Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification

Protoplasts of Porphyra yezoensis were successfully cryopreserved at liquid nitrogen (LN) temperature by vitrification and subsequently regenerated into plants. The effects of different vitrification solutions, loading and dehydration time, and thawing condition on protoplasts viability after cryopreservation were evaluated. VS6 (10% w/v dimethyl sulfoxide (DMSO), 30% w/v glycerol, 10% sucrose in seawater) seems to be a preferable vitrification solution for P. yezoensis protoplasts. The best vitrification protocol using VS6 involves: loading with 25% VS6 for 5 min at 0 °C, dehydrating with ice-cold concentrated VS6 for 3 min, immediately immersing in LN, and then warming in 40 °C water bath. Using this protocol, P. yezoensis protoplasts showed higher viability (66.5%) after cryopreservation. Although the regrowth of cryopreserved protoplasts showed a 2–3-day lag phase and recovered completely until 20 days after thawing, the growth pattern was the same as that of non-cryopreserved protoplasts. In summary, we have developed a new method for the cryopreservation of P. yezoensis protoplasts, which allows long-term maintenance of valuable genotypes.