Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

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Abstract
An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, ‘Tan-ginbozu’ dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500μg GA3-equivalents) and 12–13 (270 μg GA3-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA1, GA3, GA4 and GA8 were active in the Phaseolus bioassay but GA8-glucoside was inactive. The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA4, GA19 and GA1. The identity of GA4 in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA19 and GA1 were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.