Preliminary investigation of cryopreservation by encapsulation-dehydration technique on <i.Brassidium</i> Shooting Star orchid hybrid

Abstract

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.