Cryopreservation of Orchid Pollinia Using the V Cryo-Plate Method.

BACKGROUND: Preserving pollinia viability and fertility for pollination at specific times of the year is very important in orchid breeding. One possible method is cryopreservation using aluminium cryo-plate which is increasingly used for the long-term storage of plant genetic resources. OBJECTIVE: To determine a V cryo-plate method for the cryopreservation of orchid pollinia and apply it to some Thai orchid species for breeding. MATERIALS AND METHODS: Pollinia of Rhynchostylis gigantea (L.) Ridl. were collected from completely open flowers in the morning and then were placed on aluminium cryo-plates embedded in alginate gel, then immersed in loading solution containing 2 M glycerol and 0.4 M sucrose for 15 min at room temperature (29oC), and then dehydrated with PVS2 solution for 0-60 min at 29oC. The cryo-plates with pollinia were directly plunged into liquid nitrogen for 40 min, and rapidly warmed in 1 M sucrose for 15 min. The cryopreserved and non-cryopreserved pollinia were used for hand-pollinating flowers of the same species for producing hybrids. RESULTS: The viability of non-cryopreserved and cryopreserved pollinia dehydrated with PVS2 was 100%. The highest capsule set after pollinating flowers with cryopreserved pollinia dehydrated with PVS2 for 40 min was also up to 100%. The protocol for cryopreservation of R. gigantea (L.) Ridl pollinia using V cryo-plate method was successfully applied for the cryopreservation of nine Thai orchid species. The exposure time to PVS2 affected the pollinia viability (range 40-100 %; average 93%) and capsule set (range 20-100 %; average 78%) of the nine species. The successful capsule set and seed production after pollination with cryopreserved pollinia in all orchid hybrids were observed. Seed germinated into protocorms and developed to plantlets cultured on modified Vacin and Went (1949) agar medium. CONCLUSION: Cryopreservation of freshly collected orchid pollinia using V cryo-plate method is an efficient tool for the long-term storage of plant germplasm and for orchid breeding.

Cryopreservation of Bletilla striata mature seeds, 3-day germinating seeds and protocorms by droplet-vitrification.

Droplet-vitrification was studied for the cryopreservation of Bletilla striata mature seeds (0 day after sowing), 3-day germinating seeds and protocorms (6, 9 and 12 days after sowing). Mature seeds, 3-day germinating seeds and 6-day old protocorms were precultured in liquid medium supplemented with 0.3 M sucrose for 3 h on a shaker (110 rpm) and then dehydrated with 2 M glycerol and 0.4 M sucrose in liquid medium (loading solution) for 15 min and exposed to PVS2 solution for 60 min at 25 degree C. The plant materials were then immersed in liquid nitrogen, rewarmed rapidly and cultured on solidified ND medium supplemented with 3% sucrose for recovery. After cryopreservation, the highest germination percentage of mature seeds, 3-day germinating seeds and survival of cryopreserved 6-day old protocorms was 93%, 91% and 84%, respectively. For 9-day old protocorms, highest survival (66%) after cryopreservation was achieved after preculture with 0.5 M sucrose for 3 h on a shaker, dehydration with loading solution for 15 min, exposure to PVS2 solution for 40 min at 25 degree C, and culture on solidified ND medium supplemented with 480 mg per liter ammonium nitrate and 3% sucrose. No survival was observed in cryopreserved 12-day old protocorms.