Transcriptomic and functional analyses identify genes associated with embryogenic callus formation in Neolamarckia cadamba

Neolamarckia cadamba is an economically significant tree used for timber and pulp production, and as traditional medicine for various ailments. However, the absence of an effective in vitro plant regeneration system hampers its further development and promotion of superior varieties. Previous researches have highlighted the efficacy of embryogenic callus for both plant regeneration and genetic modification. To elucidate N. cadamba's embryogenesis, we conducted transcriptome profiling of leaf explants across three distinct tissue culture stages. This investigation revealed significant gene expression shifts, involving 12,963 genes. Notably, 7284 and 6652 genes displayed differential expression in ECI02 vs. ECI01 and ECI03 vs. ECI02, respectively. Functional categorization highlighted the differentially expressed transcription factors predominantly associated with hormone homeostasis and signal transduction, post-transcriptional regulation, and meristem development. Given the genes link to auxin and cytokinin signal pathways, and genes affiliate to embryogenesis identity play pivotal roles in embryogenic callus formation, we focused on these genes and found 36 of them showed differential expression patterns during embryogenic callus induction, indicating these genes potentially contribute to embryogenic callus formation. Further functional characterization confirmed that overexpression of NcBBM and NcARF5 in Nicotiana benthamiana induce callus formation without exogenous phytohormone; however, only NcBBM-induced callus exhibited shoot regeneration. These findings illuminated the molecular orchestration underlying embryogenic callus formation in N. cadamba, and genetic manipulation of these candidate genes may help to improve its tissue culture. © 2024 Elsevier B.V.