Cryopreservation of Vanda coerulea protocorm-like bodies by droplet-vitrification

Abstract

Vanda coerulea is the most popular Vanda species of Thailand that has become endangered because of the wild orchid trade, deforestation and environmental changes. To preserve this orchid, droplet-vitrification was studied for the cryopreservation of protocorm-like bodies. Protocorm-like bodies were developed from one protocrom in ND liquid medium supplemented with 1 mg/L BA in combination with 0.5 mg/L NAA and 30 g/L maltose. Protocorm-like bodies (3.0 mm in diameter) were precultured in ND liquid medium supplemented with 0.5 M sucrose for 1 day on a shaker, then dehydrated with loading solution for 15 min and exposed to PVS2 solution for 30 min at 25°C. The plant materials were then immersed in liquid nitrogen, rewarmed rapidly and cultured on solidified ND medium. After cryopreservation, the survival frequency of cryopreserved protocorm-like bodies was 5%. The assessment of the genetic stability of cryopreserved protocorm-like bodies was developed using RAPD markers, size and sequence of the trnL (UAA) non-coding region of cpDNA. Results showed the same RAPD patterns of non-cryopreserved and cryopreserved protocorm-like bodies. The size and sequences of the trnL (UAA) non-coding region of cpDNA for noncryopreserved and cryopreserved protocorm-like bodies were not different. There was no difference in morphology and similar patterns of ploidy in plantlets developed from non-cryopreserved and cryopreserved protocorm-like bodies.