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 investigation of cryopreservation by encapsulation-dehydration technique on

Protocorm-like bodies (PLBs) of Brassidium Shooting Star, a new commercial ornamental orchid hybrid, were cryopreserved by an encapsulation-dehydration technique. The effects of PLB size, various sucrose concentrations in preculture media and sodium alginate concentration for encapsulation were the main parameters assessed. Four-week old PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid Murashige and Skoog (MS) media supplemented with six different sucrose concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 M) for 24 h, followed by encapsulation in 2.5, 3.0 or 3.5% sodium alginate, with 0.1 M calcium chloride been used as the hardening agent. The beads formed were then osmoprotected in half-strength liquid MS media supplemented with 0.75 M sucrose and dehydrated for three hours in 50 g heat-sterilized silica gel before cryostorage in sterile cryovials. The beads were thawed in a 40 ± 2°C water bath and then directly placed in recovery media for two weeks under tissue culture conditions. After two weeks of recovery, the survival rates of the encapsulated PLBs were evaluated by the 2,3,5-triphenyltetrazolium chloride (TTC) assay. The best conditions for the encapsulation-dehydration of Brassidium Shooting Star were discovered to be the preculture of 3 to 4mm PLB in half strength semi-solid MS media supplemented with 0.8 M sucrose, followed by encapsulation in 3.5% sodium alginate. Further biochemical analysis (chlorophyll, total soluble protein and peroxidases activities) were conducted to investigate the physiological responses of the PLBs after cryopreservation.Key words: Encapsulation-dehydration, cryopreservation, Brassidium Shooting Star, protocorm-like bodies

Encapsulation-vitrification of Dendrobium sonia-28 supported by histology

Abstract In vitro protocorm-like bodies (PLBs) of Dendrobium sonia-28 were cryopreserved through an encapsulation-vitrification method. One to two and 3-4mm PLBs were precultured in half-strength semi-solid Murashige and Skoog (MS) medium supplemented with various sucrose concentrations (0, 0.25, 0.5, 0.75 and 1.0M) at different periods (0, 3, 6 and 9 days). Precultured PLBs were encapsulated and osmoprotected for 24 hours, and then dehydrated in plant vitrification solution 2 (PVS2, 0ºC) at different periods (0, 30, 60, 90, 120, 150, 180 and 210 minutes) prior to storage in liquid nitrogen (LN, for at least 24 hours. After rapid thawing (40±2ºC) for two minutes, the beads were unloaded with 1.2M sucrose and then cultured on half-strength semi-solid MS medium devoid of growth regulators. The 2,3,5-triphenyltetrazolium chloride (TTC) assay was used to determine the viability of the treated PLBs after two weeks of recovery. Histological analyses of non-cryopreserved and cryopreserved PLBs were conducted to assess the impact of the cryopreservation procedure on the in vitro PLB cultures. Observations indicated that cryopreserved PLBs underwent anatomical changes expressed as changes in the cell structure, cell wall, nucleus and cytoplasm. The optimised encapsulation-vitrification parameters involved in this study were the preculture of 3-4mm PLBs for six days in 0.5M sucrose, followed by dehydration in PVS2 at 0ºC for 150 minutes. Thus, this method was deemed promising for cryopreservation of PLBs of Dendrobium sonia-28