Molecular Plant Breeding, 2015, Vol.6, No.23, 1
-6
3
fully toxic (Figure 6). Transgenic cotton plants
expressing the cp4EPSPS gene were planted into field
containment and no application of weed removal was
applied for 3 months. After three months, when the
cotton field contained different kind of weeds,
glyphosate was sprayed at a rate of 1900 ml/acre.
Necrosis developed first on non-transgenic plants,
which were then died, and later weeds also. However,
in comparison to FH-114 plants, which survived,
CIM-598 plants were little stunted and dull even some
died (Figure 5).
Figure 1 Cry1Ac 450 bp amplification
A: M: 1 kb plus ladder, 1-6: positive plants; B: M: 1 kb plus
ladder, 1-6: positive plants.
Figure 2 Cry2A (500 bp) amplification with gene specific
primers for both cultivars
A: M: 100 bp ladder, 1-5: FH-114 Cry2A positive, N-negative
control; B: M: 100 bp ladder, 1-5: positive plants of CIM-598,
N: Negative control.
2 Discussion
There are many factors which hinder or cause
reduction in cotton production (Brévault et al., 2013)
but one of the very serious factor along with weeds is
insect pest attack (Muzaffar et al., 2015). Insects and
weeds are responsible for 20% & 25% loss in cotton
respectively (Awan et al., 2015). It has been well
documented that insect and weeds can be controlled
by gene over expression (Furlong et al., 2013). In this
study an effort was made to transfer two BT (Cry1Ac,
Cry2A) and one herbicide resistant gene (cp4EPSPS)
into two local cotton varieties named FH-114 and
CIM-598 through Agrobacterium-mediated transform-
ation. The purpose was not only to transform the
genes but mainly to compare these cultivars with
respect to their best gene expression for insect and
weed control.
Transformation of Bt and the cp4EPSPS gene was
done by using the shoot apex method as done by Latif
(Latif et al, 2015). Transformation efficiency of both
cultivars was different under similar condition.
FH-114 transformation efficiency was 1.2% and 0.7%
and CIM-598 for Cry1Ac and Cry2A was 0.9% and
0.6% respectively. For GTG, it was 1.5% and 0.8%,
respectively for each cultivar. Acclimatization
capacity of FH-114 was better than for CIM-598. This
may be due to the potential of the introduced genes
helping to withstand against worse climate conditions
(Lawlor and Tezara, 2009). These differences in
expression may due to T-DNA transfer rate, insertion
points of transferred genes, genetics and vir genes
activity during transformation (Rao et al., 2009).
Amplification of a 450 bp product for Cry1Ac, 500 bp
for Cry2A and 350 bp for GTG confirmed the
successful transformation into FH-114 and CIM-598
cotton plants (Kiani et al., 2013). The ELISA results
of Bt and GTG also confirmed their expression in
cotton plants. Quantification of Cry1Ac, Cry2A and
GTG was the maximum for FH-114 which was 1.2, 1,
1.3 ng/µl, respectively. CIM-598 was lower in
expression than FH-114 for all three genes. This may
be due to the reason of different germplasm may have
different expression capacity of foreign genes
(Pérez-Ruíz et al., 2014). The transgenic of both
cultivars were further subjected to insect bioassay and
glyphosate spraying. FH-114 plants showed more
toxic effect to insects compared to CIM-598 plants
and the negative control. CIM-598 was little bit
susceptible to insect attack and stunted growth was
observed after 1900 ml/acre spray of Glyphosate
(Roundup™). So 100% mortality of insect and dying
of weeds confirmed the action of BT and GTG in
cotton plants especially for FH-114. CIM-598 was
wavered in action despite harboring all three genes
with lower expression. The difference in expression
capacities of both cultivars may be the ancestors
