Investigation of natural auxins and growth inhibitors 



methanol, which yielded beautiful white tissues without the slightest trace of 

 browning. To improve the ether solvent, several addenda were tried. The 

 best one was Na2S204, which is practically insoluble in ether, but which 

 dissolves in the water coming out from the tissues. This powerful reducing 

 agent kept the tissues perfectly white, but the effect it might have on the 

 auxins themselves has not yet been investigated. The addition of methanol 

 to ether improved the latter solvent so much that it was not necessary to look 

 further into the matter. It was inferred, as a working hypothesis, that since 

 either methanol alone or a mixture of ether and methanol would prevent 

 browning, they would perhaps also stop the enzymatic activity of plant 

 tissues which have caused in the past so many artefacts during auxin extrac- 

 tion. If enzymatic activity is prevented (at least by the 0°C temperature and 

 the short duration of the extraction, such as 1-3 hours), then there should be 

 no danger of other artefacts, such as those arising from an enzymatic esterifica- 

 tion of indole-3-acetic acid (lAA) with methanol or ethanol. Such an 

 esterification does not seem to proceed at an appreciable rate in vitro. We 

 have left lAA in ethanol (1 mg/c.c.) for three months at 25°C in the dark, 

 and after that time we have chromatographed it and detected only one spot 

 on the paper, that of lAA. 



Of course, we not only need a solvent which does not cause artefacts to 

 develop, but also one which has a good extractive power. In order to deter- 

 mine how several organic solvents compare, as far as the actual extraction of 

 auxins is concerned, we have performed with different solvents parallel 

 extractions of aliquots of the same material. The extracts were chromato- 

 graphed, so that not only the total ciuantity, but also the nature of the 

 extracted substances would become apparent. Three examples of this study 

 will be given. The first one {Figure 1) concerns tomato fruits, harvested 20 

 days after pollination, which have been immediately frozen, lyophilized, and 

 ground to a fine powder. Aliquots of 100 mg (dry weight) of this powder 

 were extracted for 3 hours at 0°C (ice-bath) with 20 c.c. of the chosen solvent, 

 plus two rinsings. The extracts were filtered over cotton, concentrated under 

 reduced pressure, and transferred to the paper strips by means of a tuberculin 

 syringe, as previously described (Nitsch and Nitsch, 1955). The 

 chromatograms were run in the dark and at room temperature in the 

 ?>opropanol (80)-f 28 per cent ammonia (10)+H2O (10) (v/v) solvent recom- 

 mended by Stowe and Thimann ( 1 954) . When the solvent front had travelled 

 20 cm past the initial spot, the paper strips were taken out of the tubes, dried 

 in a stream of air, and cut transversely into twenty segments 1 cm wide. 

 Each of these segments was incubated with 0-5 c.c. of a buffer at pH 5-0 

 (see below) containing 2 per cent sucrose and with ten 4 mm oat coleoptile 

 sections. 



The diagrams o^ Figure 1 show the location on the chromatograms and the 

 activity in the coleoptile sections of the growth substances extracted with 

 (1) anhydrous acetone, (2) ether, (3) ethyl acetate, and (4) absolute methanol. 

 In all cases, two groups of active compounds are apparent, one around Rf 0-4, 

 the other around RfO-7. The size of the growth peaks varies with the solvent, 

 however. In addition, the number of the peaks also varies with the solvent. 

 For example, acetone seems to extract substances which remain near the 

 origin and which are not visible in the diagram corresponding to the ether 



