Absolute ethanol

Reagents of analytical grade were used as received from their commercial sources. Benzyl chloride was obtained from ACROS Organics (Morris Plains, NJ, USA), potassium hydroxide from Riendemann Schmidt (Kajang, Malaysia), carbon disulfide from Fisher Scientific (Chino, CA, USA) and copper (II) acetate from R&M Chemicals (Essex, UK). Hydrazine hydrate, iodomethane, glyoxal, 2,3-hexanedione, 2,3-heptanedione, 2,3-pentanedione, 2,3-butanedione and copper (II) chloride were obtained from Merck (Darmstadt, Germany). Absolute ethanol, 95% ethanol, dimethyl sulfoxide, acetone and dimethylformamide were obtained from RCI Labscan (Bangkok, Thailand). Elemental analysis was conducted via a Vario MACRO CUBE CHNS analyser (Germany), while IR spectra were recorded using a PerkinElmer FTIR spectrophotometer (Waltham, MA, USA). Melting points were measured using an Electrothermal IA9100 melting point apparatus (Essex, UK).
¹H NMR spectra were obtained via a Bruker FT-NMR 300 MHz (Germany). TLC was performed via 60F aluminium silica gel aluminium plates with a 254 nm fluorescent indicator from Merck (Darmstadt, Germany). UV-Vis spectroscopy was performed via a Biochrom UltroSpec 8000 spectrophotometer (Cambridge, UK).

Single colonies of S. passalidarum CMUWF1−2 and the adapted strain were inoculated in 5 ml of YPD broth as seed cultures. The seed cultures were then inoculated and cultured in YPD and YPD supplemented with 2.0 g/l FF at 30 °C with shaking at 150 rpm. During cultivation, the samples were collected at 0, 24, and 36 h and then adjusted to be obtained OD₆₀₀ = 0.2 using deionized water (DIW). The OD₆₀₀-adjusted cells were collected by centrifugation and washed one time by DIW before resuspending in 10 µl of DIW and 190 µl of absolute ethanol (RCI Labscan Ltd., Thailand). These cells were then stained with 1 µl of 2 mg/ml diaminophenylindole (DAPI) (Sigma-Aldrich, USA), gently mixed, and incubated in the dark at room temperature for 10 min. The collected cells were washed with 200 µl of DIW three times and then resuspended in 20 µl of DIW. This suspension was dropped on an adhesion slide (Scientific Co., Ltd, USA) and observed under a fluorescence microscope with an ultraviolet filter (Nikon, Japan) [19 (link)]. The percentage of cells with diffuse chromatins was calculated as follows.

Absolute ethanol, methanol (HPLC grade), glacial acetic acid, and sodium acetate were purchased from RCI Labscan Limited (Bangkok, Thailand). Polysorbate 80 (P-80), polyethylene glycol-40 hydrogenated castor oil (PEG-40H), and polyethylene glycol-400 (PEG-400) were purchased from Sigma-Aldrich (Darmstadt, Germany). Trimethylamine and phosphoric acid were purchased from Loba Chemie Pvt, Ltd. (Mumbai, India). Analytical standard nicotine (NCT), with a purity of 98.9%, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxypropyl methylcellulose E15; HPMC E15 (AnyCoat^®-C AN15, substitution type 2910, viscosity 15 mPa·s) was purchased from Lotte Fine Chemical Co., Ltd. (Seoul, Korea).

CP, triphenylphosphine, silver nitrate, monochloroacetic acid, PVP, and polysorbate 80 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dichloromethane, absolute ethanol, methanol, glacial acetic acid, and WSP were from RCI Lab-scan Co., Ltd. (Bangkok, Thailand). CP-CG was from Ultradent Product Inc. (Salt Lake City, UT, USA). Rice seeds of Thai rice, variety Saohai, were obtained from the local market (Chiang Mai, Thailand). All other chemicals and solvents were of AR grade or the highest grade available.