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Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1–26.6% compared with that of 0.4–0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality. [ABSTRACT FROM AUTHOR]