Influence of Gibberellic Acid on lAA Disappearance 629 



light from a tungsten lamp for observation. Ten g. of frozen epicotyls 

 were ground in a chilled mortar with M/50 KHoP04-Na2HP04 buffer 

 (pH 6.4), filtered through washed cheesecloth, and made up to 110 

 ml. with buffer. Storage was at — 15° C. in the dark. Enzyme activity 

 of this preparation was low, so a preliminary experiment, based on 

 a design used by Hillman and Galston (11), was carried out to deter- 

 mine the quantities of MnCU and 2,4-dichlorophenol that had to be 

 added to the reaction mixture to give a suitable rate of lAA destruc- 

 tion. A reaction mixture of lO'^M 2,4-dichlorophenol, lO-'^M MnCU, 

 0.02i\f phosphate buffer, and enzyme at X 1/10 of the prepared con- 

 centration was chosen. 



Experiments on rate of lAA disappearance from solutions contain- 

 ing stem sections were carried out with plants grown in the dark in 

 the manner described above. Ten-mm. sections were cut, one per 

 stem, from just below the first node. After randomizing, four lots 

 of 42 sections were withdrawn, and each lot was placed in 14 ml. of 

 test solution in a 250 ml. wide-necked conical flask. Two ml. was 

 previously removed for an absorption reading at zero time: two 

 flasks were controls which were prepared for reagent blanks in the 

 absorptiometer. Preliminary experiments using 0.0166i\jr phosphate 

 buffer (pH 6.4) and no other addendum gave little lAA destruction; 

 therefore lO-^M 2,4-dichlorophenol and 10-^M MnCl^ were incor- 

 porated into the test solution. Flasks were rocked at approximately 

 90 oscillations per minute at 27° C. Whenever 2 ml. aliquots of solu- 

 tion were withdrawn for lAA determinations, six sections were re- 

 moved from each flask to maintain uniform tissue/solution ratios. 

 Manipulations for experiments in the dark were carried out in weak 

 green light (Ilford G907 filter). 



Experiments with apical tissues were carried out in a similar man- 

 ner to those with stem sections except that only 0.0166M buffer was 

 used and no addendum as a suitable rate of lAA destruction was ob- 

 tained. To prepare the tissues, as many ensheathing leaves as possi- 

 ble were stripped with fine forceps from around the apex, and the 

 remaining bud together with 10 mm. of attached stem was excised. 

 Closer stripping could be carried out with experiments in the light 

 than in the dark, while dark-grown plants were easier to prepare than 

 light-grown plants. 



Plants grown in light were raised in a greenhouse and were used 

 after 21 days when five or six internodes were present. In experiments 

 carried out in light, "warm white" fluorescent tubes were used which 

 gave an intensity of 135 foot candles at the level of the plant material 

 in the conical flasks. Under these conditions, this light did not de- 

 stroy any lAA when an irradiated solution (with addenda) was re- 

 peatedly sampled over a period of 212 min. 



