Comprehensive transcriptomic and functional characterization of protoplast regeneration in Angelica gigas Nakai.

backgroundProtoplasts that are isolated from various plant sources, including leaf mesophyll tissue, callus, will continue to have the ability to take part in cell wall regeneration, division, and expression of totipotency. In model systems like Arabidopsis thaliana, it has been thoroughly established. Meanwhile, the fate of protoplasts isolated from both embryogenic (EC) and non-embryogenic callus (NEC) in other plants is unknown. Thus, we conducted transcriptome analyses of protoplasts produced from both EC and NEC in Angelica gigas in the present investigation. To achieve this, three stages of RNA sequencing were carried out: (1) EC, (2) freshly isolated protoplasts (Pt), and (3) cells undergoing cell division (CD) during A. gigas in vitro protoplast regeneration. Different gene expression programs were identified across stages through transcriptome profiling, which highlighted early stress responses, transcriptional changes, and the acquisition of stem cell identity following protoplast isolation.

resultsA pre-existing stem cell-like condition was shown by the strong expression of WUSCHEL-RELATED HOMEOBOX5 (WOX5) and CUP-SHAPED COTYLEDON2 (CUC2) genes at the EC stage. Important genes linked to stress reactions and cellular transcriptional changes, as WOX13 and WOUND INDUCED DEDIFFERENTIATION1 (WIND1), were significantly elevated at the Pt stage. Additionally, during this phase, genes associated with the cell cycle, auxin and cytokinin signaling, and cell wall regeneration were also active, indicating a dynamic shift toward regaining stem cell identity. Key regulators of stem cell maintenance and proliferation, such as LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and ENHANCER OF SHOOT REGENERATION2 (ESR2), were then substantially expressed at the CD stage, encouraging the start of cell division. The dynamic regulation of embryogenesis-related genes, including SOMACTIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 2 (SERK2), LBD29, ESR2, LBD16, D-TYPE CYCLINS (CYCD3-2), and WOX1, during protoplast culture was validated by real-time quantitative polymerase chain reaction (RT-qPCR).

conclusionsWOX5 and CUC2 genes were found to be significantly expressed at the EC stage, suggesting a pre-existing stem cell-like state. Important genes linked to stress reactions and cellular transcriptional changes, as WOX13 and WIND1, were clearly elevated at the Pt stage. The activation of genes linked to cell wall renewal, auxin and cytokinin signaling, and the cell cycle during this phase also suggested a dynamic transition toward regaining stem cell identity. Additionally, the findings previously reported show that the EC-derived protoplasts successfully underwent cell division and contributed to the development of somatic embryos. Although the NEC-generated protoplasts did not divide upon culture, these results show that embryogenic cells maintain their embryogenic potential even after protoplast isolation and culture.