Surface Response Optimization of Culture Conditions for Dendrobium nobile Lindl Protoplasts

摘要

Dendrobium nobile Lindl is a well-known endangered Chinese medicinal herb. It is therefore important to develop a germplasm resource for this species, using either cell engineering or other approaches. Our previous study found that concentrations of 2,4-dichlorophenoxyacetic acid (S-1), 6-benzylaminopurine (S-2), and 1-naphthaleneacetic (S-3), and also incubation time (S-4) were four essential parameters for liquid culturing of D. nobile protoplasts. To identify the optimal values for these four parameters, we in this study conducted experiments using three experimental designs. The experiment based on univariate analysis defined the value ranges for each parameter. The orthogonal experiment then identified 80.08% vitality rate for the cultured protoplasts was achieved at the following optimal conditions: 1.50 mg/L for S-1, 1.00 mg/L for S-2, 1.00 mg/L for, and 24 h for S-4. Finally, response surface model (RSM) experiment showed that the vitality rate was significantly increased to 85.69% and death rate was lowered below 13.37% when the values for S1-4 were 1.42 mg/L, 1.02 mg/L, 3.18 mg/L, and 19.44 h, respectively. The RSM experiment also showed that the vitality rate was significantly affected by synergistic effects between S-1 and S-2, S-1 and S-3, and S-2 and S-3. These results thus allowed us to establish a reliable method for culturing the high-quality D. nobile protoplasts. The results also showed that the RSM was the best method for analyzing results from the three experimental designs in identifying optimum values for four key parameters.