Bt Research 2024, Vol.15, No.2, 96-109 http://microbescipublisher.com/index.php/bt 103 The use of advanced techniques such as targeted genome editing can facilitate the precise and efficient stacking of traits, ensuring that the desired characteristics are consistently expressed in the resulting crops. This approach not only improves the effectiveness of gene stacking but also simplifies the breeding process, making it more manageable and scalable (Ainley et al., 2013). The comparative analysis of stacked and single-trait crops underscores the importance of regulatory frameworks that recognize the safety and benefits of stacked traits. The evidence from field trials and compositional analyses supports the need for updated regulatory guidelines that streamline the approval process for stacked GM crops, thereby promoting their adoption and commercialization (Bell et al., 2018; José et al., 2020). These lessons provide a roadmap for future research and development in gene stacking, paving the way for more resilient and sustainable agricultural practices. 6 Challenges and Limitations 6.1 Technical and biological challenges Gene stacking, while promising, faces several technical and biological challenges. One of the primary technical hurdles is the difficulty in achieving stable expression of multiple genes in transgenic plants. The expression of multiple genes can lead to unpredictable interactions and reduced efficacy of individual genes, as seen in the case where the expression of the cry1Cgene decreased after gene stacking in rice (Yang et al., 2011). Additionally, the development of transgenic plants with multiple stacked genes is complex and time-consuming, often requiring advanced molecular techniques and extensive field testing to ensure stability and effectiveness (Halpin, 2005). Biologically, the evolution of pest resistance remains a significant challenge. Despite the use of gene stacking to delay resistance, pests can still develop resistance to multiple Bt toxins over time. This is particularly concerning given the high adaptability of pests and the potential for cross-resistance, where resistance to one toxin confers resistance to another (Ainley et al., 2013). Moreover, the effectiveness of gene stacking can be influenced by environmental factors and the genetic background of the host plant, which can affect the expression and performance of the stacked genes (Xu et al., 2018). 6.2 Regulatory and ethical issues The deployment of Bt crops with stacked genes also raises several regulatory and ethical issues. Regulatory frameworks for genetically modified organisms (GMOs) vary widely across different countries, and obtaining approval for crops with multiple transgenes can be more complex and time-consuming than for single-gene modifications. This can delay the commercialization and adoption of such crops, limiting their potential benefits. Additionally, there are concerns about the long-term environmental impacts of releasing transgenic plants with stacked genes, including potential effects on non-target organisms and biodiversity (REX Consortium, 2016). Ethically, the use of gene stacking in Bt crops raises questions about the control and ownership of genetic resources. The development and commercialization of these crops are often dominated by large biotech companies, which can lead to issues of access and equity, particularly for smallholder farmers in developing countries (Dormatey et al., 2020). There is also the broader ethical debate about the use of genetic engineering in agriculture, with some stakeholders advocating for more natural and sustainable farming practices over the use of GMOs (Bailey-Serres et al., 2019). 6.3 Economic and market considerations From an economic perspective, the development and deployment of Bt crops with stacked genes involve significant costs. The research and development process is expensive, and the regulatory approval process can add further financial burdens. These costs are often passed on to farmers, who may face higher seed prices for transgenic crops with stacked genes compared to conventional or single-gene varieties (Shailani et al., 2020). Additionally, the market acceptance of GMOs varies, with some regions and consumers being more resistant to genetically modified products, which can limit the market potential for these crops (Crété et al., 2020).
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