24 Fundamentals of Auxin Action 



keep seeds moist but not wet; cover. On the day after planting, the germinating 

 seeds should be exposed to 2 hours of red light to inhibit elongation of the first 

 internode. 



Loading 



Two days after planting, when the roots are 0.5 to 2 cm. long, put each germi- 

 nated seed in a glass holder, and place in a rack with at least one root in water. 

 Be sure coleoptiles are oriented vertically, and that the water meniscus does not 

 touch the seed itself. The plants will be ready one day after loading. 



Preparation of agar 



Place mold on microscope slide over ice or ice-filled refrigerator tray. The hot 

 agar is then pipetted into the mold to make an agar plate 1 mm. thick. After 

 gelation, place the cutting form over the molded agar and cut into 12 uniform 

 squares (2X2X1 mm.) with razor blade. 



Setting up 



73-75 hours after planting, the test is started: 



1. First decapitation. Cut off terminal 1 mm. with conventional scissors. 



2. Wait 3 hours; this is a convenient time to prepare the agar blocks. 



3. Remove terminal 2-4 mm. of coleoptile without breaking the leaf inside 

 (figure 5 C). Use special decapitating scissors, or alternatively, break the 

 coleoptile section off with fine pointed tweezers. 



4. Pull primary leaf approximately halfway out. Use cork-lined tweezers or 

 other wide-nosed tweezers. 



5. Cut off leaf about one quarter inch above the coleoptile tip. 



6. Apply agar block containing auxin on one side of coleoptile tip by placing 

 it against the protruding primary leaf and drawing it down to rest firmly 

 on the coleoptile tip (figure 5 F). Use small spatula for this step. Apply one 

 treatment to a rack of 12 plants. 



7. Record the time at which the rack was treated. Wait 90 minutes. 



8. Place rack in shadowgraph box, with plants pressed close to a strip of 

 photographic projection paper. Expose to unilateral light for 3 seconds for 

 shadowgraph. Record rack number and date on back of paper. 



9. Place paper in developer solution until good contrast develops (about 1 

 minute), rinse in water, and then soak in fixing solution for 5 to 15 minutes. 

 Wash in running water for an hour or more and then lay out to dry. 



Reading 



Read with a protractor the curvatures recorded on the shadowgraph for each 

 coleoptile. Measure the maximum curvature from the straight lower region to the 

 very tip of the coleoptile. (Angle a in figure 5 C.) 



Errors introduced in the procedme for carrying out the test all 

 tend to reduce the curvature obtained. (Exception: agar block smaller 

 than standard.) Consequently, variability because of error will cause 

 a skew in distribution of the readings toward low values. For this 

 reason it is accepted as permissible to discard readings of individual 

 coleoptiles with curvatures which are much lower than the bulk of 

 the values obtained in any given treatment. Of course, all readings 



