In Vitro Propagation and Cryopreservation of Lady's Slipper Orchid (Paphiopedilum niveum (Rchb.f.) Stein)

Abstract

The snow white lady slipper orchid (Paphiopedilum niveum (Rchb.f.) Stein) is an endangered species that distributed in Southern Thailand and Northern Malaysia. The habitat destruction and over-collecting decreased the natural population. Even though P. niveum was protected by Appendix I of the CITES but the conventional propagation method provided low productivity and also take a long period of time which could not meet the commercial demand. Thus, the conservation of genetic resources of P. niveum has required the micropropagation and cryopreservation for long-term storage. This study was composed of 1) the study of the effect of plant growth regulators (PGRS) on the direct somatic embryogenesis and SEs proliferation from protocorm and 2) cryopreservation via V cryo-plate method with the application of ascorbic acid (AA). The results showed that SEs could be generated from four-month-old protocorm when culturing on modified Vacin and Went (MVW) containing 0.1 mgl1 1- Naphthaleneacetic acids (NAA) for 3 months under the dark condition. These primary SES could be proliferated into secondary SES after when culturing on free-PGRS MVW in light condition and continuously developed into plantlets after being transferred to culture on plantlet induction medium for 4 months. The uniformity of the genetic pattern between the mother plant (V1) and regenerated plants (V2 and V3) was evaluated by RAPD analysis. The results of cryopreservation using V cryo-plate method showed that the cryopreserved P. niveum SES could survive (~20%) after precondition on MVW containing 0.1 M sucrose (7 days) followed by two-step preculture on MVW containing 0.2 M and 0.6 M sucrose (each with 1 day). SES were embedded on cryo-plate with alginate gel before put into loading solution containing 2 M glycerol and 1.2 M sucrose for 30 min. The SEs was dehydrated with plant vitrification solution 2 (PVS2) for 60 min. The application of ascorbic acid (AA) in the critical step could reduce total reactive oxygen species (ROS) and malondialdehyde (MDA) production which significantly improved the survival percentage to 39% of cryopreserved P. niveum SEs.