ESTIMATION OF GLYCINE BETAINE IN PLANT TISSUES

          Glycine betaine belongs to group of compounds commonly known as quaternary ammonium compounds. It is a derivative of glycine.

It is reported to accumulate in many plant species under drought, salinity and temperature (high and low) stresses. Its precursor is choline and two enzymes viz., choline monooxygenase and betaine aldehyde dehydrogenase play crucial role in its synthesis in bacteria and plants.

It serves as an osmolyte by lowering the osmotic potential of the cell and thus prevents movement of water from the cell, as well as compatible solutes by preventing denaturation of macro-molecules like enzymes/proteins. Glycine-betaine estimation is done in dried leaf powder as per the method of Grieve and Grattan (1983).

Principle

          The assay is based on the fact that at low temperature betaine makes a betaine-periodite complex with iodide in acidic medium, which absorbs at 360 nm in UV range.

Test tubes, test tube stand, micro-pipettes (20-200 µl, 100-1000 µl and 5 ml), low temperature shaking water bath, Whatman No. 1 filter papers, UV-visible spectrophotometer.

Cold potassium iodide- iodine solution: Iodine (15.7 g) and potassium iodide (20 g) were dissolved in 100 ml of water and kept in refrigerator at 40C.

Sulphuric acid (2 N): Fifty-five ml of sulphuric acid is dissolved in distilled water and the volume made up to 1 liter.

Extract prepared by finely ground dry plant material (0.5 g) is mechanically shaken with 20 ml of deionized water for 48 h at 25oC. The samples are then filtered and the filtrate is stored in freezer until analysis.

          Thawed extracts were diluted 1:1 with 2 N sulphuric acid. Aliquot (0.5 ml) was measured into test tube and cooled in ice water for 1 h. Cold potassium iodide-iodine reagent (0.2 ml) was added and the mixture was gently mixed with vortex mixture. The samples were stored at 0-4oC for 16 h. After the expiring of the period samples were transferred to centrifuge tubes and then centrifuged at 10,000 g for 15 minutes at 0oC. The supernatant is carefully aspirated with 1 ml micropipette. As the solubility of the periodite complexes in the acid reaction mixture increases markedly with temperature it is important that the tubes be kept cold until the periodite complex is separated from acid media. The periodite crystals are dissolved in 9 ml of 1,2-dichloro ethane (reagent grade). Vigorous vortex mixing is done to effect complete solubility in developing solvent. After 2.0-2.5 h the absorbance was measured at 365 nm with UV-visible spectrophotometer.

            Reference standards of glycine-betaine (50-200 µg/ml) were prepared in 2 N sulphuric acid and the procedure for sample estimation was followed.

Greive, C.M. and Grattan, S.R. (1983). Rapid assay for determination of water-soluble quaternary amino compounds. Plant Soil, 70: 303-307.