Article ID: UTD-114
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, 1st preculture, 2nd 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 (1st preculture) before being transferred to the same medium with 0.6 M sucrose for one day (2nd 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 1st preculture) could reduce total ROS and MDA levels, leading to a high regeneration percentage (39%) of cryopreserved P. niveum SEs.