uptake and Fate of C^'<-Iabeled 2,4-D in Bean Stems 275 



In general, the second and third internodes above the primary leaf 

 were used for investigation. The stems were scrubbed in water, cut 

 into sections 7 to 10 mm. in length, and groups of five sections were 

 weighed on a Roller-Smith balance and planted, basal end down, in 

 modified "White's medium. The 2,4-D was either incorporated into 

 the medium or applied in small agar blocks to the apical ends of 

 the stems. These blocks were formed either by cutting a thin layer 

 of agar containing 2,4-D into small squares in a petri plate or by 

 drawing the liquefied agar into glass tubing of 4 mm. inside diameter, 

 extruding the agar after solidification, and cutting the agar into sec- 

 tions 2 mm. thick. Very uniform blocks were obtained in this man- 

 ner. The medium used in these experiments was a modification of 

 White's medium (27) as obtained from Dr. Jacques Lipetz of Yale 

 University. The constituents in g/1 were: Ca(N03) o -41120, 0.2; 

 NaoSO,, 0.2; KCl, 0.065; NaHoPO^, 0.017; MgS04-7H,0, 0.36; Hoag- 

 land's A-Z solution (14), 1 ml.; Klein and Manos' iron versenate solu- 

 tion (19), 3 ml.; sucrose, 30; and agar, 15. The pH was adjusted to 

 6.5 to 7.0 before autoclaving. It should be noted that vitamins and 

 glycine were omitted, since it was found that the bean stems prolifer- 

 ated in their presence even without the addition of 2,4-D. In experi- 

 ments which lasted longer than 3 days, the stems were surface steri- 

 lized by immersion in a 1:10 dilution of Clorox for 1.5 min., followed 

 by two rinsings in sterile distilled water. All succeeding operations 

 were carried out under aseptic conditions. 



The bean stems were incubated at 25 to 28° C. and 70 to 80 per 

 cent humidity in the dark for 10 days for the development of cal- 

 luses. They were then harvested and the groups of five stems weighed. 

 Gains in weight were calculated on a fresh weight basis. Preliminary 

 experiments have shown that equivalent results were obtained when 

 using either fresh or dry weights. 



The carboxyl-labeled 2,4-D had a specific activity of 22.3 mc/milli- 

 mole. It was found to be at least 96 per cent radiochemically pure by 

 isotope dilution analysis, and showed only single radioactive spots 

 when chromatographed on Whatman No. 1 paper in isopropanol-am- 

 monia water (25), butanol-propionic acid-water (18), or 90 per cent 

 aqueous butanol (26). Depending on the experiment, the 2,4-D was 

 used undiluted, or was diluted up to tenfold with unlabeled material. 

 It was stored as a lO-^M solution in acetone in the deep freeze. No evi- 

 dence of radio-decomposition could be found after 8 months. The 

 final concentration of 2,4-D used was always lO-^M unless otherwise 

 specified. At the end of the experiment the stems were rinsed quickly 

 in ice Avater, placed in test tubes, and frozen in a dry ice-acetone mix- 

 ture. AVhen necessary, they were stored at —20° C. The radioactive 



