Optimization of regeneration conditions using response surface methodology for embryogenic cell-derived protoplasts in Angelica gigas Nakai

Abstract

Abstract Background To achieve a stable protoplast regeneration system, various factors must be considered in combination. In this study, the culture conditions at each developmental stage of protoplasts isolated from embryogenic calli of Angelica gigas Nakai to their regeneration into plants were optimized using response surface methodology (RSM). Results To establish an efficient culture method, the plating efficiencies of liquid, sieve, and thin-alginate-layer (TAL) methods were investigated. The RSM was designed using three independent factors: 2,4-D, kinetin, and phytosulfokine (PSK). Optimized values ​​were predicted in the cell division, multi-cell formation, and somatic embryo (SE) stages to determine which developmental stage is favorable for regeneration. Among the culture methods tested, TAL showed the highest multi-cell formation efficiency. During the optimization of each step from protoplasts to plant regeneration, the concentrations of 2,4-D and kinetin gradually increased throughout the culture process, and a relatively high concentration of 90 nM PSK was required for multi-cell formation. Using RSM, the highest SE development was predicted to occur when 1.5 mg·L− 1 2,4-D, 1.0 mg·L− 1 kinetin, and 0 nM PSK are used, which was approximately 3.64-fold higher than those of the control. In the experiments confirming reproducibility using the conditions identified with RSM, the same trend was observed. Conclusions This study indicates that (1) different concentrations of key factors are required for each developmental stage during protoplast culture and that (2) RSM can be used to reliably optimize their required concentrations.