{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T10:36:34Z","timestamp":1787567794616,"version":"build-2736575974"},"reference-count":38,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T00:00:00Z","timestamp":1787529600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the Major Expenditure Increase and Decrease Project at the Central Level","award":["2060302"],"award-info":[{"award-number":["2060302"]}]},{"name":"the Construction of the Southern Medicinal Plant Germplasm Resource Base in Northern Guangdong","award":["2024B12120600006"],"award-info":[{"award-number":["2024B12120600006"]}]},{"name":"the China Agriculture Research System","award":["CARS-21"],"award-info":[{"award-number":["CARS-21"]}]},{"award":["CARS-21"],"award-info":[{"award-number":["CARS-21"]}],"id":[{"id":"https:\/\/ror.org\/03p5eky76","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>Cibotium barometz (L.) J.Sm., a medicinal fern, relies on three interconnected processes for in vitro propagation: spore germination, ordered gametophyte development, and successful initiation and early establishment of the sporophyte generation. However, during aseptic subculture, gametophytes frequently deviate from this developmental trajectory. The predominant abnormalities include filamentous arrest, thick fan-shaped blades, and irregular spatulate blades, all of which were associated with reduced archegonial maturation and subsequent sporophyte establishment. In this study, developmental time-course observation, paraffin-section histology, and medium-regulation experiments were integrated to establish a diagnosis\u2013recovery\u2013initiation framework for Cibotium barometz gametophytes. Spore germination and gametophyte development were divided into eight practical checkpoints: spore imbibition, spore-wall rupture, rhizoid emergence, rhizoid elongation, filamentous prothallus, lamellar prothallus, cordate gametophyte, and sporophyte formation. The ontogeny of archegonia was delineated into six histological stages, ranging from the initial-cell stage to the pre-fertilization stage. Based on an integrative assessment of population appearance, individual morphology, and anatomical sections, abnormal gametophytes were classified into three diagnostic categories. Filamentous arrest manifested as wool-like or thread-like growth patterns, featuring persistent chain-like cellular organization and failure to develop a two-dimensional blade. Thick fan-shaped blades showed pronounced enlargement and thickening; section images revealed narrow, densely packed cells, accumulation of storage compounds, and localized wall thickening, a pattern consistent with, but not sufficient to demonstrate, a preferential shift toward vegetative proliferation. Irregular spatulate blades were narrow and asymmetric and showed morphological and histological features suggestive of disrupted tissue polarity. Although archegonial initial cells or primordium-like structures were intermittently detected, they infrequently advanced to mature archegonia. Medium treatment significantly affected arrest rate, hypertrophy rate, sporophyte initiation, and mean young-sporophyte height (p &lt; 0.001). Murashige and Skoog (MS) medium supplemented with IAA, 6-BA, and activated carbon was associated with the highest arrest rate, whereas full-strength MS medium was associated with the most pronounced hypertrophy. Among hormone-free subculture treatments, 1\/2 MS + activated carbon gave the highest sporophyte initiation rate (71.45% \u00b1 9.16%); the same formula also performed well in primary hormone-free cultures (70.54% \u00b1 9.07%). Correlation analysis showed a strong positive association between sporophyte initiation rate and mean young-sporophyte height (r = 0.935, p &lt; 0.001), while arrest and hypertrophy were negatively associated with sporophyte initiation. These results support evaluating in vitro propagation using quantified developmental outcomes\u2014arrest rate, hypertrophy rate, sporophyte initiation rate, and mean young-sporophyte height\u2014rather than biomass alone. Under the tested conditions, hormone-free 1\/2 MS + activated carbon was associated with the most favorable recovery outcomes; the underlying mechanisms require validation by quantitative histology, gene-expression analysis, endogenous-hormone profiling, and direct measurement of compounds adsorbed by activated carbon.<\/jats:p>","DOI":"10.3390\/plants15172570","type":"journal-article","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T09:20:02Z","timestamp":1787563202000},"page":"2570","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Histological Diagnosis and Recovery Regulation of Gametophyte Developmental Disorders During In Vitro Propagation of the Medicinal Fern Cibotium barometz"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-4292-4190","authenticated-orcid":false,"given":"Wumei","family":"Si","sequence":"first","affiliation":[{"name":"School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-4967-9418","authenticated-orcid":false,"given":"Yunfang","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2080-0710","authenticated-orcid":false,"given":"Heng","family":"Jiang","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyi","family":"Yuan","sequence":"additional","affiliation":[{"name":"School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kunhua","family":"Wei","sequence":"additional","affiliation":[{"name":"School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7816-8362","authenticated-orcid":false,"given":"Quan","family":"Yang","sequence":"additional","affiliation":[{"name":"School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China"},{"name":"Key Laboratory of Lingnan Medicinal Materials Production and Development, National Administration of Traditional Chinese Medicine, Guangzhou 510006, China"},{"name":"Guangzhou Comprehensive Experimental Station, China Agriculture Guangzhou Research System for Chinese Medicinal Materials, Guangzhou 510006, China"},{"name":"Guangdong Engineering Technology Center for Planting and Comprehensive Development of Lingnan Traditional Chinese Medicinal Materials, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gang","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"key":"ref_1","unstructured":"Royal Botanic Gardens, Kew (2026, May 15). 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