WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix–loop–helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

Genetic transformation serves as a critical tool for gene function research and crop improvement. However, its efficiency is often low and highly dependent on species, genotypes, and explant types, significantly restricting its broader application. Many developmental factors have been proven pivotal not only for plant growth and development but also for the regulation of callus formation and shoot regeneration, which are key steps in the process of genetic transformation. Thus, this review focuses on the application of developmental factors in enhancing transformation efficiency across species. Developmental factors are classified into four regulatory pathways: morphogenesis, wound signaling, epigenetic modification, and hormone signaling. Among them, morphogenic factors have been extensively studied for enhancing transformation efficiency, while the potential of the other three pathways remains less explored in species beyond Arabidopsis. We summarize these established mechanisms and regulatory networks, providing a valuable reference for elucidating these mechanisms in other plant species. Furthermore, we also propose strategies for identifying species-specific efficient developmental factors and improving molecular mechanisms. Ultimately, this review provides a comprehensive summary of the mechanism and application of developmental factors and offers theoretical support to overcome the bottlenecks in genetic transformation.