INTRODUCTION: The wild populations of Microsorum punctatum face endangerment due to habitat degradation and low spore reproductive efficiency. Fern life cycles involve alternating gametophyte and sporophyte generations, where gametophyte development and sporophyte transition represent critical bottlenecks in in vitro propagation, heavily influenced by environmental factors and culture conditions. Although asexual propagation techniques such as green globular bodies (GGBs) have been successfully applied in some fern species, low sporophyte induction efficiency and proliferation challenges persist, hindering large-scale production. This study employed M. punctatum spores to systematically investigate sterile germination mechanisms, gametophyte proliferation, and sporophyte regeneration. A dual-pathway rapid propagation system was established, integrating high-efficiency prothallus proliferation with GGBs induction, aiming to provide both theoretical insights and practical solutions for conserving endangered fern resources and advancing industrial-scale cultivation.
RATIONALE: The unique alternation of generations life cycle in ferns, characterized by independent gametophyte survival, provides a theoretical framework for in vitro propagation. Studies have demonstrated that gametophyte homogenization culture and GGBs induction can overcome sporophyte regeneration barriers, while medium composition and phytohormone ratios critically regulate developmental phase transitions. To address the challenges of low spore propagation efficiency and habitat sensitivity in M. punctatum, this study leverages its gametophyte proliferation potential and rhizome meristematic activity in sporophytes. By optimizing aseptic systems and induction conditions, as well as mimicking the natural fertilization microenvironment, a dual-path regeneration system integrating prothallus proliferation and GGB-based propagation was established, laying a theoretical foundation for efficient conservation of endangered ferns.
RESULTS: Spore germination was optimally achieved in 1/2MS medium. Prothalli exhibited vigorous proliferation in MS medium supplemented with 0.3 mg·L^-1 6-BA and 1.5 mg·L^-1 NAA, reaching a proliferation coefficient of 9.6 after 60 days of culture. Fragmented prothalli transferred to 1/4MS medium with sterile water supplementation achieved a young sporophyte induction coefficient of 10.0 following 90 day cultivation. GGBs were successfully induced from young sporophytes in 1/2MS medium containing 1.5 mg·L^-1 6-BA and 0.1 mg·L^-1 NAA, showing 93.3% induction efficiency and a remarkable proliferation coefficient of 32.0. The GGB differentiation into plantlets was most efficient in 1/2MS medium, yielding a conversion rate of 92%. Acclimatized plantlets demonstrated over 90% survival rate post-transplantation.
CONCLUSION: This study successfully established an efficient in vitro rapid propagation system for M. punctatum spores. Optimization of sterilization duration and culture medium types significantly enhanced spore germination rates. A prothallus culture protocol with a high proliferation coefficient was developed, overcoming bottlenecks in gametophyte mass propagation. Liquid immersion-assisted fertilization technology enabled efficient induction of young sporophytes, while the GGBs induction system markedly shortened the regeneration cycle. For the first time, a dual-pathway rapid propagation strategy—“prothallus proliferation-sporophyte induction” combined with “GGBs cyclic regeneration” was proposed. The study demonstrated that the meristematic properties of M. punctatum GGBs are distinct from callus tissue, providing a robust technical framework for the conservation of endangered ferns and industrial-scale seedling production.

Formation of antheridia, archegonia, and sporophyte production in Microsorum punctatum