In this article we ill discuss about the auxin and gibberellin bioassay of phytohormones.
Auxin Bioassay of Phytohormones:
Auxins are one of the major phytohormones essential for plant growth and development. Normally, in plants, auxins are synthesized in the growing apex and then transported to various regions for physiological activities.
In plants, auxins are present in small quantities, thus chemical test of plant materials does not always provide desirable results. As such, bioassay methods are often used for detection of auxin in unknown phytohormone extracts. There are several bioassay techniques for auxin: Avena coleoptile curvature test, Root inhibition test, Wheat coleoptile test, etc.
(A) Rice-Root Inhibition Test:
Materials Required:
1. 2-day old germinated rice seedlings
2. Glass tube, blotting paper, auxin solution of known concentration, distilled water
3. Scale, graph paper, pencil etc.
Procedure:
1. Take a number of test tubes in a rack and mark them serially.
2. Put different known concentration auxins (IAA solutions) in different tubes serially and place a blotting paper along the wall of each tube. One of the tubes should contain the unknown concentration of auxin.
3. Place germinated rice seedlings on the blotting paper by forceps, after measuring the initial length of root, in different tubes containing different conc. of IAA soln. (0.0 ppm. 1 × 10-6 ppm, 1.5 × 10-6 ppm, 2 × 10-6 ppm).
4. Keep all the sets in darkness to protect photo destruction of IAA and measure the root length after 48 hours of treatment in each set separately.
5. Prepare a standard curve of root growth inhibition against different known conc. of IAA solution.
6. If any unknown sample solution is used in this expt., the concentration of such solution can be determined by comparison of data with the standard curve.
Results:
(B) Wheat Coleoptile Elongation Test:
Materials Required:
1. Wheat coleoptiles (fresh)
2. Indole acetic acid (Auxin) stock solution (100 ppm)
3. Glass tube, measuring cylinder, petridishes, pipettes etc.
4. Distilled water, Blotting paper, Graph paper, slides etc.
Procedure:
1. Prepare different concentrations of auxin from stock solution and place separately on each petridish and label serially as 100 ppm, 75 ppm, 50 ppm, 25 ppm, 10 ppm and control.
2. Place 3 coleoptide tips in each petridish. Each tip is of 3 mm length and cut from the tips leaving 2 mm at the apical part.
3. All the sets were kept for 24 hrs. in dark and then length increment of each coleoptile tip was measured separately after blotting them.
4. The percent increase of coleoptile length were plotted against each concentration on graph paper and thus a standard curve was prepared.
Results:
Gibberellin Bioassay of of Phytohormones:
The Gibberellins (GAs) are one of the major groups of growth-promoting hormones which play essential roles in the regulation of growth and development of seed plants. By comparing the response to the extract obtained from plants with a standard response curve obtained by treating dwarf peas with a range of known dosages of GA3, one can assay the quantity of GA3 present in the unknown extract.
Materials and Equipments Required:
1. Unknown extract of GA3
2. 10-day old seedlings of dwarf pea varieties
3. Standard GA3 solution
4. Micrometer burette
5. Tween 20 soln. (0.05%)
Procedure:
1. Dwarf Pea seedlings in plastic pots are used as the experimental test material.
2. Measure the shoot heights of the plants from the cotyledonary node to the highest visible node.
3. Using a micrometer burette, apply 10 |al containing 0.01, 0.1, 1.0, 10 or 100 mg of GA3 to the shoot apex of each plant.
4. Apply 10 ml of unknown extract (UE) to each plant in separate sets. Against 10 µl of extract dilute in the ratio of 1: 9 with 0.05% Tween 20 solution and apply to separate plants.
5. After one week measure the shoot height of the plants.
6. Calculate the mean increment in shoot length for each group.
7. Plot a standard curve by plotting on semi-log paper, increase in shoot length in cm versus dosage of GA3.
8. Compare the responses of the two groups of extract-treated plants and interpolate to determine the mean concentration of GA3 in the extract; express as X µg/ml. 