Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 216 these regulations requires extensive safety assessments, including evaluating potential allergenicity, toxicity, and environmental impact (Sun et al., 2019; Salonia et al., 2020). The development of marker-free systems is also essential to address public concerns and regulatory requirements regarding the presence of selectable marker genes in transgenic plants (Salonia et al., 2020). Additionally, the approval process for GMOs can be lengthy and costly, further complicating the commercialization of these advanced breeding technologies. 7.4 Economic and scalability challenges The economic feasibility and scalability of multi-gene stacking strategies in citrus breeding are significant concerns. The initial costs of developing genetically modified citrus varieties, including research, development, and regulatory approval, are substantial (Sun et al., 2019; Salonia et al., 2020). Furthermore, the scalability of these technologies to produce sufficient quantities of transgenic plants for commercial use is challenging. The long juvenility phase of citrus plants means that it takes several years before the benefits of the genetic modifications can be realized in terms of fruit production and pest resistance (Salonia et al., 2020). Additionally, the adoption of these technologies by farmers may be hindered by the high costs and the need for specialized knowledge and infrastructure to manage genetically modified crops (Sendín and Filippone, 2018). In summary, while multi-gene stacking strategies hold great promise for enhancing citrus pest resistance, several challenges and limitations must be addressed. These include technical difficulties in gene stacking and transformation, ecological and environmental considerations, regulatory and safety issues, and economic and scalability challenges. Addressing these challenges will require continued research, collaboration, and innovation in the field of citrus breeding. 8 Future Directions and Perspectives 8.1 Emerging trends and technologies in multi-gene stacking The field of citrus pest resistance breeding is rapidly evolving with the advent of new plant breeding techniques (NPBTs) and genetic engineering technologies. One of the most promising trends is the use of CRISPR/Cas9 for targeted genome editing, which has shown significant potential in conferring resistance to diseases such as citrus canker and Huanglongbing (HLB) (Jia et al., 2017; Sun et al., 2019; Salonia et al., 2020; Conti et al., 2021). The development of improved CRISPR systems, such as CRISPR/Cpf1, further enhances the precision and efficiency of these genetic modifications (Sun et al., 2019). Additionally, the integration of multiple resistance genes through hybridization and genetic transformation is becoming increasingly feasible, allowing for the stacking of genes that confer broad-spectrum resistance. This approach has been successfully demonstrated in other crops, such as potatoes, and holds great promise for citrus as well (Rogozina et al., 2021). 8.2 Integration with other pest management strategies While genetic engineering and multi-gene stacking offer powerful tools for developing pest-resistant citrus varieties, their effectiveness can be further enhanced when integrated with other pest management strategies. For instance, combining genetically modified resistant varieties with traditional methods such as biological control, cultural practices, and the use of insecticides can provide a more comprehensive and sustainable approach to pest management. Understanding the mechanisms of resistance, such as the overexpression of detoxification genes in pests like the Asian citrus psyllid, can also inform the development of more targeted and effective pest control measures (Tian et al., 2019). Moreover, the use of marker-assisted selection (MAS) in conjunction with genetic engineering can accelerate the breeding process and ensure the incorporation of desirable traits (Endo et al., 2020). 8.3 Long-term vision and potential breakthroughs in citrus pest resistance breeding Looking ahead, the long-term vision for citrus pest resistance breeding involves the creation of highly resilient citrus varieties that can withstand a wide range of biotic and abiotic stresses. This will likely involve the continued refinement of genome editing technologies and the development of more efficient transformation and regeneration protocols (Poles et al., 2020; Huang et al., 2022). The integration of genomic selection (GS) and genome-wide association studies (GWAS) can also play a crucial role in identifying and selecting for traits that contribute to pest resistance and overall fruit quality (Minamikawa et al., 2017). Additionally, the exploration of novel promoters and marker-free systems will be essential for the development of commercially viable genetically
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