Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface methodology

Abstract

The microscopic gametophytic phase of the commercial brown alga, Undaria pinnatifida, can be used for several applications, including the production of bioactive compounds, aquaculture and as germplasm bank. Therefore, gametophytes are good candidates for cellular biotechnology techniques, many of which rely on protoplasts (“naked” living plant cells). This study reports on the optimization of protoplast yield from male and female gametophytes of U. pinnatifida using different mixtures of commercial enzymes and chelation pre-treatment. Key conditions for achieving the highest protoplast yield, such as enzyme combinations, chelation pre-treatment, growth, temperature, incubation time, pH and osmolarity, were investigated. Protoplast isolation conditions were modelled by using response surface methodology (RSM) via Box-Behnken design (BBD) and subsequently experimentally verified in its predictability of protoplast production. The enzyme composition with 1% cellulase RS, 2 U mL⁻¹ alginate lyase and 1% driselase with chelation pre-treatment, at 2481–2591 mOsm L⁻¹ H₂O and adjusted to pH 5.8–6.1, produced the highest protoplast yields of 3.12 ± 0.51 × 10⁶ protoplasts g⁻¹ fresh weight for male gametophyte and 2.11 ± 0.08 × 10⁶ for female gametophyte when incubated at 20 °C for 4–7 h using cultures at mid or early exponential phase, respectively. These conditions also gave high amounts of protoplasts from other strains of Korea. Our results show the effectiveness of commercial enzymes combined with chelation pre-treatment in protoplast isolation and RSM with BBD is a useful method for rapidly producing the higher yields of protoplasts from brown alga.