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Abstract
The effects of cryoprotectants and their concentrations, prefreezing temperature, equilibration time, thawing temperature, and the regenerative efficacy of cryopreserved vegetative thalli (apical tips) of Gracilaria corticata J. Agardh., Ulva lobata Duetzing, and Hypnea musiformis (Wulfar) Lamouroux were evaluated. The apical tips were suspended in various cryoprotective solutions and slowly cooled to −40˚C over a period of 4 h. After the slow cooling step, the suspensions were immediately immersed in liquid nitrogen (LN), which were thawed after 2 days. Both U. lobata and H. musiformis survived maximally in 10% DMSO, whereas 10% glycerol was found most suitable for G. corticata. A slow cooling temperature of −40˚C and thawing temperature of 40˚C were found most suitable irrespective of the species. The equilibration time of 60 min was found most suitable for G. corticata, while 45 min was sufficient for both H. musiformi and U. lobata. Cryopreserved vegetative thalli were then successfully regenerated, though the survival was considerably reduced up to a period of 28 days after which it was stabilized.