Cryopreservation of Dendrobium candidum Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Abstract

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l -1 a-naphthalene acetic acid and 0.5 mg l -1 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 lmol m -2 s -1 ) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl 2 solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS 2 ) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.

Ex situ Conservation and Cryopreservation of Orchid Germplasm

“…The germplasm to be conserved is encapsulated in sodium alginate, immersed in a loading solution of glycerol and sucrose, and finally exposed to a cryoprotective solution such as PVS2 before storage in liquid nitrogen. The method has been successfully used to preserve the protocormlike bodies of Dendrobium candidum (Yin and Hong 2009) and Dendrobium nobile (Mohanty et al 2012). Optimizing each step of the process resulted in a survival rate of 89% for D. candidum (Yin and Hong 2009) and 78% for D. nobile (Mohanty et al 2012).…”

“…The method has been successfully used to preserve the protocormlike bodies of Dendrobium candidum (Yin and Hong 2009) and Dendrobium nobile (Mohanty et al 2012). Optimizing each step of the process resulted in a survival rate of 89% for D. candidum (Yin and Hong 2009) and 78% for D. nobile (Mohanty et al 2012). In the latter case, optimized encapsulation-vitrification resulted in significantly higher survival of the protocorm-like bodies compared with encapsulationdehydration (78% vs. 53%).…”

Microscopical analysis of in vitro Mokara Broga Giant orchids PLBs

“…PLBs are also the common target tissue for genetic transformation studies in orchids since they can proliferate rapidly and have high abilities to regenerate into complete plantlets (Liau et al, 2003;Sreeramanan et al, 2008). Furthermore, PLBs can also serve as an appropriate plant material for cryopreservation experiment (Yin and Hong, 2009). PLBs are well-differentiated tissues that are sometimes viewed as orchid embryos that develop with two discrete bipolar structures, namely, the shoot and root meristem.…”

Orchid cryopreservation

“…Orchid flowers are known for their natural beauty and for attracting the interest of various producers, making information about their cultivation and storage increasingly relevant. In addition to their ornamental potential, some genera provide food products such as vanilla, medicines and other products used in industry (Dressler, 1993;Lucksom, 2007;Zanenga-Godoy, 2003).…”