Continuous, Automated Acetylene Reduction Assays Using Intact Plants

An automated method was developed for continuous, in situ determination of acetylene reduction (N(2) fixation) by intact soybean plants (Glycine max [L.]). The culture vessel containing the roots of intact plants grown in sand culture is sealed at the surface and an air-acetylene mixture continuously injected into the root chamber. The effluent gas is automatically sampled and injected into a gas chromatograph. Continuous acetylene assay at intervals as short as 3.5 min may be made over a period of several days, without attention, except for plant watering. Adverse effects of prolonged exposure of the root system to acetylene were mitigated by pulse injection of acetylene for 20 min followed by 40 min of acetylene-free air. Bare root systems can be suspended in a reaction chamber and sprayed with water or nutrient solution; this permits periodic removal of the root system for sampling nodules.In studies lasting several diurnal cycles, acetylene reduction did not decline more than 50% of the maximum rate in light, thus nitrogenase activity depends on concomitant photosynthesis and on carbohydrate from storage pools.