Morphological and transcriptome analyses reveal mechanism for efficient regeneration of adventitious buds from in vitro leaves of Rhododendron delavayi regulated by exogenous TDZ

The efficient differentiation of adventitious buds from in vitro leaves has been of great significance for improving Rhododendron traits via genetic transformation. Yet, the actual mechanism of leaf differentiation into adventitious buds remains unclear. In this study, morphological and transcriptome analyses revealed the mechanism by which exogenous thidiazuron effectively regulates the buds’ regeneration from in vitro leaves of Rhododendron delavayi. The experimental treatment consisted of 0.36 µM thidiazuron combined with 0.05 µM 1-naphthaleneacetic acid; for the control, 0.05 µM 1-naphthaleneacetic acid was added to the medium. The results showed that s2 (15 to 22 d) was the key stage for leaf budding. At this stage, the content of endogenous zeatin riboside in leaves regulated by exogenous thidiazuron reached its peak value, and the leaves gained regeneration ability. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that differentially expressed genes associated with plant hormone signal transduction pathway, DNA replication, and amino sugar and nucleotide sugar metabolism were predominantly enriched at four developmental stages between the control and treatment. During the adventitious bud formation induced by thidiazuron, the encoding histidine kinase gene (AHK4), type a Arabidopsis response regulator genes (ARR6, ARR9, ARR17), auxin-responsive protein genes (IAA13, IAA27), and auxin influx carrier protein genes (LAX2, LAX3) were up-regulated. Those genes initiate differential expression of a series of genes regulating DNA replication, cell wall formation and repair, promotion of cell proliferation, and differentiation, thereby effectively completing bud regeneration from in vitro leaves.