International Journal of Horticulture, 2024, Vol.14, No.3, 142-155 http://hortherbpublisher.com/index.php/ijh 151 Figure 4 Injection protocols and observed rates for emerging nymphs from injected eggs (Adopted from Chaverra-Rodriguez et al., 2023) Image caption: Figure shows two protocols for injecting CRISPR-Cas9 ribonucleoproteins (RNPs) into the embryos of the Asian citrus psyllid (Diaphorina citri). The figure is divided into several panels, each displaying different injection protocols and results. A: Protocol 1: Eggs are removed from the plant, aligned on a microscope slide, injected with the RNP mix, and then incubated until nymphs hatch. Protocol 2: Injections are performed while the eggs are still attached to the plant. This involves uprooting the seedlings, mounting them on a glass slide for injection, and then replanting the seedlings. B: This panel compares the survival rates of nymphs hatched from eggs that were either not injected, injected with ultrapure water, injected with RNPw (targeting the white gene), or injected with RNPkh (targeting the kh gene). The results show a significant reduction in the survival rates of injected eggs, with the lowest survival rate observed in eggs injected with ultrapure water. C: This panel compares the survival rates of nymphs between Protocol 1 and Protocol 2. The comparison between the two protocols indicates that maintaining natural conditions during injection (keeping the eggs on the plant) can improve embryo survival rates, although further optimization is required to achieve visible and heritable mutations (Adapted from Chaverra-Rodriguez et al., 2023) 7 Future Directions and Perspectives 7.1 Emerging technologies and innovations in CRISPR/Cas9 for citrus breeding The application of CRISPR/Cas9 technology in Citrus breeding is rapidly evolving, offering new avenues for developing resistance to pests such as the Asian Citrus Psyllid (ACP). Recent advancements have optimized methods for introducing Cas9 ribonucleoprotein (RNP) into early embryos and adult females of ACP, resulting in
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