International Journal of Horticulture, 2024, Vol.14, No.3, 142-155 http://hortherbpublisher.com/index.php/ijh 148 Additionally, Jia et al. (2019) studied the use of the CRISPR/Cas9 system to edit the citrus CsLOB1 gene to enhance citrus resistance to HLB. They successfully created gene-edited citrus plants through Agrobacterium-mediated epidermal transformation, demonstrating high editing efficiency and significant improvement in disease resistance. 3.3 Development of transgenic and non-transgenic CRISPR/Cas9 citrus lines The development of both transgenic and non-transgenic CRISPR/Cas9 citrus lines is essential for creating ACP-resistant germplasm. Transgenic approaches involve the stable integration of CRISPR/Cas9 components into the citrus genome. This method allows for continuous expression of the gene-editing machinery, facilitating multiple rounds of editing or the introduction of complex traits (Jia et al., 2019). However, transgenic plants are subject to stringent regulatory scrutiny and may face market acceptance challenges. while non-transgenic methods rely on transient expression or direct delivery of CRISPR/Cas9 components，such that they do not integrate into the plant’s genome. Techniques such as Agrobacterium-mediated transformation, particle bombardment, or the use of ribonucleoprotein complexes can be employed to achieve transient expression. This approach results in plants that are free of foreign DNA after the editing process, potentially easing regulatory hurdles and improving consumer acceptance. Recent advances have shown that both transgenic and non-transgenic CRISPR/Cas9 citrus lines can be developed successfully. For instance, a DNA-free plant gene-editing technique using virus-delivered CRISPR/Cas9 has been developed. In tobacco, this method achieved high-frequency gene editing through viral infection (Ma et al., 2020). Another study demonstrated a method for creating non-transgenic mutant plants through transient expression of the CRISPR/Cas9 system and developed a fast, cost-effective high-throughput mutation screening protocol (Chen et al., 2018). These non-transgenic lines retain the desired traits while being more likely to gain regulatory approval and market acceptance. 4 Mechanisms of Resistance 4.1 Genetic pathways and resistance mechanisms targeted by CRISPR/Cas9 CRISPR/Cas9 technology has been employed to target specific genetic pathways in the Asian citrus psyllid (ACP) to impede the lifecycle and behavior of the psyllid or disrupt the transmission of Huanglongbing (HLB). The primary focus has been on genes involved in detoxification and metabolic processes, which are crucial for the insect's survival and resistance to insecticides. For instance, genes such as cytochrome P450, glutathione S-transferase (GST), and esterases have been identified as key players in the resistance mechanisms of ACP (Yu and Killiny, 2018; Tian et al., 2019; Chen et al., 2021). By using CRISPR/Cas9 to knock out or modify these genes, researchers aim to disrupt the detoxification pathways, thereby increasing the susceptibility of ACP to insecticides and reducing their ability to spread Huanglongbing (HLB) disease (Chaverra-Rodriguez et al., 2023). 4.2 Role of host plant defense genes in conferring resistance Host plant defense genes play a significant role in conferring resistance to ACP. These genes are involved in various defense mechanisms, including the production of defensive enzymes and secondary metabolites that deter insect feeding and reproduction. For example, the expression of genes encoding superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) has been shown to be modulated in response to ACP infestation (Qasim et al., 2021). By using CRISPR/Cas9 to enhance the expression of these defense genes, it is possible to create Citrus varieties that are more resistant to ACP, thereby reducing the spread of HLB (Qasim et al., 2021; Gao et al., 2021). 4.3 Enhancing resistance through multiplex gene editing Multiplex gene editing using CRISPR/Cas9 allows for the simultaneous targeting of multiple genes, which can enhance resistance to ACP more effectively than single-gene modifications. This approach can be used to knock out multiple detoxification genes or to modify several host plant defense genes at once, thereby creating a more
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