Cryopreservation of Vanda coerulea Protocorms by Encapsulation-Dehydration

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2% Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 ± 3° C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 ± 3°C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40°C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40% was observed with cryopreserved beads with 35% water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.