Carbon isotope discrimination (CID) measurements

For CID measurements, leaf samples from transgenic chickpea lines and control were collected at stress level 1 and 4. Harvested leaves were dried to 80 °C in a hot air oven for 3 d and finely powdered in a mill. Carbon isotope composition was determined on 1.0 mg sample with Isotopic Ratio Mass Spectrometer (IRMS, Model Thermo Finnigan, Bremen, Germany) at the University of Agricultural Sciences, Bangalore, India. The IRMS was interphased with a combustion device (Flash EA 1112) and worked on a continuous flow mode. Samples were applied to a flash combustion element analyzer (EA-1112, Carlo Erba, Milan, Italy). An adequate quantity of CO₂ gas separated and purified by the element analyzer was introduced to an isotope ratio mass spectrometer (Delta XP Plus, Thermo Finnigan, Hamburg, Germany) to estimate the ratio of the isotopic composition expressed as δ13C (¹³CO₂/¹²CO₂) with the standard error of 0.1%. Standard potato starch calibrated against Pee Dee Belemnite carbonate was used for comparison. CID of the samples was calculated as described earlier [74].