Gametophyte Development and Sporophyte Regeneration of Alsophila spinulosa

Alsophila spinulosa (Wall. ex Hook) Tryon is one of the few remaining tree ferns on Earth. As a living Mesozoic fern fossil, it has great significance for studies on paleo-climate and paleo-environmental changes. A. spinulosa also has considerable ornamental and medicinal value. This study assessed in vitro spore germination, gametophyte development and sporophyte regeneration in A. spinulosa and established efficient protocols for gametophyte proliferation, callus induction and regeneration from sporophytes. The effects of several plant growth regulators (PGRs) and their concentrations on gametophyte development and sporophyte formation were also investigated. No morphological variations were observed on PGR-free Murashige and Skoog (MS) medium although the presence of PGRs in MS medium induced some variations during the development stages of filaments and prothallia. When filaments formed on the surface of each prothallium, prothallia could be proliferated. Highest prothallium proliferation coefficient (PPC) was 6.6 within 30 days on MS medium with 1.0 mg L −1 6-benzyladenine. Sporophytes readily formed roots on PGR-free MS medium or in MS medium with 0.2–1.0 mg L −1 indole-3-butyric acid and/or α-naphthaleneacetic acid, with a minimal 93% rooting. After young rooted sporophytes were transferred to a vermiculite and peat substrate, survival percentage was 92.7% within one month (and 88.7% after two months). Callus was successfully induced and adventitious shoots formation from sporophyte base on MS medium with 0.2 mg L −1 2,4-dichlorophenoxyacetic acid and 0.5 mg L −1 thidiazuron. Each callus cluster could differentiate into mean 13.6 sporophytes.