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.