Towards understanding the protection afforded by cryoprotectants commonly used in the cryopreservation of zygotic explants of recalcitrant seeds

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. The post-cryo success achieved with the excised zygotic explants used for cryopreservation is variable, and for some species dependent on the use of cryoprotectants and rapid drying. However, the efficacy of different cryoprotectants seems to be species dependent and there is a paucity of information on the mechanisms via which particular cryoprotectants protect zygotic embryos from recalcitrat seeds during partial drying and cryogenic cooling. The present study used Differential Scanning Calorimetry (DSC) and Cryo- Scanning Electron Microscopy (Cryo-SEM) to characterise the behaviour of selected cryoprotectants in zygotic embryos during drying and cooling to cryo- genic temperatures. These studies were carried out on a range ofrecalitrant-seeded species (Castanospermum australe, Trichilia dregeana, Amaryllis belladonna, Strychnos gerrardii) that show differential tolerance to cryoprotectants and cryo- preservation. Exposure to glycerol or sucrose did not change the behaviour of water in zygotic explants of most of the species studied. DSC revealed little to no difference between fresh samples and those cryoprotected, showing very sim- ilar enthalpy of melting transitions (300 J/gH 2 O) and amount of unfrozen water (0.30 gH 2 O/gdm); except for Atropa belladonna treated with glycerol that showed a slightly higher amount of unfrozen water (0.45 gH 2 O/gdm). However, Cryo- SEM images showed important changes in the mechanical properties of the cryo- protected tissue during rapid drying. In general, glycerol allowed for less destruc- tive compaction of the zygotic tissues during desiccation, particularly in A. belladonna. Glycerol was also observed to help in membrane stabilization of Corynosoma australe, particularly during rehydration of the tissue. The results are discussed in relation to other studies on the mechanisms of cryoprotection in shoot tips and will inform the future cryopreservation protocols for zygotic germplasm of recalcitrant-seeded species.