RESUMEN
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 water per g dry mass; however, the use of chemical cryoprotectants before flash drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g water per g dry mass, reducing survival to 10% and 0% after exposure to -196 degree C and -210 degree 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 degree C per min facilitated survival of 70% of axes with attached cotyledonary segments at 0.28 g water per g dry mass after exposure to -70 degree C, with 45% viability retention when further cooled at 15 degree C per min to -180 degree 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.
