RE-ESTABLISHMENT OF SYMBIOSIS TO Anabaena-FREE Azolla

Symbiosis to Anabaena-free Azolla has been re-established by placing indusium containing Anabaena onto the Anabaena-free megasporocarps. Anabaena-free megasporocarps were obtained by removing the indusium and the apical membrane of the megaspore apparatus of normal Azolla. The recovery of symbiosis in artificially reconstituted Azolla-Anabaena association was confirmed using the scanning electron microscope, monoclonal antibody test (McAb-C16), and nitrogen fixation capacity （ARA）.Laboratory-grown Anabaena azollae cells inoculated subsequently to the decapitated megaspore apparatus were observed to have entered the leaf cavities of sporophyte, although some portions of Anabaena filaments were found outside the leaf cavity. Plants invaded by artificially inoculated Anabaena did not have sufficient N2-fixing activity to allow the growth in the N-free medium.