At the end of the elongation and rooting period
the remaining medium in each vessel was collected to
determine pH, EC, and the content of NO3
- and NH4
+.
For pH and EC measurements, spent medium has been
centrifuged before analysis. Nitrate and ammonium
concentrations were analyzed in the liquid fraction of
substrate which was obtained by the medium centrifugation
for 20 min at 15000 × g and filtration through
Whatman filter. Nitrate and ammonium concentrations
in the liquid fraction of the medium were analyzed by
spectrophotometry (Helios Beta, Spectrophotometer,
Thermo Electron Corporation, England). Nitrate was
assayed using the salicylic acid-sulfuric acid method
(Cataldo et al. 1975). Ammonium was determined by
the phenol-hypochlorite reaction (Weatherburn 1967).
Nitrate and ammonium uptake by plantlets was calculated
by measuring the difference between nutrient
residual after 20 and 40 days of culture and nutrient
supplied by MS medium and the uptake was expressed
on the base of plant dry weight (Adelberg et al. 2010).