**2.1.5 Rehydration**

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 a dual photoperiod.

Cryopreserving Vegetatively Propagated Tropical Crops

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

hydrolysate at pH 5.7 ± 0.1), or further conditioned for cryopreservation.

**2.2.3 Silica gel dehydration and cooling of explants** 

to 16 h photoperiod (40 μ mol m-2 s-1) at 25±1°C.

three minutes for all treatments.

**2.2.4 Rehydration** 

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 489

(Murashige and Skoog 1962) with 2.5 μM kinetin, 20 mg l-1 L-cysteine, 2% (0.056 M) sucrose,

Yam shoot tips (~1 - 2 mm) were excised from cultures grown on MS pregrowth medium [as above, except containing 3% (0.09 M) sucrose instead of 2% sucrose] for five weeks, placed on sterilised nylon mesh, which was then positioned, explant cut side down, on fresh medium, overnight. Explants were then transferred on the mesh to semi-solid medium with higher sucrose concentrations (0.3, 0.5, 0.7 and 1.0 M, the control material continuing to be exposed to 0.09 M sucrose) in 90 mm Petri dishes for one, three, five or seven days, each followed by transfer either to growth-enhancing medium (MS complete salts with vitamins, 3% sucrose, 5 μM kinetin, 20 mg l-1 L-cysteine, 0.8% agar, 1% filter-sterilised casein

Yam explants dehydrated using the same methodology as described above for Frafra potato. Dehydrated explants were placed in cryovials, which were plunged into, and maintained in, liquid nitrogen for one hour, or cooled at 1ºC min-1 in a Nalgene cryo freezing container (Mr Frosty™), to -70°C, and maintained for at least four hours at this temperature. Rewarming was effected immediately on retrieval from the cryogen, in a water bath at 40°C for two to

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

Explants that had been pregrown and precultured were exposed to 1 ml plant vitrification solution 2 (PVS2) as designed by Sakai and colleagues (Sakai et al., 1990), but modified as follows (MPVS2): basic MS medium, 30% glycerol, 15% ethylene glycol, 15% DMSO (v/v), 0.4 M sucrose (w/v), 0.1 M CaCl2.2H2O and 1% D-raffinose at pH 5.7±0.1. Inclusion of calcium chloride and raffinose has been found to be beneficial in promoting recovery after cryopreservation in other species (Mycock, 1999). Explants were treated for 0, 10, 20, 30 or 40 minutes in cryovials. The vitrification solution was decanted and the explants washed three times in 1 ml rehydration solution (as described above) for 30 minutes, then cultured on growth medium and incubated in the dark. Cultures were transferred to the alternating light/dark conditions once signs of growth and development were observed. Prior to being cooled to -70 or -196ºC, cryoprotected explants were suspended in fresh 0.5 ml MPVS2 in

**2.2.5 Cryoprotection with modified plant vitrification solution 2(MPVS2)** 

0.7% agar, and maintained under a 16 h photoperiod (40 μ mol m-2 s-1) at 25±1°C.
