Cold storage and cryopreservation by encapsulation-dehydration of Cephalanthera rubra (L.) Rich., a threatened orchid species

Abstract

Cephalanthera rubra (L.) Rich. is an orchid species in danger of extinction. In vitro conservation of this species has not been reported earlier. The aim of this study was to evaluate the effect of encapsulation-dehydration pretreatment on the survival percentage of cold-preserved and cryopreserved shoot tips. Plant cryopreservation at ultra-low temperatures in liquid nitrogen (−196 °C) is an effective, lowcost, long-term, and proper conservation method for many plant species, particularly those included in the red list, such as orchids. The use of different pretreatments of plant germplasms to withstand against liquid nitrogen (LN) is a prerequisite of cryopreservation. In vitro preservation techniques, especially cold storage and cryopreservation, are two reliable methods to preserve genetic resources of orchids. The use of protective pretreatments against cold and freezing stress plays an irrefutable role in maintaining the germination of germplasms after cold storage and cryopreservation. One of the most important and widely used pretreatments, especially for ornamental plants, such as orchids, is encapsulation-dehydration. The encapsulation-dehydration method was used as pretreatment for cold storage and cryopreservation of C. rubra (L.) Rich. germplasm. In situ-derived shoot tips were used as explants and encapsulated in sodium alginate-beads. Encapsulated shoot tips were treated in Murashige and Skoog liquid medium containing 0.75 M sucrose for 120 min and then dehydrated under a sterile air-flow of laminar hood cabinet for 120 min. Shoot tips were successfully cold preserved in a refrigerator at 4 °C and cryopreserved by ultra-rapid freezing in LN and thawing in controlled environmental conditions with encapsulation-dehydration. The highest recovery level (82.33 and 83.66%) after cold storage and direct immersion in LN was obtained with the encapsulation technique based on 120 min dehydration, respectively. The lowest recovery level (37.66% and 33.66%) was recorded in cold-preserved and cryopreserved shoot tips without pretreatments (control).