with double-layer brown bags to prevent light from getting into the root
systems, were placed in a Conviron chamber for plant growth under
conditions described by Brown et al. (2011).
After two days in the growth chamber, roots were inoculated with
R. leguminosarum bv. viciae 3841 (at 2 × 106 CFU per plant) to induce
root nodulation. The flasks were shaken twice daily to minimize nanoparticle
aggregation and resuspend settled nZnO particles. Nodules
were collected at 7 and 14 days post-inoculation (dpi) and photographed
with a Nikon D700 camera. Nodule sizes were measured
using the ImageJ® software and the ultra-structure of nodules was observed
using light microscopy and TEM.