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To increase oil accumulation in the high-biomass vegetative organs of tobacco ( Nicotiana tabacum L.) plants for renewable production of biofuels, VgDGAT1a isolated from developing seeds of Vernonia galamensis L. was ectopically overexpressed in tobacco leaves using a constitutive promoter. The transgenic tobacco leaves showed a 3.5–5.0-fold increase in oil content compared to the control, with a maximum increase of 9.2% (DW). The transgenic leaves also showed a substantial change in fatty acid composition, with significant enhancement of linoleic acid (18 : 2) and notable reduction of -linolenic acid (18 : 3). The overexpression of VgDGAT1a exhibited no deleterious effect on other phenotypes in the tobacco plant. These results will facilitate development of a novel system for lipid metabolic engineering in vegetative organs of plants, as well as provide a platform for the production of biofuels using the vegetative organs of commercial non-food crops.