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The present study was intended to produce a protocol for the conservation of two endangered ferns of southern Western Ghats of India using in vitro spore culture. In addition this study reports spore germination, gametophyte development, changes in the reproductive phases and sporophytes formation of the medicinally important ferns Pronephrium triphyllum (Sw.) Holttum and Sphaerostephanos unitus (L.) (Holttum). Matured spores of the two selected ferns were harvested, filtered through 40μM nylon membrane and sterilized with 0.1% mercuric chloride for 3 to 5 min and rinsed with sterile distilled water for 15 min showed less frequency of mortality and a high percentage of spore germinations. For Pronephrium triphyllum, the spores sown on the Knop’s basal agar medium showed the highest percentage (38.3 plus or minus 1.13) of germination. Highest percentage (52.3 plus or minus 1.43) of sporophyte formation was observed in Knop’s liquid medium. For Sphaerostephanos unitus, the highest percentage (36.8 plus or minus 1.31) of spore germination was observed in the Knop’s basal agar. The highest percentage of sporophyte formation was observed only in Knop’s medium (76.8 plus or minus 1.41), other media failed to induce sporophyte formation. The in vitro raised plantlets were hardened and established in the natural habitat and distributed to various botanic gardens as a part of ex situ conservation. Cytological and isoperoxidase analysis confirmed the genetic uniformity between mother plants and in vitro raised sporophytes / plants. The established protocol of the present study will be useful for the multiplication and conservation of the two threatened ferns of the Western Ghats. The same protocol may also be applicable to similar threatened ferns.

Ex situ conservation of two threatened ferns of the Western Ghats through in vitro spore culture