Quantitative determination of indole-3-acetic Acid in sugarbeet leaves using a double standard isotope dilution gas chromatographic assay

Quantitative levels of indole-3-acetic acid (IAA) were determined in leaf blades of two sugarbeet cultivars by a double standard isotope dilution assay using column chromatography followed by reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography with nitrogen thermionic detection. The double standard method was validated as a quantitative tool by gas chromatography/selected ion monitoring mass spectrometry using 2,′,4′,5′,6′,7′-d₅-IAA as the internal standard. Progenies of one breeding line that had been selected for a high taproot to leaf weight ratio were used to correlate IAA levels with varying leaf and plant size at day 31 from germination. In spite of size differences, no significant difference in IAA levels per unit leaf weight could be found. The possible relationship between day 31 leaves and IAA content at an earlier stage of development is discussed in the text. A second analysis used four developmental leaf stages, classified as expanding, recently mature, aging, and senescing leaves. Expanding leaves contained the most IAA, senescing leaves contained the least IAA, with recently mature leaves and aging leaves containing intermediate amounts. The DNA content of each of the four developmental leaf stages was determined and DNA levels per gram fresh weight were found to be constant at all developmental stages.