Effects of Ascorbic Acid on PVS2 Cryopreservation of Dendrobium Bobby Messina’s PLBs Supported with SEM Analysis

Abstract

Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3-4mm of PLBs size precultured in 0.2M sucrose for 1day, treated with a mixture of 2M glycerol and 0.4M sucrose supplemented with half-strength liquid MS media at 25°C for 20min and subsequently dehydrated with PVS2 at 0°C for 20min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40°C for 90s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2% (w/v) sucrose without any growth regulators and resulted in 40% growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10% when 0.6mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture.