Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 215 for resistance to bacterial canker using the CsLOB1 gene showed promising results. The GM citrus plants exhibited significantly lower disease incidence and better overall health compared to non-GM controls (Salonia et al., 2020). Trial 3: Another study focused on the use of CRISPR/Cas9 technology to develop citrus varieties resistant to huanglongbing (HLB). The field trials indicated that the GM citrus plants had reduced HLB symptoms and improved fruit quality (Sun et al., 2019). Trial 4: Research on stacked transgenic traits for pest and herbicide resistance highlighted the importance of using multiple genes targeting the same pest species. The field trials demonstrated that such stacking could delay the evolution of pest resistance and improve overall pest management (Gressel et al., 2017). Trial 5: A genome-wide association study (GWAS) and genomic selection (GS) in citrus breeding populations showed that these advanced genomic tools could enhance the detection of beneficial traits and improve the accuracy of selecting pest-resistant varieties (Minamikawa et al., 2017). 6.3 Criteria and metrics for evaluating pest resistance in genetically modified citrus Evaluating pest resistance in GM citrus involves several criteria and metrics. Pest incidence and severity: The primary metric is the incidence and severity of pest infestations. This includes counting the number of pests per plant and assessing the extent of damage (Sun et al., 2019; Li et al., 2020). Plant health and vigor: Metrics such as plant height, leaf area, and overall vigor are used to assess the health of GM plants compared to non-GM controls (Gressel et al., 2017; Salonia et al., 2020). Yield and fruit quality: Yield parameters, including the number of fruits per plant and total fruit weight, are critical. Fruit quality traits such as size, juiciness, and acidity are also evaluated (Minamikawa et al., 2017). Resistance durability: Long-term studies are conducted to assess the durability of pest resistance over multiple growing seasons. This includes monitoring for any signs of pest adaptation or resistance breakdown (Gressel et al., 2017). Environmental impact: The impact of GM citrus on non-target organisms and overall ecosystem health is also considered. This includes studies on beneficial insects, soil health, and biodiversity (Sun et al., 2019; Salonia et al., 2020). By adhering to these methodologies and criteria, researchers can effectively evaluate the performance of GM citrus varieties and their potential for commercial use in pest resistance breeding programs. 7 Challenges and Limitations 7.1 Technical challenges in gene stacking and transformation The application of multi-gene stacking strategies in citrus pest resistance breeding faces several technical challenges. One of the primary issues is the complexity of the citrus genome, which includes high heterozygosity, polyembryony, and long juvenility phases, making traditional breeding methods laborious and time-consuming (Sendín and Filippone, 2018; Salonia et al., 2020). Genetic transformation techniques, such as Agrobacterium tumefaciens-mediated transformation, have been employed to introduce resistance genes like Bs2 for citrus canker resistance. However, the efficiency of these transformations and the subsequent regeneration of plants remain significant bottlenecks (Sendín and Filippone, 2018; Salonia et al., 2020). Additionally, optimizing protocols for genome editing tools like CRISPR/Cas9 and CRISPR/Cpf1 systems is crucial for achieving precise and efficient gene stacking (Sun et al., 2019). 7.2 Ecological and environmental considerations The ecological and environmental impacts of deploying genetically modified citrus varieties with stacked resistance genes must be carefully evaluated. There is a potential risk of unintended effects on non-target organisms and the broader ecosystem. For instance, the introduction of multiple resistance genes could lead to changes in pest populations and the emergence of new pest species or strains that can overcome the resistance (Li et al., 2020). Moreover, the long-term sustainability of these genetically modified crops in diverse environmental conditions needs thorough assessment to ensure they do not negatively impact biodiversity or lead to ecological imbalances (Sun et al., 2019). 7.3 Regulatory and safety issues Regulatory frameworks for genetically modified organisms (GMOs) vary significantly across different countries, posing a challenge for the widespread adoption of multi-gene stacked citrus varieties. Ensuring compliance with
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