An improved protocol for protoplast production, culture, and whole plant regeneration of the commercial brown seaweed Undaria pinnatifida
José Ávila-Peltroche, Boo Yeon Won, Tae Oh Cho
Abstract
Protoplasts (i.e., living plant cells devoid of cell wall) represent an important biotechnological tool with utility for the conduction of in vitro studies and crop improvement in economic cultivars. Undaria pinnatifida is an annual brown kelp commonly used in Asian cuisine and several industries because of its biological properties. It is one of the major seaweed cultivars used worldwide. This study establishes a simple and effective protocol for protoplast isolation, culture, and whole plant regeneration of U. pinnatifida. The results showed that high protoplast yields (2–4 × 107 protoplast g−1 of fresh weight) were obtained using the commercially available enzymes cellulase “Onozuka” RS (1 %) and alginate lyase (4 U mL−1) without any pre-treatment and with a short incubation time (2–4 h). The isolation protocol described herein presented with more effective results with meristematic explants and was superior to the protocols reported for U. pinnatifida and other brown algae. Additionally, the enzymatic mixture could be reused up to six times. Whole plant regeneration was accomplished after 3 months in culture at 20 °C using two culture media, and a mixture of antibiotics (penicillin G, streptomycin, and chloramphenicol) was added once at the beginning of the culture. Our regeneration protocol was less laborious and more cost-effective, exhibiting final plating efficiency higher than previously reported and demonstrating the potential to be used to produce up to 3 × 104 sporophytes g−1 of explant. The established protocol can be easily used for genetic manipulation, protoplast fusion, or micropropagation, thus enabling the expansion of the use of protoplasts in algae.