Tree Genetics and Molecular Breeding 2024, Vol.14, No.3, 132-143 http://genbreedpublisher.com/index.php/tgmb 135 in the host (Liu et al., 2020). Transgenic plants or tissues expressing the candidate genes are subjected to pathogen challenge assays. These involve exposing the plants to specific pathogens to evaluate the effectiveness of the resistance genes in conferring protection. The response of the transformed plants is assessed through molecular techniques like qPCR, RNA-Seq, and Western blotting to measure gene expression levels. Phenotypic analyses, including disease symptom scoring and growth assessments, provide additional validation of gene function (Rellstab et al., 2019). To confirm laboratory findings, field trials are conducted where transgenic conifers are planted in natural settings and monitored for resistance to diseases under real-world conditions. These trials help determine the practical applicability of the resistance genes in forestry management (Xie et al., 2023). 5.2 Success stories of gene function verification in laboratory and field conditions Case study 1: NBS-LRRgene in pine species A notable example is the verification of the NBS-LRR gene in various pine species. Researchers cloned and expressed the NBS-LRR gene, which was identified as a key player in resistance against pine wilt disease (PWN) caused by the nematode Bursaphelenchus xylophilus. Xie et al. (2023) demonstrated that PmNBS-LRR97 can significantly activate the expression of reactive oxygen species (ROS)-related genes in Pinus massoniana, participating in the defense response against pine wood nematode (PWN). Due to the lack of a successful genetic transformation system for Pinus massoniana, Xie et al. (2023) transformed PmNBS-LRR97 into Nicotiana benthamianato verify its function (Figure 1). Figure 1 Quantitative verification and phenotypic difference analysis of PmNBS-LRR97-overexpressing tobacco (Adopted from Xie et al., 2023) Image caption: (A) Relative expression of PmNBS-LRR97 in WT and transgenic tobacco; error bars represent three biological replicates ± SD. (B) Plant height analysis of WT and PmNBS-LRR97-overexpressing tobacco at 30, 60, and 90 d; error bars represent three biological replicates ± SD. Student’s t-test was used to test the significance (*, p < 0.05; **, p < 0.01). (C) Differences in the flowering number and branch number between WT and PmNBS-LRR97-overexpressing tobacco at 90 d; error bars represent three biological replicates ± SD. (D) Different phenotypes of WT and PmNBS-LRR97-overexpressing tobacco. Scale bar = 7 cm (Adopted from Xie et al., 2023)
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