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Soybean is a valuable protein and oilseed crop ranked among the most significant of the major crops. Field experiments were carried out in 2016–2019 in South-East Poland. The influence of soybean cultivars (Aldana, Annushka), nitrogen fertilizer (0, 30, 60 kg∙ha−1 N) and inoculation with B. japonicum (control, HiStick® Soy, Nitragina) on the content of fatty acids (FA) in soybean seeds was investigated in a three-factorial experiment. This study confirms the genetic determinants of fatty acid composition in soybean seeds and their differential accumulation levels for C16:0, C16:1, C18:1n9, C18:2, C18:3, and C20:0 as well saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids. Increasing the rate from 30 to 60 kg ha−1 N did not produce the expected changes, suggesting the use of only a “starter” rate of 30 kg ha−1 N. Inoculation of soybean seeds with a strain of Bradyrhizobium japonicum (HiStick® Soy, BASF, Littlehampton, UK and Nitragina, Institute of Soil Science and Plant Cultivation–State Research Institute, Puławy, Poland) is recommended as it will cause a decrease in SFA and C16:0 acid levels. This is considered nutritionally beneficial as its contribution to total fatty acids determines the hypercholesterolemic index, and it is the third most accumulated fatty acid in soybean seeds. The interaction of cultivars and inoculation formulation on fatty acid content of soybean seeds was demonstrated. An increase in the value of C16:0 content resulted in a decrease in the accumulation of C18:1, C18:2, and C18:3 acids. The content of each decreased by almost one unit for every 1% increase in C16:0 content. The dominant effect of weather conditions on the FA profile and C18:2n6/C18:3n3 ratio was demonstrated. This suggests a need for further evaluation of the genetic progress of soybean cultivars with respect to fatty acid composition and content under varying habitat conditions.

Effect of Nitrogen Fertilisation and Inoculation with Bradyrhizobium japonicum on the Fatty Acid Profile of Soybean (Glycine max (L.) Merrill) Seeds