The primary sources of nitrogen for plants have been suggested to be nitrate (NO3-) and ammonium (NH4+). However, when both nitrate and ammonium are simultaneously available to plants, it is very difficult to differentially quantify NO3-/NH4+ utilization in culture media or soil. Consequently, the contribution of NO3-/NH4+ to total inorganic nitrogen assimilation cannot be determined.ResultsWe developed a method called the bidirectional stable nitrogen isotope tracer to differentially quantify the nitrate and ammonium utilization by Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets in vitro. The utilization efficiency of nitrate was markedly lower than the utilization efficiency of ammonium for plantlets of both Ov and Bn. In both Ov and Bn, the proportion of NO3-/NH4+ utilization did not show a linear relationship with inorganic nitrogen supply. The Ov plantlets assimilated more nitrate than the Bn plantlets at the lowest inorganic nitrogen concentration.ConclusionsQuantifying the utilization of nitrate and ammonium can reveal the differences in nitrate and ammonium assimilation among plants at different inorganic nitrogen supply levels and provide an alternate way to conveniently optimize the supply of inorganic nitrogen in culture media.