Cryopreservation of Paphiopedilum niveum (Rchb. f.) Stein calli using encapsulation-vitrification and vitrification methods

Abstract

© 2017, International Society for Horticultural Science. All rights reserved. Paphiopedilum niveum (Rchb. f.) Stein has been listed in the CITES Appendix I. To evaluate the success of calli cryopreservation of P. niveum, the encapsulation-vitrification and vitrification techniques were compared. Six-month-old callus clumps were precultured in modified VW liquid medium containing various sucrose concentrations (0, 0.25, 0.50, 0.75 and 1.00 M) for 5 d with daily increasing sucrose concentration and dehydrated in PVS2 at different periods (0, 20, 40, 60, 80 and 100 min). Non-cryopreserved calli were used as a control. The encapsulation-vitrification technique, which provided the best result, exhibited the highest viability absorbance value (0.237±0.011) and low moisture content (MC) at 27.34±0.96% when calli were precultured in 0.5 M sucrose followed by a 100-minute exposure to PVS2. Histological and histochemical observations also showed normal cell with a large nucleus, a dense cytoplasm, non-disrupted starch grains and protein. Although the cryopreserved calli via vitrification presented high viability (0.216±0.009) and low moisture content (23.93±2.05%), the vitrification-based calli displayed the disruption of both starch grains and protein. These cryopreserved calli exhibiting no change in ploidy level provided the survival rates at 29.63±10.31% (encapsulation-vitrification) and 22.22±7.85% (vitrification).