Tuatara: Volume 6, Issue 1, January 1956
A Note on the Bio-assay of Auxins as a Class Experiment
A Note on the Bio-assay of Auxins as a Class Experiment
The technique described in this note is essentially the same as the Avena straight-growth method developed by Bentley and Housley (1954); modifications have been introduced to make the technique suitable for class experiments. Advantages of the method are:—
1. |
No special apparatus is required. |
2. |
In contrast to the coleoptile curvature test, satisfactory results can be obtained consistently without previous experience in the technique. |
3. |
The linear growth responses are more simply measured than the curvatures in the coleoptile curvature test or the pea test (van Overbeek and Went, 1937). |
Oat grains are soaked overnight (the variety Onwards, kindly supplied by Crop Research Division, D.S.I.R., has been found satisfactory). These are then sown fairly thickly on the surface of moistened ‘vermiculite’ in a seed box and are covered thinly with a mixture of approximately equal volumes of vermiculite and sand. The substitution of vermiculite for sand (as used by Bentley and Housley) has two advantages. Firstly, the amount of water used in the initial wetting of the substrate is far less critical since the margin between providing sufficient water for growth on the one hand, and avoiding water-logging with consequent loss of aeration on the other, is greatly increased. Secondly it disposes of the necessity for subsequent waterings during the growth period.
In order to maintain the high humidity necessary for the growth of the test seedlings, the seed box is supported above free water contained in a deep tin, which substitutes for the constant humidity room used by Bentley and Housley. Blotting paper dipping into the water, lines the sides of the tin and a sheet of glass is used for a cover (Fig. 1a). The tin is placed in the dark in an incubator at a temperature of about 25° C.
When the coleoptiles emerge through the surface a red photographic safety lamp supported above the glass plate is switched on. This prevents excessive growth of the first internode. When the coleoptiles are 1.4-1.5 cm. long (about 80 hours after sowing the grain under the conditions described) the seed box is removed and coleoptile sections are prepared in a dark room using a red light which is phototropically inactive.
page 25For the preparation of the sections the following are required:—
1. |
A cutter made from two safety razor blades screwed to either side of a piece of wood, 1 cm. in thickness. (Fig. 1b). |
2. |
A microscope slide on the underside of which is stuck a rectangular piece of white paper so that one of its edges demarks a line 3 mm. from one of the longer edges of the slide. |
Fig. 1 — A: humidity chamber for growing the oat seedlings; a, glass cover; b, blotting paper lining walls; c, tin container; d, seed box; e, vermiculite; f, stand; g, water. B: cutter; a, razor blade screwed to wood. C: coleoptiles ready for cutting; a, 3 mm. line on slide.
The sections are floated on the solutions to be assayed which are contained in petri dishes. These are incubated in the dark at a temperature of 25° C. for 24 hours. At the end of this period the length of the coleoptile sections is measured to the nearest mm. using a flexible celluloid rule.
References
Bentley, J. A., and Housley, C. — (1952) Journ. Expt. Bot. 3, 393. (1954) Physiol. Plant. 7, 405.
Van Overbeek, J., and Went, F. W. (1937) — Bot. Gaz. 99, 22.