Hormones and hormone precursors 



reduced pressure. The aqueous extract remaining was extracted with ether 

 and the ethereal sohition separated into acid and neutral fractions with 

 sodium bicarbonate. Some of the aqueous fraction was distilled under 

 reduced pressure until a thick syrup remained. 



(b) Excised and seedling roots of tomato [var. Best-of-All) — Excised roots were 

 obtained by growth of 10 mm clonal tips, derived from clonal sector cultures, 

 in a modified White's medium for 10-12 days at 27°C. The method of 

 culture has been descriljed by Street (1954). The average yield of material 

 was 18 g fresh weight per 100 roots. 



Seedling roots were grown in sand in a greenhouse, watered daily with the 

 culture medium (less sucrose, vitamins, and Fe2(S04)3), and harvested 

 after 4-5 weeks. Harvested roots were stored at — 15°C until sufficient 

 quantities had been collected. 



The extraction technicjue was essentially the same as for the cabbage, 

 except that sometimes, where stated, ether was used for extraction instead of 

 alcohol. 



{c) Maize roots and seeds — Seed of maize (var. Great White Horse Tooth) 

 was germinated in sand and the roots and seeds collected when the coleoptiles 

 were approximately 2 cm long. The material was well washed, frozen, and 

 extracted with ether. 



Paper chromatography technique 



The descending method was used with Whatman No. 1 papers. Loading 

 was usually in the form of a strip, and controls of lAA and IAN (approx. 

 40 //g) were run simultaneously with each extract. Chromatograms were 

 developed in the dark at room temperature without prior equilibration, in 

 either 4 vol. zVopropanol : 1 vol. 0-15 N ammonia, or «-butanol saturated 

 with 1-5 N ammonia. They were developed for about 20 hours, by which 

 time the solvent front normally reached a distance of 20-30 cm, dried in a 

 current of air at room temperature for 20 min and examined in filtered u.v. 

 light (2537 A transmitted) for fluorescence. Chromatograms were usually 

 divided for bio-assay according to the fluorescence pattern. Each division 

 was eluted in 12 ml water. Chromatographic solvents were removed in 

 vacuo with air drawn through the solution at a bath temperature of 35-40°C. 



Spray reagents 



Chromogenic reactions were developed by drying the papers at 35°C, and 

 then heating at 50-60°C for 1-5 min. The following sprays were used: 

 ferric chloride/perchloric acid, 2 ml 0-05M FeClg plus 100 ml 5 per cent 

 HCIO4; nitrous/nitric acid, 1 g KNOg in 200 ml HNO3 (sp. gr. 1-42 diluted 

 XlO); /j-dimethylaminobenzaldehyde, 2 g in a mixture of 20 ml HCl 

 (sp. gr. 1-18) and 80 ml absolute ethanol. 



Bio-assay techniques 



The method of Avena straight-growth assay has been reported (Bentley, 

 1950), and the technicjue described in detail (Bentley and Housley, 1954). 

 10 mm sections were cut from coleoptiles of either 1-4-1 -6 cm, 1-6-1 -8 cm, or 

 1-8-2-0 cm, floated on 10 ml test solution in 2 in. Petri dishes, and their length 

 measured after approximately 1 7 hours. Ten replicates per treatment were 

 used except when otherwise stated. 



V^ 41 



