Molecular Plant Breeding 2024, Vol.15, No.2, 81-89 http://genbreedpublisher.com/index.php/mpb 83 complex with crRNA and cleaves the foreign DNA near the PAM region. This targeted cleavage mechanism allows for precise genetic modifications in poplar, enabling researchers to knock out detrimental genes or insert beneficial ones to enhance drought tolerance. The use of CRISPR/Cas9 in poplar demonstrates its potential for improving genetic traits, contributing to the development of more resilient tree varieties capable of withstanding environmental stresses. Figure 1 Mechanism of CRISPR/Cas9 action (Adopted from Arora and Narula, 2017) 3.2 Key drought resistance genes in poplar Several key genes have been identified in poplar that play significant roles in conferring drought resistance. These include genes involved in the biosynthesis of abscisic acid (ABA), a hormone that regulates stomatal closure and other drought responses. Additionally, genes encoding for dehydrins, late embryogenesis abundant (LEA) proteins, and aquaporins are crucial for maintaining cellular hydration and protecting cellular structures during drought stress. The identification and functional characterization of these genes have been facilitated by advanced genomic and transcriptomic studies, which have provided insights into the complex genetic networks underlying drought resistance in poplar (Chen and Lu, 2020; Badhan et al., 2021; Park et al., 2022). 3.3 Traditional methods for enhancing drought resistance Traditional methods for enhancing drought resistance in poplar trees have primarily relied on selective breeding and hybridization. These approaches involve selecting and cross-breeding individuals with desirable traits, such as deep root systems, efficient water use, and robust stress responses. While these methods have been successful to some extent, they are often time-consuming and labor-intensive. Moreover, the genetic basis of drought resistance is complex and involves multiple genes, making it challenging to achieve significant improvements through traditional breeding alone. Recent advancements in molecular biology and genetic engineering, such as CRISPR/Cas9 technology, offer new opportunities to enhance drought resistance in poplar more efficiently and precisely (Chandrasekaran et al., 2016; Arora and Narula, 2017; Borrelli et al., 2018; Chen et al., 2019). 4 Using CRISPR/Cas9 to Edit Drought-Resistance Genes in Poplar 4.1 Selection of target genes for drought resistance The selection of target genes is a crucial step when applying CRISPR/Cas9 technology to enhance the drought resistance of poplar. Genes associated with drought resistance typically play roles in various physiological and biochemical pathways, such as lignin biosynthesis, reactive oxygen species (ROS) scavenging, and transcriptional regulation. For example, the 4-coumarate-CoA ligase (4CL) gene involved in the lignin biosynthesis pathway was
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