Long‐term stability of RNAi‐mediated gene silencing in high‐oleic soybean lines

Abstract

In Brazil, renewable biofuel is mainly produced from soybean [ Glycine max (L.) Merr.] oil. Typically, soybean‐derived oil contains approximately 25% of the monounsaturated oleic acid, 13% of the saturated fatty acid palmitic, and 55% of the polyunsaturated fatty acid (PUFA) linoleic acid. The high PUFA profile negatively affects biofuel performance, reducing oxidative stability, whereas saturated fatty acid content reduces viscosity and cold flow performance. Increasing the content of oleic acid at the expense of PUFA concentration is a desirable trait for biofuel quality. In an earlier study conducted by our group, using RNA interference (RNAi) technology, a cellular mechanism responsible for post‐transcriptional gene silencing, generated a high‐oleic, low‐palmitic acid soybean line, designated event 65‐25. Now, in this report, we show the stability of the RNAi line 65‐25 for 15 generations (10 years). Small interfering RNA (siRNA) originating from the RNAi construction was detected in the generation of the original 65‐25 event. Additionally, analyses of the expression of fatty acid biosynthesis genes, particularly the genes encoding for delta‐12 desaturase (FAD2), delta‐15 desaturase (FAD3), and the fatty acyl‐acyl carrier protein thioesterase B (FATB), were compared to the wild‐type (WT) soybean line, Conquista from Embrapa, with the high‐oleic lines. In the analyzed lines derived from 65‐25, FAD2 , FAD3 , and FATB genes were downregulated compared to WT plants. Oil profile analysis showed that the RNAi lines retained a more than twofold increase in oleic acid seed content relative to WT line. Our data confirm that the RNAi transgene remained stable for over 15 generations in soybean seeds. These RNAi lines consistently downregulate key genes in the fatty acid biosynthesis pathway— FAD2‐1 , FAD2‐2 , FAD3 , and FATB1 —leading to enhanced oleic acid content in mature seeds.