S04 - Session P4 - Regeneration of plants from embryogenic callus-derived protoplasts and its comparative transcript profiling by RNA-seq in Angelica gigas

Authors: Hansol Lee *, So-Young Park, Jong-Eun Han, Suk-Hyun Kwon

Somatic embryogenesis is an important process to study developmental pathways, and embryogenic cells are one of the stem cells in the plant. Recently these embryogenic cells have reported that biosynthesize important physiological metabolites and are being studied as important tools for genome editing. However, its embryogenic capacity is reduced by long-term culture, and somatic clonal mutation also often occurs in many species. In this study, high purity embryogenic stem cells were obtained by isolated protoplasts from embryogenic callus (EC) of Angelica gigas , which is valuable medicinal plant, and regeneration protocol was established from isolated protoplasts. To determine the protoplast isolation conditions, the enzyme incubation time, culture media, and methods were adjusted. For the continuous cell division, the growth regulators and culture methods were investigated. Additionally, the comparative transcripts were analyzed during protoplast isolation and re-activation of cell divisions by RNA-seq. The results show that the optimal incubation time of the EC in enzyme solution was 7 hours, and the suitable medium was MS medium for the cell division. As for the culture method, the thin alginate layer was effective. After three months of culture, many micro-calli were obtained in high concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyl adenine (BA), and kinetin, but subsequently many somatic embryos were developed in low concentrations of 2,4-D. It reveals that low conc. of 2,4-D was essential to maintain the capacity of embryogenic stem cells during this process. RNA-seq results showed that many genes related to stem cell identification lost their expression during the protoplast isolation process. The results obtained in this study could be used to understand how embryonic capacity could be maintained and manipulated in Angelica gigas .