Somatic embryogenesis and plant regeneration in Medicago arborea L.

An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxy-acetic acid (2,4-D; 9 muM) and kinetin (9 muM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 muM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 muM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0 +/- 0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0 +/- 0.89%) when cultured on basal MS medium with 5 muM indolebutyric. acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N-6-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 muM.