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.
Highlights
To improve the ex situ conservation of plant biodiversity, new strategies had to be developed (Engelmann and Engels 2002)
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
We describe an efficient method for the in vitro propagation of the fern species O. regalis and the cryopreservation of its gametophytes for ex situ conservation purposes
Summary
To improve the ex situ conservation of plant biodiversity, new strategies had to be developed (Engelmann and Engels 2002) These include in vitro technologies and cryopreservation for mass propagation and long-term storage of plant germplasm. Most have focused on crop plants of agricultural, horticultural or pharmaceutical value (Cruz–Cruz et al 2013) Application of these technologies is still restricted to a limited number of rare and threatened species (Pence 2014; Kaczmarczyk et al 2011). Similar research into both in vitro and liquid nitrogen (LN) techniques for the conservation of ferns is described in only 19 Page 2 of 12. There is urgent need for further research to improving and implementing novel methods for the conservation of fern biodiversity
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