Molecular Plant Breeding 2024, Vol.15, No.3, 144-154 http://genbreedpublisher.com/index.php/mpb 149 6 Functional Verification of Edited Genes 6.1 In vitro assays In vitro assays are crucial for the initial functional verification of edited genes. These assays allow researchers to observe the direct effects of gene edits in a controlled environment. For instance, the PmPR10-3.1 gene from Pinus monticola was expressed in Escherichia coli, and the purified recombinant protein exhibited inhibitory effects on the spore hyphal growth of fungal pathogens such as Cronartium ribicola, Phoma exigua, and P. argillacea. This demonstrates the potential of the edited gene in conferring disease resistance. Additionally, in vitro assays can help identify the physiological roles of pathogenesis-related (PR) proteins, which are essential in plant defense responses (Liu et al., 2021). 6.2 Transgenic pine models Creating transgenic pine models is a critical step in verifying the functionality of edited genes in a living organism. These models help in understanding how the edited genes perform in the complex biological systems of pine trees. For example, the use of genome editing tools like CRISPR has enabled the development of transgene-free, disease-resistant crop varieties by targeting susceptibility (S) genes (Zaidi et al., 2018). In the context of pine trees, transgenic models can be used to study the expression and impact of edited genes such as PmPR10-3.1 in response to pathogen infection, providing insights into their role in quantitative disease resistance (Weiss et al., 2020; Liu et al., 2021). 6.3 Field trials and environmental assessments Field trials and environmental assessments are essential for evaluating the real-world applicability and effectiveness of edited genes. These trials help in understanding how the edited genes perform under natural environmental conditions and in the presence of various biotic and abiotic stressors. For instance, the genomic architecture of quantitative disease resistance in white pine species has been studied to facilitate marker-assisted disease resistance breeding (Weiss et al., 2020). Field trials can validate the effectiveness of edited genes like PmPR10-3.1 in providing resistance to diseases such as white pine blister rust, thereby confirming their potential for practical applications in forestry and agriculture (Liu et al., 2019; Weiss et al., 2020; Liu et al., 2021). 7 Case Studies and Success Stories 7.1 Successful gene edits conferring disease resistance Recent advancements in gene editing have demonstrated significant success in conferring disease resistance in pine species. One notable example is the use of the CRISPR/Cas9 system, which has been applied extensively in tree genetic studies to develop new disease-resistant cultivars. This system has shown great potential in regulating lignin biosynthesis and shortening the breeding cycle of forest trees, thereby enhancing their resistance to various diseases (Chen and Lu, 2020). Another successful application of gene editing is the identification and functional characterization of the PmPR10-3.1 gene in western white pine (Pinus monticola). This gene was found to play a crucial role in quantitative disease resistance (QDR) to white pine blister rust. The purified recombinant protein of PmPR10-3.1 exhibited inhibitory effects on the growth of fungal pathogens, providing valuable insights into the genetic architecture underlying QDR in conifers (Liu et al., 2021). Prime editing, a novel genome editing technology, has also shown promise in correcting genetic defects and conferring disease resistance. For instance, prime editing has been used to generate precise in-frame deletions in the CTNNB1 gene, mimicking mechanisms of disease development and functionally recovering disease-causing mutations in organoid models. This technology offers greater precision than traditional methods and holds therapeutic potential for various plant diseases (Schene et al., 2020). 7.2 Comparative analysis of edited vs. non-edited pines Comparative studies between edited and non-edited pines have provided compelling evidence of the benefits of gene editing in enhancing disease resistance. For example, a study on sugar pine (Pinus lambertiana) revealed that single nucleotide polymorphisms (SNPs) associated with the Cr1Rgene, a major gene for resistance to white pine
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