Triticeae Genomics and Genetics, 2025, Vol.16, No.3, 120-129 http://cropscipublisher.com/index.php/tgg 124 enough. The resistance measures that were previously relied on quickly became ineffective in the face of it, especially the defense line that relied on only one gene, which almost collapsed at the slightest touch. The spread rate and mutation ability of this strain have forced people to reconsider the breeding approach-broad-spectrum and persistence are no longer "options" but "must" (Zhang et al., 2017; Yu et al., 2022). 5.2 Review of the stacked gene construct and its field-ready potential The real turning point actually does not lie in "a certain magical gene", but in the construction of a five-gene box. This combination of multiple stem rust resistance genes, when placed at the same site, can form an effective barrier for wheat from the seedling stage to the mature plant stage. This build contains both ASR and APR, among which four have been confirmed to have effects. More importantly, such a single locus superposition greatly simplifies the genetic process. What originally required generations of hybridization to achieve can now be accomplished with a single transfer. Field trials also provided positive feedback: in the face of highly invasive isolates including Ug99, these strains demonstrated significant high resistance (Luo et al., 2021; Jost et al., 2023). However, to be honest, some Pgt isolates have already been able to break through these combinations, which also reminds us that we cannot "put it once and for all". Continuous monitoring and dynamic adjustment of the portfolio strategy are the keys to going down this path. 5.3 Global implications and lessons learned What does this case prove? It is demonstrated that modern genomic tools and transgenic strategies can indeed be useful when dealing with complex agricultural diseases. But this does not mean that the problem has been solved. The pathogen of rust disease is still evolving, and the regulatory threshold for genetically modified crops is not low either. In different countries and regions, how to implement these gene combinations and how to carry out personalized deployment based on the distribution of diseases still rely on long-term strategies for support. There is experience: the combination can be strong, but not rigid. It has to be used in conjunction with agricultural management methods and flexibly adjusted according to regional differences. Otherwise, even the best genes will one day be broken through (Zhang et al., 2021). 6 Challenges and Limitations of Gene Stacking in Wheat 6.1 Technical challenges in multigene engineering Although the idea of superimposing multiple genes on a crop like wheat, which has a large and complex genome, is good, it is not so smooth to implement. Not only do multiple resistance genes have to be inserted into a specific site, but they also need to be expressed stably. This in itself is quite "tormenting". Although there have been successful cases of five gene superpositions, larger constructs, such as those with eight genes and a size of 63 kb, have not yet been thoroughly tested. No one can guarantee exactly how much can be contained in wheat. Moreover, can a set of combinations perform well in all varieties and various climates? This is also a question mark. Sometimes, a certain gene that can resist diseases in one variety may not work in another. Whether the resistance is strong or not depends not only on the genes themselves but also on whether the "compatibility" with the wheat variety and the "personality" of the local pathogenic bacteria match. 6.2 Regulatory and biosafety concerns Technically, it can be advanced, but whether it can be widely promoted is another matter. Especially in the field of genetically modified organisms, wheat has not gone as smoothly as some other crops. The review process for genetically modified organisms in many countries is quite strict, especially when it comes to new varieties with multiple gene superpositions, the approval process is even more lengthy. Even if it has been scientifically verified to be safe, the regulatory environment in some regions will still slow down the deployment progress. Moreover, the issue of gene loss is not an unfounded worry. Some people are worried that the genes transferred in might be accidentally passed on to wild relatives or affect non-target organisms. Therefore, it is fundamentally difficult to obtain approval without conducting long-term monitoring and a comprehensive risk assessment. In addition, issues such as the ownership of intellectual property rights and public acceptance, which are "beyond technology", also make commercial promotion not so easy.
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