In vitro culture of arbuscular mycorrhizal fungus and Frankia for inoculation of micropropagated Casuarina equisetifolia L.

Micropropagated, rooted, and calli explants of Casuarina equisetifolia L. w ere inoculated with Frankia UGL 020605S and the arbuscular mycorrhizal fung us (AMF) Glomus mosseae, in single and dual co-culture, in vitro. Different micropropagation media formulations were evaluated for their capacity to s timulate germination of G. masseae spores and growth of Frankia. Murashige and Skoog basal nutrient (half strength) medium, supplemented with 6-benzyl aminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), and pyruvate was selected for the in vitro co-culture of C. equisetifolia callus explants, G. mosseae, and Frankia. This medium (M4) supported 70% AMF spore germinati on with 44 and 34% of the germinating spores producing single and branched hyphal strands, respectively. Hoaglands (quarter strength, modified by Hoag lands and Arnon (1950)) nutrient medium (M5) with no supplements was select ed for the in vitro co-culture of rooted C. equisetifolia explants, G. moss eae, and Frankia and supported 57% AMF spore germination with 29 and 40% of the germinating spores producing single and branched hyphal strands, respe ctively. Both media supported significant growth of Frankia. In both cases agar was substituted with Terragreen(R) AMF appressoria and intercellular h yphae were observed in rooted C. equisetifolia at 28 days; arbuscule format ion occurred at 56 days postinoculation. Frankia infection was evident afte r 28 days. This was observed in both dual and single in vitro co-cultures. No specific immunofluorescent or immunogold reactions to monoclonal antibod ies (mABs) anti-Frankia < 8C5 > and anti-G. mosseae < F5G5 > were evident i n C. equisetifolia callus explants.