Trichome Density Quantification in Tef Leaves

Tef plants from the three accessions were grown for 1 month (no aphids were applied on these leaves). Then, 2 cm sections were sampled from the widest part of three leaves: (i) lower leaf (a first leaf from the base), (ii) middle leaf, and (iii) upper leaf. The three leaves were dissected, bleached in 80% (v/v) ethanol, boiled at 90°C for 20 min, and washed with distilled water as previously described (Batyrshina et al., 2020b (link)). For trichome visualization, leaves were mounted on microscope slides with the adaxial side facing up, covered with glass coverslips. A digital camera connected to an Axioplan 2 Upright Light Microscope (Zeiss, Oberkochen, Germany) was used for imaging. For each tef accession, five biological replicates with two pictures per leaf were taken. For density quantification, trichomes were counted using ImageJ software¹ and normalized per mm².