In vitro regeneration of the medicinal woody plant Phellodendron amurense Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm(2) sections of about 10-day-old leaves on MS medium enriched with 4.4 muM BAP and 1.0 muM NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 muM TDZ and 4.0 muM 2,4-D or 4.0 muM NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 muM BAP and 1.0 muM NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2-4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 muM IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2-6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.