Molecular Entomology 2024, Vol.15, No.2, 52-60 http://emtoscipublisher.com/index.php/me 55 laboratory and field conditions, significantly reducing pest populations and damage (Iqbal et al., 2021). However, the evolution of resistance in some pest species remains a challenge, necessitating the development of strategies to manage and mitigate resistance (Tabashnik et al., 2023). 4.2 RNA interference (RNAi) for targeted pest control RNA interference (RNAi) is another powerful tool for enhancing insect pest resistance in sugarcane. This technique involves the expression of double-stranded RNA (dsRNA) that targets and silences specific genes essential for pest survival. RNAi offers high target specificity and minimal environmental impact compared to traditional chemical insecticides. Studies have demonstrated the effectiveness of RNAi in downregulating detoxification genes in pests, leading to reduced pest viability and increased susceptibility to plant defenses (Price and Gatehouse, 2008; Eakteiman et al., 2018; Chung et al., 2021). Despite its potential, the commercial application of RNAi in transgenic plants is still limited, and further research is needed to optimize gene targets and delivery methods (Chung et al., 2021; Halder et al., 2022). 4.3 CRISPR-Cas9 and its potential in developing resistant varieties The CRISPR-Cas9 genome editing system has emerged as a revolutionary tool for developing insect-resistant sugarcane varieties. By precisely targeting and modifying specific genes, CRISPR-Cas9 can create mutations that confer resistance to insect pests. For instance, CRISPR-mediated knockout of ABC transporter genes in pests has been shown to confer high levels of resistance to Bt toxins (Guo et al., 2019; Fabrick et al., 2021). This technology allows for the rapid development of resistant strains and provides insights into the genetic mechanisms underlying pest resistance. The potential of CRISPR-Cas9 in sugarcane genetic engineering is vast, offering a flexible and efficient approach to enhance pest resistance. 4.4 Stacked traits for enhanced resistance Combining multiple resistance traits, or "stacking," is a strategy to enhance the durability and effectiveness of pest-resistant sugarcane. Stacked traits can include the expression of multiple Bt toxins, RNAi constructs, and other resistance genes within a single plant. This approach aims to provide broad-spectrum resistance and reduce the likelihood of pests developing resistance to any single trait. For example, transgenic sugarcane lines expressing both Bt toxins and herbicide tolerance genes have shown strong resistance to pests and improved agronomic performance under field conditions (Wang et al., 2017; Iqbal et al., 2021). The integration of stacked traits represents a comprehensive strategy to achieve sustainable pest management in sugarcane cultivation. By leveraging these genetic engineering mechanisms, researchers aim to develop sugarcane varieties with robust and long-lasting resistance to insect pests, thereby improving crop yield and reducing reliance on chemical pesticides. 5 Case Study 5.1 Overview of a specific genetic engineering initiative in sugarcane One notable genetic engineering initiative aimed at enhancing sugarcane resistance to insect pests involved the introduction of the cry1Ac gene, which encodes an insecticidal protein from Bacillus thuringiensis (Bt). This initiative was undertaken to address the significant yield losses caused by the sugarcane stem borer (Diatraea saccharalis), a prevalent pest in sugarcane cultivation. The cry1Ac gene was selected due to its proven efficacy in other crops and its ability to produce a protein that is toxic to certain insect pests but safe for humans and other non-target organisms (Weng et al., 2006; Zhou et al., 2018; Qamar et al., 2021). 5.2 Implementation and outcomes of the case study The implementation of this genetic engineering initiative involved several key steps. First, the cry1Ac gene was synthetically optimized to match the codon usage of sugarcane, enhancing its expression in the plant. This synthetic gene was then introduced into sugarcane varieties using Agrobacterium-mediated transformation and microprojectile bombardment techniques (Weng et al., 2006; Dessoky et al., 2020). The transformed sugarcane lines were subjected to rigorous testing to confirm the integration and expression of the cry1Ac gene. Molecular analyses, including PCR and Southern blotting, verified the presence of the gene,
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