Szx16 stereomicroscope

Specimens were examined and photographed using a Nikon D810 digital camera, an Olympus szx16 stereomicroscope attached with a DP74 digital camera, and a Zeiss Axioscope A1 microscope attached with an Axiocam 506 digital camera. Diameters of the bead and halo were averaged between the maximum and the minimum dimensions. Both the distance between the centroids of adjacent beads (inter-bead distance)^{25 (link)} and the spacing (or gap) between adjacent beads^{8 (link)} were measured to overcome the uncertainty of beads with fuzzy and irregular edges^{25 (link)}.

For analyzing the activity of the MtNF-YB17 promoter, a 1.25-kbp fragment upstream of the start codon was amplified using primer pairs listed in Supplementary Table 1 and was cloned into pBGWFS-RedRoot which was kindly provided by Dr. Shu-Yi Yang (National Taiwan University, Taiwan).
Medicago roots were transformed with MtNF-YB17pro:GUS and composite roots were collected at 6 weeks after transplanting for staining. The roots were fixed in ice-cold 90% acetone for 30 min and washed three times with phosphate buffered saline (PBS). Then, the roots were incubated in GUS staining buffer (5 μM EDTA, 0.5 mM potassium ferricyanide. 0.5 mM potassium ferrocyanide and 0.5 mg ml^-1 5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt) at 37°C for 6 h and washed with PBS to stop the reaction. The stained roots were observed under an Olympus SZX16 stereomicroscope and a Zeiss Axio Imager M2 light microscope (Zeiss Microscopy, Germany).