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Detection of Isoflavones in Seedling Subterranean Clover

Univ. of California Cooperative Ext. Servi., Bishop, CA 93514
Texas A&M Univ. Agric. Res. and Ext. Ctr., Overton, TX 75684

^* Corresponding author.

Reproductive disorders in sheep (Ovis ones) have been linked to ingestion of subterranean clovers (Trifolium brachycalycinum Katzn. & Morley, T. subterraneum L., and T. yanninicum Katzn. & Morley) containing high levels of the phytoestrogenic isoflavones formononetin (7-hydroxy-4-methoxyisoflavone; FM), genistein (5,7,4-trihydroxyisoflavone; GE), and biochanin A (5,7-dihydroxy-4-methoxyisoflavone; BA). Breeding programs involving subterranean clovers should include selection for low isoflavone concentrations. Our objective was to develop a seedling assay that would predict field levels of FM, GE, and BA in subterranean clover. Five subterranean clover cultivars were grown in the field at Overtoil, TX, over a 3-yr period (1985, 1986, and 1987); leaf samples were analyzed for FM, GE, and BA. The same cultivars were used to test potential seedling assays. Six seedling growth stages were used: ungerminated seed (UGS), swelled seed (SWS), seedlings after 48 h germination with testa removed (2DAYS), seedlings grown in distilled water for 8 d (8DAYS), seedlings grown in the greenhouse for 42 d (42DAYS), and regrowth harvested 21 d later (REGRO). Tissue samples were extracted with methanol (7 d at 5 °C) and analyzed using high performance liquid chromatography HPLC techniques. Field-grown samples of each of the cultivars were consistent with previously known values for FM, GE, and BA. Only trace amounts of isoflavones were detected in the UGS and SWS treatments. The 2DAYS and 8DAYS seedling treatments were highly variable and inconsistent in regard to isoflavone content compared with field data for the cultivars. Regression equations using the 42DAYS and REGRO treatments were accurate in their prediction of FM, BA, and total isoflavones in field-grown subterranean clover. The 42DAYS and REGRO treatments for determining isoflavone content of subterranean clover seedlings could be used to rank germplasm for potential phytoestrogen effects.

Received for publication June 4, 1990.