Determination of proline content

1 g of fresh plant tissue was crushed in 3 % aqueous sulphosalicylic acid. Thereafter, the homogenates were centrifuged at 10,000×g for 10 min, the supernatant was collected and reaction was started by adding acid-ninhydrin solution (1.25 g ninhydrin in 30 ml glacial acetic acid). Reaction mixtures were heated at 95 °C for 1 h after getting cooled; they were extracted with 4 ml toluene by vortexing for 1 min. The upper toluene layer was then pipetted out and absorbance was read at 520 nm, using toluene as a blank and expressed as µM proline/g of fresh tissue (Bates et al. 1973).