In vitro propagation and long-term observation of acclimated plants in endangered tree fern Alsophila costularis

Abstract

Alsophila costularis Barker (Cyathea costularis), an endangered tree fern with tree-like erect stem, attracts gardening enthusiasts as a special ornamental plant. In vitro propagation can be advantageous for germplasm conservation and commercial application of A. costularis. Here, we described in vitro propagation of A. costularis via spore culture and green globular bodies (GGBs) system, as well as the long-term observation of acclimated plants regenerated from GGBs. In spore culture, the low concentration of mineral salt (1/8 MS) was beneficial for sporophyte formation on gametophytes, but sporophytes per conical flask was only 8 plantlets. In GGB system, cytokinin thidiazuron (TDZ) was essential for GGB induction and multiplication. The maximum of GGB induction frequency (93.33%) was obtained on 1/2MS medium with 2.0 mg/l TDZ by using juvenile sporophytes as explants, and the same medium was optimal for GGB multiplication. 1/4 MS supplemented with 0.1% (w/v) activated carbon (AC) was appropriate for plantlet regeneration from GGB, GGB differentiation frequency was 100%, and 42.40 plantlets could be regenerated from one piece of GGBs. The maximum of plantlet height (4.64 cm) was obtained on 1/2 MS with 0.1% (w/v) AC. After 6 years of acclimatization cultivation for plantlets regenerated from GGBs, plants in the plastic pots with diameter of 60 cm showed the excellent vegetative and reproductive growth, and the mature spores of these plants could produce sporophytes. Morphological and histological observation demonstrated that A. costularis GGBs was a green structure consisting of multiple single GGBs with hair-like structures. One single GGB could develop into one plantlet.