Improved micropropagation protocol for leatherleaf fern (Rumohra adiantiformis) using rhizomes as donor explant

A new micropropagation protocol for leatherleaf fern (Rumohra adiantiformis (G. Forst.) Ching) was successfully established using rhizomes as the donor explant, following appropriate sterilization. Murashige and Skoog (MS) medium containing 0.25 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 0.2 mg/l α-naphthalene acetic acid (NAA), 1.0 mg/l 6-benzyl adenine (BA), and 0.5 mg/l thidiazuron (TDZ) with 30 g/l sucrose (IM-4) was the most appropriate medium for culture initiation. When entire rhizomes harvested from mother stock plants were cultured on a simple paper bridge containing liquid IM-4 medium, culture initiation improved with 3.4 rhizomes regenerated per sub-culture. The average multiplication rate of newly regenerated rhizomes increased to 5.7/rhizome on half-strength MS medium supplemented with 0.05 mg/l IAA, 0.25 mg/l BA, 0.5 mg/l Kin, 1 g/l activated charcoal and 20 g/l sucrose (MM-5). The level of multiplication peaked in the fifth subculture and retained high quality until the sixth subculture. From the seventh subculture onwards, the quality of regenerated fronds was reduced. The regenerated rhizomes rooted easily on MM-5 and could be acclimatized ex vitro with 97–100% survival.

► A new micropropagation protocol for leatherleaf fern (Rumohra adiantiformis) was established using rhizomes as the donor explant. ► MS medium containing 0.25 mg/l 2,4-D, 0.2 mg/l NAA, 1.0 mg/l BA and 0.5 mg/l TDZ with 30 g/l sucrose was the best medium for culture initiation. ► On a simple paper bridge containing liquid medium, culture initiation improved to 3.4 rhizomes regenerated per sub-culture. ► 5.7 new rhizomes/rhizome formed on half-strength MS medium + 0.05 mg/l IAA, 0.25 mg/l BA, 0.5 mg/l Kin, 1 g/l activated charcoal, 20 g/l sucrose. ► Regenerated rhizomes rooted easily and could be acclimatized ex vitro with 97–100% survival.