Molecular Plant Breeding 2024, Vol.15, No.2, 81-89 http://genbreedpublisher.com/index.php/mpb 84 targeted in chickpeas to understand its role under drought stress (Badhan et al., 2021). Similarly, the MYB transcription factor Reveille 7 (RVE7), which regulates the circadian rhythm, was edited to study its effect on drought resistance (Badhan et al., 2021). These examples highlight the importance of selecting genes that play significant roles in the plant's response to drought conditions. 4.2 CRISPR/Cas9 vector construction and delivery methods The construction of CRISPR/Cas9 vectors and their delivery into poplar cells are pivotal for successful gene editing. The CRISPR/Cas9 system typically involves the use of a Cas9 endonuclease and a single guide RNA (sgRNA) that directs the Cas9 to the specific genomic location. Various methods have been employed to deliver these components into plant cells, including Agrobacterium-mediated transformation, which has been successfully used in poplar (Fan et al., 2015). Additionally, the use of ribonucleoproteins (RNPs) composed of Cas9 and sgRNA has been shown to reduce off-target effects and avoid integrational mutagenesis (Arora and Narula, 2017; Mout et al., 2017). Nanoparticle-based delivery approaches are also emerging as efficient methods for direct cytosolic delivery of Cas9-RNP complexes, achieving high gene editing efficiencies (Mout et al., 2017). 4.3 Case studies of successful gene editing in poplar for drought resistance Some studies have demonstrated the successful application of CRISPR/Cas9 technology in editing various traits in poplar trees, including drought resistance. For example, editing the phytoene desaturase gene (PDS) in an interspecific hybrid poplar (Populus davidiana × P. bolleana) achieved high mutation efficiency and generated albino phenotypes, indicating successful gene knockout (Wang et al., 2020). The research team focused on a hybrid poplar variety called Shanxin yang, known for its good cold and drought resistance. Through Agrobacterium-mediated transformation, recombinant plasmids containing the Cas9 expression cassette and two sgRNA cassettes were introduced into poplar leaves. The results showed that transgenic poplars with PDS gene mutations exhibited various phenotypes, including complete albinism, variegation, and light green phenotypes. The study demonstrates that the CRISPR/Cas9 system can efficiently edit the poplar genome, providing a new approach for improving drought resistance in poplar trees (Figure 2). Figure 2 Representative Shanxin yang phenotypes resulting fromPDSgene mutations (Adopted from Wang et al., 2020) Image caption: Total albino (A), variegated (B), and pale green (C) phenotypes of the regenerated shoots. (D) Wild-type (WT) Shanxin yang shoots. (E) Comparison between the 1-month-old albino and WT seedlings (Adopted from Wang et al., 2020) By analyzing Figure 2, it can be seen that poplar regeneration buds with PDS gene mutations exhibit varying
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