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Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to the non-transgenic control plants. Evaluation of effect of temperature and soil nutrients availability on transgene expression in cotton plants was done at two different cotton growing regions, Multan and Lahore, Pakistan and results suggested that despite of higher temperature in Multan field, an increased level of Cry and cp4EPSPS proteins was recorded due to higher soil organic matter availability compared to Lahore field. Before commercialization of any transgenic variety its biosafety study is mandatory so, a 90 days biosafety study of the transgenic cotton plants with 40% transgenic cottonseeds in standard diet showed no harmful effect on wister rat model when studied for liver function, renal function and serum electrolyte. [ABSTRACT FROM AUTHOR]