The development of an in vitro propagation and conservation system for the endangered serpentine fern Asplenium cuneifolium Viv.

Key messageProtocols for the long-term storage of spores, proliferation, and cryopreservation of gametophytes, induction of syngamy, and multiplication of sporophytes through somatic embryogenesis have been developed for Asplenium cuneifolium Viv. Asplenium cuneifolium Viv. is an endangered fern species restricted to European serpentine rocks, which face a risk of being easily damaged by both natural and anthropogenic agents. Establishing a comprehensive system of effective micropropagation and long-term storage of this species is therefore desirable. Freshly collected spores of A. cuneifolium were subjected to direct storage at 5 degrees C and in liquid nitrogen (LN). The viability of spores stored at 5 degrees C decreased by 10% after 1 year, whereas storage in LN did not change the initial spore viability even after 3 years. For the initiation of the gametophyte culture, disinfected spores were sown onto half-strength Murashige and Skoog medium (1/2MS) supplemented with 0.06 M sucrose. Following 6 months, abundant proliferation of secondary gametophytes was achieved. For the cryopreservation of gametophytes, encapsulation-vitrification and encapsulation-dehydration, were compared. Of these two methods, encapsulation-vitrification ensured survival of gametophytes of 64.5-93%, while encapsulation-dehydration guaranteed their 100% viability. The acceleration of syngamy on secondary gametophytes was achieved using a medium with macro- and micronutrients reduced to the 1/8 of the MS free of NH4NO3 and vitamins. The syngamic sporophytes could be multiplied by somatic embryogenesis (SE) induced on etiolated stipe explants in an 1/2MS medium devoid of any plant growth regulators. Adding 0.3 M of sucrose to the medium almost quadrupled the efficiency of the SE. The genetic stability of gametophytes after cryopreservation, as well as sporophytes obtained from them, was confirmed by flow cytometry, amplified fragment length polymorphism, and inter-simple sequence repeat markers.

Automated summary

Protocols for long-term storage of spores, proliferation and cryopreservation of gametophytes, induction of syngamy, and multiplication of sporophytes through somatic embryogenesis in Asplenium cuneifolium Viv. have been developed. The species faces a risk of being easily damaged by natural and anthropogenic agents, thus establishing an effective micropropagation and long-term storage system is desired. Storage of spores at 5°C resulted in a 10% decrease in viability after 1 year, while storage in liquid nitrogen did not affect viability even after 3 years. Encapsulation-dehydration ensured 100% viability of cryopreserved gametophytes, while encapsulation-vitrification ensured survival of 64.5-93% of gametophytes. Syngamy on secondary gametophytes was accelerated by reducing macro- and micronutrients to 1/8 of MS medium and excluding NH4NO3 and vitamins. Somatic embryogenesis on etiolated stipe explants in an 1/2MS medium without any plant growth regulators allowed multiplication of syngamic sporophytes. Adding 0.3 M sucrose to the medium increased the efficiency of somatic embryogenesis fourfold. Flow cytometry, AFLP, and ISSR markers confirmed the genetic stability of cryopreserved gametophytes and sporophytes derived from them.