Let us make an in-depth study of: 1. Bioassay for Auxins 2. Bioassay for Gibberellins and 3. Bioassay for Kinetin (or Cytokinins).
Quantitative estimation of a known or suspected biologically active substance (such as a hormone or drug) by measuring its effect on a living organism in standard conditions is known as bioassay.
Bioassay for Auxins:
Several, bioassays have been devised for auxins such as Avena curvature test, Avena section test, split pea stem curvature test, cress root inhibition test etc.
A brief account of the Avena curvature test is given below:
Avena Curvature Test:
Principle:
This test is based on the polar transport of the auxin in Avena coleoptile. The auxin applied on one side of the cut coleoptile stump will diffuse down that side only and will cause that side to grow more resulting in curvature of the coleoptiles. Within limits this curvature is directly proportional to the amount of auxin applied.
Procedure:
(i) Avena grains are germinated and grown in total darkness. The seedlings are exposed to short periods (2-4 hrs.) of red light two days after germination.
(ii) When the roots are about 2mm. long, the seedlings are planted in special glass holders, using the water culture method (Fig. 17.37 A).
(iii) The straight coleoptile are selected (Fig. 17.37 B).
(iv) The tips of the coleoptile (about 1 m.m.) are removed and placed on agar-agar (Fig. 17.37 C, D).
(v) The agar is cut into blocks of standard size (usually 1mm3),-(Fig. 17.37 E) which now contain auxin.
(vi) After about 3 hours a second decapitation of the coleoptile (about 4 m.m.) is made to remove the tip which might have regenerated and the first leaf of the seedling is pulled so that its connection from the base is broken (Fig. 17.37 F).
(vii) An agar block containing auxin is now placed on one side of the cut coleoptile. The projecting primary leaf gives support to the auxin block (Fig. 17.37 G).
(viii) After about 90 minutes the shadowgraphs of the seedlings are taken and the angle of curvature (α) is measured by drawing a vertical line and a line parallel to the curved portion of the coleoptile (Fig. 17.37 H).
(ix) Within limits the curvature of the coleoptile is directly proportional to the concentration of auxin in agar block. In case of Indole-3-Acetic Acid (IAA) the maximum response is at about 0.2 mg/litre (Fig. 17.37 I).
Bioassay for Gibberellins:
A number of bioassays are known for gibberellins such as pea test, dwarf corn test, lettuce hypocotyl test, cucumber hypocotyl test, barley endosperm test, etc. The relative activity of the different gibberellins is different in different bioassay systems.
For instance, the relativity of GA1 to GA9 in dwarf corn (strains d1, d3 and d5) is in the following order:
d1—GA1 > GA3 > GA4 > GA7 > GA5 > GA6 > GA8 > GA9
d3—GA5 > GA7 > GA9 > GA3 > GA4 > GA1 > GA6 > GA2
d5—GA5 > GA7 > GA3 > GA4 > GA9 > GA1 > GA6 > GA2 > GA8
A brief account of the dwarf corn test is given below:
Dwarf Corn Test:
Principle:
This bioassay is based on the fact that gibberellins cause elongation of the internodes, and in case of corn they also increase the length of the leaf sheaths which surround the internodes.
Procedure:
The procedure of this bioassay is simple. A measured amount of the test solution in a suitable wetting agent is applied into the first unfolding leaf of corn seedlings when they are 6 to 7 days old. The seedlings are allowed to grow for 6 or 7 more days till the first and the second leaves are fully developed. The increase in the length of the first leaf sheath is measured and is plotted against the concentration of gibberellin applied. Within limits, a direct relationship is observed between the two.
Barley Endosperm Test:
Principle:
This bioassay is probably the most widely used and is based on the fact that during germination of grains such as barley, wheat and oats, gibberellin is released from the embryo which moves to the aleurone cells where it induces synthesis of the enzyme α-amylase (Fig. 17.17). This enzyme hydrolyses starch into simple reducing sugars.
Procedure:
(i) Barley seeds are cut in half transversely and the halves containing embryo discarded, (ii) Endosperm halves are sterilized and allowed to imbibe water for three days. (iii) Endosperm halves are now transferred to a flask containing buffer, CaCl2 and the test solution. Chloramphenicol is added to check bacterial contamination, (iv) After 24 hours of incubation at 25°C with shaking, the seeds are filtered off and the supernatant is analysed for a-amylase activity (i.e., disappearance of starch and build up of reducing sugars), (v) Within limits, a direct relation is observed between conc. of gibberellin and a-amylase.
This bioassay is specific for gibberellic acid (GA3) and will detect a minimum of 0.2 nanogram of GA3.
Bioassay for Kinetin (or Cytokinins):
A number of bioassays have also been devised for cytokinins which are based on their specific physiological activities. They are Cell division tests, Chlorophyll preservation tests. Cell enlargement tests, Germination tests and Differentiation tests. A brief account of one of the cell division tests which are based on the induction of cell division in cytokinin-requiring tissue cultures is given below:
Carrot Root Phloem Bioassay:
(i) Mature roots of cultivated carrot (Daucus carota var. sativus) are peeled and their surfaces sterilized (Fig. 17.38 A).
(ii) With the help of a special cutter they are cut into thin slices about 1-2 m.m. thick (Fig. 17.38. B).
(iii) With the help of a canula, secondary phloem explants are removed from a distance about 1.2 m.m. away from the cambium (Fig. 17.38 C, D).
(iv) The secondary phloem explants weigh about 2.5—3 mg. (Fig. 17.19 E).
(v) About three explants are inoculated into a culture tube containing 10 ml. of medium (Fig. 17.38 F).
(vi) The culture tubes are placed on a wheel which turns on a horizontal axis at 1 rpm (Fig. 17.38. G) so that the explants are alternatively exposed to medium and the air.
(vii) After a specified time (about 18-21 days) the explants are removed, weighed, macerated and the cells are counted. The number of the cells is converted into milligrams (1 mg being approximately equal to 10,000 cells) and is plotted against the time in days (Fig. 17.38 H).
(The classical methods for bioassays of plant growth hormones auxins, gibberellins etc. have now been replaced with modern methods of separation and quantification such as high performance liquid chromatography (HPLC) and gas chromatography (GC) which are then followed by mass spectrometry (MS) to provide proof of structure. Immunoassay methods are also used which are more rapid and thousand times more sensitive than bioassays).
