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.

市售褐藻Undaria pinnatifida的微观配子生体阶段可用于多种应用，包括生物活性化合物的生产，水产养殖以及作为种质库。因此，配子体是细胞生物技术的良好候选者，其中许多依赖原生质体（“裸露”的活植物细胞）。这项研究报告了优化的pinnatifida的男性和女性配子体原生质体产量使用不同的商业酶混合物和螯合剂进行预处理。研究了获得最高原生质体产量的关键条件，例如酶组合，螯合预处理，生长，温度，孵育时间，pH和摩尔渗透压浓度。原生质体分离条件通过Box-Behnken设计（BBD）使用响应表面方法（RSM）进行建模，随后通过实验验证了其原生质体生产的可预测性。酶成分为1％的纤维素酶RS，2 U mL ^-1藻酸盐裂解酶和1％的果胶酶经螯合预处理，在2481–2591 mOsm L ^-1 H ₂ O下调节至pH 5.8–6.1，产生最高的原生质的3.12±0.51×10 ⁶原生质体g ^-1在指数期中期或早期分别在20°C孵育4–7 h时，雄配子体的新鲜重量和雌配子体的2.11±0.08×10 ⁶。这些条件还导致来自韩国其他菌株的大量原生质体。我们的结果表明，商业化酶与螯合剂预处理相结合在原生质体分离中的有效性，带有BBD的RSM是从褐藻类快速生产更高产量原生质体的有用方法。