Cryopreservation of Protocorm-Like Bodies of the Hybrid Orchid Bratonia (Miltonia flavescens × Brassia Longissima)

In this study, cryopreservation of Bratonia (Miltonia flavescens (Lindl.) Lindl. × Brassia longissima (Reichb.) Nash), a hybrid tropical orchid, was achieved using protocorm-like bodies (PLBs) multiplied in vitro. Cryopreservation was performed using a vitrification protocol including pretreatment of PLBs with a loading solution (LS, 2.0 M glycerol + 0.4 M sucrose) for 15 min followed by treatment with modified PVS2 vitrification solution (containing PEG instead of ethylene glycol) for 1 h. Increasing benzyladenine (BA) concentration in the recovery medium to 5.0 or 10.0 mg l⁻¹ during the initial 3 weeks after rewarming provided 20.4% post-cryopreservation regrowth. By contrast, preliminary culture of PLBs with abscisic acid (ABA) and high sucrose concentrations (up to 0.3 M) as well as addition of reduced glutathione during the preculture, loading and post-culture steps were not beneficial. Forty to 45 plants were regenerated from each PLB which withstood cryopreservation. No morphological differences were observed between plants regenerated from cryopreserved and untreated PLBs. Investigations into the functional activity of photosystems I and II in PLBs suggest that electron transport was retained in the reaction centers of both photosystems shortly after cryopreservation.