Unlike animals, plants are sessile organisms that cannot move freely in response to fluctuating and complex environments. As a result, plant development follows post-embryonic processes, enabling flexible developmental strategies to adapt to changing environment. The WUSCHEL-related homeobox (WOX) gene family plays a crucial role in regulating these post-embryonic processes in plants. In this study, we performed an evolutionary analysis of the WOX gene family across 29 plant species, isolating a total of 330 WOX family genes. Our study identified a fern protein with similar length and conserved motifs to WUS gene of spermatophyte, suggesting that the modern clade of the WOX family may have already diverged in ferns. Furthermore, we conducted a pan-genome analysis of the WOX family in Malus, examining the number and gene characteristics of WOX family members across eight varieties. The promoter elements of WUS-1, WUS-2, WOX5-1, and WOX5-2 in different Malus varieties were analyzed further. Additionally, we examined the expression patterns of modern clade WOX family members in developing tissues and during leaf-borne shoot regeneration of Malus. We developed the transgenic lines with inducible overexpression of MdWUS-1 or MdWOX5-1, which revealed that mild upregulation of MdWUS-1 significantly promoted leaf-borne shoot formation, while strong upregulation of MdWUS-1 led to browning and death of explants, likely due to oxidative stress. These findings provide new insights into the evolution of the WOX gene family from ferns into seed plants and lay the foundation for further studies on the spatiotemporal regulation of gene expression during shoot regeneration.

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