Cryopreservation of axillary buds of grape (Vitis vinifera) in vitro plantlets

Axillary buds sampled from in vitro plants of four grape varieties could withstand cryopreservation using the encapsulation-dehydration technique. Regrowth percentages ranged between 15 and 40%. Buds were encapsulated in alginate beads containing 0.5M sucrose, precultured at 5 degrees C on media containing daily increasing sucrose concentrations from 0.1 to 1.0M, then desiccated to 21% moisture content (fresh weight basis) and frozen slowly (pre-freezing to -40 degrees C at 0.2 degrees C/min followed by immersion in liquid nitrogen). The physiological state of the in vitro mother-plants and the freezing procedure employed dramatically influenced the results. Regrowth after cryopreservation was achieved only when buds were sampled from mother-plants that had been kept without subculture for 3 to 4 months. An additional one-month period of cold-acclimation of mother-plants at 5 degrees C and a two-step freezing procedure improved growth recovery. The multiplication rate of shoots produced from cryopreserved buds was lower than that of controls during the first subcultures after thawing. However, the rooting ability of control and cryopreserved plantlets became similar from the fourth subculture onwards.