## 4 Discussion  
During the last two decades, commercial transgenic Bt cotton plants have been developed for insect pest control. These plants have descended from a common parent, \"Monsanto-531\", which was obtained by agro-transformation. The latter plants harbor the expression cassette containing the Cry1 Ac gene driven by the CaMV35S promoter. The CaMV35S drives the expression of Cry1 Ac protein in nearly all parts of the cotton plant, and it is by far the most widely-used promoter for plant transformation, especially for dicotyledonous species, including cotton [38; 39; 40]. Although the CaMV35S promoter has been highly useful for driving constitutive gene expression in transgenic plants, several studies have shown that expression levels may vary throughout the growing season, possibly due to interactions associated with particular physiological stages of plant growth and/or environmental factors [41; 42]. There are no available means of regulating candidate gene expression temporally by growth stage or spatially, in plant parts, given that cellular energy is required for all processes but cannot be harnessed and modulated [10; 43]. Thus,  
Figure 4: Temporal expression of Cry1 Ac gene in transgenic cotton plants. Error bars represent standard deviations. Figure 5: Spatial expression of Cry1 Ac in the anthers, bolls, leaves, squares and roots of transgenic cotton plants. identifying alternative promoters to drive gene expression in green tissues of plants has become increasingly desirable. _Agrobacteriumum_-mediated transient transformation offers a powerful tool for rapid evaluation of transgenes [33; 44; 45], promoters [19], and gene editing in crop plants [46]. In a previous study, the LIR of the CLCuKoV-Bu was characterized and shown to function as an effective bidirectional promoter in agroinfiltration assays using GUS as the reporter gene [19]. In this study, the LIR was subcloned from a previously available CLCuKoV-Bu genomic clone [29] and the bidirectional promoter activity to drive Cry1 Ac gene expression was verified using a transient assay, prior to stably transforming cotton plants with the promoter driving expression of the Cry1 Ac gene. The purpose of this study was to construct a cassette with a single gene, Cry1 Ac, and demonstrate the potential effectiveness of two begomoviral bidirectional promoters to drive robust Cry1 Ac expression transiently, and/or in transgenic cotton, compared to the CaMV35S promoter. To achieve robust expression in cotton, the binary vector pCAMBIA-1301, previously used to achieve high levels of gene expression in cotton, was selected as the plasmid vector for expressing Cry1 Ac driven by the bidirectional CLCuKoV-BuCl and CLCuKoV-BuV1 promoters by Agrobacteriumum-mediated transformation (Figure 1). The Cry1 Ac transient expression assays were carried out 72 h post-infiltration using a commercially-available immunos strip assay. No Cry1 Ac protein was observed in the CLCuKoV-BuV1-Ac agroinfiltrated leaves (Figure 2), a result that was inconsistent with a previous report. These results observed in this study system indicate that CLCuKoV-BuV1 is a weak promoter, or unable to drive Cry1 Ac expression in the agroinfiltrated leaves, and support those reported from a previous study that evaluated the robustness of the CLCuKoV-Bu bidirectional promoter [19]. PCR detection of insect resistant genes and event in transgenic cotton is most widely used an efficient and highly reliable method. It is based on the stable nature of the genomic DNA and sensitivity of the PCR methodology [47; 48]. PCR amplification from transgenic cotton plants using gene-specific internal primers of Cry1 Ac-CEMB has validated the results. A prerequisite for a successful regulatory framework and commercial approval process is the biosafety evaluation of transgenic events, with molecular characterization as an important component [49]. The expression of Cry1 Ac gene among cotton cultivars can vary throughout the growing season in Pakistan [8].