Photomicroscope 2

Mitotic chromosomes were investigated using root tips taken from living plants cultivated in the greenhouse of SCBG (Table S4). Actively growing root tips were pretreated with 0.002 mol/L 8-hydroxyquinoline solution while shielded from light for about 1.5 h at 4°C, and then washed with distilled water, fixed in freshly prepared cold Carnoy’s Fluid (ethanol: acetic acid solution = 3:1) in a mixture of water and ice for 2–3 h at 4°C, washed with distilled water three times, and later hydrolyzed in 1 mol/L hydrochloric acid at 60°C for 7–8 min. After three more rinses in distilled water, root tips were stained in 1% aceto-orcein solution for more than 15 min, and then squashed on the slide for light microscopy. Chromosome counts were made using cells at metaphase division and observed under Zeiss Axioscope. Photographs were taken with a Zeiss Photomicroscope II. The chromosomes of at least ten metaphase cells were counted and the measurements of three cells were made.

Samples were fixed in 2.5% glutaraldehyde in 0.03 M phosphate buffer for 24 hours, then dehydrated in an ethanol series (15, 30, 50, 70, and 95%) for 90 minutes each. The method of sample preparation was developed and validated by Deng et al. [23 (link)]. The basic structure was determined using the paraffin section method and starch grains were dyed by I-KI staining, and observed by using a Zeiss Photomicroscope II (Carl Zeiss Jena, Germany). Phloem plugging was detected using toluidine blue and epifluorescence photomicrographs that were captured using an Olympus BX51 epifluorescence microscope (Olympus Inc., Tokyo, Japan).