Molecular Plant Breeding 2024, Vol.15, No.2, 81-89 http://genbreedpublisher.com/index.php/mpb 82 ensuring their sustainability and productivity in changing climates. This study not only provides new insights for improving drought resistance in poplars but also offers valuable references for the study of stress tolerance in other plants. 2 CRISPR/Cas9 Technology: Mechanism and Applications 2.1 Basics of CRISPR/Cas9 gene editing CRISPR/Cas9, which stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9, is a revolutionary gene-editing technology that allows for precise modifications at specific locations within the genome. The system comprises two key components: the Cas9 enzyme, which acts as molecular scissors to cut DNA, and a single guide RNA (sgRNA) that directs Cas9 to the target DNA sequence. This technology has been widely adopted due to its simplicity, efficiency, and versatility in various organisms, including plants (Badhan et al., 2021; Park et al., 2022). 2.2 CRISPR/Cas9 applications in plant science CRISPR/Cas9 has been extensively utilized in plant science to enhance traits such as disease resistance, stress tolerance, and yield improvement. For instance, in chickpea, CRISPR/Cas9 was used to edit the 4CL and RVE7 genes, which are associated with drought tolerance, demonstrating high-efficiency editing and providing insights into drought stress mechanisms (Badhan et al., 2021). Similarly, in rice, the OsSAP gene was edited to study its role in drought stress, showing that CRISPR/Cas9 can significantly reduce breeding cycles and improve stress-related traits (Park et al., 2022). Additionally, CRISPR/Cas9 has been employed to develop virus-resistant cucumber plants by targeting the eIF4E gene, resulting in non-transgenic plants with enhanced resistance to multiple viruses (Chandrasekaran et al., 2016). In forest trees, CRISPR/Cas9 has been applied to develop new drought-resistant cultivars and regulate lignin biosynthesis, showcasing its potential in tree genetic studies and breeding (Chen and Lu, 2020). 2.3 Advantages of CRISPR/Cas9 over traditional breeding techniques CRISPR/Cas9 offers several advantages over traditional breeding techniques. It allows for precise and targeted modifications without the need for extensive backcrossing, which is time-consuming and labor-intensive. For example, the development of virus-resistant cucumber plants using CRISPR/Cas9 did not require long-term backcrossing, unlike traditional methods (Chandrasekaran et al., 2016). CRISPR/Cas9 can significantly shorten breeding cycles, as demonstrated in rice, where the technology enabled rapid generation of drought-tolerant cultivars (Park et al., 2022). CRISPR/Cas9 can be used to edit multiple genes simultaneously, providing a more comprehensive approach to trait improvement. This was evident in the chickpea study, where both 4CL and RVE7 genes were edited to enhance drought tolerance (Badhan et al., 2021). Overall, CRISPR/Cas9 represents a powerful tool for plant breeders and researchers, offering unprecedented opportunities for crop and tree improvement (Chandrasekaran et al., 2016; Chen and Lu, 2020; Badhan et al., 2021; Park et al., 2022). 3 Drought Resistance in Poplar Trees 3.1 Physiological and molecular responses to drought stress Poplar trees, like many other plant species, exhibit a range of physiological and molecular responses to drought stress. These responses are crucial for their survival and adaptation in water-limited environments. Physiologically, drought stress in poplar trees leads to reduced leaf water potential, stomatal closure, and decreased photosynthetic rates. These changes help to minimize water loss and maintain cellular turgor pressure. On a molecular level, drought stress triggers the expression of various stress-responsive genes, including those involved in the synthesis of osmoprotectants, antioxidants, and stress-related proteins (Figure 1). These molecular responses are part of a complex regulatory network that helps the plant to cope with and adapt to drought conditions (Arora and Narula, 2017; Zhu et al., 2020; Wang et al., 2022). The study by Arora and Narula (2017) demonstrates the mechanism of CRISPR/Cas9 in modifying the poplar genome. The process begins with the acquisition phase, where foreign DNA is integrated into the CRISPR locus of the bacterial genome. This locus is then transcribed into a primary transcript and processed into crRNA with the help of tracrRNA during crRNA biogenesis. During the interference phase, the Cas9 endonuclease forms a
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