The frozen plant material is homogenized in 3% aqueous sulfosalicylic acid (0.01g/0.5ml) and the residue is removed by centrifugation at 12000 rpm for 10 min.
1 ml of homogizad tissue reacts with 1 ml of ninhydrin acid and 1 ml of glacial acetic acid in a test tube for 1 hour at 100oC and the reaction is stopped in an ice bath
The reaction mixture is extracted with 2 ml of toluene, mixed vigorously and left at room temperature for 30 minutes until the separation of the two phases
The toluene containing chromophore (1 ml upper phase) is warmed to room temperature and its optical density is measured at 520 nm using toluene for blank.
Proline levels are determined from a standard curve using D-Proline
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