Setreo discovery v20

Trichome density was quantified on the abaxial surface of the terminal leaflets of leaves 3–5 (counted from the apical meristem). Trichomes were visualized using a stereomicroscope (SetREO Discovery. V20, Zeiss, Germany). Images were recorded using Axio Vision 4.7 software (Carl Zeiss Ltd., Herts., United Kingdom). The trichome number was counted manually across a measured leaf area (automatically calculated by the software), and the average (mean) trichome density was then calculated for the whole leaf.

The morphology and distribution of the GTs were evaluated using a stereomicroscope (SetREO Discovery. V20, Zeiss, Jena, Germany) and SEM (SU3500, Hitachi, Tokyo, Japan). The tissues were fixed with a slide and observed using a stereomicroscope. For the SEM, leaves were fixed in glutaraldehyde (2.5% (v/v) in 0.1 M phosphate buffer (pH 7.3) for 12 h at 4 °C. They were then washed with a phosphate-buffered saline solution (Thermo Fisher, Shanghai, China) three times for 15 min each and dehydrated with an acetone dilution series (30, 50, 70, 90, and 100%). Acetone was replaced with tert-butanol overnight. The material was dried overnight in a freeze dryer (FreeZone 2.5 Liter Benchtop Freeze Dry System, Labconco, Kansas, USA). The specimens were then gold-coated (EM ACE200, Leica Microsystems, Leica Microsystems, Wetzlar, Germany). The coated specimens were then viewed and photographed using an SEM.

The morphology and distribution of trichomes on ten leaves collected from ten individual plants were evaluated using a stereomicroscope (SetREO Discovery, V20, Zeiss, Jena, Germany). For each leaf, ten observation fields were randomly selected on both the adaxial and abaxial surfaces, with a size of 1 × 1 mm, using a stereomicroscope. The dimensions of all types of GTs in each observation field were measured. The dimensions of GT head cells were measured using ZEN Microscopy Software (Zeiss, Jena, Germany).