and fresh biomass compared to
KCl-treated plants in the study. NaCl also showed less of an
effect on plant regeneration in the present study. This can
be attributed to differing plant regeneration responses to
NaCl, as reported in previous studies conducted on quince
and tomato, respectively (D’Onofrio et al., 2002; Liza et al.,
2013), in which CaCl2 and NaCl negatively affected plant
regeneration.
MS medium was supplemented with activated
charcoal in shoot and root induction medium. Activated
charcoal may provide irreversible adsorption of inhibitory
compounds in the culture medium and substantially
decreases toxic metabolites, phenolic exudation, and
brown exudates accumulation (Thomas et al., 2008;
Barpete et al., 2015b).
Full- and half-strength MS medium were evaluated for
root induction. The results of the present study showed
that application of half-strength MS medium containing
0.50 mg/L IBA seemed more appropriate for rooting of
cotton shoots, as it exhibited greater effects in terms of
root length and lateral root development. These results are
in agreement with Ochatt et al. (2013) and Barpete et al.
(2014a, 2014b), who suggested half-strength MS medium
for efficient root induction. The present results are also in
agreement with Huang and Liu (2014), who showed that
IBA has a pivotal role in shoot induction of rice during
osmotic stress.
834
The suitability of the LB4404 strain harboring various
plasmids for the transformation of cotton has been reported
by various researchers who obtained 0.4%, 0.26%, 0.7%,
and 0.55%, respectively (Khan et al., 2011; Rao et al., 2011;
Bajwa et al., 2013; Bakhsh et al., 2014, 2015). In the present
study, we used 1000 embryo explants for each treatment;
the best and highest transformation (1.10%) efficiency in
primary transformants resulted from KCl treatment, while
transformation efficiency in NaCl and control remained
0.70% and 0.40%, respectively.
The putative transformed plants were subjected to
PCR to confirm the integration of cry1Ac and bar genes.
The amplification of 412 bp and 310 bp confirmed the
presence of cry1Ac and bar genes, respectively. To confirm
expression of insecticidal genes under wound-inducible
promoter, leaf biotoxicity assays were performed using
Spodoptera exigua larvae. The levels of cry1Ac protein
accumulated were sufficient to confer protection against
the targeted pest up to 72 h of incubation. These results are
in agreement with Breitler et al. (2004), Gulbitti-Onarci
et al. (2009), and Kumar (2009), who found increased
mortality of targeted pests by using wound-inducible
promoter in rice, tobacco, and cotton, respectively.
Leaf bioassays against lepidopterans in transgenic
Bt cotton expressing cry1Ac under the control of 35S
promoter are well documented (Tohidfar et al., 2008;
Bakhsh et al., 2010; Khan et al., 2013). The number of
dead larvae in each treatment was variable, and that can be
attributed to variation in expression of cry1Ac gene. The
variation in expression of cry1Ac protein in cotton leaves
has been reported by Chen et al. (2000), Mahon et al.
(2002), Bakhsh et al. (2011, 2012), and Khan et al. (2015).
In conclusion, KCl treatment may have affected
transmembrane potentials and pH gradients, long
distance transport, ribosomal function, and changes
in enzyme activities in treated plants, which may have
positively affected transformation, resulting in enhanced
transformation efficiency. However, the detailed
mechanism governing osmotic stress regulation of
cytokinin–auxin signaling and carbohydrate metabolism
needs further study.
Acknowledgments
The authors are grateful to the Scientific and Technological
Research Council of Turkey (TÜBİTAK) for financial
support of the research work (TÜBİTAK project no.:
111O254). The authors are also grateful to Prof Khalid
Mahmood Khawar for his valuable suggestions for the
improvement of the manuscript.
BARPETE et al. / Turk J Biol
References
Ahmed HA, Hajyzadeh M, Barpete S, Özcan S (2014). In vitro plant
regeneration of Iraqi cotton (Gossypium hirsutum L.) cultivars
through embryonic axis. J Biotechnol Res Cent (special
edition) 8: 90-94.
Bajwa KS, Shahid AA, Rao AQ, Kiani MS, Ashraf MA, Dahab AA,
Bakhsh A, Latif A, Khan MAU, Puspito AN et al. (2013).
Expression of Calotropis procera expansin gene CpEXPA3
enhances cotton fiber strength. Aus J Crop Sci 7: 206-212.
Bakhsh A, Anayol E, Özcan S (2014). Comparison of transformation
efficiency of five Agrobacterium tumefaciens strains in
Nicotianatabacum L. Emir J Food Agr 26: 259-264.
Bakhsh A, Emine A, Özcan SF, Hussain T, Aasim M, Khawar KM,
Özcan S (2015). An insight into cotton genetic engineering
(Gossypium hirsutum L.): current endeavors and prospects.
