Abstract: Agriculture is the backbone of economy of Pakistan
and cotton is the major agricultural export and supreme source of raw
fiber for our textile industry. To combat severe problems of insect
and weed, combination of three genes namely Cry1Ac, Cry2A and
EPSPS genes was transferred in locally cultivated cotton variety
MNH-786 with the use of Agrobacterium mediated genetic
transformation. The present study focused on the molecular screening
of transgenic cotton plants at T3 generation in order to confirm
integration and expression of all three genes (Cry1Ac, Cry2A and
EPSP synthase) into the cotton genome. Initially, glyphosate spray
assay was used for screening of transgenic cotton plants containing
EPSP synthase gene at T3 generation. Transgenic cotton plants which
were healthy and showed no damage on leaves were selected after 07
days of spray. For molecular analysis of transgenic cotton plants in
the laboratory, the genomic DNA of these transgenic cotton plants
were isolated and subjected to amplification of the three genes. Thus,
seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A
gene) and all twenty (EPSP synthase gene) were produced positive
amplification. On the base of PCR amplification, ten transgenic plant
samples were subjected to protein expression analysis through
ELISA. The results showed that eight out of ten plants were actively
expressing the three transgenes. Real-time PCR was also done to
quantify the mRNA expression levels of Cry1Ac and EPSP synthase
gene. Finally, eight plants were confirmed for the presence and active
expression of all three genes at T3 generation.