Adding Ascorbic Acid to Reduce Oxidative Stress during Cryopreservation of Somatic Embryos of Paphiopedilum niveum (Rchb.f.) Stein, an Endangered Orchid Species

Abstract

Paphiopedilum niveum (Rchb.f.) Stein, an endangered species, has been listed in CITES Appendix I and its germplasm conservation is required. To improve the regeneration of cryopreserved somatic embryos (SEs), adding 0.1 mM ascorbic acid (AsA) at a critical step during cryopreservation was investigated. The reactive oxygen species (ROS) and malondialdehyde (MDA) contents were also assessed during five steps (preconditioning, 1^st preculture, 2^nd preculture, osmoprotection, and dehydration) of a developed V cryo-plate technique as described briefly. Two-month-old SEs were preconditioned on modified Vacin and Went medium (MVW) containing 0.1 M sucrose for seven days. These SEs were precultured on MVW containing 0.2 M sucrose for one day (1^st preculture) before being transferred to the same medium with 0.6 M sucrose for one day (2^nd preculture). Precultured SEs were embedded on a cryo-plate, incubated in loading solution (LS) with 1.2 M sucrose for 30 min at 25°C and dehydrated with plant vitrification solution 2 (PVS2) for 60 min at 25°C. It was found that applying AsA on day 7 after culture (before the 1^st preculture) could reduce total ROS and MDA levels, leading to a high regeneration percentage (39%) of cryopreserved P. niveum SEs.