Contents

Abstract............................................................................................................................................2

Introduction......................................................................................................................................2

MATERIAL AND METHODS.......................................................................................................2

Materials......................................................................................................................................2

Method.........................................................................................................................................3

RESULTS & DISCUSSIONS.....................................................................................................4

Abstract
This paper is a report on the estimation of Proline content in chick pea (Cicer arietinum) and
mung bean (Vigna radiata) under stressed and unstressed conditions. The stress is induced in
the seeds using NaCl and the estimation is done colorimetrically. The effect of the stress induces
an increase in the Proline content of the seeds.

Introduction
What happens to seeds when they are germinated in high stress conditions? Studies show that
under high salinity there is an accumulation of Proline which acts as an osmolyte for osmotic
adjustments and also helps in stabilizing sub-cellular structures such as membranes and proteins.
This study tries to understand the variation in proline content in chick pea (Cicer arietinum) and
mung bean (Vigna radiata) under stressed and unstressed conditions. Firstly proline is
precipitated using sulpho-salicylic acid and treated with Ninhydrin (2,2-dihydroxyindane-1,3-
dione) which is extensively used in many amino acid assays. The intensity of the colour complex
due to the reaction between proline and Ninhydrin is measured using a Photocolorimeter and
finally discusses the variation in proline content.
MATERIALS AND METHODS

Materials
Acid ninhydrin: warm 1.25 g ninhydrin in 50 mL glacial acetic acid and 20 mL 6 M phosphoric
acid with agitation until dissolved. Store at 4 degree C and use within 24 hours 3 % aqueous
sulpho-salicylic acid, glacial acetic acid, Toluene, proline sample, test tubes, pipettes, separating
funnel, glass rods, water bath, colorimeter, chick pea (Cicer arietinum) and mung bean (Vigna
radiata) germinated in normal water and in 50mM NaCl solution.

Method

1. Extract 1 g of germinated seed material by homogenizing in 10 ml of 3% aqueous sulpho-salicylic acid

2. Filter the homogenate through whattman's filter paper no 2.

3. Take 2 ml of filtrate in a test tube and add 2 ml of glacial acetic acid and 2 ml of acid ninhydrin. Heat it
in boiling water bath for 1 hr.

4. Terminate the reaction by placing the tube in ice bath.

5. Add 4 ml toluene to the reaction mixture and stir well 20 -30 seconds.

6. Separate toluene layer and warm to room temperature.

7. Measure the red colour intensity at 520 nm.

8. A series of standard with pure proline in a similar way and prepare a standard graph is prepared.

9. The amount of proline is estimated using the standard graph.

RESULTS & DISCUSSIONS

Table 1

Plant material used OD value obtained at 520 nm Concentration of Proline as

from colorimeter per the standard graph
(micrograms)
Chick pea unstressed 0.92 24.88
Chick pea stressed 1.18 31.70
Mung bean unstressed 0.64 17.54
Mung bean stressed 0.97 26.19

1.4

1.2

0.8 absorbance
Absorbance

Linear (absorbance )
chick pea stressed
0.6
chikpea unstressed
mung bean stressed
0.4 mung bean unstressed

0.2

0
0 5 10 15 20 25 30 35
Concentration (micrograms)

Figure 1
BIBLIOGRAPHY