**2.1.6 Cryoprotection**

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.