Acta Physiol Plant 37: 171-179.
Bakhsh A, Rao AQ, Shahid AA, Husnain T, Riazuddin S (2010).
Camv 35S is a developmental promoter being temporal and
spatial in expression pattern of insecticidal genes (Cry1ac &
Cry2a) in cotton. Aust J Basic Appl Sci 4: 37-44.
Bakhsh A, Shahzad K, Husnain T (2011). The variation in spatio
temporal expression of insecticidal genes in transgenic cotton.
Czech J Genet Plant Breed 47: 1-9.
Bakhsh A, Siddiq S, Husnain T (2012). A molecular approach
to combat spatio-temporal variation in insecticidal gene
(Cry1Ac) expression in cotton. Euphytica 183: 65-74.
Barampuram S, Allen G, Krasnyanski S (2014). Effect of various
sterilization procedures on the in vitro germination of cotton
seeds. Plant Cell Tiss Organ Cult 118: 179-185.
Barpete S, Aasim M, Khawar KM, Özcan SF, Özcan S (2014a).
Preconditioning effect of cytokinins on in vitro multiplication
of embryonic nod of grass pea (Lathyrus sativus L.). Turk J Biol
38: 485-492.
Barpete S, Anayol E, Ahmed HA, Özcan SF, Köm D, Delpasand S,
Aasim M, Khawar KM, Özcan S (2013). Optimization of invitro culture condition for genetic transformation of cotton
(Gossypium hirsutum L). In: The International Plant Breeding
Congress (IPBC), Antalya, Turkey, pp. 248.
Barpete S, Khawar KM, Özcan S (2014b). Differential competence
for in vitro adventitious rooting of grass pea (Lathyrus sativus
L.). Plant Cell Tiss Organ Cult 119: 39-50.
Barpete S, Oğuz MC, Özcan SF, Anayol E, Ahmed HA, Khawar
KM, Özcan S (2015a). Effect of temperature on germination,
seed vigor index and seedling growth of five Turkish cotton
(Gossypium hirsutum L.) cultivars. Fresenius Environ Bull 24:
1-7.
Barpete S, Özcan SF, Aasim M, Özcan S (2015b). In vitro high
frequency regeneration through apical shoot proliferation of
Hemianthus callitrichoides ‘Cuba’—a multipurpose ornamental
aquatic plant. Turk J Biol 39: 493-500.
Breitler JC, Vassal JM, Catala MDM, Meynard D, Marfa V, Mele E,
Royer M, Murillo I, San Sequndo B, Guiderdoni E et al. (2004).
Bt rice harbouring cry genes controlled by a constitutive
or wound-inducible promoter: protection and transgene
expression under Mediterranean field conditions. Plant
Biotechnol J 2: 417-430.
Chen S, Wu J, Zhou B, Huang J, Zhang R (2000). On the temporal
and spatial expression of Bt toxin protein in Bt transgenic
cotton. Acta Gossypii Sin 12: 189-193.
D’Onofrio C, Morini S (2002). Increasing NaCl and CaCl2
concentrations in the growth medium of quince leaves: II:
effects on shoot regeneration. In Vitro Cell Dev Biol - Plant
38: 373-377.
Daud M, Ali S, Variath MT, Zhu SJ (2012). Antioxidative enzymes
status in upland cotton callus culture under osmotic stresses. In:
The International Conference on Computational Techniques
and Artificial Intelligence (ICCTAI-2012), Penang, Malaysia,
pp. 279-282.
Doyle J (1991). DNA protocols for plants-CTAB total DNA isolation.
In: Hewitt GM, Johnston A, editors. Molecular Techniques in
Taxonomy. Berlin, Germany: Springer-Verlag, pp. 283-293.
Gould J, Magallanes-Cedeno M (1998). Adaptation of cotton shoot
apex culture to agrobacterium-mediated transformation. Plant
Mol Biol Rep 16: 1-10.
Gulbitti-Onarici S, Zaidi MA, Taga I, Ozcan S, Altosaar I
(2009). Expression of Cry1Ac in transgenic tobacco
plants under the control of a wound-inducible
promoter (AoPR1) isolated from Asparagus officinalis to
control Heliothis virescens and Manduca sexta. Mol Biotechnol
42: 341-349.
Han GY, Wang XF, Zhang GY, Ma ZY (2009). Somatic embryogenesis
and plant regeneration of recalcitrant cottons (Gossypium
hirsutum). Afr J Biotechnol 8: 432-437.
Huang WL, Lee CH, Chen YR (2012). Levels of endogenous abscisic
acid and indole-3-acetic acid influence shoot organogenesis in
callus cultures of rice subjected to osmotic stress. Plant Cell
Tiss Organ Cult 108: 257-263.
