Bioscience Methods 2024, Vol.15, No.3, 139-148 http://bioscipublisher.com/index.php/bm 142 3.3 CRISPR-Cas9 and genome editing 3.3.1 Overview of CRISPR technology in sugarcane CRISPR-Cas9 is a powerful genome-editing tool that allows precise modifications of specific genes. This technology has been increasingly applied in crop improvement, including sugarcane, to enhance traits such as insect resistance. CRISPR-Cas9 enables the targeted disruption or modification of genes associated with susceptibility to insect pests, thereby conferring resistance. 3.3.2 Targeting insect resistance genes In sugarcane, CRISPR-Cas9 can be used to target and edit genes that play a role in insect resistance. For instance, genes involved in the production of secondary metabolites or proteins that deter insect feeding can be upregulated or modified to enhance resistance. Although specific examples in sugarcane are still emerging, the potential of CRISPR-Cas9 to create insect-resistant varieties is promising, as demonstrated in other crops (Iqbal et al., 2021). 3.4 Plant-microbe interactions 3.4.1 Endophytes and biocontrol agents Plant-microbe interactions, particularly with endophytes and biocontrol agents, offer an alternative strategy for enhancing insect resistance in sugarcane. Endophytes are microorganisms that live within plant tissues and can confer resistance to insect pests by producing bioactive compounds or inducing systemic resistance in the host plant. The use of biocontrol agents, such as beneficial bacteria and fungi, can also help manage insect populations by outcompeting or directly antagonizing pests. 3.4.2 Enhancing resistance through symbiotic relationships Symbiotic relationships between sugarcane and beneficial microbes can be leveraged to enhance insect resistance. For example, certain endophytic bacteria and fungi can produce insecticidal compounds or enhance the plant's own defense mechanisms. These symbiotic interactions can be harnessed through inoculation of sugarcane with selected microbial strains, providing a sustainable and environmentally friendly approach to pest management. In conclusion, biotechnological strategies, including genetic engineering, RNAi technology, CRISPR-Cas9 genome editing, and plant-microbe interactions, offer promising avenues for enhancing insect resistance in sugarcane. These approaches can significantly reduce yield losses due to insect pests and contribute to sustainable sugarcane production. 4 Case Study 4.1 Background of the case study Sugarcane (Saccharum officinarum) is a vital crop for sugar production globally, but it faces significant threats from various insect pests, including cane borers and weevils, which lead to substantial yield losses and reduced sucrose content. Traditional breeding methods have struggled to develop insect-resistant varieties due to the complex genetic makeup of sugarcane and the absence of naturally resistant genes in its genome. Consequently, biotechnological approaches have been explored to enhance insect resistance in sugarcane (Zhou et al., 2018; Iqbal et al., 2021; Qamar et al., 2021). 4.2 Application of biotechnological approaches in the selected case This study focused on the application of genetic engineering to introduce insect resistance in sugarcane. Several strategies have been employed, including the overexpression of cry proteins, vegetative insecticidal proteins (vip), lectins, and proteinase inhibitors (PI). Notably, the cry1Ac gene fromBacillus thuringiensis has been widely used to develop transgenic sugarcane lines resistant to stem borers (Figure 2) (Zhou et al., 2018; Dessoky et al., 2020; Iqbal et al., 2021). Additionally, advanced techniques such as host-induced gene silencing (HIGS) and CRISPR/Cas9 have been explored for sustainable pest control. 4.3 Results and impact on insect resistance The introduction of the cry1Ac gene into sugarcane has shown promising results. Transgenic lines expressing cry1Ac demonstrated significant resistance to stem borers, with some lines achieving up to 100% mortality of
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