CRYOPRESERVATION OF HORSE-CHESTNUT (AESCULUS-HIPPOCASTANUM L.) SOMATIC EMBRYOS USING 3 DIFFERENT FREEZING METHODS

Cryopreservation of somatic embryos of Aesculus hippocastanum L. cultu red on nutritive media containing abscisic acid (ABA) at concentration s 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 w ith cryoprotectants, and (iii) fast freezing with desiccation techniqu es. The 'cryoprotectant' freezing techniques included the embryo pretr eatment on ABA containing medium for 4 days, followed by cryoprotectiv e 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 de grees C at 1 degrees C/min, held for 30 min at this transfer temperatu re and then immersed in liquid nitrogen (LN) had the best embryo recov ery (43%). The 'desiccation' method involved an air drying step of sim ilar ABA-pretreated, non-cryoprotected embryos followed by rapid cooli ng. Embryos precultured on 0.75 mu M ABA, then subjected to a 4 h peri od of air desiccation (water content reduction to 13%) showed about th e same level of survival (46%) as found with the 'cryoprotectant' slow freezing technique. The air-dry 'desiccation' method is easier to app ly than the more complicated 'cryoprotectant' method.