Functional analysis of RsWUSb with Agrobacterium-mediated in planta transformation in radish (Raphanus sativus L.)

The WUSCHEL-related homeobox (WOX) family genes are involved in cell division and differentiation throughout various stages of plant growth and development. Till now, the function of WOX genes in radish remains unclear. In this study, a total of 23 RsWOX genes were identified from the radish genome. RT-qPCR analysis showed that the expression level of RsWUSb was significantly increased with exogenous cytokinin treatment. Overexpression of RsWUSb in Arabidopsis resulted in apical shoot clustering. Notably, the RsWUSb- overexpressed radish plants were generated by Agrobacterium-mediated in planta transformation approach. Compared with the wide-type (WT) plants, the RsWUSb-OE plants displayed clustered apical shoots, more leaves, and weak apical dominance. Moreover, yeast one-hybrid (Y1H) and dual-luciferase assay (DLA) showed that the RsRR18-1 (RESPONSE REGULATOR 18-1) bound to the promoter of RsWUSb and upregulated its transcription. Furthermore, the expression of RsRR18-1 was significantly activated by exogenous cytokinin, suggesting that cytokinin signaling might regulate the formation and development of radish shoots via RsRR18-1-RsWUSb module. Collectively, these findings indicated that in planta transformation approach is a simple and effective method for generating transgenic plants and gene function validation in radish.