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Plant Regeneration from Protoplast Isolated from Embryogenic Cells of Angelica gigas
Han-Sol Lee,So-Young Park,Jong-Eun Han,Jae-Heyuk Shin,Heyuk-Jun Kwon 한국원예학회 2021 한국원예학회 학술발표요지 Vol.2021 No.10
Protoplasts, cells lacking the polysaccharide wall, allow a technical complexity in cell-based study of plant. This study was carried out to investigate the effect of various factors on embryogenic cell-derived protoplast cultures to establish a reliable regeneration protocol in Angelica gigas. Firstly, the method of protoplast isolation was stablished by determination of enzyme solution. In next step, protoplast culture was conducted by investigation of the basal media, growth regulators, culture methods [liquid culture, sieving, thin-alginate layer (TAL) method]. Finally, micro-calli induced from protoplast cultures were transferred to hormone-free medium for the further development. High yield of the protoplast was successfully isolated from embryogenic cells, and those were classified into 4 groups according to their size. The highest frequency of cell division was achieved at 7 h of enzyme incubation treatment and the MS medium. The initial cell division was firstly observed in the Group 1(< 20 μm). As the culture period passed, the large groups gradually increased, and the small groups tend to decrease in number. The largest micro-calli were observed in 0.1 - 0.5 mg·L-1 2,4-D single treatments while the vigorous cell division was observed in the medium containing 1.0 mg·L<SUP>-1</SUP> 2,4-D, 0.5 mg·L<SUP>-1</SUP> kinetin. Among three culture method, the TAL showed the highest frequency of cell division compared to other methods. After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets with high efficiency from TAL. Somatic embryos developed into well-rooted plants on hormone-free MS medium. In study, high frequency cell divisions were occurred from small in size, small vacuoles and cytoplasm-rich protoplasts, and these protoplasts were considered to embryonic stem cells. In future studies, we intend to analyze the differential gene expression to identify the protoplast types and molecular changes during protoplast recovery and development into plants.
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