**4. Conclusions**

Undoubtedly, cryopreservation has high potential for the long-term storage of vegetative explants. It is, however, vital to achieve appropriate tissue water content and the quality of the initial material. This study has shown that vitrification-based cryopreservation protocol is useful for yam explants. However, Frafra potato is extremely sensitive to the vitrification based protocol while explants of the latter easily become hyperhydric and are impossible to dehydrate sufficiently for cryopreservation

The findings are relevant for cryopreservation of a range of yam germplasm (Quain et al., 2009) and also provide a sound basis for further attempts to cryopreserve Frafra potato genetic resources. The technique represents developed simple, cost-effective and potentially reliable methodology that does not require sophisticated equipment. Such procedures should be adapted for germplasm conservation of other species, using limited resources in laboratories in sub-Saharan Africa.

Findings in this present study suggest that in order to achieve an optimal recovery of cryopreserved explants, the donor plants should be adequately conditioned and the recovery media enriched while testing the different cryogenic procedures. Although encapsulation vitrification and encapsulation dehydration procedures have been used in cryopreservation procedure, for the yams used in this study, the use of non-encapsulated explant proved to be a better option. The encapsulation of explants after cryopreservation in the production of e.g. synthetic seeds (Naidoo, 2006; Perán *et al.,* 2006), is, however, worth exploring.

The key to successful cryoprotection has been suggested as inducing tolerance to vitrification solutions and the ability of explants to tolerate dehydration treatment by cryoprotectants has been hypothesised by several researchers as the determining factor for successful cryosurvival (Langis & Steponkus, 1990; Reinhoud *et al*., 1995). It is still not conclusive whether having been hardened, explants being used in procedures would survive cryopreservation. However, Frafra potato explants were not amenable to conditioning by any of these pretreatments. The requirements for successful cryopreservation differ for different species. There is the possibility that the optimum developmental stage of the explant for successful cryopreservation varies from species to species. Therefore there is the need to ascertain and test many parameters on the basis of each species. However, culture conditions especially those that will obviate hyperhydricity, are of paramount importance, as presently indicated for Frafra potato.

It can be concluded from the experiments that:


500 Current Frontiers in Cryobiology

Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for *D. rotundata* shoot tips treated with MPVS2 vitrification solution, and cooled to -70ºC, was 16% for 15 min treatment and 44% for 40 min. Explant rehydration was in 1.0M sucrose supplemented solution. After the 40 min MPVS2 treatment the TTZ test indicated 88% viability retention of explants cooled to -70ºC, and 44% at -196ºC. Plantlet development was obtained for -70ºC-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with silica gel dehydration techniques. It was concluded that vitrificationsolution based cryopreservation presently offers the best option for conservation of this

Undoubtedly, cryopreservation has high potential for the long-term storage of vegetative explants. It is, however, vital to achieve appropriate tissue water content and the quality of the initial material. This study has shown that vitrification-based cryopreservation protocol is useful for yam explants. However, Frafra potato is extremely sensitive to the vitrification based protocol while explants of the latter easily become hyperhydric and are impossible to

The findings are relevant for cryopreservation of a range of yam germplasm (Quain et al., 2009) and also provide a sound basis for further attempts to cryopreserve Frafra potato genetic resources. The technique represents developed simple, cost-effective and potentially reliable methodology that does not require sophisticated equipment. Such procedures should be adapted for germplasm conservation of other species, using limited resources in

Findings in this present study suggest that in order to achieve an optimal recovery of cryopreserved explants, the donor plants should be adequately conditioned and the recovery media enriched while testing the different cryogenic procedures. Although encapsulation vitrification and encapsulation dehydration procedures have been used in cryopreservation procedure, for the yams used in this study, the use of non-encapsulated explant proved to be a better option. The encapsulation of explants after cryopreservation in the production of e.g. synthetic seeds (Naidoo, 2006; Perán *et al.,* 2006), is, however, worth

The key to successful cryoprotection has been suggested as inducing tolerance to vitrification solutions and the ability of explants to tolerate dehydration treatment by cryoprotectants has been hypothesised by several researchers as the determining factor for successful cryosurvival (Langis & Steponkus, 1990; Reinhoud *et al*., 1995). It is still not conclusive whether having been hardened, explants being used in procedures would survive cryopreservation. However, Frafra potato explants were not amenable to conditioning by any of these pretreatments. The requirements for successful cryopreservation differ for different species. There is the possibility that the optimum developmental stage of the explant for successful cryopreservation varies from species to species. Therefore there is the need to ascertain and test many parameters on the basis of each species. However, culture conditions especially those that will obviate hyperhydricity,

are of paramount importance, as presently indicated for Frafra potato.

*Dioscorea* species.

**4. Conclusions** 

exploring.

dehydrate sufficiently for cryopreservation

laboratories in sub-Saharan Africa.


Frafra potato explants easily becomes hyperhydric, and are impossible to dehydrate sufficiently for cryopreservation, this provide a sound basis for further attempts to cryopreserve Frafra potato genetic resources. These observations therefore make available information for further investigation towards development of cryopreservation protocol.
