Early investigation on cryopreservation of Dendrobium sonia-28 using encapsulation-dehydration with modified Evan blue assay

This study was conducted to determine the potential of cryostoring and regenerating Dendrobium sonia-28 protocorm-like bodies (PLBs) using the encapsulation-dehydration technique. The parameters tested for this study included the PLB size range (1 to 2 and 3 to 4 mm), preculture using six different sucrose concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 M) and encapsulation using three different sodium alginate concentrations (2.5, 3.0 and 3.5%). Based on initial trials, 1 to 2 mm PLBs that were precultured in 1.0 M sucrose were selected for further studies as they produced the best viability as indicated by the Evans blue (EB) staining method. Subsequently, the PLBs were subjected to a 30 min encapsulation experiment involving the three sodium alginate concentrations. Finally, the chlorophyll content and total soluble protein of cryopreserved, non-cryopreserved and untreated PLBs were determined.Key words: Orchid, protocorm-like bodies, cryopreservation

Preliminary analysis of cryopreservation of Dendrobium Bobby Messina orchid using an encapsulation-dehydration technique with Evans blue assay

In vitro grown protocorm-like bodies (PLBs) of Dendrobium Bobby Messina hybrid were cryopreserved in liquid nitrogen (LN) at -196°C by an encapsulation-dehydration technique. PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid MS media supplemented with six different concentrations of sucrose (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 M). The PLBs were then encapsulated to form the beads in halfstrength liquid MS media supplemented with different concentrations of sodium alginate (2.5, 3.0 and 3.5%). The beads were placed in 2 ml cryovials and plunged into LN for 24 h. The beads were then thawed in a 40°C water bath for 90 s and were placed in recovery media composed of half strength semisolid MS media supplemented with 2% sucrose for four days under dark condition. After 12 days, the Evans blue dye assay was carried out to determine the viability of the PLBs. The highest viability was found in 1 to 2mm PLBs precultured in half strength semi-solid MS media supplemented with 1.0 M sucrose and encapsulated in 2.5% sodium alginate. Biochemical content analyses (chlorophyll, total soluble protein and peroxidase activities) were done to investigate the physiological responses of the PLBs after cryopreservation.Key words: Orchid, protocorm-like bodies, Dendrobium Bobby Messina, encapsulation-dehydration