Bt Research 2024, Vol.15, No.2, 96-109 http://microbescipublisher.com/index.php/bt 101 Zhao et al. (2023) compared the growth and development of transgenic plants (TG) expressing three transgenes (AVP1, OsSIZ1, and Fld) and single transgene (AVP1, OsSIZ1, or Fld) with non-transgenic wild-type (WT) control plants. The results showed that the expression of the three transgenes significantly enhanced plant growth performance, including higher biomass and stronger root development. The transgenic plants also exhibited significantly better tillering, internode number, and internode length compared to the wild-type control plants. Moreover, the multi-gene engineering approach significantly improved plant growth and development, providing a new strategy for increasing crop yield. Furthermore, the integration of Bt genes into crops has shown substantial benefits in terms of yield increase and reduction in insecticide usage. Bt crops like Bt corn and cotton have demonstrated high levels of protection from major insect pests, leading to increased yields and reduced reliance on chemical insecticides. By enhancing plant health and yield through gene stacking, the overall sustainability and productivity of agricultural systems can be improved, ensuring food security and environmental conservation. 5 Case Studies of Successful Gene Stacking 5.1 Examples from major Bt crops Gene stacking has been widely adopted in major Bt crops such as maize, cotton, and rice to enhance resistance against pests and diseases. For instance, in rice, the introduction of two Bacillus thuringiensis (Bt) genes through sexual crossing has shown significant success. The resulting transgenic rice lines exhibited higher resistance to pests like the striped stem borer and leaffolders compared to single-gene lines, demonstrating the commercial potential of this approach (Yang et al., 2011). Similarly, in maize, the use of targeted genome editing techniques such as zinc finger nucleases (ZFNs) has enabled the precise integration of multiple herbicide resistance traits, resulting in crops that can withstand various herbicides and thus offer better weed management (Ainley et al., 2013). In cotton, the combination of Bt genes has been shown to provide durable resistance against a range of pests. Field trials conducted over several years have demonstrated that stacked Bt cotton varieties not only offer enhanced pest resistance but also maintain agronomic and phenotypic characteristics similar to their single-trait counterparts, thereby ensuring no compromise on yield or quality (Figure 3) (José et al., 2020). These examples underscore the effectiveness of gene stacking in major Bt crops, providing a robust strategy for pest and disease management. José et al. (2020) studied the agronomic and volunteer plant characteristics of cotton stacks (Cotton stacks 1-4) and single events. The results showed that, compared to the control group, the cotton stack materials exhibited significant differences in agronomic traits. The stack materials outperformed single genetic engineering materials and conventional controls in certain traits, such as plant height and yield. Tests conducted at different locations further supported these findings, indicating that gene stacking technology has the potential to improve cotton yield and agronomic traits. This study provides important experimental data and theoretical support for genetic engineering breeding in cotton. 5.2 Comparative analysis of stacked vs. single traits Comparative studies between stacked and single-trait genetically modified (GM) crops have consistently shown the superiority of stacked traits in terms of pest resistance and overall crop performance. For instance, a comprehensive study involving soybean, maize, and cotton revealed that stacked GM crops exhibited negligible differences in agronomic and phenotypic characteristics compared to their single-trait counterparts. This indicates that stacking does not introduce additional risks but rather enhances the benefits of GM crops (José et al., 2020). Moreover, the durability of resistance conferred by stacked traits is significantly higher than that of single traits. In rice, for example, the pyramiding of two Bt genes resulted in higher resistance levels and better field performance against pests compared to single-gene lines (Yang et al., 2011). Similarly, the use of gene stacking in maize through targeted genome editing has shown that stacked traits can be precisely integrated and maintained
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