Protoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D. pacifica and S. lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. Protoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL−1), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D. pacifica. Under optimal conditions, the protoplast yields from D. pacifica and S. lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 106 protoplasts g−1 fresh weight, respectively. The values obtained for S. lomentaria were 2–3 orders of magnitude higher than previously reported. Our results show that high protoplast yields can be obtained from D. pacifica and S. lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D. pacifica. The method proposed here can help to expand protoplast technology in more brown algal species.

中文翻译：

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使用简单的商业酶制剂从 Dictyopteris pacifica 和 Scytosiphon lomentaria 中分离原生质体

原生质体（即裸植物细胞）可用于具有经济价值的栽培品种的体外操作和遗传改良。在过去的十年中，经济褐藻的原生质体研究很少，并且通常受到使用非商业酶或粗提取物分离原生质体的阻碍。Dictyopteris pacifica 是褐藻属的一部分，以其广泛的化学多样性和生物学特性而闻名。Scytosiphon lomentaria 是一种可食用的棕色海藻，具有抗氧化、抗肿瘤和抗病毒特性。到目前为止，还没有针对这两种经济褐藻使用商业酶的原生质体分离方案。在这项研究中，我们使用市售酶从 D. pacifica 和 S. lomentaria 的培养样品中获得了原生质体。此外，我们研究了 Driselase 包含和 Ca 螯合预处理对原生质体产量的影响，以优化原生质体制备的条件。使用市售的 Cellulase Onozuka RS (1%) 和藻酸盐裂解酶 (4 U mL-1) 和短孵育时间 (4 h) 从 Dictyopteris pacifica 和 Scytosiphon lomentaria 中分离原生质体。Driselase 对两种物种的原生质体生产均未显示出显着影响。Ca 螯合预处理仅增加了 D.pacifica 中原生质体的数量。在最佳条件下，D. pacifica 和 S. lomentaria 的原生质体产量分别为 4.83 ± 2.08 和 74.64 ± 32.49 × 106 个原生质体 g−1 鲜重。获得的 S. lomentaria 值比以前报告的高 2-3 个数量级。我们的结果表明，使用商业酶（纤维素酶 RS 和藻酸盐裂解酶）的简单混合物和短孵育时间（4 小时），可以从 D. pacifica 和 S. lomentaria 中获得高原生质体产量。这项工作也代表了在 D. pacifica 中分离原生质体的第一份报告。这里提出的方法可以帮助扩展更多褐藻物种的原生质体技术。