Evaluation location: Minnesota, United States
Cultivars were acquired from commercial nurseries. Trees were planted during the summer of 2014 in the nursery fields at the University of Minnesota, St. Paul, MN, and were watered as needed. In 2015, trees were 3 years old and 1–3 cm diameter at 0.5 m above the ground. A portion of stem was collected approximately 0.60 m above the ground. Samples were then placed in a −20°C freezer until sectioned. Four pieces (approximately 2 mm wide) spaced 90° apart with a random starting point were cut from the stem segment using a high-profile microtome blade. Pieces were soaked in 100% TFM™ tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA) for approximately 16 hr. An IEC Minotome® cryostat (International Equipment Co.) at −20°C was used to cut 15- to 30-μm-thick transverse sections. After sectioning, the sections were cleared with water and subsequently stained with 0.1% safranin O (dye content ≥85%) (Sigma-Aldrich®) (w/v) solution for 20 s. After removing excess safranin O with a paper towel, sections were mounted in water by placing a drop of DI water onto the surface of the sample and then subsequently covering the sample with a coverslip. Sections were allowed to air-dry at room conditions for 24 hr. Images were taken at 40× using a Nikon Eclipse E600 microscope (Nikon Instruments Inc.) with a Nikon Digital Camera DXM 1200F (Nikon Instruments Inc.). Many samples were larger than the field of view for the low magnification of the microscope objective and field of view for the digital documenting system, so multiple images of the same section were stitched together in Photoshop™ (Adobe Systems Inc.) using the photomerge feature. Focus stacking was performed as needed in Photoshop™. A 500-μm-wide area of the xylem following the same ray parenchymal cells of the annual ring of interest was analysed. This area included earlywood and latewood vessels. Vessel elements were manually traced or selected using the magic wand in Photoshop™. Images were subsequently analysed using the thresholding feature in ImageJ (Schneider et al., 2012) to generate a mask of vessel elements with a D (equivalent circle diameter) ≥15 μm. The masked images were then analysed using ROXAS 3.0 (von Arx et al., 2013). Jansen et al. (2009) found that the total intervessel cell wall thickness (mean ± SD) in Ulmus americana was 2.946 ± 0.665 μm; therefore, the double cell wall thickness was set to 4 μm to ensure most connected vessels would be included when analysed for vessel aggregation. Mean number of vessels per group were counted, where solitary vessels are also considered a group (Carlquist, 2001)