Natural auxins 



forth above (i.e. the free lAA was extracted with a single extraction and 

 tryptophan was not converted to lAA during extraction). Therefore, the 

 following extraction and chromatographic method was adopted for routine 

 use in determining indole compounds in photoinduced tissues: 



I. Extraction 



1. 300 g plant tissue harvested and frozen instantly. 



2. Frozen tissue ground to fine powder without thawing. 



3. Frozen ground tissue extracted with absolute ethanol for 12 to 24 hours 

 at -10°C. 



4. Ethanol evaporated off in vacuo leaving aqueous fraction, 



5. Aqueous fraction acidified to pH 4-0 with 0-1 N H3PO4. 



6. Acidified aqueous fraction saturated with glucose and extracted three 

 times with ethyl ether. 



7. Ether extract concentrated to 2-5 ml or less. 



II. QjLiantitative Paper Chromatography 



8. 2-5 jul of extract were removed and applied to Whatman No. 1 paper 

 as described previously (Vlitos and Meudt, 1953). 



9. 0-1, 0-25, 0-5, 1-0, 2-5, and 5-0 micrograms of lAA (or any other indole 

 compound) were applied to Whatman No. 1 paper. 



10. After development of papers, density measurements of spots were used 

 to construct standard calibration curve (Vlitos and Meudt, 1953). 



1 1 . The amount of indole compound in extract was calculated by referring 

 to a standard calibration curve. 



All the operations pertaining to extraction were performed in a cold 

 room ( — 10°C). The standard calibration curve is based on five replicate 

 paper chromatographs run simultaneously. 



If smaller quantities of tissue are extracted it is of course possible to rely 

 upon qualitative paper chromatography followed by elution of the substance 

 from the paper 'and subsequent biological assays {Avena coleoptile tests, 

 root-growth measurements, etc.). The latter procedure introduces additional 

 steps in the method and, no doubt, reduces the quantitative precision of the 

 technique. Therefore it is desirable to use at least 300 g of tissue per extraction 

 and maintain the quantitative aspect of the method. 



INFLUENCE OF ENVIRONMENTAL VARIABLES ENCOUNTERED DURING 

 EXTRACTION ON THE STABILITY OF lAA 



In addition to avoiding the conversion of precursors to I A A during extrac- 

 tion, it was necessary to assay the influence of the environmental variables 

 which might affect the recovery of free lAA from plant tissues. The effects 

 of pH, light, and temperature on the stability of lAA were evaluated only in 

 so far as they might influence the recovery of lAA for the short periods of 

 time encountered during extraction and chromatography of the extracts. 



The influence of pH on the stability of lAA in a series of phthalate buffers 

 adjusted to pH 1-5 to 9-0 with HCl and NaOH is recorded in Table 4. 



If an extraction of free lAA from plant tissues is not completed within 

 a few hours there is a possibility of inactivation of the compound at low pH 

 values. However, in experiments of this type an acidified extract is usually 



60 



