Cryopreservation of Gracilaria tikvahiae (Rhodophyta) and other macrophytic marine algae

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Abstract
Sporelings and apical segments of mature thalli of the marine red alga Gracilaria tikvahiae McLachlan were frozen and stored alive successfully in liquid nitrogen for 4 years. A cryoprotectant was necessary for survival during the freeze–thaw cycle and dimethylsulfoxide (DMSO) was excellent for this purpose. Plants were pretreated with the DMSO a few minutes before freezing. A DMSO concentration of 1.5 M gave the best results. At this concentration the duration of pretreatment from 5 to 90 min was not important; however, the temperature during pretreatment was. Improved viability was obtained with pretreatment at room temperature (22–23°C) rather than at 0°C in an ice bath. Plants were frozen in a two-step procedure having an initial slow freezing rate followed by a rapid rate when samples were plunged into liquid nitrogen. Cooling rates of 0.2°C min−1 to 32°C min−1 gave similar results, indicating that freezing rate was not a critical variable within this range. Slow cooling had to be continued to temperatures below −25°C before immersion in liquid nitrogen for consistently good survival. The genotype and physiological condition of plants also had a large influence on their survival. Conditions resulting in high survival rates for G. tikvahiae were found to be adequate for successful cryopreservation of a number of other macrophytic marine algae.

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