Articles
Development of cryopreservation protocols for endangered wild orchids in Korea
Article number
1262_7
Pages
43 – 52
Language
English
Abstract
Biodiversity conservation is a global issue, and preventing the extinction of endangered wild species is challenging.
Cryopreservation is considered the only feasible option for the long-term conservation of endangered species that are vegetatively propagated or have unorthodox or short-live seeds.
In this study, we explore the feasibility of using cryopreservation to conserve endangered Korean orchid species.
Following the introduction of propagules in vitro and their multiplication, several desiccation- and vitrification-based protocols were applied to diverse materials including the rhizomes, protocorms, protocorm-like bodies (PLBs), and seeds. Dendrobium moniliforme seeds were successfully cryopreserved using both desiccation and vitrification.
For desiccation, the seeds were dried over silica gel for 0-25 h and then stored in liquid nitrogen in cryovials.
For vitrification, the seeds were cryopreserved in cryovials following precultural, osmoprotection, and cryoprotection treatments.
The cryovials were rewarmed in a 40°C water bath for 1.5 min and the samples incubated with 30% sucrose for 40 min for unloading.
Both methods resulted in 80% germination.
Although survival was observed following all three cryopreservation techniques tested on Cymbidium kanran rhizomes, i.e., preculture-desiccation, vitrification, and droplet-vitrification, only the rhizomes cryopreserved via preculture-desiccation developed into in vitro plants.
The protocol applied to the PLBs of D. moniliforme was as follows: a progressive preculture with 10% sucrose for 31 h, 17.5% sucrose for 17 h, and 25% sucrose for 4-5 h; osmoprotection with C4-35% (17.5% glycerol + 17.5% sucrose) for 30-50 min; cryoprotection with PVS3 (50% glycerol + 50% sucrose) for 40-120 min; cooling and rewarming using cryovials or aluminium foil strips; and unloading with 30% sucrose for 30-60 min.
Vitrification was successfully applied to Habenaria radiata protocorms with approximately 70% post-cryopreservation regrowth.
The cryopreservation protocols developed in this study can be applied for the safe long-term conservation of these endangered orchid species.
Cryopreservation is considered the only feasible option for the long-term conservation of endangered species that are vegetatively propagated or have unorthodox or short-live seeds.
In this study, we explore the feasibility of using cryopreservation to conserve endangered Korean orchid species.
Following the introduction of propagules in vitro and their multiplication, several desiccation- and vitrification-based protocols were applied to diverse materials including the rhizomes, protocorms, protocorm-like bodies (PLBs), and seeds. Dendrobium moniliforme seeds were successfully cryopreserved using both desiccation and vitrification.
For desiccation, the seeds were dried over silica gel for 0-25 h and then stored in liquid nitrogen in cryovials.
For vitrification, the seeds were cryopreserved in cryovials following precultural, osmoprotection, and cryoprotection treatments.
The cryovials were rewarmed in a 40°C water bath for 1.5 min and the samples incubated with 30% sucrose for 40 min for unloading.
Both methods resulted in 80% germination.
Although survival was observed following all three cryopreservation techniques tested on Cymbidium kanran rhizomes, i.e., preculture-desiccation, vitrification, and droplet-vitrification, only the rhizomes cryopreserved via preculture-desiccation developed into in vitro plants.
The protocol applied to the PLBs of D. moniliforme was as follows: a progressive preculture with 10% sucrose for 31 h, 17.5% sucrose for 17 h, and 25% sucrose for 4-5 h; osmoprotection with C4-35% (17.5% glycerol + 17.5% sucrose) for 30-50 min; cryoprotection with PVS3 (50% glycerol + 50% sucrose) for 40-120 min; cooling and rewarming using cryovials or aluminium foil strips; and unloading with 30% sucrose for 30-60 min.
Vitrification was successfully applied to Habenaria radiata protocorms with approximately 70% post-cryopreservation regrowth.
The cryopreservation protocols developed in this study can be applied for the safe long-term conservation of these endangered orchid species.
Publication
Authors
E. Popova, H.H. Kim
Keywords
desiccation, droplet-vitrification, protocorm-like body, rhizome, seed, vitrification
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