Somatic embryogenesis and plant regeneration in Medicago arborea L.

An efficient plant regeneration system employing cotyledons, hypocotyls, pe tioles 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 tw o-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxy-ac etic acid (2,4-D; 9 muM) and kinetin (9 muM), and followed by transfer to k inetin-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 c ulture were critical for enhanced production of somatic embryos. The best e xplants 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 convert ed into normal plantlets (8.0 +/- 0.89%) when cultured on basal MS medium w ith 5 muM indolebutyric. acid. No somatic embryos were obtained when thidia zuron was used in the culture media. Using petioles as explants and N-6-ben zyladenine (BA), embryogenesis was induced in the second step of culture wh en BA was removed from the medium and the concentration of 2,4-D was decrea sed to 2.25 muM.