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Abscisic add (ABA) concentrations and growth rates of developing soybean (Glycine max [L.] Merr. cv. Wye) seeds and pod wails were determined from anthesis to maturation using high pressre liquid chromatographic techniques. Developing soybean seeds contain up to 12,200 ng/g fresh weight of ABA compared to 330 ng/g fresh weight for pod wails. In the developing seeds ABA levels correlated with growth rates, being the highest during the most active growth period of seed enlagement, and then decreasing to less than 10 ng/g fresh weight at maturity. Higher levels of ABA were found to occur in the cotyledons and seed coats than the root-shoot axes at 21 days postanthesis. The time required for excised root-shoot axes to initiate growth in liquid culture decreased as seed development progressed and ABA levels of the seeds declined.Abscisic acid (ABA) is recognized as a naturally occurring plant hormone of major importance in the regulation of plant growth and development. It has been implicated in such plant growth processes as senescence; abscission of leaves, flowers, and fruits; rest and dormancy of seeds, buds, and tubers; and inhibition of vegetative growth (1,(10)(11)(12)16). In mature seeds, relatively high levels have been found in some species and its occurrence implicated in dormancy and the inhibition of germination (1, 4, 10-12, 15, 16, 18, 19, 21). In developing seeds, little is known of the concentration changes and the physiological role of ABA. In this investigation, we examined the ABA content and growth rates of the developing seeds and pod walls and the growth of root-shoot axes excised from the developing seed at different developmental stages. A preliminary report of this work has been presented (14).

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A method for the analysis of lndole-3-acetic acid (IAA) in (23), and GC coupled with mass spectrometry (2,11,24). In most instances the GC methods require extensive purification of the plant extracts, derivative formation for volatility, and selective detectors. Although the absolute sensitivity of the GC and GC-MS methods is very high, the total sample sensitivity is often limited by the size of the sample injectable into the instruments. The high cost of GC-MS instrumentation makes these methods out of range for routine analysis by most workers.A sensitive spectrofluorometric assay for IAA was proposed (15,29) in which IAA is converted to indole-a-pyrone by a reaction with acetic anhydride and trifluoroacetic acid. An evaluation of the assay (7) Radioimmuno techniques are useful in assays of proteins, drugs and related hormones; however, the IAA molecule may be too small a hapten to impart specificity and sensitivity to the radioimmunoassay (9).The possible utility of HPLC for the determination ofcytokinins in plant extracts was suggested (18) and HPLC was used for the separation and/or determination of plant hormones (3,5,6,20,32). The HPLC-UV254 detector used to determine ABA levels (20, 32) detected ng quantities from a wide variety of plant extracts; however, the determination of IAA is complicated by the low A of IAA at 254 nm and the difficult separation of IAA from other plant components. This report outlines the conditions for the HPLC separation of IAA using several column-mobile phase systems and two sensitive, selective detectors for the quantitation of IAA in plant extracts. The HPLC method has been used to determine the IAA level in various plant tissues and these are compared with published data. The plant parts were dissected, weighed, frozen in liquid N2, and stored at -40 C until extraction for IAA analysis. MATERIALS AND METHODSPreparation of Plant Extracts. All reagents and solvents were ACS grade. The ether was further purified by passing through an aluminum oxide (Woelm, basic) column just before use. The over-all scheme for the extraction of free IAA, and the clean-up, was similar to that outlined (32). Plant material (0.1-5 g) was removed from the freezer and extracted in a blender with 75 ml of cold 80% methyl alcohol, to which was added a known aliquot of 3-indolyl [1-_4C]

for 1M anion and 1M solvating agent concentrations. The varying magnitudes of these extraction constants may be attributed to the increasing acidity of the proton of the solvating species in the order chloroform < -amyl alcohol < pzerz-butylphenol.Effect of ZVjiV-Dimethylcaprylamide on Solvating Agent.It was of interest to determine if the schematic model postulated in Case I was reasonable. This was done by utilizing an electron donor solvating agent. This agent would not be expected to enhance partitioning of the ion pair. An experiment was carried out using fixed concentrations of dextromethorphan, bromide ion, and /z-zerz-butylphenol but adding increasing amounts of dimethylcaprylamide to the organic phase. The results shown in Figure 8 indicate that the addition of the amide actually reduced the partition coefficient. These results would suggest that the amide did not act as a solvating agent even when present in rather high concentrations. Further, the decrease in partitioning at constant p-ZerZ-butylphenol could be expected, since in previous studies in these laboratories it has been shown that phenols formed strong association complexes with disubstituted aliphatic amides (11). Therefore, the addition of the amide resulted in the complexation of the phenolic solvating agent and essen-(11) E. G. Shami, unpublished Ph.D. thesis, University of Wisconsin, 1964.