
Anemia rotundifolia, a rare fern species was studied to determine that can explants of gametophyte be used to multiply the plants (gametophytes and sporophytes) and also to understand the reproductive barriers. Explants were sown on Parker’s & Thompson’s culture media. Study demonstrated that explants produced secondary regenerates which matured into cordate gametophytes and bear only archegonia. These regenerates gave rise numerous tertiary gametophytes that bear female and male gametes intermixed showing monoecious sexuality. Secondary regenerates had option for intergametophytic selfing, as it bears only archegonia. Tertiary regenerate exhibiting female and male gametes performed both inter and intragametophytic selfing, as a result each tertiary regenerates produced multiple sporophytes. Secondary gametophytes did not produce any antheridia and sporophyte, indicating that establishment of sporophytes on tertiary regenerates has reciprocal impact on secondary gametophytes development. Study concluded that apical explants of gametophyte in A. rotundifolia may be used for mass multiplication of gametophytes and sporophytes, and also to ensure its in vitro conservation. Expression of archegonia on secondary regenerates and presence of both the gametes on tertiary regenerates confirmed that the moderate heterozygosity and homozygosity could be cause of reproductive barriers and success in consecutive regenerates.