Tree Genetics and Molecular Breeding 2024, Vol.14, No.3, 144-154 http://genbreedpublisher.com/index.php/tgmb 146 3.2 Criteria and methods for selecting candidate genes The selection of candidate genes for salt tolerance involves several criteria and methods. One primary criterion is the differential expression of genes under salt stress conditions. For example, the NAC13 gene was selected based on its significant up-regulation in response to salt stress in poplar (Zhang et al., 2019a). Another criterion is the functional validation of candidate genes through overexpression and suppression studies. The PeERF1 gene was functionally verified by transforming it into Populus alba × Populus glandulosa, where overexpressed plants exhibited better growth and physiological characteristics under salt stress compared to wild-type plants (Ge et al., 2022). Additionally, the physiological and biochemical responses of transgenic plants are evaluated to confirm the role of candidate genes in salt tolerance. For instance, transgenic poplar overexpressing PtGSTF1 showed improved shoot growth, wood formation, and optimized ion homeostasis under salt stress, confirming its role in enhancing salt tolerance (Figure 1) (Gao et al., 2022). Figure 1 Overexpression of PtGSTF1 enhanced salt tolerance in transgenic plants (Adopted from Gao et al., 2022) Imagine caption: (a) Callus induction analysis. Leaf explants from four-week-old wild type (WT) and PtGSTF1 transgenic plants were cultured on callus induction medium supplemented with 0 (control) or 150 mM NaCl for 1 week. (b) Growth phenotypes of WT and transgenic plants. Four-week-old plants grown in a greenhouse were treated with 0 (control) or 150 mM NaCl for another 4 weeks. (c–f) Plant heights, fresh and dry weights, and growth inhibitions before and after the salt treatment. WT, wild type; OE2, OE4 and OE5, different transgenic lines. Values are the mean ± SD from three independent experiments (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Adopted from Gao et al., 2022) Gao et al. (2022) illustrates the impact of PtGSTF1 overexpression on salt tolerance in transgenic poplar plants compared to wild type (WT). Panel (a) shows callus formation in WT and transgenic leaf explants under normal and 150 mM NaCl conditions, highlighting better callus induction in transgenic lines under salt stress. Panel (b) displays the growth phenotypes of WT and transgenic plants before and after 150 mM NaCl treatment, with transgenic lines (OE2, OE4, OE5) exhibiting superior growth and less severe damage. Panels (c-f) quantify plant height, fresh weight, dry weight, and growth inhibition, respectively. The data reveal that transgenic lines significantly outperform WT in all measured traits under both normal and salt stress conditions, demonstrating that PtGSTF1 overexpression enhances salt tolerance in poplar plants. 3.3 Case studies: successful identification of salt tolerance genes in poplar Several case studies highlight the successful identification and functional verification of salt tolerance genes in poplar. One notable example is the PeERF1 gene from Populus euphratica, which was identified using a combination of WGCNA and GWAS. Transgenic Populus alba × Populus glandulosa plants overexpressing PeERF1 exhibited enhanced salt tolerance, demonstrating the gene's potential for improving salt tolerance in poplar breeding (Ge et al., 2022). Another example is the PsnHDZ63 gene, identified through RNA-seq analysis. Transgenic Populus simonii × P. nigra plants overexpressing PsnHDZ63 displayed better morphological and
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