Developing a cryopreservation protocol for the forest-forming seaweed Phyllospora comosa as a tool for restoration

Global declines in seaweed forests are reducing genetic diversity and, consequently, the resilience of these habitats to changing environmental conditions. To conserve genetic diversity and secure these important coastal habitats, new preservation strategies are needed, such as cryopreservation. We aimed to develop a cryopreservation protocol for sperm and germlings of the fucoid seaweed Phyllospora comosa, a species highly vulnerable to anthropogenic stressors. We tested the cytotoxicity of cryoprotectant agents commonly used in seaweed cryopreservation (dimethyl sulfoxide, ethylene glycol, glycerol, methanol and propylene glycol). Non-lethal cytotoxic effects of cryoprotectants on germlings and sperm were observed, including a reduction in length (germlings), motility and concentration (sperm). Freezing trials resulted in post-thaw sperm survival. Best results were observed with 10% glycerol (final, v/v) under a freezing rate of -42.4 ± 5.7 °C/min, displaying 18.7% post-thaw motility and 1.5 × 10⁶ sperm/mL, compared to an initial motility of 44.9% and 6.4 × 10⁶ sperm/mL in untreated sperm samples. Germlings could not be revived after thawing, despite testing several freezing rates, cryoprotectant concentrations (5-30%), equilibration times (10-60 min) and added sugars. Our results demonstrate that live Phyllospora sperm can be recovered following cryopreservation, providing an opportunity to store diverse genotypes for breeding and enhancing genetic diversity in distant populations. However, further work is required to improve the efficiency of cryopreservation protocols and apply them in conservation practices. Our findings provide guidelines for future trials with Phyllospora and other fucoid species to incorporate cryopreservation techniques that help secure genetic diversity in the face of climate change.