A HIGHLY-SENSITIVE RICE SEEDLING BIOASSAY FOR THE DETECTION OF FEMTOMOLE QUANTITIES OF 3-BETA-HYDROXYLATED GIBBERELLINS

A very sensitive and specific bioassay using prohexadione calcium [BX- 112, which blocks 2beta- and 3beta-hydroxylation of gibberellins (GAs) ] with uniconazole (which blocks oxidation of ent-kaurene, ent-kaureno l and ent-kaurenal) in a microdrop assay was developed for several ric e (Oryza sativa L.) varieties, including cv. Waito-C, which is already specific to 3beta-hydroxylated GAs. The sensitivity and specificity o f cvs. Waito-C, Tan-ginbozu and Koshihikari to 3beta-hydroxylated GAs was greatly enhanced by treatment of the seeds with a combination of 4 0 mM prohexadione calcium and 80 muM uniconazole. The minimum detectab le doses of 3beta-hydroxylated GAs (GA1, GA3, GA4 and GA7) in the thre e cultivars treated with both chemicals were 1 to 10 fmol (i.e. ca. 35 0 fg to 3.5 pg) per plant. This is equal to 30-fold more sensitive tha n Waito-C treated with uniconazole alone, and 30 to 1000-fold more sen sitive 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 fmo l per plant. This is about equal to the activity in Waito-C treated wi th uniconazole alone. Application of this assay to an extract from Rap hanus sativus was compared with the data by gas chromatography/mass sp ectrometry (GC/MS), confirming the conclusions reached using authentic test GAs, namely that use of uniconazole plus BX-112 appreciably enha nced the detection sensitivity to fractions shown by GC/MS to contain GA1 and GA4, both 3beta-hydroxylated GAs.