Protocol for: ESTIMATION OF GLYCINE BETAINE IN PLANT TISSUES
Abstract or Description:
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.
CH3 N+- CH2-COO
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).
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.
Instruments and glassware
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
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.
Citation: Shekhar C Bisht, VPKAS, Almora, India