Cryopreservation of protocorms of dendrobium cariniferum Rchb. f.

Abstract

Dendrobium cariniferum Rchb. f. is a native orchid wildly distributed in northern Thailand, Assam and northern Myanmar. Conservation of protocorms of D. cariniferum in liquid nitrogen (LN) by encapsulation-dehydration and encapsulation-vitrification were studied. Only encapsulation-vitrification method was successful as follows: 2-4 mm protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.3 M sucrose at 25±2°C on the shaker at 110 rpm under daylight fluorescent lamps at the intensity of 37 μmol m-2s-1for 16 hours per day for 3 days. Then the protocorms were embedded in a loading solution (LS) containing 2% sodium alginate, 2 M glycerol and 0.4 M sucrose for 1 h. The mixture was slowly added to modified VW liquid medium supplemented with 0.1 M calciumchloride and LS. After 1 h, the beads were dehydrated with plant vitrification solution 2 (PVS2) containing 30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide in 2 ml cryotubes for 60 min at 25±2°C. The protocorms were then plunged rapidly in LN. After rapid warming, the PVS2 was replaced with 0.5 ml of 1.2 M sucrose in modified VW solution (unloading solution) and kept at 25±2°C for 20 min prior to transfer on modified VW agar medium. The survival rate of cryopreserved protocorms was about 15% and they were able to develop into whole plantlets.