Cryopreservation of Dendrobium cruentum Rchb. F. seeds by D cryo-plate and V cryo-plate techniques

Dendrobium cruentum Rchb. f. is one of native Thai orchid species that faced extinction problem because of their attractive characteristics. Consequently, conservation of this species is urgently needed. In this study, cryopreservation technique was applied to D. cruentum Rchb. f. seeds for long-term conservation. A successful protocol for D. cruentum Rchb. f. seed cryopreservation was developed by using D cryo-plate and V cryo-plate techniques. Seed viability was tested by TCC solution and 93.8 % of dyed seeds were shown. For cryo-plate technique, seeds were encapsulated over the cryo-plate by using 2 % (w/v) sodium alginate and polymerized with 100 mM CaCl₂. Encapsulated seeds were desiccated by using a laminar air-flow and PVS2 solution treatment with the same exposure time (0, 30, 60, 90 and 120 min). After cryopreservation, encapsulated seeds were cultured on modified VW agar medium. From the results, the maximum germination and regrowth percentage were observed; D cryo-plate technique with 60 min of dehydration time gave the highest germination (68.9 %) and regrowth (57.8 %). Thus, the excessive of dehydration may cause the reduction of germination and plant regeneration. In conclusion, D cryo-plate technique proved to be an appropriate for D. cruentum Rchb. f. seeds cryopreservation.