**3.1.5 Cryopreservation**

Although explants treated with mannitol and dehydrated over activated silica gel for 90 minutes had water content 0.24 gg-1 and survival was 70 % (Table 1), when exposed to liquid nitrogen, survival was nil. Ultrastructure indicated extensively degraded cells with withdrawn and broken plasmalemma, cytoplasm and nucleoplasm were all damaged (Plate not shown). Explants from all dehydration treatments did not survive on exposure to liquid nitrogen as well as ultra-cold liquid nitrogen (slash), although, it has been reported that rapid cooling enhance cryosurvival (Wesley-Smith *et al*., 1992). Having dehydrated explant to water content of 0.11gg-1, it is obvious from ultrastructure (not shown) that the prolonged stress exerted decreased explant ability to withstand freezing since there is a level below which dehydration stress is increasingly apparent (Wesley-Smith *et al*., 1992). Unlike the loss of viability in *S. rotundifolius*, explant at higher water contents have been reported to survive on exposure to liquid nitrogen (Berjak *et al.*, 1995; Kioko *et al*., 1998 and 2000)

During cryopreservation all metabolic processes cease, it is possible that mannitol treated explants were too active metabolically judging from the high number of mitochondria occurring in the cytoplasm (Plate 1b). Hence bringing the systems to a halt caused a breakdown in all the plant metabolic systems causing cytoplasm to lose its viability since following dehydration, only few organelles could be observed in cytoplasm (Plate 1f). It is also possible that with the occurrence of high number of small vacuoles in mannitol treated explant (Plate 1c) which is a characteristic whereby, large vacuoles volumes are reduced by redistributing them into smaller vesicles on exposure to mannitol (Gnanapragasam & Vasil, 1992), being an advantage for survival since water contents are relatively low (Reinhoud *et al.,* 1995). However, it is probably that, the water present in the vacuoles did not have high viscosity, which would prevent the formation of ice crystals during cooling and thawing hence causing degeneration of plant cell integrity.

Sucrose treated explant, ultrastructure indicated cytoplasmic breakdown at all stages of treatment. Although the plant cell were not in a high metabolic state prior to exposure to liquid nitrogen, cellular degeneration had already set in and may have had a major role to play, leading to loss of viability on exposure to liquid nitrogen.

The above and all associated factors need to be investigated further. These will help optimise plant cell structure prior to cryopreservation. Based on the ultrastructural studies carried out, it is obvious that the use of mannitol for pregrowth and preculture treatment, the plant tissues develop capability to tolerate other stress (desiccation). *S. rotundifolius* tissues besides yielding high explant survival, results in stable ultrastructure for further plant growth and development. However the treatment does not necessarily result in survival on exposure to cryopreservation. The use of higher concentration of mannitol may enhance cryotolerance. Other critical factors that have to be investigated include maturation of explant supported by constitution of ultrastructure and related water content which play crucial rôle in cryopreservation (Chandal *et al*., 1994; Berjak *et al*., 1993). However, the extremely high water content (18.7 – 9.64 g/g dry wt) of plant even in the greenhouse (graph not shown), may still make it difficult to cryopreserved tissues of local accessions of *S. rotundifolius.* Several attempts were made to adequately harden Frafra potato (Table 5) prior to subjecting explants to various treatments and then cooling however, none of them resulted in explants survival after cooling.


Table 5. Other attempts to acclimatize the new Frafra potato accession 99/053 to lower water content that might enhance cryosurvival.
