HcBBM promotes callus differentiation and shoot regeneration by coordinating auxin signaling in Hemsleya chinensis

ABABY BOOM (BBM), an APETALA2/ERF-like transcription factor (TF), plays an important role in promoting plant genetic transformation and regeneration. However, the hormonal regulatory networks, transcriptional mechanisms and the basis of their morphogenetic regulation underlying callus differentiation and shoot regeneration of the cucurbit medicinal plant H. chinensis are still unclear. Here, this study analyzed the dynamics of the three callus differentiation stages in H. chinensis by integrating hormonomics and transcriptomics analyses.

The results reveal dynamic changes in hormone levels during callus development. In the early stages of callus differentiation, auxin and cytokinin accumulates significantly, promoting callus differentiation. Co-expression network analysis further demonstrated that members of the AP2/ERF gene family were significantly associated with these two hormones signaling pathways. Therefore, the study cloned the HcBBM gene from H. chinensis, and functional validation showed that HcBBM overexpression significantly enhanced callus proliferation and improved adventitious shoot regeneration efficiency while maintaining normal phenotypic characteristics. Furthermore, transcriptome analysis revealed that HcBBM coordinated the balance between auxin and cytokinin signaling pathways by regulating the expression of key genes, such as ARF, GRF, and B-ARR, and integrating other hormone signals including ethylene and brassinolide, constructed a molecular network that responds to multiple hormone signals, including auxin, cytokinin, and ethylene, thereby precisely regulating callus differentiation.

This study demonstrates that the HcBBM gene in H. chinensis callus integrates auxin signalling to synergistically regulate key factors such as ARF and GRF, thereby establishing a finely tuned molecular network that controls callus differentiation and significantly enhances regeneration efficiency. This study fills part of the gaps in the research on the regeneration molecular mechanism of H. chinensis and other cucurbitaceous medicinal plants. The established HcBBM-mediated system provides an efficient tool for the genome editing of cucurbitacin biosynthesis genes in H. chinensis and the improvement of transformation efficiency in recalcitrant cucurbit medicinal plants, also offers new insights into the quality breeding and genetic improvement of other medicinal plants in the Cucurbitaceae family that are difficult to propagate.