Chapter 2 52 2.4.3.5 Vascular bundle microscopy The 14-day old tomato plants of both cultivars were treated with either WL or WL+FR for 7 days. After the 7 days, hand cross sections were made of the mid-region of the first internode and stored in 70% ethanol. The sections were stained using a 0.02% (w/v) Toluidine blue solution for 5 minutes (Pradhan Mitra and Loqué, 2014). After staining, the samples were rinsed with 70% ethanol and stored in 4°C for later analysis. The samples were mounted on a microscope slide and treated with a droplet of 3% (w/v) phloroglucinol solution (Pradhan Mitra et al., 2014). After application of the phloroglucinol, the sections were imaged with a light microscope. the pictures were acquired with Olympus BX50-WI microscope using 4X, 10X and 20X magnifications. 2.4.3.6 Microscopy analysis The sections to visualize vascular bundles were cut with a vibratome with the section thickness 150 mm or with razor blade by hand. And all samples were fixed by 4% PFA (pH= 6.9) for minimum 4 hours, and washed by 30%, 50%, 70%, 95% series of ethanol washes for 20 min per step, and put back to 70% ethanol. Then they were stained for 2 minutes with 0.1% (w/v) safranin (sigma) and washed with MQ until water was clear. All the sections were mounted with 50% glycerol on microscope slides, the cover glass was sealed with nail polish, each sample was stored in 4°C. 2.4.3.7 Epidermis imprinting After 7 days of WL or WL+FR treatment, the epidermis cell layers were peeled off from the whole internode, and using Present B.V. dental paste mix to fix them immediately. After the dental paste was fully dried, we used clear nail polish to imprint the structure. We imaged the imprints under Axioscope 2 Zeiss microscope. 2.4.4 Statistical analyses 2.4.4.1 Root measurements To measure primary root length, number, hypocotyl length and length of lateral roots, ImageJ Smartroot plugin (Lobet et al., 2011) was used to analyse the pictures. The data were analysed by Graphpad prism 8, the significant differences between WL and FR+WL were calculated by student’s t-test.
RkJQdWJsaXNoZXIy MTk4NDMw