Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification/one-step freezing

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min. incubation in PVS2 and conservation at -196 °C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e. the torpedo stage) gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45°C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse-chestnut, avoiding repeated subculturing.