Secondary Somatic Embryogenesis of Pimpinella brachycarpa

For the induction of somatic embryogenic callus, shoot-tip explants of pimpinella brachycarpa were cultured on MS medium supplemented with 2.5μM2,4-D and 0.5 μM BAP in the dark for 4 weeks. Embryogenic calli were cluster of small and dense cells termed proembryogenic masses (PEMs), whereas non-embryogenic calli were friable. These PEMs developed into somatic embryo when transferred to MS medium with 2.5μM 2,4-D and 0.5 μM BAP. After three weeks of culture, globular, heart and torpedo-stage embryos appeared and these somatic embryos were transferred ot MS liquid medium with 0.5 μM NAA at a density of 20 embryos/mL and developed into plantlets. Secondary somatic embryogenesis took placed in subculturing primary embryos on MS medium with 2.5 μM 2,4-D and 0.5 μM NAA at a frequency of 72%. After four weeks of culture in the liquid medium, these somatic embryos developed into plantlets showing cotyledons, hypocotyls and radicles. This results suggest a novel protocol for the conditions necessary to induce secondary embryogenesis and micropropagation of P.brachycarpa.