Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 219 5.3 Strategies to overcome resistance To counteract the evolution of resistance, several strategies have been proposed. One approach involves the use of multiple Bt toxins with different modes of action to reduce the likelihood of resistance development. Another strategy is the implementation of refuges, where non-Bt crops are planted alongside Bt crops to maintain a population of susceptible insects. Ongoing monitoring and genomic studies can help in the early detection of resistance, allowing for timely management interventions. The use of CRISPR/Cas9 technology to study and potentially modify resistance genes also holds promise for overcoming resistance. 5.4 Case studies on resistance development Case studies on resistance development provide valuable insights into the practical challenges of managing Bt resistance. For example, the diamondback moth, Plutella xylostella, has developed high levels of resistance to Cry1Ac toxins through mutations in the ABCC2 and ABCC3 genes. These mutations result in truncated proteins that fail to bind the toxin effectively, leading to resistance (Figure 2). Another notable case is the European corn borer, Ostrinia nubilalis, where field and laboratory studies have documented resistance evolution due to mutations affecting midgut receptor binding (Jurat-Fuentes et al., 2021). These case studies underscore the need for integrated pest management strategies to sustain the effectiveness of Bt crops. Figure 2 Cry1Ac immunodetection. (A) Larvae exposed to a high dose of Cry1Ac (7 µg) for 20 min. The green fluorescence shows Cry1Ac trapped in the peritrophic matrix (PM) and bound to the brush border membrane (BBM). (B) Control larvae not exposed to the toxin. An Alexa Fluor 488-anti-rabbit antibody was used as secondary antibody (Adopted from Pinos et al., 2021) 6 Genomic Techniques for Bt Strain Improvement 6.1 CRISPR-Cas9 and gene editing approaches CRISPR-Cas9 has emerged as a revolutionary tool in the field of genome editing, offering unprecedented precision and efficiency in modifying genetic material. This technology utilizes a guide RNA (gRNA) to direct the Cas9 endonuclease to specific DNA sequences, enabling targeted gene knockouts, insertions, and modifications. The versatility of CRISPR-Cas9 has been demonstrated across various organisms, including plants and insects, making it a powerful tool for Bt strain improvement. For instance, CRISPR-Cas9 has been successfully employed to enhance crop resistance to diseases and environmental stresses, which can be analogously applied to optimize Bt strains for better insecticidal activity (Arora and Narula, 2017). Despite its potential, challenges such as off-target effects and delivery methods need to be addressed to fully harness the capabilities of CRISPR-Cas9 in Bt strain improvement (Manghwar et al., 2020). 6.2 Comparative genomics for identifying novel genes Comparative genomics involves the analysis of genetic material from different organisms to identify genes that are unique or highly conserved. This approach can be instrumental in uncovering novel genes responsible for the insecticidal properties of Bt strains. By comparing the genomes of highly effective Bt strains with those of less effective ones, researchers can pinpoint specific genes that contribute to enhanced insecticidal activity. These identified genes can then be targeted for further study and manipulation using advanced genomic techniques like
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