Leaf Disk Assay

The leaf disk assay described by Sanan-Mishra et al. (2005) was conducted with minor modification. Four independent lines of each type of transgenic plant were used. To generate sufficient leaf disks for all treatments, three clonal plants of each independent transgenic line (T0) and WT (regenerated through tissue culture) (8 weeks old) were used. The third mature leaf (healthy and fully expanded) was collected from each plant. Leaf disks of 1.8-cm diameter were excised from the central portion of the lamina either side of the midrib. For each treatment, one leaf disk from four independent lines of each type of plant was used. The disks were floated on 5 mL of NaCl solution at 100 mM or 200 mM, or on distilled water (experimental control) for 48 h at 22°C under white light (150 or 450 μmol m^–2 s^–1) provided by a cool white LED panel with a 12 h photoperiod. Wild type N. tabacum leaf disk treated with 100 mM or 200 mM NaCl for 3 days in a leaf disk senescence assay showed mild and severe senescence, respectively, (Sanan-Mishra et al., 2005), so this concentration was used in salt stress tests. Pigment content was measured on each leaf disk after the treatment.

To simulate the light filter effect of betacyanins, another set of WT and EV leaf disks floated on the same concentration of NaCl solution was covered by a red polycarbonate filter (Rosco Supergel #346 Tropical Magenta, KEL-LPS, Auckland, New Zealand) with a similar absorption spectrum to betacyanins (530–550 nm) (Azeredo, 2009). The maximum quantum efficiency of photosystem II (F_v/F_m) was determined on each leaf disk after treatment using a Walz 2500 (Effeltrich, Germany) pulse amplitude modulated fluorometer (PAM) according to the manufacturer’s operating instructions².