Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 210 This study aims to provide a comprehensive overview of the application of multi-gene stacking strategies in citrus pest resistance breeding. By synthesizing current research and advancements in this field, the study seeks to highlight the theoretical foundations, practical implementations, and future prospects of multi-gene stacking in citrus. The significance of this study lies in its potential to inform and guide future research and breeding programs aimed at developing pest-resistant citrus varieties. By addressing the challenges and opportunities associated with multi-gene stacking, this study aims to contribute to the sustainable improvement of citrus cultivation, ensuring the long-term viability and productivity of this economically important crop. 2 Background on Pest Resistance Breeding 2.1 Traditional breeding methods for pest resistance Traditional breeding methods for pest resistance in citrus have primarily relied on classical techniques such as introduction, selection, and hybridization. These methods involve the production of variability in the plant population followed by the selection of desirable types. Historically, the evolutionary selection of resistant types resulted in land races, and with the rediscovery of Mendelian laws, breeding for resistance continued as a major approach to increasing total yield (Wenzel et al., 1985). However, these conventional methods are often bottlenecked by inadequate and lengthy breeding procedures, making them less efficient in addressing the rapid emergence of new pest threats (Rauf et al., 2013). 2.2 Advances in genetic engineering and molecular breeding The advent of genetic engineering and molecular breeding has revolutionized the field of pest resistance breeding. Techniques such as genome editing, transgenesis, and marker-assisted selection (MAS) have been employed to introduce resistance genes into citrus varieties more efficiently. For instance, genome editing in citrus via transgenesis has successfully induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1 (Salonia et al., 2020). Additionally, fast-track breeding systems integrating early flowering transgenic plants with MAS have significantly shortened the breeding duration for disease resistance, as demonstrated in the incorporation of citrus tristeza virus (CTV) resistance into citrus germplasm (Endo et al., 2020). These biotechnological approaches have shown tremendous potential in improving citrus against various diseases, making the breeding process faster and more precise (Dutt et al., 2020). 2.3 Concept and advantages of multi-gene stacking in breeding programs Multi-gene stacking, also known as gene pyramiding, involves the assembly of multiple resistance genes into a single plant variety to provide broad-spectrum and durable resistance against various pests and diseases. This strategy has been successfully employed in other crops, such as rice, where a transgene stacking system was used to develop rice lines with multi-resistance to glyphosate, borers, brown planthopper, bacterial blight, and rice blast (Li et al., 2020). The advantages of multi-gene stacking in breeding programs include enhanced resistance durability, reduced risk of resistance breakdown, and improved agronomic traits. In citrus, gene stacking through techniques like CRISPR-based genome editing holds promise for generating varieties resistant to multiple pathogens, thereby ensuring sustainable crop improvement (Sun et al., 2019; Dormatey et al., 2020). The integration of multiple resistance genes can also help overcome the limitations of single-gene resistance, which often leads to the rapid evolution of virulent pathogen strains (Yang et al., 2011). By leveraging the advancements in genetic engineering and molecular breeding, multi-gene stacking strategies can significantly enhance the efficiency and effectiveness of pest resistance breeding in citrus, providing a robust solution to the challenges posed by biotic stresses. 3 Theoretical Foundations of Multi-Gene Stacking 3.1 Definition and principles of multi-gene stacking Multi-gene stacking, also known as gene pyramiding, involves the introduction of multiple genes into a single organism to confer a combination of desirable traits, such as pest resistance, disease resistance, and herbicide tolerance (Figure 1). This strategy is more effective than single-gene approaches because it targets multiple pathways or mechanisms within the pest or pathogen, thereby reducing the likelihood of resistance development (Gressel et al., 2017; Shehryar et al., 2019; Salim et al., 2020). The principles of multi-gene stacking include the
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