Dioarfing Genes in Zea mays and Relation to Gibberellins 493 



total amount of gibberellins in normal seedlings may be difficult to 

 evaluate if the active substances represent a mixture of different kinds 

 of gibberellins, some of which are intermediates in the pathway con- 

 trolled by the normal forms of the dwarfing genes. In this event, the 

 choice of the mutant used for bioassay becomes critical. It is also pos- 

 sible that the dwarf mutants may be accumulating gibberellin inter- 

 mediates which would be inactive when assayed on the accumulating 

 mutant, yet active on one or more of the other GA^-responding mu- 

 tants. The dwarf controlled by the mutant gene farthest back in the 

 presumptive pathway would be the best choice for determining total 

 amount of gibberellins. In the final analysis, quantitative crossfeed- 

 ing studies with all five GAg-responding mutants are necessary for 

 the proper evaluation of amounts and kinds of native gibberellins 

 from normal and mutant seedlings. 



Experimental tests of the above predictions require the use of a 

 specific and quantitative assay for the detection of gibberellins and 

 gibberellin-like substances, and for the estimation of their relative 

 activities. The dwarf mutants of Zea mays have been used for such 

 a purpose because of their specificity, sensitivity, and rapidity of re- 

 sponse to the gibberellins (10, 11). For quantitative studies, standard 

 procedures are used for growing, treating, and measuring the growth 

 of the assay plants. Ten mutant seedlings are used for each dosage 

 level of a particular compound or preparation to be tested for ac- 

 tivity. Mutant seedlings are treated by placing 0.1 ml. of the material 

 to be assayed into the first unfolding leaf as it emerges from the cole- 

 optile. The test plants are grown at temperatures ranging from 25° C. 

 to 35° C. for a period ranging from seven to ten days. The length of 

 the first leaf sheath or the sum of the lengths of the first and second 

 leaf sheaths is used as a measure of response. Dosage-response curves 

 for GA3 have been found to be linear over the range of 0.001 ^g/plant 

 to 1.0 ^g/ plant when the logarithm of the response is plotted against 

 log dosage (Figure 3). The bioassay is used for quantitative studies 

 only when the curves for the standard and unknown(s) are straight 

 and parallel to each other. Estimations of relative activities are made 

 from graphic analyses of the response curves. The statistical signifi- 

 cance of differences in relative activities is determined from analyses 

 of variance of the original response data. Differences in activity of 

 20 per cent can be shown to be statistically significant at the 5 per 

 cent level. For the qualitative use of the bioassay, single plants are 

 treated repeatedly with the unknown; growth responses 25 per cent 

 over the largest dwarf control are considered as evidence for activity. 

 All unknowns are run in triplicate. The variables of light, photo- 

 period, and temperature are minimized by running the standard and 



