International Journal of Horticulture, 2024, Vol.14, No.3, 142-155 http://hortherbpublisher.com/index.php/ijh 146 2.2 Applications of CRISPR/Cas9 in plant science CRISPR/Cas9 has been widely adopted in plant science for various applications, including crop improvement and functional genomics. This technology allows for precise genetic modifications to enhance traits such as yield, quality, disease resistance, and tolerance to environmental stresses. For example, researchers have successfully used CRISPR/Cas9 to develop rice resistant to bacterial blight, wheat resistant to powdery mildew, and tomatoes with extended shelf life (Zafar et al., 2020; Wan et al., 2020; Pramanik et al., 2021). By CRISPR/Cas9-mediated gene editing, researchers edited the OsSWEET14 gene in rice, thereby enhancing resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Zafar et al., 2020). Through CRISPR/Cas9 technology, researchers targeted mutations in the MLO gene in wheat, successfully developing varieties with enhanced resistance to powdery mildew. The edited mutants exhibited significant disease resistance without any negative impact on plant growth (Wan et al., 2020). Using CRISPR/Cas9 technology, researchers edited the SlPelo and SlMlo1 genes in tomatoes, successfully extending the shelf life of tomatoes while enhancing resistance to Tomato Yellow Leaf Curl Virus (TYLCV) and powdery mildew (Pramanik et al., 2021). In citrus, CRISPR/Cas9 is being employed to target genes associated with susceptibility to HLB and ACP, aiming to create resistant cultivars. By knocking out or modifying specific genes, scientists can develop citrus plants that either repel the psyllid or hinder the bacterium's ability to infect and spread within the plant. For instance, Chaverra-Rodriguez et al. (2023) developed CRISPR/Cas9 gene-editing methods for specific modification of the ACPgenes, demonstrating that these methods are suitable for psyllid gene editing and hold potential for future use in gene-editing strategies to control HLB. Wang et al. (2019) used CRISPR/Cas9 to edit the CsWRKY22 gene to reduce susceptibility to citrus canker. The study showed that CRISPR/Cas9-mediated gene editing could successfully modify the CsWRKY22 gene in citrus, thereby enhancing its resistance to citrus canker. This indicates that CRISPR/Cas9 is an effective tool for improving disease resistance in citrus. 2.3 Advantages of CRISPR/Cas9 over traditional breeding methods CRISPR/Cas9 offers several advantages over traditional breeding methods. Traditional breeding involves crossing plants and selecting offspring with desirable traits, which can be a lengthy and imprecise process. CRISPR/Cas9 allows for the precise targeting of specific genes, reducing the likelihood of off-target effects and ensuring that only the desired genetic changes are made (Gupta et al., 2019; El-Mounadi et al., 2020). Traditional breeding is often time-consuming, labor-intensive, and less precise, relying on the natural occurrence of desirable traits and their subsequent selection over multiple generations. In contrast, CRISPR/Cas9 enables targeted modifications at specific genomic locations, significantly accelerating the breeding process and increasing precision (Mao et al., 2019; Zhu et al., 2020; Montecillo et al., 2020). Traditional breeding often faces challenges in simultaneously improving multiple traits due to genetic linkage and recombination limitations. CRISPR/Cas9 can be used to edit multiple genes simultaneously, enabling the concurrent enhancement of various desirable traits. Traditional breeding may introduce unwanted genetic material and traits from the donor parent. In contrast, CRISPR/Cas9 allows for the modification of endogenous genes without introducing foreign DNA, reducing the risk of unintended consequences and regulatory hurdles (Li et al., 2023). Moreover, CRISPR/Cas9 is applicable to a wide range of plant species and can be used to address various agricultural challenges, from disease resistance to environmental stress tolerance. These advantages make CRISPR/Cas9 a transformative tool in plant breeding, offering new possibilities for developing robust, high-yielding, and disease-resistant crops. In the context of citrus, CRISPR/Cas9 provides a promising avenue for creating ACP-resistant germplasm, potentially mitigating the devastating effects of HLB and ensuring the sustainability of the citrus industry. 3 Advances in Developing ACP-Resistant Citrus Germplasm 3.1 Identification of target genes for resistance to ACP and HLB The identification of target genes for resistance to Asian citrus psyllid (ACP) and Huanglongbing (HLB) is a critical step in developing resistant citrus germplasm. Recent studies have focused on understanding the genetic
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