Growth and regeneration of Alfalfa callus lines after freezing in liquid nitrogen

Alfalfa ( Medicago sativa L.) callus culture lines established from the cotyledons of aseptically germinated seeds were treated with a cryoprotective mixture containing 10% (w/v) polyethylene glycol, 8% (w/v) glucose and 10% (w/v) dimethylsulfoxide (DMSO), frozen at 1°/min to −30°C, and transferred to liquid nitrogen (LN). Samples in glass tubes were thawed rapidly, rinsed with a simplified Murashige-Skoog medium and placed on a modified Blaydes agar medium for growth comparisons. Variations of experimental treatments, including thawing-bath temperature, use of glass or polypropylene freezing tubes, and exposure time of callus in cryoprotectant solution, were tested. Following treatments, W74RS callus was stored 1 year in LN and, when thawed, grew at approximately the same rate as unfrozen callus. Two other lines survived LN but the cultures grew more slowly, probably indicating some killing of frozen cells. One of these, Regen S, differentiated and grew into normal-appearing, seed-producing plantlets after thawing; the other, a salt-tolerant line, retained its salt-tolerant character.