Cryotechniques for the long-term conservation of embryogenic cultures from woody plants

Since its development in the 1960s, plant cryopreservation is considered an extraordinary method of safe long-term conservation of biological material, as it does not induce genetic alterations and preserve the regeneration potential of the stored material. It is based on the storage of explants at cryogenic temperatures, such as the one of liquid nitrogen (-196 °C), where the metabolism within the cells is suspended; thus, the time for these cells is theoretically "stopped". Cryopreservation is particularly important for embryogenic cultures, as they require periodic subculturing for their maintenance, and this, in turn, increases the risk of losing the material, as well as its embryogenic potential. Periodic re-initiation of embryogenic cultures is possible; however, it is labor intensive, expensive, and particularly difficult when working with species for which embryogenic explants are available only during a limited period of the year. Among various methods of cryopreservation available for embryogenic cultures, slow cooling is still the most common approach, especially in callus cultures from softwood species. This chapter briefly reviews the cryopreservation of embryogenic cultures in conifers and broadleaf trees, and describes as well a complete protocol of embryogenic callus cryopreservation from common ash tree (Fraxinus excelsior L.) by slow cooling.