**5. Conclusions**

For species where plant transformation, regeneration and genetic backgrounds are challenging, VIGS provides a powerful tool for transient gene function studies. We demonstrated that the TRV-based VIGS system can effectively silence genes in *L. chinensis* and established a TRV-based VIGS system that could successfully induce yellow leaf phenotypes by silencing *LcCLA1* and *LcPDS*, respectively. According to the influencing factors such as *Agrobacterium* strain GV3101, tip needle injection method and inoculated plant growth temperature of 26 ◦C, the relative expression level of *LcCLA1* and *LcPDS* can reach about 25–50%, respectively. The TRV-mediated VIGS system developed and used in this study provides an alternative tool for functional gene studies in *L. chinensis*. This study also could provide a reference for the development of a rapid, transient and stable transformation system for *Lycoris*. In addition, we have improved the injection method for blades with waxy surfaces, which greatly improves the injection efficiency and ease of operation. This can provide new ideas for other plants that are not easily penetrated. These results help to lay the foundation for gene expression analysis in *Lycoris* and better studying of the molecular mechanism of related traits.

**Author Contributions:** F.Z. conceived the project and designed the experiments; G.C. and X.S. performed the experiments and analyzed the data; N.W. and Z.W. prepared and collected the samples; G.C. and F.Z. wrote and revised the manuscript; Z.W. and N.W. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (31801900); the Natural Science Foundation of Jiangsu Province (BK20180310); the Open Fund of Jiangsu Key Laboratory for the Research and Utilization of Plant Resources (JSPKLB202205); the Jiangsu Provincial Crop Germplasm Resource Bank for Conservation (2022-SJ-015); Jiangsu Provincial Forestry Science and Technology Promotion Project (LYKJ[2022]08).

**Data Availability Statement:** The data are available from the corresponding author upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.
