**3.2** *Dioscorea rotundata*

498 Current Frontiers in Cryobiology

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 Ultrastructure indicated extensively degraded cells with withdrawn and broken plasmalemma, cytoplasm and nucleoplasm were all damaged (Plate not shown).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)

hence causing degeneration of plant cell integrity.

resulted in explants survival after cooling.

play, leading to loss of viability on exposure to liquid nitrogen.

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

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

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

**3.1.5 Cryopreservation** 

Comparatively, yam explants cultured on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g g-1 dry mass to between 4.8 and 5.5 g g-1 dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Following cryoprotection with MPVS2 the Plate (6) below indicated the growth of nodal explants.

Plate. 6. Growing cultures of yam explant subjected to pregrowth on 0.09 sucrose supplemented medium for 5 weeks, precultured on medium containing 0.3 m sucrose for 3 or 5 d, treated with MPVS2 for varied duration and unloaded with rehydration solution containing 0.3 or 1 M sucrose and cultured on regeneration medium for six week

Cryopreserving Vegetatively Propagated Tropical Crops

It can be concluded from the experiments that:

protocol.

The procedure incorporates

adequately conditioned.

**5. Acknowledgements** 

pp. 223-228.

**6. References** 


weeks,

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 501

Successful cryopreservation of *Dioscorea rotundata* is possible using a simple vitrification

For the first time successful cryopreservation of *Dioscorea rotundata* accession 'Pona'

The technique represents developed simple, cost-effective and potentially reliable

Procedures can be adapted for germplasm conservation of other species, using limited

To achieve an optimal recovery of cryopreserved explants the donor plants should be

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.

The authors wish to acknowledge financial support received from the UNU/INRA and the TWOWS. They also wish to thank Mrs B. Asante (University of Ghana Legon) and Mrs P,

[1] Ashun MD (1996). *In vitro studies on micropropagation of various yam species* (*Dioscorea*

[2] Berjak, P., Mycock, DJ, Walker, M, Kioko, JI, Pammenter NW, and Wesley-Smith J (1999).

[3] Berjak P, Mycock DJ, Wesley-Smith J, Dumet D. & Watt MP (1996) Strategies for *in vitro*

[4] Berjak, P, Mycock DJ, Watt P, Wesley-Smith J and Hope B (1995). Cryopreservation of

[5] Berjak, P, Vertucci CW and Pammenter N.W. (1993). Effects of developmental status and

recalcitrant seeds of *Camellia sinensis*. *Seed Science Research* 3, 155-166.

Conservation of Genetic Resources Naturally occurring as recalcitrant seeds. In M. B., K.J. Bradford and J Vazquez-Ramus (eds) *Seed Biology Advances in Applications*

conservation of hydrated germplasm. M. N. Normah *et al*., (eds). In vitro

Pea (Pisum sativum L.). In Y.P.S. Bajaj (ed) Biotechnology in Agriculture and

dehydration rate on the characteristics of water and desiccation-sensitivity in

species) M.Phil. Thesis submitted to University of Ghana – Legon.



methodology that does not require sophisticated equipment.

Frafra potato is extremely sensitive to the vitrification based protocol.

Maartens (University of Kwa-Zulu Natal) for technical assistance.

*conservation of plant genetic resources* 19-52.

Forestry (32) Springer: 293-307.

which is an elite variety in Ghana has been achieved.

resources in laboratories in sub-Saharan Africa.


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 *Dioscorea* species.
