Two-stage system for micropropagation of several Genista plants producing large amounts of phytoestrogens.

A two-stage method for in vitro propagation of six Genista species from shoot tips was developed. Multiple microshoot cultures were obtained by growing the shoot tip explants on Schenk and Hildebrandt medium supplemented with 9.84 microM 6-(gamma,gamma-dimethylallylamino)-purine and 0.99 microM thidiazuron. The best shoot elongation was achieved on Schenk and Hildebrandt medium containing 4.92 microM indole-3-butyric acid. The rooting of shoots brought best effects (100%) on Schenk and Hildebrandt medium with 2.68 microM 1-naphthaleneacetic acid. HPLC analysis indicated that six-month-old regenerated plants as well as the herb of intact plants produced a rich set of simple flavones (derivatives of luteolin and apigenin) and isoflavones (derivatives of genistein, daidzein, formononetin and biochanin A). Multiple microshoot cultures of all species produced no simple flavones at all. In vitro shoots accumulated selectively a rich group of phytoestrogens in the form of aglucones, glucosides and esters (derivatives of genistein and daidzein). Cultures obtained in vitro synthesized many times more isoflavones than the intact plants. In all shoots which were micropropagated the dominating compound was genistin (e.g. shoots of G. tinctoria--ca 3281.4 mg per 100 g dry weight). Possible influence of tissue differentiation on isoflavone content under in vitro and in vivo conditions is discussed.