**1. Introduction**

484 Current Frontiers in Cryobiology

Withers, L.A., 1991. Biotechnology and plant genetic resources Conservation. In: *Plant* 

Yamuna, G. 2007. *Studies on Cryopreservation of Spices Genetic Resources* Ph. D Thesis,

Yamuna, G, Sumath,i V., Geetha, S. P., Praveen, K., Swapna, N. and Nirmal Bab, K. 2007.

Paroda and R.K. Arora. IBPGR, New Delhi, pp. 273–297.

University of Calicut, Kerala, India.

*CryoLetters*.28(4):241-252

*Genetic Resources Conservation and Management: Concepts and Approaches*, (Eds.) R.S.

Cryopreservation of *In Vitro* grown shoot of Ginger (*Zingiber officinale* Rosc).

Root and tuber crops in the Sub-Saharan African region play a major role in daily diet, accounting for over 50% of the total staple. *Dioscorea* spp. and *Solenostemon rotundifolius* are among the tuber staples in West Africa. *Solenostemon rotundifolius* (Poir) J.K. Morton is an edible starchy tuber crop known to have originated in tropical Africa (Schippers, 2000). It occurs in western, central, eastern and southern Africa. In Ghana, it is popular in the northern part of the country and its common name is Frafra potato (Tetteh and Guo, 1993). In South Africa, it occurs mainly in coastal KwaZulu-Natal, eastern Mpumalanga and northwestern Cape and it is commonly known as Zulu round potato (Schippers, 2000). It is used to combat famine as it has high protein content, and has medicinal and social values (Kay, 1973). It flowers profusely, yet has rare seed production and is therefore propagated vegetatively by means of vine cuttings and tuber sprouts. Storage of the tuber in hot climates is a problem. In Ghana, it is stored in dry places or left on the ground under trees where conditions are cool. The tuber is stored buried in the ground to maintain the good quality for about two months. Otherwise the tuber sprouts within a shorter period. However, in South Africa, the tuber stores well through the winter months (Schippers 2000).

The germplasm is endangered because although field and *in vitro* gene banks are being used for conservation, these serve short to medium term purposes, and are expensive. Efforts to conserve the germplasm in the longer-term under slow growth *in vitro* are hampered by the relatively rapid growth of the cultures. Cryostorage which is recognised as the very safe cost effective option for the long-term conservation of genetic resources, especially vegetatively propagated species and crops with recalcitrant seeds (Engelmann & Engels., 2002) therefore

Cryopreserving Vegetatively Propagated Tropical Crops

need critical investigation are discussed.

**2. Materials and methods 2.1** *Solenostemon rotundifolius*

**2.1.1 Source of explant** 

25oC +1oC.

25°C+1°C.

conditions.

**2.1.5 Rehydration** 

a dual photoperiod.

**2.1.4 Silica gel dehydration** 

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 487

procedures and their ability to survive after exposure to cryogenic temperatures is investigated here, with the ultimate aim of developing a simple protocol for long-term conservation of the germplasm of *Dioscorea* species via cryopreservation. Parameters that

*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

Nodal cuttings were cultured on MS medium supplemented with either 0.058 M (2%) or 0.1 M sucrose or 0.1 M mannitol and 0.8% agar. Cultures were incubated for two to three weeks

*Solenostemon rotundifolius* nodal cuttings consisting two buds (having lateral buds which are microscopically globular and covered by leaf primodia as described by Niino *et al*., 2000) were obtained from pregrown cultures, positioned on sterile nylon mesh cut side down and placed on fresh pregrowth media overnight. Explants were then transferred on mesh to media with higher sucrose concentrations (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 M) sucrose for one to seven days. Media supplemented with 0.1 and 0.3 M mannitol were also used as pregrowth treatment. Incubation was under a 16 h photoperiod (40µM/m2/s-1), at

Explants were placed on oven-sterilised aluminium foil and dehydrated over approximately 35 g activated silica gel in covered 90 mm glass Petri dishes for 30 min to 16 h under sterile

Explants were rehydrated following cryoprotection treatment, silica gel dehydration and cooling. This was carried out in cryovials containing liquid MS medium supplemented with 0.1 M sucrose, 1mM MgCl2.6H2O and 1 µM CaCl2.2H2O for 30 minutes. Re-hydrated buds were cultured on growth medium, incubated under continuous dark conditions till signs of growth and development were observed (at least one week) before they were transferred to

**2.1.2 Conditioning donor plant material in culture (pregrowth)** 

**2.1.3 Conditioning excised explants in culture (preculture)** 

after which uniformly developed plantlets were used for various experiments.

provide a viable alternative to the long-term storage, and ensure recovery of stable germplasm (Gonzalez-Arnao *et al*., 1999).

*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 deteriorative processes, is a worthwhile option to be explored.

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

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 procedures and their ability to survive after exposure to cryogenic temperatures is investigated here, with the ultimate aim of developing a simple protocol for long-term conservation of the germplasm of *Dioscorea* species via cryopreservation. Parameters that need critical investigation are discussed.
