Molecular Plant Breeding, 2015, Vol.6, No.23, 1
-6
2
breeding methods for the improved resistance against
insects or weeds (Li et al., 2012). Genes encoding
antifungal proteins, such as endochitinase, β-1,
3-glucanases and glucose oxidase, or components of
signaling pathways involved in the defense response,
have for example been used to generate transgenic
plants resistant to various plant pathogens (Ijaz and
Khan, 2012). Indeed genetic engineering is the major
tool for improving crop yield and reducing insect pest
damage and other crop related problem. Finding best
cotton and other crop varieties for genetic transformation
is however vital for any success to use transgenic
insect or weed-resistant cotton (Furbank et al., 2015).
Selection of such best cotton varieties with respect to
transformation efficiency, acclimatization and with
better expression of the transgenic is therefore
required to save time, money and man power (Zhao et
al., 2011).
In this study we transformed three genes, CEMB
(Centre of Excellence in Molecular Biology) double
BT (Cry1Ac and Cry2A) and herbicide resistance
gene (cp4EPSPS).The main aim of this study was to
measure transformation efficiency, acclimatization
capacity and expression between two cultivars of
cotton and finding the best germplasm for further
modification of problematic traits.
1 Results
1.1 Regeneration efficiency of both cultivars
Approximately one thousand embryos of each cultivar
were transformed with both constructs (Bt and
cp4EPSPS gene) using the Agrobacterium mediated
transformation method. The overall regeneration
efficiency of FH-114 was 71% and CIM-598 was 62%.
After two month of continuous kanamycin selection,
efficiency was reduced to 20% for FH-114and 14% for
CIM-598.
1.2 Acclimatization therapy
After continuous one month kanamycin selection,
putative transgenic cotton plants were grown on
selection free medium for further one month. The
acclimatization therapy was started initially for 15 min
and then continuous increase of fifteen minutes up-to
one month. During the first five days, plants of both
cultivars were stable with little bit dehydrated
conditions. Between 10-20 days, further loss of 4%
plants was observed for FH-114 and 12% for
CIM-598.The remaining plants were shifted to soil.
FH-114 plants were more physically healthy than
CIM-598 at the end of acclimatization process.
1.3 Confirmation of putative transgenic plants
through PCR
Genomic DNA was isolated from both cultivars and
PCR was performed with gene specific primers. An
expected 450 bp product for Cry1Ac and a 500 bp
product for Cry2A were obtained in transgenic plants of
both cultivars (Figure 1 and 2). The cp4EPSPS gene
was also amplified with gene specific primers and a
350 bp amplification product was identified on an
agarose gel stained with ethidium bromide (Figure 3).
1.4 Transformation efficiency
After transformation into
˜
one thousand embryos, only
12 were positive for Cry1Ac in FH-114 and seven in
CIM-598. FH-114 was positive for nine plants and
CIM-598 for six as for as Cry2A was concerned.
Fifteen plants of FH-114 were positive for GTG and 8
of CIM-598 (Only best are shown in the Figure 1, 2
and 3). Transformation efficiency of FH-114 for Cry1Ac
was 1.2% and 0.7% for CIM-598. For Cry2A,
efficiency was 0.9% and 0.6%, respectively. GTG
transformation efficiency was 1.5% and 0.8% respectively
(Figure 5). Both varieties were transformed under similar
conditions and mediums.
1.5 Expression quantification through ELISA
The best positive plants for each gene were subjected
to total protein isolation. Protein specific antibodies
were used for protein expression in an ELISA
detection system for protein quantification (Muzaffar
et al, 2015). Highest Cry1Ac, Cry2A and GTG
amounts measured were 1.2, 1, 1.3 ng/µl respectively
for FH-114 and 0.9, 0.5, 0.9 ng/µl respectively for
CIM-598 (Figure 4).
1.6 Insects and Glyphosate analysis
Mortality percentage of
Heliothus armigera
2
nd
instar
larvae was variable after 30, 60 and 90 days due to
variation in the level of gene expression. At different
time points during plant growth, CIM-598 had lower
gene expression and was therefore more susceptible to
chewing insect. In comparison, FH-114 plants were
