Germination and subsequent development was assessed after seeds of Vanda tricolor were stored in liquid nitrogen. Mature seeds, harvested 7 months after self-pollination, were directly plunged into liquid nitrogen. Germination of cryopreserved seeds on solid New Dogashima (ND) medium supplemented with 1 mg/l 6-benzyladenine, 0.5 mg/l 1-naphthaleneacetic acid and 2% sucrose was faster than non-cryopreserved seeds (28 days versus 60 days after sowing). Immature seeds, harvested 6 months after self-pollination, were treated with or without loading solution (LS) of 2 M glycerol and 0.4 M sucrose in liquid ND medium, pH 5.4 at 25°C for 15 min and dehydrated with PVS2 solution for 0-210 min on ice and then cryopreserved by vitrification. The results showed that the germination percentage of cryopreserved seeds treated with LS was higher than without LS. After 90 days of sowing, the highest germination percentage of cryopreserved seeds was 13.6% which was higher than non-cryopreserved seeds (10.5%) when seeds were treated with LS for 15 min and then dehydrated with PVS2 solution for 180 min. After 150 days of sowing, protocorms of non-cryopreserved and cryopreserved seeds were able to form new protocorms (budding protocorms) and developed into shoots after 180 days of sowing. There were no significant differences between growth and development of protocorms derived from noncryopreserved and cryopreserved seeds.

Vanda coerulea is a popular albeit endangered blue orchid of Thailand, which would be desirable to propagate using regeneration methods. Here, we studied the effects of culture media (Vacin and Went, and Murashige and Skoog), sucrose concentrations (0-30 g/l), and plant growth regulators (BA, TDZ, and NAA) on adventitious shoot regeneration from shoot tip culture of V. coerulea. Shoot tips cultured on modified Vacin and Went (VW) medium supplemented with 10 g/l sucrose showed higher shoot and root formations, as well as plantlet height than those cultured on Murashige and Skoog medium. Addition of 1 mg/l BA to the modified VW medium induced the best shoots and roots after 3 months of culture. The combination of 0.5 mg/l NAA and 2 mg/l TDZ gave the optimal number of roots per explant and plantlet height after 3 months of culture. Survival rate of plantlets cultured in the greenhouse was 100% after 3 months of culture. There were no differences in morphology or patterns of ploidy level between stock plants and regenerated plants.

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