Use of DNA/DNA hybridization techniques to authenticate the production of newAzolla-Anabaenasymbiotic associations

Abstract

The establishment of Azolla‐Anabaena‘recombination’ symbioses by grafting the Anabaena‐containing indusium (cap) from the donor megasporocarp onto a decapitated megasporocarp from an Anabaena‐free recipient has been recently reported. We have generated strain‐ and species‐specific DNA probes for unequivocally establishing the identity of Anabaena azollae strains isolated from Azolla‐Anabaena symbioses (whether new, i.e. heterosymbioses or natural i.e. homosymbioses). Eight out of nine heterosymbioses tested proved to contain the expected heterosymbiont while one recombined association was shown to contain both its own homosymbiont and a heterosymbiotic Anabaena strain. In addition, a species‐specific plant probe was isolated from the total DNA of Azolla microphylla roots and used to establish the identity of a host plant used for recombination experiments.

Domestication of the Floating Fern Symbiosis Azolla

“…A key stage for "swapping" N. azollae is the germinating sporeling of a "decapitated" megasporocarp, from which the indusium cap has been removed and, then, to which a new indusium cap from another species has been added. The germinating N. azollae resting stages, the akinetes, under the new indusium cap will apparently invade the developing sporeling shoot apex to generate hybrid fern cyanobacterial symbioses (Lin and Watanabe 1988;Plazinski et al 1989). The hybrids are viable suggesting that the N. azollae functions and mechanisms of recognition/immunity are conserved between Azolla species.…”

Isolation of Agrobacterium sp., strain from the Azolla leaf cavity

“…A strain of Azolla filiculoides Lam. was collected in Slack's Creek in the Snowy Mountains Region of New South Wales, Australia, and cultured under laboratory conditions as previously described [11]. Two cultures of Anabaena-free ferns, Azolla filiculoides 136 and A. pinnata 97 were kindly provided by the National Azolla Research Centre, Fuzhou, China.…”

Secondary metabolites that shape the permanent symbioses between Azolla ferns and nitrogen-fixing cyanobacteria

“…Genetic tools to leverage the information garnered from the (meta)genomes of Azolla, however, are entirely missing, and they pose a significant challenge to develop. Although some filamentous cyanobacteria from the genus Nostoc are amenable to transformation (Koksharova & Wolk, 2002), N. azollae is an obligate endosymbiont, and once lost, the symbiosis could never be reestablished (Plazinski et al, 1989). The fern symbioses can therefore not be sterilized for growth on sugar-rich media typically used in tissue culture.…”