uptake and Fate of C"'-Iabeled 2,4-D in Bean Stems 277 



a naphthoresorcinol spray (8) and amino acids with a 0.2 per cent 

 ethanolic ninhydrin spray. 



RESULTS AND DISCUSSION 



The Effect of 2,4-D Concentration, Exposure Time, and the Origin of 

 the Bean Stem Internode Used 



In all the previously reported studies on the induction ot callus 

 formation in bean stems in vitro, the tissue was maintained in the 

 presence of the growth regulator for the full duration of the experi- 

 ment (2, 12,22). Since it would be impossible to conclude when in- 

 duction of proliferation took place under those conditions, it was 

 important to establish the minimum effective concentration of 2,4-D, 

 and hoAv long the exposure to 2,4-D had to last. In initial qualita- 

 tive experiments it was found that the shortest exposure time tested, 

 1 day, was sufficient for the induction of fully developed proliferations, 

 if a concentration of IQ-^M 2,4-D was supplied. When the 2,4-D con- 

 centration was decreased to lO-^M an exposure of 3 days was necessary. 



While it is recognized that the calluses which are formed as a re- 

 sult of the application of 2,4-D to the bean stems are not true tumors, 

 in that they are not auxin autonomous, it was, nevertheless, of in- 

 terest to determine if any potentiation of the callus formation took 

 place when the tissue was cut and planted for varying lengths of 

 time prior to the application of 2,4-D. For this purpose, stem sec- 

 tions were first planted on plain White's medium, exposed to 2,4-D 

 for 1 day, and then returned to White's medium. Preliminary results 

 indicate an increase in proliferation when the stems are pre-incubated 

 for 1 to 3 days in this fashion prior to the application of 2,4-D. 



Figure 1 shows the time course of the uptake of l-Ci^-2,4-D. Each 

 point is the average of four determinations. The radioactivity of the 

 stem increased very rapidly and reached a plateau 2 to 3 hrs. after 

 application. At this time about 10 to 20 per cent of the radioactivity 

 initially present in the agar blocks had been taken up into the stems. 

 The over-all concentration of the 2,4-D in the sap of the stems was 

 about 3 X ^0'''M at that time. When the agar blocks were removed at 

 the end of 3 hrs. (arrow on the figure) and the stems allowed to stand 

 for a continued period of time, the results depicted by the dotted 

 line in Figure 1 were obtained. It will be noted that while the initial 

 drop in radioactivity was very rapid, further changes were very slow. 

 In a separate experiment it was found that at the end of 18 clays 

 some 60 per cent of the radioactivity initially present was still found 

 in the stems. 



It was of interest to identify the factors controlling the uptake 

 of 2,4-D. Table 1 summarizes the results of these experiments. Ten- 



