**2.2.4 Rehydration**

488 Current Frontiers in Cryobiology

Liquid medium (MS) supplemented with 0.2, 0.4 M, 0.07 M, 0.14 M, sucrose, 2.0 M, 0.64 M, 1.28 M, 3.23 M glycerol, 2.42 M ethylene glycol, 0.017 M raffinose, 0.64 M, 1.28 M and 1.92 M DMSO, in varied combinations, plant vitrification solution II (PVS2), (Sakai *et al*., 1990) and half strength PVS2 were used to cryoprotect nodal cutting explants for 5, to 40 minutes. Explants used were obtained from shoots grown on 0.1 M mannitol for 2-3 weeks. Excised explants were cultured on 0.3 M mannitol for 72 hours, and cryoprotected in cryovials, using 1 ml cryoprotectant solution (the 1 ml cryoprotectant was decanted and replace with 0.5 ml during cryoprotection). To enhance explant cryoprotection, dehydration over activated silica gel for 60 minutes either before or after cryoprotection was also investigated. Following cryoprotection, the cryoprotectant solution was decanted and explants were washed three times with rehydration solution (described above). However, explants subjected to cooling, (LN or freezing to -70°C) were immediately on retrieval, rewarmed in water bath at 40oC for two - three minutes. Following rewarming, tissues were then allowed to stay for 30 min in the rehydration solution before blotting dry, and cultured on growth medium. Incubation was in continuous dark conditions till signs of growth and development were observed. Developing cultures were transferred to 16 h photoperiod.

Individually weighed explants were oven-dried at 80°C for 48 hours to determine dry mass. Water content was determined individually for 5-10 explants, and expressed on a dry mass

Explant survival was assessed weekly for three weeks after culturing. Generally, 8-10 explants were used per treatment and experiments were replicated three times. Surviving

A standard glutaraldehyde-osmium fixation method was used, followed by dehydration through an acetone series embedding in a low viscosity epoxy resin (Spurr, 1969). Sections of the meristematic regions of axillary buds and shoot tips were collected on 200 mesh hexagonal copper 3.05 mm grids. Sections were post-stained with uranyl acetate and lead citrate, washed with distilled water, and viewed and photographed with a JEOL 100-S

*In vitro* cultures of *Dioscorea rotundata* ("Pona"), accession number PS 98 013 were obtained from the *in vitro* gene bank of the Department of Botany, University of Ghana, Legon, where the plants were maintained under long-term slow growth conditions at 18ºC. Cultures were multiplied and sub-cultured at six-week intervals on Murashige and Skoog (MS) medium

explants were those which showed shoots with buds, leaves, and root development.

**2.1.6 Cryoprotection** 

**2.1.7 Frafra potato assessments 2.1.7.1 Water content determination** 

**2.1.7.3 Transmission electron microscopy** 

transmission electron microscope.

**2.2** *Dioscorea rotundata* **2.2.1 Source of explant** 

(g H2O g-1 dry mass) basis.

**2.1.7.2 Survival** 

The rehydration solutions consisted of MS complete salts with vitamins, 2.5 μM kinetin, 20 mg l-1 L-cysteine, 1mM MgCl2.6H2O, 1μM CaCl2.2H2O, 1% casein hydrolysate (filtersterilised), and 1 M sucrose, at pH 5.7±0.1, magnesium and calcium chlorides having been shown to enhance explant recovery of date palm somatic and pea zygotic embryos (MyCock 1999). Rehydration was for 30 minutes. Rehydrated buds were blotted dry and cultured on growth medium (as above). Cultures were incubated under continuous dark conditions at 24±1°C until signs of growth and development were observed, before they were transferred to 16 h photoperiod (40 μ mol m-2 s-1) at 25±1°C.
