Molecular Plant Breeding 2025, Vol.16, No.3, 202-210 http://genbreedpublisher.com/index.php/mpb 208 Solutions include: optimizing the composition of the culture medium (adding appropriate hormones and balancing salts), selecting suitable explants (such as young stem nodes or inflorescence tissues), and improving resistance to stress bacteria to prevent contamination. In addition, new transformation technologies can be tried, such as the aforementioned virus-mediated in vivo editing method- directly inoculating plant viruses carrying editing elements into golden pitaya plants to induce gene editing mutations (Baumann et al., 2020). 6.2 Challenges of polyploidy and heterologous genomes The genome of the bird’s nest fruit has undergone a whole genome duplication (WGD) event (Chen et al., 2021). Although it is diploid, it may have some gene family redundancy or multi-copy homologous sequences. This will bring challenges to precise editing: when there are multiple homologous copies of the target gene, all copies need to be knocked out at the same time to show the phenotype, otherwise the unedited copies may compensate for the function. In this regard, a multi-sgRNA parallel editing strategy can be adopted to design multiple sgRNAs at one time to target each homologous sequence, thereby increasing the probability of mutation of each copy (Tiwari et al., 2023). Another point is to prevent off-target effects caused by high sequence similarity between different copies. To this end, sgRNA design should avoid conservative sequence regions as much as possible, and select variant sites to guide editing to achieve differential editing. For example, the citrus CitAN1 and CitPH4 genes belong to the MYB family. When designing sgRNAs, their specific sequences can be targeted to avoid off-target effects (Miao et al., 2024). 6.3 Multi-gene regulation characteristics of flavor traits The flavor metabolic network is complex, and multi-gene editing may bring unexpected metabolic effects. For example, reducing a certain organic acid may cause carbon flow to shift to other metabolites, causing changes other than flavor (De Carvalho et al., 2017; Holt et al., 2018). Solving such problems requires comprehensive analysis of the edited strains through metabolomics to detect abnormalities early. If negative effects occur, consider refining the editing strategy, such as using promoter editing (reducing expression rather than knocking out) to achieve mild regulation rather than completely shutting down the pathway. By comparing gradient mutation materials, find the best balance between flavor enhancement and growth performance. 7 Future Outlook 7.1 New model of multi-omics co-breeding Gene editing breeding in the future will rely more on big data support. By integrating genome, transcriptome, metabolome and phenolic spectrum analysis, we will have a more comprehensive understanding of the complex network of golden pitaya flavor formation. By using machine learning and other means, we can dig out the key factors affecting flavor from massive data and improve the accuracy of target gene selection (Kaur et al., 2023). In particular, for the metabolism of aromatic substances, which is currently underrecognized, new regulatory genes are expected to be discovered with the help of multi-omics. For example, we may find enzymes that determine the unique floral fragrance of golden pitaya or transporters that affect aroma release. These new discoveries will continue to expand the editable target library, making flavor improvement more targeted and creative. 7.2 Coordination of flavor improvement with other traits Excellent varieties need to have excellent comprehensive performance in flavor, yield, resistance and other aspects. In the future, gene editing is expected to improve multiple traits at the same time through "superimposed editing". For example, while improving the sweetness of golden pitaya, superimposing the editing of disease-resistant genes, we can cultivate varieties that are both delicious and disease-resistant. This requires sophisticated design and balance, but some successful cases have emerged: for example, flavor and storage-resistant genes were edited simultaneously in tomatoes, achieving the best of both worlds (Ortega-Salazar et al., 2024). With the development of multi-gene editing technology, scientists can tailor multi-trait optimization solutions for golden pitaya, so that flavor enhancement does not sacrifice agricultural traits, and truly achieve a “flavor-agronomy win-win”.
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