Engineering Plants to Produce Petrochemical Alternatives in Vegetative Tissues

Background: In a world of dwindling non renewable energy resources, it is vital we find environmentally friendly alternatives. By using genetically altered plants that can accumulate oils throughout the entire biomass (leaves, stems, roots), we can greatly increase the production of certain oils that are considered high value. We hypothesized that, under the control of a constitutive promoter, transformation of Arabidopsis thaliana with HA-FADX, a gene that codes for conjugated α-Eleostearic fatty acid (from exotic Tung tree), will result in progeny that accumulate α-Eleostearic acid in vegetative tissue. Methods: We sequenced the plasmid of our gene of choice to ensure proper alignment and used segregation analysis to ensure single insertion homologous lines . We then tested for expression of our gene within the plant. By using PCR of mRNA and Thin Layer Chromatography, we were able to confirm transcription of HA-FADX within several of our lines. We then observed and selected lines based on morphology to be homogenized and trans-methylated for analysis. The seed oil of lines was run through a Gas Chromatograph (Flame Ionization Detector) [GC(FID)] before homogenizing, trans-methylating, and running the associated vegetative tissue samples through GC(FID). We then confirmed the presence of α-Eleostearic acid in vegetative tissue by  cross referencing our same samples through GC Mass Spec to confirm that the molecular composition matches that of our positive control. Results: Plants with genetic alteration were observed to be stunted in growth compared with wild type plants, with notable yellowing of leaves and wrinkling in the seeds of certain lines. Vegetative tissue analysis showed detectable amounts of α-Eleostearic acid; however, the seed analysis had no positive results. After analyzing the same samples through GC-MS, we verified the molecular composition of our sample as α-Eleostearic acid. Finally, lines with high amounts of α-Eleostearic acid showed high expression of our target gene’s mRNA. In our analysis, we found that 12 of 17 transformed lines produced detectable amounts of α-Eleostearic acid. Conclusion: By engineering plants that produce novel fatty acids throughout the entire biomass of the crop, we can address issues related to the use of non-renewable energy sources. The α-Eleostearic acid that some of our transformed plant lines produced can be used in formulations of jet fuel, lubricants, inks, dyes, coatings, resins, and plastics, and as such, represents an important replacement for the petrochemical industry.