Cryopreservation of Salix species using sections from winter vegetative scions

Twigs of Salix species are candidates for cryopreservation procedures because they become tolerant of freezing temperatures during mid-winter. We examined several variables in developing a two-step cryopreservation procedure for sections from these twigs. Samples of Salix triandra cooled to-30°C or-35°C and then transferred to the vapor phase over liquid nitrogen gave the greatest percent shoot formation. Cooling rate to-35°C had a major influence on shoot formation. Samples cooled at rates greater than 10°C/hr showed no shoot formation. The highest percent of shoot formation was achieved by cooling at 0.21°C/hr. Cooling rate from-35°C to liquid nitrogen did not influence shoot formation. Warming procedures affected shoot formation. Transferring samples from-160°C either to a +2°C cold room or to-3°C methanol gave similar levels of shoot formation. No shoot formation occurred either with warming in +40°C water or very slowly in a Styrofoam box. Eight of eleven Salix taxa tested using the established protocol had significant levels of shoot formation after cryogenic treatment.