Synchronization of somatic embryogenesis at high frequency using carrot suspension cultures: model systems and...

Materials and methods for the high frequency induction and synchronous somatic embryogenesis from cultured cells of higher plants are described, using carrot suspension cultures as a model system of higher plants. The following four synchronous systems of somatic embryogenesis, which were established in our laboratories, are reported: (1) Somatic embryogenesis from single cells. a) Small spherical single cells, obtained from suspension cultures in the presence of 2,4-D, zeatin and mannitol by sieving, density gradient centrifugation in Percoll solutions and manual picking up, form embryogenic cell clusters, which differentiate to embryos at high frequency, when embryogenic cell clusters are transferred to a medium lacking 2,4-D. b) Explants of hypocotyls of regenerated plantlets from somatic embryos were cultured after treatment with 2,4-D for 12-24 h, and then transferred into a fresh medium lacking 2,4-D. Single cells are released from hypocotyl explants and differentiated into embryos at high frequency. In this system, a large number of single cells and embryogenic cells can be collected. (2) Somatic embryogenesis from embryogenic cell clusters, which are obtained from suspension cultures by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed, differentiate synchronously to globular embryos at high frequency. Plantlets are formed from globular embryos. (3) Embryogenic cell clusters obtained according to the procedure described in (2) are cultured at cell densities of 2x10(3) cell clusters ml(-1). Globular embryos differentiate to torpedo-shaped embryos and subsequently to plantlets at high frequency when they are cultured at densities below 150 globular embryos ml(-1).