International Journal of Horticulture, 2024, Vol.14, No.3, 142-155 http://hortherbpublisher.com/index.php/ijh 149 robust resistance mechanism. For instance, co-silencing of multiple GST genes in ACP has been shown to significantly increase their susceptibility to insecticides (Yu and Killiny, 2018). Similarly, targeting multiple cytochrome P450 genes can disrupt various detoxification pathways, leading to higher mortality rates in ACP populations (Chen et al., 2018; Tian et al., 2019). This strategy not only enhances the effectiveness of resistance but also reduces the likelihood of resistance development in ACP. In summary, the mechanisms of resistance conferred by CRISPR/Cas9-mediated gene editing involve a multifaceted approach targeting key genetic pathways, host defense genes, and the use of multiplex editing to enhance overall resistance. These strategies are essential for developing citrus germplasm that can withstand the persistent and evolving threats posed by ACP and HLB, ensuring the sustainability of citrus production. 5 Challenges in Developing CRISPR/Cas9 Citrus Germplasm 5.1 Technical challenges in CRISPR/Cas9 delivery and efficiency One of the primary technical challenges in developing CRISPR/Cas9 citrus germplasm is the efficient delivery of the CRISPR/Cas9 components into citrus cells. The thick cell walls of citrus plants can impede the penetration of CRISPR/Cas9 complexes, making it difficult to achieve high transformation efficiency. The regeneration of whole plants from transformed cells is often inefficient and time-consuming, further complicating the process (Chen et al., 2021). Additionally, ensuring the precise and efficient editing of target genes is a significant challenge. The effectiveness of CRISPR/Cas9 depends on the design of guide RNAs (gRNAs) that can accurately target the desired DNA sequences without affecting other regions of the genome. Developing reliable and reproducible protocols for gRNA design, delivery, and expression in citrus tissues is critical for the success of gene editing efforts (Tian et al., 2019). 5.2 Off-target effects and genetic stability Off-target effects, where the CRISPR/Cas9 system inadvertently edits unintended genomic sites, pose a significant risk to genetic stability. Such off-target modifications can lead to unintended phenotypic consequences, potentially affecting plant growth, development, or resistance traits. Kimberland et al. (2018) pointed out that the CRISPR/Cas9 system can produce off-target effects and biological variations in genome editing experiments, which can confound experimental results. Reducing off-target effects requires the careful design and validation of gRNAs and the use of high-fidelity Cas9 variants with reduced off-target activity. Hajiahmadi et al. (2019) summarized various methods for reducing off-target effects of the CRISPR/Cas9 system in plants, such as using dCas9 and paired Cas9 nickases. They found that using paired sgRNAs can significantly reduce the frequency of unintended mutations. Furthermore, maintaining the genetic stability of edited plants over successive generations is crucial. Somaclonal variation, which can occur during the tissue culture process, may introduce additional genetic changes that complicate the breeding and evaluation of CRISPR/Cas9-edited citrus lines. Ensuring that the edited traits are stably inherited and do not revert or produce undesirable side effects is essential for the practical application of CRISPR/Cas9 technology in citrus breeding. 5.3 Regulatory, ethical, and public acceptance issues The development and deployment of CRISPR/Cas9-edited citrus germplasm face significant regulatory, ethical, and public acceptance challenges. Regulatory frameworks for gene-edited crops vary widely between countries, and there is ongoing debate about how these plants should be classified and managed. Some regions may regulate CRISPR/Cas9-edited plants similarly to genetically modified organisms (GMOs), while others may adopt more lenient approaches if no foreign DNA is introduced. Ethical concerns regarding the manipulation of plant genomes and potential ecological impacts also need to be addressed，particularly regarding the potential ecological impacts of releasing gene-edited citrus plants into the environment. Concerns about biodiversity, gene flow to wild relatives, and unintended consequences on
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