Vitrification-based cryopreservation of protocorm-like bodies of an endangered lady's slipper orchid: Paphiopedilum niveum (Rchb.f.) Stein.

BACKGROUND: Paphiopedilum niveum (Rchb.f.) Stein, an endangered lady's slipper orchid, is listed in CITES Appendix I and thus requires conserving. OBJECTIVE: The study aimed to cryopreserve protocorm-like bodies (PLBs) by a vitrification technique. MATERIALS AND METHODS: Four-month-old PLBs were precultured in modified Vacin and Went liquid medium with various sucrose concentrations for 24 h and 5 d with daily increasing sucrose concentrations followed by different exposure times to plant vitrification solution 2 (PVS2). RESULTS: Precultured PLBs in 0.75 M sucrose for 5 d with stepwise increase of sucrose and PVS2 for 90 min provided the highest viability after drying to low moisture content. The cryopreserved PLBs exhibited fewer dark stained nuclei and accumulated starch grains than those of the control. Cryopreserved PLBs had some shrunken epidermal cells, no change in ploidy level, a survival level of 22 % and the regenerated plantlets grew well. CONCLUSION: This study is the first report evaluating the success of cryopreserved PLBs of P. niveum via a vitrification protocol.

Anthocyanins in the bracts of Curcuma species and relationship of the species based on anthocyanin composition.

Five anthocyanins, delphinidin 3-O-rutinoside, cyanidin 3-O-rutinoside, petunidin 3-O-rutinoside, malvidin 3-O-glucoside and malvidin 3-O-rutinoside, were identified. Three anthocyanins, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside and pelargonidin 3-O-rutinoside, were putatively identified based on C18 HPLC retention time, absorption spectrum, including λmax, and comparisons with those of corresponding standard anthocyanins, as the compounds responsible for the pink to purple-red pigmentation of the bracts of Curcuma alismatifolia and five related species. Cluster analysis based on four major anthocyanins formed two clusters. One consisted of only one species, C. alismatifolia, and the other consisted of five. Each cluster further formed sub-clusters depending on either species or habitats.

Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration.

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2 percent Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 +/- 3 degree C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 +/- 3 degree C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40 degree C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40 percent was observed with cryopreserved beads with 35 percent water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.