Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface meth
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23RD INTERNATIONAL SEAWEED SYMPOSIUM, JEJU
Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface methodology Jose Avila-Peltroche 1 & Boo Yeon Won 1 & Tae Oh Cho 1 Received: 29 July 2019 / Revised and accepted: 28 February 2020 # Springer Nature B.V. 2020
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. Keywords Phaeophyceae . Commercial enzymes . Undaria pinnatifida . Gametophytes . Protoplast isolation . Response surface methodology
Introduction Protoplasts are living plant cells from which cell walls have been removed, usually by digesting with enzymes. These cells are potentially totipotent and represent an important biological tool for genetic improvement, tissue culture and physiological studies (Reddy et al. 2008; Baweja et al. 2009). Although protoplast isolation and culture techniques have been investigated for more than five decades (Cocking 1960), their utility in genome-editing and gene silencing technologies has led to a reemergence of protoplast systems over the past few years (Burris et al. 2016). The development of this type of systems * Tae Oh Cho [email protected] 1
Department of Life Science, Chosun University, Gwangju 61452, Korea
is based on the establishment of reproducible protocols for protoplast isolation and culture (Bhojwani and Razdan 1996). Undaria pinnatifida (Harvey) Suringar is one of the most important economic se
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