Non-cryogenic preservation of thalli, germlings, and gametes of the green seaweed Ulva rigida

There is a need to develop reliable and cost-effective preservation techniques that support the growing demand for Ulva cultivation. Using combinations of two dimethyl sulfoxide concentrations (DMSO; 10 and 15%), three non-cryogenic temperatures (4, − 20 and − 80 °C), and one- and two-step cooling, we evaluated the effectiveness of seven methods for preserving Ulva thalli, germlings, and gametes for periods of up to 184 days. Preservation success was assessed using post-thaw regrowth and a pigmentation index. Refrigeration at 4 °C without DMSO successfully preserved thalli for 184 days with little loss of plant quality. In contrast, the freezing methods produced low regrowth success and poor quality thallus tissue, with few plants surviving beyond 30 storage days. Freezing temperatures were lethal to germlings after storage for only one day, but germlings preserved at 4 °C had regrowth rates comparable to thalli preserved under the same conditions. In contrast, Ulva gametes were successfully preserved at freezing temperatures for 184 days although viability was relatively low (7.0-18.7% at − 20 °C and 3.5-12.1% at − 80 °C). Gamete viability at 4 °C decreased from 94.74 ± 4.30% to 26.12 ± 3.97% when the storage time was extended from one to 184 days. DMSO (10%) reduced gamete viability both at − 20 °C and − 80 °C. We demonstrate for the first time, the contrasting responses of Ulva thalli, germlings, and gametes to different non-cryogenic preservation methods.