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. 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 2nd 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. 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.

Cotton crop is considered valuable for its fiber and seed oil. Cotton fiber is a single-celled outgrowth from the ovule epidermis, and it is a very dynamic cell for study. It has four distinct but overlapping developmental stages: initiation, elongation, secondary cell wall synthesis, and maturation. Among the various qualitative characteristics of cotton fiber, the important ones are the cotton fiber staple length, tensile strength, micronaire values, and fiber maturity. Actin dynamics are known to play an important role in fiber elongation and maturation. The current review gives an insight into the cotton fiber developmental stages, the qualitative traits associated with cotton fiber, and the set of genes involved in regulating these developmental stages and fiber traits. This review also highlights some prospects for how biotechnological approaches can improve cotton fiber quality.

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.

Actin dynamics is pivotal in controlling cotton fiber elongation and the onset of secondary wall biosynthesis. We report that overexpression of GhACTIN1 under fiber fiber-specific promoter, GhSCFP, improves cotton fiber length, strength, and micronaire value. However, the effect of transgene has a more positive effect on fiber strength and micronaire value than fiber length. F-actin quantification and cellulose contents measurement in transgenic developing cotton fiber during the elongation phase showed an increase of up to 8.7% and 4.7% respectively. Additionally, physiological factors such as water use efficiency showed no significant change in transgenic cotton lines, while stomatal conductance and photosynthetic rate were significantly increased. Moreover, agronomical data determined that lint percentage (GOT) and seed cotton yield also increased up to 4.6% and 29.5% respectively, in transgenic cotton lines compared to the control lines. Our data demonstrate that the GhACTIN1 gene is a strong candidate gene for cotton fiber and yield improvement.