Physiological parameters and differential expression analysis of N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea-induced callus of Eucalyptus urophylla × Eucalyptus grandis

In this study, we analyzed differences in the enzyme activities and transcriptomes of embryogenic and non-embryogenic calli to gain insights for improving the success of tissue culture-based breeding. A total of 2,856 differentially expressed genes (DEGs; 1,632 up-regulated and 1,224 down-regulated) were identified based on RNA sequencing and verified by reverse transcription quantitative polymerase chain reaction. Gene set enrichment analysis revealed that many of the up-regulated DEGs in embryogenic callus were enriched in the photosynthesis processes. Furthermore, the enzyme activity, hormone content, and cytokinin oxidase/dehydrogenase (CKX) gene expression analyses were found to be consistent with the transcriptome results. Cytokinin biosynthesis in N-phenyl-N'-[6-(2-chlorobenzothiazol)-yl] urea (PBU)-induced embryogenic callus increased owing to CKX repression. Measurement of endogenous hormones by high-performance liquid chromatography revealed that, compared with non-embryogenic callus, in embryogenic callus, the indole-3-acetic acid, abscisic acid and trans-zeatin riboside content had significantly higher values of 129.7, 127.8 and 78.9 ng/g, respectively. Collectively, the findings of this study will provide a foundation for elucidating the molecular mechanisms underlying embryogenic callus differentiation and can potentially contribute to developing procedures aimed at enhancing the success of callus-based plant regeneration.