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  Section: Plant Lab Protocols
 
 
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Methodology for Plant Hormone

 
     
 
Indoleacetic acid
 
Indole-3-acetic acid (IAA) is an important hormone involved in plant growth and development. As this phytohormone is intimately involved in the biochemical, physiological and genetical functions of plants, information on the exact amount of IAA is essential. It occurs in minute quantities. The determination of hormonal amounts in plant tissues is generally a time-consuming and laborious procedure. Bioassays take longer time and are less accurate and liable to interference by other substances. Although a few physicochemical methods are available for extraction and estimation of IAA, they are either time consuming or less reliable. A fluorimetric method developed by Stoessl and Venis1 and improved by Knegt and Bruinsma2 is relatively rapid and measures at nanogram level. The method is described below.
 
 



Principle

IAA is reacted with trifluoroacetic acid and acetic anhydride to convert it into indole-a-pyrone which is measured fluorimetrically.
 
 
Materials
Methanol (re-distilled)
Trifluoroacetic Acid Acetic Anhydride Reagent: Mix equal volume of each liquid, pre-cooled to 0°C. Freshly prepare and store on ice until use.
Spectrophotofluorimeter: Primary filter 440nm and secondary filter 490nm (for low concentration measurement).
 
 
Extraction of IAA
Freeze a known quantity (5g) of plant material in liquid nitrogen and grind to a fine powder using a pestle and mortar. Continue grinding with 10mL methanol (redistilled) to a fine suspension. Filter the homogenate through a G4 glass filter under suction into a 100mL flask. Extract the material on the filter twice by adding 10mL methanol and then once with 5mL. Evaporate the filtrate in a rotary evaporator at 30°C to an aqueous residue.
 
To the aqueous residue add 10mL of cold 0.5M K2HPO4 solution so that pH reaches to about 8.5. Transfer to a suitable separating funnel and shake with 10mL light petroleum ether. Repeat this step again and then shake with 10mL diethyl ether each time discard the lipid fraction. Adjust the aqueous layer to pH 3 by adding about 3mL of 2.8M phosphoric acid. Extract IAA with 10mL diethyl ether.
 
The 10mL diethyl ether is then extracted with 10mL cold 50mM K2HPO4 solution. The pH of the solution is adjusted to 3 with phosphoric acid (0.28M) and the IAA is passed into a final 10mL diethyl ether. The ether is then evaporated in a few min under reduced pressure. Dissolve the residue in a known volume (5mL) of cold redistilled methanol.
 
 
Procedure
1.
Pipette out 1mL of the above methanolic extract each in four different test tubes.
2.
To each tube add 1mL of methanol containing 0,10, 20 or 30ng of IAA respectively.
3.
Dry the contents in each tube completely under reduced pressure and cool to 0°C.
4.
To each flask add 0.2mL of ice-cold trifluroacetic acid-acetic anhydride reagent and mix.
5.
Place the tubes on ice for exactly 15 min to ensure the complete conversion of IAA into indole-a-pyrone. Stop the reaction by adding 3mL water.
6.
A blank may be prepared occasionally by adding first 3mL water to one of four aliquots and 0.2mL reagent after 15 min.
7.
Take the readings in a spectrophotofluorimeter using an excitation at 440nm and emission at 490nm for low concentration samples.
8.
Calculate the amount in unknown.
 
 



Notes

The reagent components and the reagent should be cooled on ice before use. Reaction should also be carried out on ice to avoid irregular results.
 
Chlorophylls and carotenoids interfere with the measurement. Avoid these contaminations during extraction by collecting only clear layers.
 
 



References

1. Stoessl, A and Venis, M A (1970) Anal Biochem 34 344.
2. Knegt, E and Bruinsma, J (1973) Phytochem 12 753.
 
     
 
 
     




     
 
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