Molecular Plant Breeding, 2015, Vol.6, No.23, 1
-6
5
Figure 6 Bioassay of transgenic and non-transgenic control plants
A: Non-transgenic plant almost fully damaged; B: Transgenic FH-114 variety stayed healthy, no insect attack; C: CIM-598 transgenic
variety, a portion damaged by insects.
Table 1 Primers with sequences used in this study
Primer
Name
Sequence (5’-3’)
Product
Size
Cry2A-F
AGATTACCCCAGTTCCAGAT
500 bp
Cry2A-R
GTTCCCGAAGGACTTTCTAT
GTG-F
CCCTGGTGACAAGTCCATCT
350 bp
GTG-R
CTGCACACCCATCTCTCTGA
Cry1Ac-F
ACAGAAGACCCTTCAATATC
1 Kbp
Cry1Ac-R
GTTACCGAGTGAAGATGTAA
Transgenic plants were screened on the antibiotic
kanamycin (50 mg/ml) added to the medium and
cultivated in the presence of the antibiotic for
selection. Putative transgenic plants were the placed in
drug free medium for shoot and root formation as
reported by Muzaffar (Muzaffar et al, 2015).
4.3 Plant acclimatization
Two month old putative transgenic plants were shifted
to pots from tubes and were exposed to light for 15
min on the first day and then with 15 min increases to
light exposure/day over one month. During
acclimatization plants were exposed to light from 10
am in the morning and were watered daily.
4.4 Confirmation of gene insertion in plants
through PCR
Genomic DNA from putative transgenic plants was
isolated according to Lenin (2001). Reaction mixture
for PCR contained 100 ng DNA (2 µl), 10×PCR
buffer (2 µl), 2.5 mM MgCl
2
(2 µl), 1 mM dNTPs (2
µL),1 pico moles of each primer (2 µl) and 2.5 U Taq
DNA polymerase in a total volume of 20 µl. Gene
specific primers are shown in table 1. Reactions were
carried out in an ABI 9700 thermocycler under the
following conditions, initial DNA denaturation at
94
o
C for 5 min then 35 cycles of denaturation at 94
o
C
for 1 min, primer annealing at 51
o
C for Cry2A and
GTG, while 50
o
C for Cry1Ac for 1 min followed by
DNA extension at 72
o
C for 3 min. After amplification,
products were resolved on a 2% agarose gel and
visualized under ethidium bromide staining.
4.5 Enzyme Linked Immune SorbentAssay (ELISA)
An Envirologix Kit (Cat # 051) was used for the
Enzyme Linked Immune Sorbent Assay for Cry1Ac,
Cry2A and cp4 EPSPS expression. Leaf samples (1 g)
for transgenic cotton plants were subjected to grinding
and total crude protein was isolated by using protein
isolation buffer (0.5 M EDTA, Glycerol, 5 M NaCl, 2
M Tris-Cl, NH
4
Cl, PMSF, DTT).
4.6 Confirmation of Cry1Ac and Cry2A toxicity
through leaf bioassay
Transgenic plants were subjected to 2
nd
instar larva of
Helicoverpa armigera
to check their toxic level. A
total of three leaves from upper, middle and lower part
of transgenic cotton plants of 25, 55 and 85 day old
were exposed to
Helicoverpa armigera
larvae after
2-3 days exposure, insect mortality was determined
due to feeding larvae on transgenic and non-transgenic
cotton plant material.
