A highly-sensitive rice seedling bioassay for the detection of femtomole quantities of 3?-hydroxylated gibberellins

Abstract

A very sensitive and specific bioassay using prohexadione calcium [BX-112, which blocks 2β- and 3β-hydroxylation of gibberellins (GAs)] with uniconazole (which blocks oxidation of ent-kaurene, ent-kaurenol and ent-kaurenal) in a microdrop assay was developed for several rice (Oryza sativa L.) varieties, including cv. Waito-C, which is already specific to 3β-hydroxylated GAs. The sensitivity and specificity of cvs. Waito-C, Tan-ginbozu and Koshihikari to 3β-hydroxylated GAs was greatly enhanced by treatment of the seeds with a combination of 40 mM prohexadione calcium and 80 μM uniconazole. The minimum detectable doses of 3β-hydroxylated GAs (GA1, GA3, GA4 and GA7) in the three cultivars treated with both chemicals were 1 to 10 fmol (i.e. ca. 350 fg to 3.5 pg) per plant. This is equal to 30-fold more sensitive than Waito-C treated with uniconazole alone, and 30 to 1000-fold more sensitive than Waito-C with no growth retardant soak. Minimum detectable doses of 3-nonhydroxylated GAs (GA9, GA19 GA20) and GAs with very low biological activity (GA8 and GA17) were equal to or more than 1000 fmol per plant. This is about equal to the activity in Waito-C treated with uniconazole alone. Application of this assay to an extract from Raphanus sativus was compared with the data by gas chromatography/mass spectrometry (GC/MS), confirming the conclusions reached using authentic test GAs, namely that use of uniconazole plus BX-112 appreciably enhanced the detection sensitivity to fractions shown by GC/MS to contain GA1 and GA4, both 3β-hydroxylated GAs.