Effects of droplet-vitrification on Brassidium Shooting Star’s orchid protocorm-like bodies (PLBs)

Abstract

In the present study, protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid was successfully cryopreserved using droplet-vitrification method. Seven (7) parameters have been optimised such as PLB size, sugar type, preculture concentration, preculture incubation duration, plant vitrification solution 2 (PVS2) incubation duration, thawing method and unloading duration. Assessments of viability were evaluated by using TTC spectrophotometric at 490nm absorbance and growth recovery of regenerated PLBs. The results showed that survival of cryopreserved plants were superior when 3–4mm PLBs precultured on sucrose enriched media. Decrease in survival was observed when cryopreserved PLBs exposed to media higher than 0.25M sucrose. However, extended preculture duration to 7 days improved the growth recovery of non-cryopreserved PLBs to 70%. The present study proved that PVS2 treatment promotes growth recovery of cryopreseved PLBs. Regenerated PLBs observed when PLBs treated with PVS2 for 40min before plunging into liquid nitrogen. The highest recovery of 30% was obtained with 3–4mm PLBs precultured on semi-solid media supplemented with 0.25M sucrose for 7 days, followed by loading treatment for 20min, dehydration with PVS2 solution for 40min prior to store in liquid nitrogen and rinsing with unloading solution for 15min. Addition of ascorbic acid and α-tocopherol which was reported improved regrowth of cryopreserved in several plants did not affect the regrowth of Brassidium Shooting Star orchid’s PLBs. However, inclusion of α-tocopherol at preculture, loading, unloading and recovery steps enhances faster recovery of cryopreserved PLBs which observed three weeks after treatment.