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

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