Bioscience Methods 2025, Vol.16, No.6, 289-298 http://bioscipublisher.com/index.php/bm 292 saves time, and makes breeding decisions more grounded (Salgotra and Stewart, 2020). Moreover, a series of practical markers have been developed for some key resistant traits (such as vesicular disease) (Wang et al., 2020). If the transcriptome, metabolome and other data can be combined for analysis, the ability of MAS to analyze complex traits will be further enhanced. 3.3 Breeding strategies for pyramiding multiple resistance genes Although a single resistance gene can bring short-term effects, in today's era of rapid evolution of pathogenic bacteria, this strategy is often not sustainable enough. Therefore, breeders are increasingly inclined to "package" multiple resistance genes into the same strain, which is commonly referred to as "gene aggregation" or "compound resistance". Traditional breeding has struggled in this regard, but with molecular marker-assisted technology, it becomes more feasible to superimpose QTL or multiple disease-resistant genes. Some studies have also pointed out that this compound strategy can significantly reduce the probability of pathogen escape or resistance failure (De Almeida et al., 2021). In terms of operational methods, marker-assisted backcrossing, cyclic selection, or even gene box design and gene editing techniques can be used for precise operations. However, to be fair, even if the technology is mature and truly effective aggregation is achieved, the right gene combinations still need to be selected to ensure that they can also work synergistically in similar genetic backgrounds. 4 Molecular Tools for Improving Tea Plant Resistance 4.1 Mining resistance genes through transcriptomics and genomics Often, the resistance of tea plants is not led by a single gene, but by a complete network working together. Research at the transcriptome and genomic levels has long been involved in this matter. Proteins such as the flavonoid pathway and NB-ARC domain are highly active in resisting diseases and low temperatures, and the relevant data are also increasing (Li et al., 2025). In some special scenarios, BAHD acyltransferase and members of the ABA receptor family (PYL) have also been observed to be significantly expressed after pest and disease stress (Qiao et al., 2024), and these genes have now become potential breeding targets. Interestingly, after aggregating and analyzing different transcriptome data, tens of thousands of differentially expressed genes (DEGs) could be screened out, involving the MAPK signaling pathway, various plant hormone pathways (SA, JA, ET), and secondary metabolic pathways (Hazra et al., 2023; Xu et al., 2025). These results not only indicate that the regulation is very complex, but also show that there are actually many clues to follow in resistance breeding. 4.2 Application of RNA interference (RNAi) in pest control When it comes to precise pest control, RNA interference technology does offer a relatively "clean" approach. Unlike pesticides that strike indiscriminately, RNAi can target specific genes of pests and has an extremely low off-target rate. At present, there are more than one method. Besides transgenic methods to enable tea plants to express double-stranded RNA (dsRNA) by themselves, there are also non-translational methods such as trunk injection and foliar spraying (Cagliari et al., 2019). However, in the final analysis, these Dsrnas can only function if they can exist stably and be effectively absorbed. In this regard, the addition of nanoparticle technology has been of great help and improved the delivery efficiency (Yan et al., 2020; Ahmad et al., 2025). However, it should be said that although RNAi is good, there are still some obstacles before it can be truly promoted. Issues such as its stability in the environment, how to use it on a large scale, and regulatory reviews all remain to be addressed (Chen and De Schutter, 2024). 4.3 Potential of gene editing tools in functional validation and trait improvement The CRISPR/Cas9 tool has now almost become a "standard configuration" in the field of breeding, but in perennial crops like tea trees, its direct application is still in the trial stage. However, many researchers have already regarded it as a good helper for verifying the functions of candidate resistance genes. For instance, members of the NB-ARC, PYL, and BAHD families can be analyzed for their specific roles in the resistance response through CRISPR knockout or insertion (An et al., 2023). Another advantage of the editing tool is that it can help superimpose multiple resistant traits without having to worry about introducing redundant traits as in traditional hybridization. When gene discovery technology and editing technology truly "join hands", in the future, to cultivate highly resistant varieties in tea trees, it may no longer require so many generations of breeding.
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