Overexpression of TPS2 gene to increase thymol content using Agrobacterium tumefaciens-mediated transformation in Trachyspermum ammi (Qom ecotype)

Trachyspermum ammi is a well-known pharmaceutical plant in Iran and has many medicinal properties including anti-digestive, anti-spasm, antimicrobial, antiviral, and relieving rheumatic pains. This plant is important for its essential oil and its main constituent; i.e., thymol. Several properties of this plant are related to this valuable constituent. This research was conducted to increase thymol content of this plant by genetic transformation using Agrobacterium tumefaciens. To enhance thymol production, we used TPS2 gene (Terpene Synthase 2), which is a key enzyme involved in thymol synthesis pathway. In this research, an effective genetic transformation system was developed for Trachyspermum ammi with Agrobacterium tumefaciens. To overexpress TPS2 gene in Trachyspermum ammi, a gene construct called p8I121-TP harboring the neomycin phosphotransferase (nptII) gene as a plant-selected marker was designed. This work was performed using three strains of Agrobacterium tumefaciens (LBA4404, GV3101, and EHA101), hypocotyl explants, and factors including inoculation time (5, 10, 15, and 20 min) and co-cultivation time (1-3 days). Polymerase chain reaction (PCR) and Southern blot analysis were performed to confirm the integration of TPS2 and nptII transgenes into the plant genome. Based on these findings, the EHA101 strain in 5-min inoculation and 1-day co- cultivation showed a higher efficiency compared to two other strains. Transformation efficiency varied between 6.12 and 18.36% based on the Agrobacterium strains for transformation. The qPCR results demonstrated that the TPS2 gene was more expressed in the transgenic plants than non-transgenic plants. HPLC analysis showed that the aerial part of transgenic plants has the greater thymol compared to the non-transgenic plants. To the best of our knowledge, this study demonstrates, for the first time, a suitable and efficient method for overexpression TPS2 gene to enhance thymol by Agrobacterium-mediated transformation method in Trachyspermum ammi.