Integration of tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunda regalis L.

Abstract

A simple and reliable technique for in vitro multiplication and long-term preservation using liquid nitrogen was developed for gametophytes of Osmunda regalis. The effect of Knop’s medium and various concentrations (1/2, 1/4, 1/8) of mineral salts provided in Murashige and Skoog (MS) basal medium, together in the presence or absence of both ammonium nitrate and a full complement of vitamins on gametophyte proliferation and sporophyte production was determined. Moreover, the effectiveness of gametophyte cryopreservation by vitrification and encapsulation–vitrification techniques was assessed. Maximum gametophyte proliferation (89 %) occurred on the ammonium nitrate- and vitamin-free [(-NH4NO3)(-vit)] MS medium with 1/2 or 1/4 strength mineral salts. The maximum production of sporophytes (30 plantlets per gametophyte clump) required 1/8MS (-NH4NO3)(-vit) medium. The flow cytometric analysis revealed that the sporophytes contained twofold more pg DNA than gametophytes. This confirmed that the sporophytes were obtained by sexual reproduction. The vitrification protocol and PVS2 solution were ineffective for cryopreservation. The greatest survival rate (81.6 %) following cryo-exposure occurred following treatment of encapsulated gametophytes with PVS3 solution for 3 h. This protocol allowed the recovery of gametophyte cultures following 6 weeks after rewarming. Finally, 100 % of sporophytes produced in vitro were successfully acclimated to ex vitro conditions. Application of the in vitro and cryopreservation methods made it possible to improve the number and time of O. regalis sporophyte production. Whole system of micropropagation can be completed in approximately 1 year. The protocols open new avenues for the mass propagation, germplasm conservation and resource management of the species.