Trifluralin

The application of Trifluralin was tested on the variety Tammarin Rock. This variety was screened in an earlier study, in which it exhibited good green plant regeneration but a relatively low frequency of chromosome doubling (30%) [11 (link)]. Eight donor plants were sown over two sowing dates (Table 5). Spikes 1–10 were harvested from the first sowing date, and spikes 11–20 were harvested from the second sowing date. Spikes were harvested over 6 days and stored at 4 °C for up to 2 days. The experimental design and protocol was the same as Experiment 1 with anthers from spikes 1–10 exposed to three treatments (control, 1 µM Trifluralin for 24 h and 1 µM Trifluralin for 48 h) and anthers from spikes 11–20 exposed to different treatments (control, 3 µM Trifluralin for 24 h and 3 µM Trifluralin for 48 h) (Table 7). As per Experiment 1, anthers were exposed to Trifluralin immediately following mannitol and n-butanol pretreatment at the start of the induction phase. Trifluralin (N13689, Sigma-Aldrich) was dissolved in sterile dimethyl sulfoxide (DMSO) (D2650, Sigma-Aldrich), and 10 or 30 µL of Trifluralin stock was added to 4 mL LIM to provide final concentrations of 1 or 3 µM, respectively.

High-performance liquid chromatography (HPLC)-grade methanol, acetonitrile and pH buffers (4.0, 7.0 and 10.0) were purchased from VWR (Atlanta, GA, USA). Analytical standards for lindane, diazinon, linuron, trifluralin, paraquat and ammonium acetate were purchased form Sigma Aldrich (St. Louis, MO, USA). Sulfuric acid (H₂SO₄, 95–98%), PCP and 2,4,6-TCP were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Ethylene glycol and formic acid (HCOOH, 88%) were purchased from Thermo Fisher (Waltham, MA, USA). CM was obtained from BASF (Lampertheim, Germany) with a total surface area of approximately 850 m²/g, an external surface area of 70 m²/g and cation exchange capacity equal to 97 cmol/kg [28 (link)]. SM clay was obtained from the Source Clay Mineral Repository at the University of Missouri-Columbia with an estimated cation exchange capacity equal to 75 cmol/kg. The generic formula for these clays is: (Na,Ca)_0.3(Al,Mg)₂Si₄O₁₀(OH)_2⋅nH₂O. Clays were sieved through 45 μm to achieve uniform particle size [28 (link),29 (link)]. Ultrapure deionized water (18.2 MΩ) was generated in the lab using an Elga™ automated filtration system (Woodridge, IL, USA) and used in all experiments.