Root cryopreservation to biobank medicinal plants: a case study for Hypericum perforatum L.

Abstract

In this study, an effective root-based cryopreservation method was developed for Hypericum perforatum L., an important medicinal species, using in vitro plants. A systematic approach was applied to determine effective combinations of protocol steps such as preculture, osmoprotection, vitrification solution treatment, and unloading, followed by protocol optimization using a single-factor approach. The effects of root section type (root tips, middle sections, or basal sections), duration of root section culture after excision, and donor plant age were also investigated. In a wild genotype, middle and basal root sections excised from 8-wk-old plants and cryopreserved at the age of 10 d after excision showed the highest plant regrowth after cryopreservation. In the optimized protocol, root sections were precultured in 10% (w/v) sucrose for 17 h, osmoprotected with a solution composed of 17.5% (w/v) glycerol and 17.5% (w/v) sucrose for 20 min, followed by a vitrification solution of 40% (w/v) glycerol and 40% (w/v) sucrose for 30 min, and cryopreserved using aluminum foil strips (droplet-vitrification). After rewarming in preheated 25% (w/v) sucrose solution and 30-min unloading, root segments were recovered on medium supplemented with 1.0 mg L−1 gibberellic acid and showed 78% plant regrowth. This cryopreservation method was successfully adapted for five elite lines of H. perforatum with a 45 to 87% regrowth rate after cryopreservation. These results suggest that root cryopreservation may be an effective method for medicinal plant conservation and should be tested with a broader range of species.