为加强野生蕨类植物的保护与开发, 优化星蕨(Microsorum punctatum)孢子萌发方法和条件, 比较分析了不同因素对原叶体增殖、孢子体诱导、绿色球状小体(GGBs)诱导及其发育为幼孢子体的影响, 建立人工高效快繁技术体系。以成熟孢子为材料, 分别以MS、1/2MS、1/3MS和1/4MS为基本培养基在无菌条件下萌发; 通过L₉(3⁴)正交试验, 研究无机盐浓度、植物生长调节剂及其质量浓度对原叶体发生和增殖的影响。当原叶体增殖到一定数量时, 再以MS、1/2MS、1/3MS和1/4MS为基本培养基筛选适宜诱导孢子体的培养基; 随后, 以幼孢子体为材料诱导GGBs发育为新的幼孢子体并炼苗移栽。适宜孢子萌发的培养基为1/2MS, 原叶体在MS+0.3 mg·L^-1 6-BA+1.5 mg·L^-1 NAA培养基中大量增殖, 60天后增殖系数约为9.6; 将原叶体切割后接入1/4MS培养基, 加无菌水培养90天后, 幼孢子体发生系数约为10.0; 幼孢子体在1/2MS+1.5 mg·L^-1 6-BA+0.1 mg·L^-1 NAA培养基中可诱导出GGBs, 诱导率达93.3%, GGBs在此培养基中的增殖系数达32.0; 在1/2MS培养基中GGBs的分化成苗率较高, 最高约为92%; 试管苗经炼苗移栽成活率在90%以上。该研究建立了原叶体-受精-孢子体和幼孢子体-GGBs-幼孢子体2个技术体系, 尤其是GGBs的产生, 极大缩短了植株的再生周期。研究结果可为优质种苗及其它蕨类植物的人工繁育提供技术支撑。

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