Estimation of proline from plant tissue:-

*Principle:-
Proline estimation is based on the formation of brick red colored praline-ninhydrin complex in acidic medium. This complex is soluble in toluene and thus can be separated from aqueous phase. This ensures that there is no interference with other amino acids, which also form blue colored complex with ninhydrin.

Accumulation of proline in higher plants is an indication of disturbed physiological condition, triggered by biotic or abiotic stress condition. Free proline content can increase upon exposure of plants to drought, salinity, cold, heavy metals, or certain pathogens. Determination of free proline levels is a useful assay to monitor physiological status and to assess stress tolerance of higher plants. Here we describe three methods suitable for determination of free proline content. 
-The isatin paper assay is simple and is suitable to assay proline content in large number of samples.
 -The colorimetric measurement is quantitative and provides reliable data about proline content. 
-The HPLC-based amino acid analysis can be employed when concentration of all amino acids has to be compared

*Protocol :-

1. By homogenizing 0.5 g of plant material in 10 ml of 3% aqueous sulphosalicyclic acid, the extract was made.
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2. The homogenate is filtered through Whatman No. 2 filter paper.
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3. 2 ml of filtrate is taken in a test tube and 2 ml of glacial acetic acid and 2 ml acid-ninhydrin are added in a sequence.
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4. The mixture is heated it in the boiling water-bath for 1 h.
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5. The reaction is stopped by placing the tube in ice-bath.
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6. 4 ml toluene is added to the reaction mixture and stir well for 20-30 sec.
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7. The toluene layer is separated and warm to room temperature.
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8. The red colour intensity is measured at 520 nm.
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9. A series of standard with pure proline in a similar way is made and prepare a standard curve. 
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10. Thus the amount of proline in the test sample is determined from the standard curve.

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