Tree Genetics and Molecular Breeding 2024, Vol.14, No.5, 218-228 http://genbreedpublisher.com/index.php/tgmb 224 facilitating the study and manipulation of gene functions in various biological processes, including cambium regulation. CRISPR-Cas9, in particular, has been widely adopted due to its simplicity, efficiency, and versatility in editing genes across different species (Shen et al., 2021). Figure 3 ARF6 and ARF8 expression pattern overlaps with RGA and GAI during hypocotyl secondary growth (Adopted from Ben-Targem et al., 2021) Image caption: (A–D) Hypocotyl vibratome cross-sections at 0, 8 and 20 d after flowering (daf). Left and middle panels: confocal images of sections cleared with ClearSee and stained with Calcofluor White showing GFP signal in the nuclei (red arrows). Right panels: GUS assay on vibratome sections stained with phloroglucinol (red arrows indicates the phloem). (A) RGA:NLS-GFP-GUS (left and middle panels) and RGA:GUS (right panel). (B) GAI:NLS-GFP-GUS (left and middle panels) and GAI:GUS (right panel). (C) ARF6:NLS-3xGFP (left and middle panels) and ARF6:GUS (right panel). (D) ARF8:NLS-3xGFP (left and middle panels) and ARF8:GUS (right panel). White scale bar: 20 μm and black scale bars: 20 μm (Adopted from Ben-Targem et al., 2021) 6.2 Applications in modifying cambium-specific genes Gene editing has been successfully applied to modify cambium-specific genes to understand their roles in secondary growth and wood formation. For instance, the CRISPR-Cas9 system was used to edit the PdBRI1 genes in Populus, which are involved in brassinosteroid signaling. The edited lines exhibited significant changes in cambial activity and wood development, highlighting the importance of these genes in regulating cambium function (Wang et al., 2022a). Similarly, the expression of the co-transcriptional regulator PanNOOT1 in Parasponia andersonii was manipulated using CRISPR-Cas9, demonstrating its essential role in controlling secondary growth (Shen et al., 2021). 6.3 Potential for improving tree stress resistance Gene editing technologies hold great potential for enhancing tree stress resistance by targeting genes involved in stress response pathways. For example, the manipulation of cytokinin signaling pathways through the expression of a constitutively active cytokinin receptor variant in poplar increased cambial activity and stem growth, which could potentially improve the tree's resilience to environmental stresses (Riefler et al., 2022). Additionally, the regulation of brassinosteroid biosynthesis via the overexpression of PagDET2 in poplar promoted cambium cell division and xylem differentiation, suggesting a role in stress adaptation (Wang et al., 2022b).
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