Transcriptome and metabolome analyses reveal the efficiency of in vitro regeneration by TDZ pretreatment in mulberry

Thidiazuron (TDZ) is a plant growth regulator that is widely used as a defoliant and cytokinin-like compound. Additionally, mulberry is an economically important crop. However, regeneration is difficult in most mulberry species, and the regulatory mechanism controlling the regeneration process remains unclear. In this work, an efficient in vitro shoot regeneration system for mulberry was established by TDZ application. The results showed that the optimum adventitious bud induction, 70.83% in tetraploid M. notabilis cv. Chuanbei1 (CB1) and 96.82% in M. abla cv. Yunhua (YH), was obtained by the shoot pretreatment method using petiolate leaves as explants. The high-frequency regeneration petiolate leaf explants of YH treated with TDZ (T8) and the control (CK) were subjected to multiomic analyses. The comparison of the TDZ-treated with the CK groups identified 494 upre-gulated differentially expressed genes (DEGs) and 293 downregulated DEGs. GO analysis indicated that these DEGs were related mostly to integral component of membrane, transcription factor activity, and phosphorelay signal transduction system. Metabolomic analysis identified 1,889 compounds, including 205 differentially accumulated metabolites (DAMs). Of these DAMs, the number of lipids and lipid-like molecules was highest and accounted for 27.8% of all DAMs. Transcriptome profile, metabolomic, and phytohormone integrated analyses revealed that TDZ rearranged adenine-type cytokinins (CTKs) and negatively regulated endogenous CTK signal transduction. Moreover, TDZ treatment enhanced auxin signal transduction by increasing the expression of auxin flux carrier genes and downstream regulated genes without changing auxin biosynthesis in mulberry. In sum-mary, this study provides a new method for mulberry in vitro regeneration and contributes to a better under-standing of the transcriptional and metabolic changes that occur in mulberry after TDZ treatment.