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Bryophytes, particularly mosses of the family Bryaceae, represent an evolutionarily significant group with considerable medicinal potential. Despite their ecological, evolutionary and pharmacological importance, in vitro studies on bryophytes remain limited when compared to those on vascular plants. To address this gap, the present review synthesizes existing protocols and methodologies for the in vitro propagation of Bryaceae, with a focus on species prevalent in India. It highlights key factors influencing successful culture, including sterilization techniques, temperature, photoperiod, culture vessels, media composition, sugar types and hormonal supplements. Sodium hypochlorite has emerged as the most effective sterilizing agent, while optimal growth temperature ranges from 22–27 °C. Standard photoperiods of 16/8 and 12/12 hr light/dark have been widely adopted. Petri plates are the preferred culture vessels due to ease of handling and observations. Among growth media, Knop and Murashige and Skoog media, sometimes in modified formulations, have shown the highest efficacy for protonema and gametophyte development across species like Bryum argenteum, Rhodobryum giganteum and Anomobryum filiforme. Hormonal responses are highly concentration-dependent and exhibit interspecific variability. Along with plant growth regulators, auxin and cytokinin are the two principal hormones that play a critical role in regulating bud initiation and gametophyte development. Additionally, sugars such as glucose and sucrose promote growth in certain Bryaceae species but may inhibit others. This review highlights critical research gaps in the understanding of light intensity and spectral quality (i.e., specific wavelength suitable for growth and development), temperature tolerance and nutrient formulations, underscoring the need for species-specific in vitro protocols. The findings will lay a foundation for advancing Bryaceae propagation for conservation and bio-industrial applications.