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Background  Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.    Results  Successful cloning was achieved by the amplification of  Cry2A ,  Cry1Ac , and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that  Cry1Ac  and  Cry2A  were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of  Cry1Ac - Cry2A  and  Tp2  showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of  Cry1Ac  and 0.568 μg/g tissue of  Cry2A  was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2 nd  instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.    Conclusion  Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton