CONSERVING A LIFE CYCLE - SIMULTANEOUS CRYOPRESERVATION OF ORCHID SEEDS AND MYCORRHIZAL FUNGI

Abstract

Effective ex situ conservation of terrestrial orchids requires conservation of both seed and the fungi necessary for the seed to germinate. A potential method for conserving the seed and fungi simultaneously is encapsulation-dehydration. This technique was tested on four threatened orchids from New South Wales, Australia (Pterostylis saxicola, Diuris arenaria, D. flavescens and D. tricolor) and their mycorrhizal fungi (Ceratobasidium sp. for P. saxicola and Tulasnella calospora for all Diuris spp.). Seed and fungal hyphae were encapsulated in 2% sodium alginate, pre-treated for 24 h in 0.75 M sucrose, then dried for 18 h on sterile absorbent paper in a laminar flow cabinet. Beads containing P. saxicola or D. arenaria and their compatible fungi were stored at 23, 4, -18 and -196°C for 0, 3, 6, 12 and 24 months. Beads containing D. flavescens or D. tricolor and fungi were stored at -18°C for zero and three months only. Fungal recovery for both the Ceratobasidium sp. and T. calospora declined to ≤2% following storage at temperatures ≥4°C; however, no significant decline in fungal recovery was observed for either species following storage at -18°C or -196°C for up to 24 months (P > 0.4 in all cases). Seed germination did not occur in the absence of fungal growth therefore the trends in seed germination generally followed those of fungal recovery. Germination for P. saxicola and D. arenaria gradually declined to 0% for beads stored at 23 or 4°C, but there was no significant decline in germination of P. saxicola from beads stored at -18°C or -196°C (P = 0.527 and 0.547, respectively), or of D. arenaria from beads stored at -196°C (P = 0.542), for up to 24 months. Similarly, no decline in fungal recovery or germination was observed from beads containing D. flavescens or D. tricolor and stored at -18°C for three months. All seedlings produced normal plants that transferred easily from in vitro culture to potting mix. Simultaneous encapsulation-dehydration may therefore be useful for short-term storage of orchid seeds and compatible fungi at -18°C, or longer term storage at -196°C.