## 4 Discussion
PCR analyses of transgenic cotton plants confirmed the amplification of a 565 bp product corresponding to Cry1 Ac gene (Patent No. K140649-aroA) verifying the presence of Cry1 Ac gene in the genome of cotton test plants (Figure 3). One month-old PCR positive-cotton plants were assessed relative accumulation of the CEMB-Cry1 Ac toxin using the ELISA assay. By 90 days post-transformation, Cry1 Ac toxin accumulation was three-fold higher in the cotton leaves expressing the transgene was driven by the CLCuKoV-BuC1 promoter, compared to CaMV35S driven expression in cotton plants (Figure 4). This is consistent with the results of a previous study, in which the CaMV35S promoter showed low Cry1 Ac toxin levels in transgenic cotton leaves [10]. inconsistent and/or to express lower than desirable levels of Cry9C in Bt transgenic cotton at certain times during the growing season [51]. In this study, the use of a begomoviral bidirectional promoter to enhance expression of the Cry1 Ac gene was explored for the first time in a field-ready cotton cultivar. The CLCuKoV-BuC1 promoter was shown to drive Bt toxin expression in green tissues of cotton, including in leaves, bolls, squares, and anthers, while at the same time Bt protein was minimally detectable in cotton roots (Figure 5), and represents a feature that may aid in alleviating public concerns about the environmental safety of transgenic crop protection strategies. The results of this study demonstrate that the CLCuKoV-BuC1 promoter is capable of robust expression of the Cry1 Ac gene and its accumulation in leaves and bolls of cotton plants for at least eight weeks post-transformation. The observation may pave the way to an understanding of plant physiology and development, particularly with respect to the mechanisms involved in modulating spatiotemporal variations in transgene expression, not only of Cry1 Ac, but of other transgenes, as well, during different plant growth stages. Conceptualization, A.A.S. and T.H. ; methodology, M.A.A., A.A.S., A.Q.R., J.K.B. and T.H. ; software, M.A.A., A.Q.R. and J.K.B. ; validation, M.A.A., A.A.S., A.Q.R. and T.H. ; formal analysis, A.A.S. and T.H. ; Investigation, M.A.A. ; resources, A.A.S., T.H. and J.K.B. ; data curation, A.A.S., A.Q.R. and T.H. ; writing--original draft preparation, M.A.A., A.A.S., A.Q.R., J.K.B. and T.H. ; writing--review and editing, M.A.A., A.A.S., A.Q.R., J.K.B. and T.H. ; visualization, M.A.A., A.A.S. and T.H. ; supervision, A.A.S. and T.H. ; project administration, M.A.A., A.S., A.Q.R. and T.H. ; funding acquisition, A.A.S., T.H. and J.K.B. All authors have read and agreed to the published version of the manuscript. The authors are indebted to other members of their laboratories for discussion and support. The authors gratefully acknowledge funding received from the Higher Education Commission of Pakistan-Indigenous 5000 PhD Fellowship Scheme (17-5-4 (Av4-170) HEC/Ind/Sch/2007) (PIN: 074-Av4-170) and International Research Support Initiative Program (1-8/HEC/HRD/2011/1755) (PIN: IRSIP 17 BMS 18). Not applicable. The authors are thankful to HEC, Pakistan for providing the IRSIP grant to [PERSON] for research visit to the laboratory of respected [PERSON], (The School of Plant Sciences, The University of Arizona, Tucson, AZ, USA). The acknowledgment is also due to [PERSON] (IAGS, University of the Punjab) for providing GV1301 strain of _Agrobacteriumtumefaciens_, to [PERSON] (University of Maryland, College Park, MD, USA) for assistance in construct preparation, to [PERSON] from Chak No.59/F-Hasilpur for providing tobacco (_N. tabucum_) seedlings and to [PERSON] (CEMB) for assistance in agroinfiltration assay. The authors declare no conflict of interest.