Index >> Actinorhizal Plants (Frankia Induced Nodulation) >> Isolation and Characteristics of Frankia

Older actinorhizal nodules are generally big and are subjected to con­taminants which interfere with the isolation of pure cultures of Frankia. This was the reason why earlier workers had missed real Frankia and ended up by isolating contaminants. Young nodules that are not suberized are preferred to older ones. The contaminants are excluded by surface sterilization with osmium tetroxide under mild vaccum in a fume hood to avoid injury by the toxic nature of the surface sterilant. The nodules are then washed repeatedly and disrupted or dissected to expose nodular cells to release the sparse number of Frankia cells. The process of macera­tion or disruption of nodules may often release toxic phenolic compounds that may suppress Frankia growth. Hence some workers have passed the homogenates through activated charcoal to get rid of the polyphenolic compounds. To concentrate Frankia cells, the homogenates are passed through nylon membranes or subjected to sucrose-density centrifugation.

The vials are incubated at 25-28°C upto 2 months discarding those vials that develop growth of fast growing contaminants. When Frankia growth becomes dearly noticeable in some nodule lobes, such colonies are subcultured repeatedly and tested for nodulation on aseptically grown seedlings of Casuarina.

Diem and Dommergues from France provide the following protocol for the isolation of Frankia endophytes from root nodules: Clear the nodule of extraneous organic matter, soil and dirt under running water by fre­quent examination under a dissecting microscope. Fragment the nodule into individual lobes, sterilize the lobes by immersion into a 3.0 per cent aqueous solution of osmium tetroxide for 1-4 min. according to the nodule mass and age, wash in sterile distilled water several times and cut the nodule lobes into 0.1-0.5 mm³ pieces with the help of a sterile scalpel.