**2.1.1 Source of explant**

486 Current Frontiers in Cryobiology

provide a viable alternative to the long-term storage, and ensure recovery of stable

*Dioscorea* species, colloquially known as yams, of family Dioscoreaceae are perennial monocotyledonous climbers with underground tubers which, in some species are edible and serve as major staples in sub-Saharan Africa. Propagation is routinely vegetative, using either the tubers or vine cuttings. Farmers ensure the production of true-to-type crops by using clonal planting material, because of the social and staple importance attached to yams in sub-Saharan Africa. Hence the conservation of clonal germplasm of yam is extremely important. *Dioscorea* spp. has about 700 species within the family, nine of which are medicinal plants that accumulate steroid saponins in their rhizomes. Six species of Dioscorea *D*. *bulbifera*, *D*. *cayenensis*, *D*. *dumentorum*, *D*. *prahensilis*, *D. alata* and *D. rotundata* contain mealy starch with a good level of vitamin C and other nutritive substances, which serve as major staples in sub-Saharan Africa. *Dioscorea rotundata* is native of West Africa, where it plays important role in the socio cultural life of the people. *Dioscorea alata* is the most widespread worldwide and is most cultivated in Southeast Asia, the Caribbean and West Africa. *Dioscorea rotundata* is now utilised in other parts of the world, and it has become a foreign exchange earner particularly in Ghana. *In vitro* slow growth tissue culture methods have been used in conserving the germplasm (Ashun 1996; Ng & Daniel 2000; Ng & Ng 1991). Although this method usefully complements the traditional form of conservation, it serves only short- to medium-term storage purposes. Thus cryopreservation, which imposes a stasis on metabolic and

Explant treatment to attain low water content which is critical for cryopreservation has in some protocols been by exposing tissues to stress, which enhance desiccation and cold tolerance (Withers 1985; Jitsuyama *et al*., 2002). Such stress has been induced by abscisic acid, sugars, mannitol and sorbitol (Mastumoto *et al*., 1998; Jitsuyama *et al*., 2002; Veisseire *et al*, 1993; Panis *et al*., 2002; Walter *et al*., 2002). The use of cryoprotectants, which exert osmotic stress and lead to loss of free water from tissues and vitrification when frozen has also been induced by using reagents such as sucrose, glycerol, DMSO, ethylene glycol, proline and many others (Engelmann et al., 1994; Harding & Benson, 1994; Matinez-Montero *et al*, 1998; Plessis *et al*., 1993; Nishizawa *et al*., 1993). Desiccation of tissues on activated silica gel (Hatanaka *et al*., 1994; Cho *et al*., 2002), in laminar air flow cabinets (Gonzalez-Benito & Pezez, 1994; Thammasiri, 1999) and flash driers (Berjak *et al*., 1999; Pammenter *et al*., 1991; Wesley-Smith *et al.*, 1992; Walter *et al*., 2002; Potts & Lumkin 1997) have all been used to appreciably reduce water content to enhance cryotolerance. Although these treatments have all been reported to be successful in enhancing cryopreservation of some tissues, there are differences in response to known protocols which have been mainly attributed to specie and variety specificity (Gonzalez-Benito *et al*., 2002; Martinez-Montero *et al*., 1998; Panis *et al*.,

The prevention of the formation of lethal ice crystals when tissue is exposed to sub-zero temperatures is essential for successful cryopreservation, of vegetatively propagated germplasm. This chapter looks at the various attempts made to cryopreserve germplasm of *Solenostemon rotundifolious* and possible underlying mechanism that might have led to failure of tissues to respond to all methods utilized. Tissue survival, water contents and ultrastructure are used as parameters for analyzing response to various treatments. Also, response of yam *in vitro*-grown explants (shoot tips and axillary buds) to various desiccation

germplasm (Gonzalez-Arnao *et al*., 1999).

deteriorative processes, is a worthwhile option to be explored.

2002; Gonzalez-Arnao *et al*., 1999).

*In vitro* cultures of *Solenostemon rotundifolius* accession number UWR 002 was obtained from the *in vitro* gene bank that had been maintained under slow growth conditions at 18oC. *In vitro* cultures were multiplied on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 2% sucrose and 0.7% agar. Subculturing was carried out at four-weeks intervals. Cultures were maintained under a 16 h photoperiod (40 µM /m2/s1) at 25oC +1oC.