Huang WL, Liu LF (2002). Carbohydrate metabolism in rice during
callus induction and shoot regeneration induced by osmotic
stress. Bot Bull Acad Sin 43: 107-113.
Huang WL, Wang YC, Lee PD, Liu LF (2006). The regenerability
of rice callus is closely related to starch metabolism. Taiwan J
Agric Chem Food Sci 44: 100-107.
James C (2014). Global status of commercialized biotech/GM crops:
ISAAA Brief No. 49 ISAAA, Ithaca, NY, USA.
Jimenez VM (2005). Involvement of plant hormone and plant growth
regulators on in vitro somatic embryogenesis. Plant Growth
Regul 47: 91-110.
Khan GA, Bakhsh A, Ghazanfar M, Riazuddin S, Husnain T (2013).
Development of transgenic cotton pure lines harboring a
pesticidal gene (cry1Ab). Emir J Food Agr 25: 434-442.
835
BARPETE et al. / Turk J Biol
Khan GA Bakhsh A, Riazuddin S, Husnain T (2011). Introduction
of cry1Ab gene into cotton (Gossypium hirsutum) enhances
resistance against Lepidopteran pest (Helicoverpa armigera).
Span J Agr Res 9: 296-300.
Molassiotis AN, Sotiropoulos T, Tanou G, Kofidis G, Diamantidis
G, Therios I (2006). Antioxidant and anatomical responses
in shoot culture of the apple rootstock MM 106 treated with
NaCl, KCl, mannitol or sorbitol. Biol Plantarum 50: 61-68.
Khan ZA, Abdin MZ, Khan JA (2015). Functional characterization
of a strong bi-directional constitutive plant promoter isolated
from Cotton leaf curl Burewala virus. PLoS ONE 10(3):
e0121656.
Murashige T, Skoog F (1962). A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant 15:
473-97.
Kumar M, Shukla AK, Singh H, Tuli R (2009). Development of
insect resistant transgenic cotton lines expressing cry1EC gene
from an insect bite and wound inducible promoter. J
Biotechnol 140: 143-148.
Lee ST, Huang WL (2014). Osmotic stress stimulates shoot
organogenesis in callus of rice (Oryza sativa L.) via auxin
signaling and carbohydrate metabolism regulation. Plant
Growth Regul 73: 193-204.
Liza LN, Nasar ANM, Zinnah KMA, Chowdhury MA, Ashrafuzzaman
M (2013). In vitro growth media effect for regeneration of
tomato (Lycopersicon esculentum) and evaluation of the salt
tolerance activity of callus. J Agr Sustain 3: 132-143.
Mahon R, Finnergan J, Olsen K, Lawrence L (2002). Environmental
stress and the efficacy of Bt cotton. Aust Cotton Grower 22:
18-21.
Ochatt SJ, Conreux C, Jacas L (2013). Flow cytometry distinction
between species and between landraces within Lathyrus species
and assessment of true-to-typeness of in vitro regenerants.
Plant Sys Evol 299: 75-85.
Rao AQ, Bakhsh A, Nasir IA, Riazuddin S, Husnain T (2011).
Phytochrome B mRNA expression enhances biomass yield and
physiology of cotton plants. Afr J Biotechnol 10: 1818-1826.
Satyavathi VV, Prasad V, Gita LB, Sita LG (2002). High efficiency
transformation protocol for three Indian cotton varieties via
Agrobacterium tumefaciens. Plant Sci 162: 215-223.
Snedecor SW, Cochran WG (1967). Statistical Methods. Ames, IA,
USA: The Iowa State University Press, pp. 327-329.
Thomas TD (2008). The role of activated charcoal in plant tissue
culture. Biotechnol Adv 26: 618-631.
Mengle K (1980). Effects of potassium on assimilate conduction to
storage tissue. Ber Beutsch Bot GCs 93: 353-362.
Tohidfar M, Ghareyazie B, Mosavi M, Yazdani S, Golabchian R
(2008). Agrobacterium-mediated transformation of cotton
(Gossypium hirsutum) using a synthetic cry1Ab gene for
enhanced resistance against Heliothis armigera. Iranian J
Biotechnol 6: 164-173.
Mishra R, Wang HY, Yadav N, Wilkins T (2003). Development of a
highly regenerable elite Acala cotton (Gossypium hirsutum cv.
Maxxa)—a step towards genotype-independent regeneration.
Plant Cell Tiss Organ Cult 73: 21-35.
Watanabe HY (1970). Effects of nitrogen phosphorus and potassium
on rice in relation to ascorbic acid and NAA on phosphorylation
in rice in relation to photosynthetic rate of single leaves. Soil
Sci Plant Nutr 16: 163-166.
Marschner H (1995). Mineral Nutrition of Higher Plants. 2nd
edition. San Diego, CA, USA: Academic Press, pp. 889.
836