The Effects of Desiccation and Exposure to Cryogenic Temperatures on Embryonic Axes of Landolphia kirkii

The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates on survival, assessed by the ability for both root and shoot development, of embryonic axes excised with a small portion of each cotyledon, from mature, recalcitrant seeds of Landolphia kirkii. All axes withstood rapid (flash) drying to a water content of c. 0.28 g g⁻¹ (dry mass basis); however, the use of chemical cryoprotectants before flash drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g g⁻¹, reducing survival to 10% and 0% after exposure to −196°C and −210°C, respectively. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent rewarming and rehydration. Cooling at 1°C min⁻¹ facilitated survival of 70% of axes with attached cotyledonary segments at 0.28 g g⁻¹ after exposure to −70°C, with 45% viability retention when further cooled at 15°C min⁻¹ to −180°C. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached.