Molecular Plant Breeding 2024, Vol.15, No.3, 144-154 http://genbreedpublisher.com/index.php/mpb 146 Liu et al. (2021) illustrates the impact of PmPR10-3.1 protein on spore germination of fungal pathogens, specifically Phoma exigua and Cronartium ribicola. Micrographs (a) through (e) show conidiospores of P. exigua treated with varying concentrations of PmPR10-3.1 for 18 hours, displaying inhibited hyphal growth and swelling at hyphal tips, especially at higher concentrations (b to e). Similarly, images (f) and (g) depict urediniospores of C. ribicola treated for 24 hours, with the treated spores (g) showing reduced hyphal growth compared to the control (f). These observations highlight PmPR10-3.1's potential as an antifungal agent, effectively hampering fungal development and providing a promising avenue for enhancing plant disease resistance. 3 Genome Editing Technologies for Pine Disease Resistance 3.1 CRISPR/Cas9 system 3.1.1 Principles and mechanisms The CRISPR/Cas9 system, derived from the adaptive immune system of bacteria, has revolutionized genome editing due to its simplicity, efficiency, and precision. The system uses a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it creates a double-strand break. This break can then be repaired by the cell's natural repair mechanisms, leading to targeted modifications such as insertions, deletions, or substitutions (Manghwar et al., 2019; Rodriguez-Rodriguez et al., 2019; Sharma et al., 2020). The CRISPR/Cas9 system's ability to make precise edits has made it a powerful tool for genetic research and therapeutic applications (El-Mounadi et al., 2020; Li et al., 2021). 3.1.2 Applications in plant genomics CRISPR/Cas9 has been widely adopted in plant genomics for its ability to introduce precise genetic modifications. It has been used to enhance disease resistance in various crops by targeting and modifying susceptibility genes. For instance, CRISPR/Cas9 has been employed to develop resistance against viral, fungal, and bacterial diseases in model plants and crops such as rice, tomato, wheat, and citrus (Borrelli et al., 2018; Charlesworth et al., 2018; Ahmad et al., 2020). The technology's versatility and efficiency make it an ideal tool for improving disease resistance in pine trees, potentially leading to more resilient forestry practices. 3.2 Other genome editing tools (TALENs, ZFNs) In addition to CRISPR/Cas9, other genome editing tools such as Transcription Activator-Like Effector Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs) have been used for precise genetic modifications. TALENs and ZFNs function by creating double-strand breaks at specific DNA sequences, similar to CRISPR/Cas9, but they use different mechanisms for DNA recognition and binding. TALENs use customizable DNA-binding domains derived from transcription activator-like effectors, while ZFNs use zinc finger domains to recognize specific DNA sequences (Borrelli et al., 2018; Charlesworth et al., 2018). Although these tools have been effective in various applications, CRISPR/Cas9 has largely overtaken them due to its ease of design, higher success rate, and lower cost (Charlesworth et al., 2018; Manghwar et al., 2019). 3.3 Delivery methods for genome editing in pines Effective delivery of genome editing components into pine cells is crucial for successful genetic modifications. Various delivery methods have been explored, including Agrobacterium-mediated transformation, biolistic particle delivery (gene gun), and protoplast transfection. Agrobacterium-mediated transformation is commonly used for its efficiency in delivering DNA into plant cells, although it is more challenging in gymnosperms like pines. Biolistic particle delivery involves shooting DNA-coated particles into plant tissues, which can be effective for species that are recalcitrant to Agrobacterium transformation. Protoplast transfection, which involves the introduction of DNA into isolated plant cells without cell walls, offers another alternative but requires efficient regeneration protocols to produce whole plants from edited cells (Charlesworth et al., 2018; Ahmad et al., 2020; Li et al., 2021). Each method has its advantages and limitations, and the choice of delivery method may depend on the specific requirements of the pine species and the desired genetic modification.
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