Optimization of protoplast isolation and subsequent regeneration from the economically important brown alga Ecklonia cava using response surface methodology

Abstract

Ecklonia cava is an edible kelp species distributed in Korea and Japan with diverse uses in ecological restoration, traditional medicine, and the pharmaceutical and abalone industries. These features make E. cava a good candidate for cellular biotechnology research, which is often dependent on protoplasts. To date, however, there have been no studies that have focused on the isolation of protoplast from E. cava. In this study, we sought to optimize the yield of protoplasts from cultured young sporophytes of E. cava using commercial enzymes, with a particular focus on identifying the key conditions for achieving high protoplast yield, including enzyme concentration, chelation pre-treatment, growth, temperature, incubation time, pH, and osmolarity. Protoplast production conditions were modeled using response surface methodology (RSM) via Box–Behnken design (BBD), and the practical utility of these conditions was experimentally verified. We found that an enzyme composition of 1.67 % cellulase RS and 4 U mL−1 alginate lyase without chelation pre-treatment, at 1570 mOsm L−1 H2O and pH 6 produced the highest protoplast yield of 4.45 ± 2.16 × 107 protoplasts g−1 fresh weight when incubated at 20 °C for 4 h using young (2-week-old) cultures. Our data revealed a strong positive correlation between growth rate and protoplast yield, thereby indicating that growth rate could serve as an index for determining the optimal timing of protoplast isolation from cultured material. The optimized protocol resulted in protoplasts capable of cell wall formation, division, and regeneration. These findings indicate the efficacy of commercial enzymes in protoplast isolation and that BBD-mediated RSM is a useful approach for the production of viable protoplasts from kelp sporophytes.