E β farnesene

For identification of volatiles the following analytical standards were purchased from Sigma-Aldrich (Milan, Italy): α-pinene, β-pinene, p-cymene, limonene, γ-terpinene, linalool, trans-pinocarveol, terpinen-4-ol, α-terpineol, myrtenol, nerol, carvone, geraniol, geranial, eugenol, (E)-caryophyllene, α-humulene, (E)-β-farnesene, (E)-β-ionone, (E)-nerolidol, caryophyllene oxide, n-pentacosane, n-heptacosane; (E)-Phytol was previously isolated from Onosma echioides [43 (link)]. For retention-index (RI) determination, a mixture of hydrocarbons, ranging from octane (C₈) to triacontane (C₃₀) (Supelco, Bellefonte, PA, USA) was used and run under the experimental conditions reported below. All compounds were of analytical standard grade. Analytical-grade hexane solvent was purchased from Carlo Erba (Milan, Italy); it was successively distilled through a Vigreux column before use.

The plasmids listed were transformed via electroporation into the Agrobacterium tumefaciens GV3101::pMP90 strain, as described by reference [76 ], and plated onto selective media. Agrobacterium-mediated transient expression in Nicotiana benthamiana was performed, as described by reference [20 (link)], with three leaves from a single plant pooled after four days for the subsequent analysis. A mock infiltration using MMA buffer and non-infiltrated wild-type leaves served as controls. Headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) was performed as described by reference [77 (link)], with the following modifications: adsorption of analytes was done at 30 °C for 20 min and an initial oven temperature of 100 °C. Sesquiterpenes were identified using the authentic standards (E)-β-farnesene and (E)-β-caryophyllene from Sigma-Aldrich, USA and/or the Wiley 275 mass spectral library. For extracted ion chromatograms (EIC), the cumulative mass (m/z) response for 161, 189, and 204 was used.

2-ethyl-3,5(6)-dimethyl pyrazine (EDP, a mixture of the 3,5- and 3,6-dimethyl isomers, herein referred to as pheromone isomers), E-β-Farnesene (EBF), 2,4,6-trimethylpyridine (TMP), and ylang ylang oil were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). The purities of these four chemicals were >95%.