Cryopreservation of Eisenia bicyclis (Laminariales, Phaeophyta) in liquid nitrogen

Gametophytic cells of Eisenia bicyclis (Kjellman) Setchell were cryopreserved in liquid nitrogen (LN). Ethylene glycol was the most effective cryoprotectant when used alone, and in combination with 10% (w/v) proline improved the survival. Viability of female and male cryopreserved cells after thawing reached 62.0% and 52.6% immediately after thawing, but these levels decreased to 31.1% and 27.2% after 4 days postthawing culture. The optimal prefreezing temperature was -40 degreesC, and cells prefrozen to temperatures >-40 degreesC were damaged mainly by intracellular ice crystal formation during immersion in LN, while those prefrozen to temperatures <-40 degreesC were damaged mainly by excessive dehydration during prefreezing. The survival rates were not affected after storage in LN for at least 200 days.