Cryopreservation of horse-chestnut (Aesculus hippocastanum L.) somatic embryos using three different freezing methods

Cryopreservation of somatic embryos of Aesculus hippocastanum L. cultured on nutritive media containing abscisic acid (ABA) at concentrations of 0.75 mu M, 7.5 mu M and 75.0 mu M was evaluated for three cooling methods: (i) slow freezing with cryoprotectants, (ii) fast freezing with cryoprotectants, and (iii) fast freezing with desiccation techniques. The 'cryoprotectant' freezing techniques included the embryo pretreatment on ABA containing medium for 4 days, followed by cryoprotective treatment in liquid medium containing 0.5 M dimethylsulfoxide, 0.5 M glycerol, 1.0 M sucrose, and cooled at slow, and rapid rates. Embryos pretreated on a medium containing 0.75 mu M ABA, and cooled to -35 degrees C at 1 degrees C/min, held for 30 min at this transfer temperature and then immersed in liquid nitrogen (LN) had the best embryo recovery (43%). The 'desiccation' method involved an air drying step of similar ABA-pretreated, non-cryoprotected embryos followed by rapid cooling. Embryos precultured on 0.75 mu M ABA, then subjected to a 4 h period of air desiccation (water content reduction to 13%) showed about the same level of survival (46%) as found with the 'cryoprotectant' slow freezing technique. The air-dry 'desiccation' method is easier to apply than the more complicated 'cryoprotectant' method.