18 Fundamentals of Auxin Action 



taken advantage of this fact in framing a very simple technique for 



obtaining free auxin: 



1. Freeze plant material on CO2 ice. 



2. Slice bulky tissues into 2-5 mm. slices. 



3. Extract with peroxide-free ether at 0° C for two half-hour 

 intervals. 



4. Combine the ether extracts and reduce volume by evaporation 

 to a few ml. 



5. Transfer quantitatively to agar for Avena assay. 



This technique has the advantages of being extremely simple, avoid- 

 ing interference by inhibitors which would appear upon boiling, and 

 by-passing the requirement for a lyophilization apparatus. 



The techniques described above have all been designed for the 

 extraction of free auxin. Extraction of bound auxin can be carried on 

 using either of the latter two methods by carrying on the extraction 

 over a period of time. Van Overbeek et al (1947) have determined 

 free auxin by short time extraction and bound auxin by long term 

 extraction (20 to 281 hours) in various pineapple tissues. They have 

 demonstrated that their short term extraction of free auxin yields 

 results practically identical with Gustafson's boiling method. Their 

 comparisons of free and bound auxins in various plant tissues demon- 

 strate the extreme usefulness of this technique. 



BIOASSAYS FOR AUXINS 



The Avena Test 



The original assay for auxin as described by Went (1928) is given 

 in great detail and thoroughness in the book, "Phytohormones" (Went 

 and Thimann, 1937, p. 24-51). The following description of the test 

 is essentially a summary of their presentation. 



The physiological basis for the Avena or oat test lies in the strict 

 polar transport of auxins in the Avena coleoptile. The strict polarity 

 and rapidity of auxin transport result in a difference in growth rate 

 between the side of the coleoptile to which auxin is applied and the 

 side to which none is applied. The differential growth causes curvature 

 of the coleoptile, which is proportional to the amount of auxin ap- 

 plied. It should be remembered that chemicals which are not swept 

 along by the active transport system will not have any effect in the 

 test. Thus salts, metallic ions and sugars have little effect upon 

 the curvature. The same is true of auxins which are poorly transported, 

 even though they may show growth activity in other tests. 



