Bt Research 2024, Vol.15, No.2, 96-109 http://microbescipublisher.com/index.php/bt 99 Another approach in genetic engineering is the use of site-specific recombination systems, such as the Cre-lox system, which allows for the precise integration of multiple genes into specific genomic locations. This method ensures the co-inheritance of the stacked genes, providing stable expression of the desired traits across generations (Srivastava, 2018). The high efficiency and precision of recombinase-mediated gene stacking make it a powerful tool for developing crops with complex trait combinations, such as improved tolerance to both drought and salinity. These advancements in genetic engineering have significantly expanded the possibilities for gene stacking, enabling the creation of crops with multiple, stacked traits that are difficult to achieve through traditional breeding alone. 3.3 Advanced biotechnological methods Advanced biotechnological methods have further enhanced the capabilities of gene stacking by introducing more sophisticated techniques for gene integration and expression. One such method is the use of modular gene stacking systems, which allow for the assembly of large, complex gene constructs that can be introduced into plants in a single transformation event. These systems, such as the GNS system, utilize advanced cloning techniques to create multi-gene expression cassettes that can be efficiently integrated into the plant genome (Qin et al., 2022). This approach not only simplifies the process of gene stacking but also increases the likelihood of successful integration and expression of the stacked genes. Another promising biotechnological method is the use of genome editing tools, such as CRISPR/Cas9, to precisely modify plant genomes and introduce multiple genes at specific locations. This technique allows for the targeted insertion of genes, reducing the risk of off-target effects and ensuring the stable expression of the stacked traits (Schaart et al., 2016). Genome editing has been used to create crops with enhanced resistance to diseases and environmental stresses by stacking multiple resistance genes into a single locus (Shailani et al., 2020). The precision and flexibility of genome editing make it an ideal tool for gene stacking, enabling the development of crops with complex trait combinations that are tailored to specific agricultural needs. 4 Mechanisms of Enhancing Durability 4.1 Multiple modes of action One of the primary strategies to enhance the durability of Bt crops is through the incorporation of multiple modes of action. This involves stacking different genes that produce various insecticidal proteins, each targeting different aspects of pest physiology. For instance, the development of transgenic rice expressing a fusion protein of Cry1Ab and Vip3A has shown significant resistance to major rice pests such as the Asiatic rice borer and rice leaf folder, without compromising agronomic performance (Xu et al., 2018). This approach ensures that pests are less likely to develop resistance simultaneously to multiple toxins, thereby prolonging the effectiveness of the Bt crops. Moreover, the combination of different Bt genes, such as Cry1Ab and Cry2A, in a single crop variety has demonstrated higher efficacy against pests compared to single-gene varieties. This gene stacking strategy not only enhances pest control but also maintains the yield and quality of the crops under field conditions (Yang et al., 2011). By employing multiple modes of action, the durability of Bt crops can be significantly improved, reducing the reliance on chemical insecticides and promoting sustainable agricultural practices. 4.2 Reducing resistance development Reducing the development of resistance in pest populations is crucial for maintaining the long-term effectiveness of Bt crops. One effective method is gene pyramiding, which involves stacking multiple resistance genes within a single plant. This strategy has been shown to suppress the emergence of virulent pathogen isolates and enhance the durability of resistance genes (Djian-Caporalino et al., 2014; REX Consortium, 2016). For example, pyramiding different resistance genes in pepper and lettuce crops has been experimentally proven to control root-knot nematodes more effectively than other strategies such as cultivar mixtures or gene rotation. Additionally, rotating and stacking resistance genes can slow the evolution of virulent pathogen strains. A model predicting the effects of different rotational management strategies indicated that rotating cultivars with different
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