Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification

This study reports on the first use of a vitrification procedure for the successful cryopreservation of embryogenic cultures of a coniferous species. Using Picea mariana embryogenic cultures, we obtained the highest survival by first preculturing embryogenic masses on semi-solid medium containing 0.8 M sorbitol for 48 h followed by incubation in PVS2 cryoprotective vitrification solution at 0°C for 30 min and direct immersion in liquid nitrogen. The replacement of sorbitol with 1.6 M glycerol also resulted in high survival. When sorbitol and glycerol were used in preculture treatments, 9 of 11 embryogenic lines survived liquid nitrogen treatments. We also demonstrated that 100% post-liquid nitrogen survival of mature somatic embryos could be obtained without pretreatments. A brief desiccation pretreatment, however, increased uniformity of somatic embryo germination.