Cryopreservation of invitro-propagated protocorms of Caladenia for terrestrial orchid conservation in Western Australia

Cryopreservation is an important tool for the exsitu preservation of endangered plants. In this article, we describe the development of a cryopreservation protocol for orchid protocorms using the terrestrial Australian species Caladenia latifolia. Protocorms of C. latifolia generated asymbiotically each month on Murashige and Skoog (MS) medium containing 10µM N6-benzyladenine (BAP) provided explant sources for cryopreservation. Three size classes of protocorms were used as source explant material [small (S, =1mm); medium (M, >1<4mm); large (L, =4mm)] in combination with five desiccation treatments, i.e. 0, 0.4, 0.6, 0.8 and 1.0M glycerol. After 2days on desiccation medium, protocorms were treated with two cryoprotectant solutions (PVS2 and PVS4 at 0°C for 15, 20, 25 and 30min) before immersion in liquid nitrogen for 1day. Protocorms were then removed from liquid nitrogen storage, warmed rapidly (in a 40°C waterbath) and placed on three recovery media: half-strength MS with 0.5µM BAP, 0.5µM 6-furfurylaminopurine (kinetin) or 0.5µM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ). Protocorms on recovery media were incubated at 25°C under dark conditions and potential protocorm survival was observed at 60 and 90days using a fluorescein diacetate (FDA) test for protocorm viability. Protocorm survival was correlated significantly with explant size. Large cryopreserved protocorms had the highest potential survival rate (>90%) relative to small (<10%) and medium (70-80%) protocorms. Different desiccation media treatments did not affect significantly the survival percentage (74-92%). Similarly, changing the cryoprotectant solution and time of incubation at 0°C did not affect significantly potential protocorm survival (76-96%). Potential protocorm survival on various recovery media was not significantly different among treatments (88-100% survival). The study indicates that the cryopreservation of terrestrial orchid protocorms is technically feasible and provides a new and potentially highly beneficial tool in terrestrial orchid conservation where seed may be limited (because of species rarity), or as a means of storing and later utilizing the large surpluses of protocorms generated in propagation programmes