14 Fundamentals of Auxin Action 



tioned before. Consequently it appears that the free and bound forms 

 are in a dynamic state, and the measurement of one strictly separated 

 from the other is often difficult. 



METHODS OF OBTAINING AUXIN 



Diffusion 



The simplest method of obtaining the growth hormone from 

 plant material is by diffusion into agar. The usual procedure followed 

 is simply to sever the growing tip or other organ to be tested under 

 conditions which discourage transpiration, and place the cut surface 

 for a period of an hour or so on a block of agar, usually 1.5%. This 

 technique yields auxin immediately available for growth and, in 

 etiolated seedlings, it can be shown in many different ways that growth 

 is proportional to the auxin obtained by diffusion. 



Three main difficulties can arise in the use of diffusion techniques: 



1. The excessive loss of water, or a negative tension in the vascular 

 system — as for example in leaves which have been recently exposed to 

 sunlight — can prevent the accumulation of diffusate in the agar block. 

 This difficulty can sometimes be alleviated by carrying out the diffu- 

 sion in a high relative humidity, or even wath the diffusion source 

 under water. How'ever, many types of leaves and stems will not yield 

 diffusible auxin quantitatively under such conditions. 



2. The destruction of auxin at the cut surface frequently inter- 

 feres with the quantitative yield. Such destruction is apparently en- 

 zymatic, in some cases attributed to polyphenol oxidase and in others 

 to peroxidase. Browning of the cut surface may indicate danger of 

 destruction by polyphenol oxidase. Several methods of reducing this 

 destruction have been used. The cut surface may be pressed onto wet 

 filter paper to reduce the amount of enzyme (van Overbeek, 1938). 

 Another method is the incorporation of 10-^ M ascorbic acid into the 

 agar as an alternative substrate for the enzyme (Wetmore and Morel, 

 1949). A promising method appears to be the use of 0.005 M potassium 

 cyanide in the wet filter paper first used to blot the cut surface, and 

 as a droplet on the surface of the agar block as well. Then before the 

 blocks are used for the Avena test, a drop of 0.005 M ferrous sulfate is 

 placed upon the block to precipitate the cyanide ions present. Use of 

 the poison causes no interference with the bioassay (Steeves et al, 

 1954). 



3. The existence of growth inhibitors common in many green 

 tissues will prevent the effective use of diffusion techniques. No means 

 of separating auxins from inhibitors have been worked out for quanti- 



