58 D. G. Crosby and A. J. Vlitos 



new types of naturally occurring nonindolic growth substances ^vhich 

 easily might be confused with lAA and IAN on paper chromatograms. 



AUXINS IN MARYLAND MAMMOTH' TOBACCO 



During the period of 1954 to 1956, Vlitos and co-workers (10, 11) 

 studied, with the aid of paper chromatography, the relationship of 

 naturally occurring indole compounds to flowering in photoinduced 

 plants. It was found that a substantial increase in the levels of lAA 

 and indole-3-pyruvic acid occurred after photoinduction of a short- 

 day soybean (Glycine max, 'Biloxi') . However, extension of the in- 

 vestigation to a short-day variety of tobacco (Nicotiana tahacum, 

 'Maryland Mammoth') revealed that neither lAA nor IAN could be 

 detected in leaves and apical tissue of these plants. A material similar 

 to, but not identical with, IAN in its chromatographic and colorimetric 

 characteristics was observed, and bioassay of chromatogram areas con- 

 taining this substance indicated the presence of a growth stimulant. 



Several factors were responsible for the decision to attempt isola- 

 tion of the growth-promoting substances of 'Maryland Mammoth' to- 

 bacco. The unusual vigor and rapid growth rate of this variety have 

 long been intriguing. Inability to detect the classical auxins, and the 

 presence of the unidentified indole, suggested that an unusual type 

 of hormonal growth regulation might be in operation. 



In order to obtain sufficient material for the isolation, seedling 

 tobacco plants were transplanted to a farm near Winfield, VV^est Vir- 

 ginia. After 3 months of growth, the leaves and apical tissue were 

 harvested, frozen in solid carbon dioxide, and transported to the re- 

 search laboratories of the Union Carbide Chemicals Company. The 

 frozen material was ground in a large, cooled mill, extracted with 

 absolute alcohol, and the extract was treated by a modification of the 

 methods reported previously (9,11). The total fresh weight of the 

 tobacco used was 2,300 lbs. 



The bioassay method of Nitsch and Nitsch (7) was used for meas- 

 urement of growth-promoting activity. Sections of first internodes 

 of dark-grown oat seedlings (Avenn sativa, 'Brighton') , 4 nun. in 

 length, were rotated for about 20 hours in citrate-phosphate buffer 

 (pH 5.0) containing 2 per cent sucrose. The final length of the sec- 

 tions was measured with the aid of a photographic enlarger, and the 

 resulting data were subjected to statistic al analysis — a step which 

 was found to be very important in obtaining significant hormone pro- 

 files. The shaded areas on histograms drawn from these data indicate 

 responses significant at the 1 per cent level. 



Hormone profiles of 'Maryland Mammoth' tobacco were obtained 

 by extraction of the homogenized leaves and apical tissue in the usual 



