Improvement of somatic embryogenesis in Medicago arborea

A number of medium constituents were evaluated in an attempt to improve somatic embryo production in Medicago arborea ssp. arborea, using cotyledons, petioles and leaves as explants. Two culture steps were applied: in the first stage (2 months), Murashige-Skoog (MS)medium was used, containing 2,4 dichlorophenoxyacetic acid (9µM 2,4-D)and kinetin (9 µM KIN)together with different nitrogen sources (alanine, glutamine, proline or tryptophan (2.5 and 5 mM); casein hydrolysate (100, 500 and 1000 mg l-1; nitrate (4.69 and 9.39 mM)or casein hydrolysate (100 mg l-1)and nitrate (4.69 mM)), polyalcohols (mannitol at 164 and 328 mM or sorbitol at 219 and 438 mM), sucrose (43.8 and 175.4 mM)or calcium (1.5 and 6 mM). In the second stage (3 months of cultivation), calli were transferred to a kinetin-free MS medium with 2,4-D (2.25 µM)only. The inclusion of proline (2.5 mM)was the most effective treatment for the induction of somatic embryos, with the petiole being the best explant. Treatment with casein hydrolysate (100 mg l-1)also improved the embryonic efficiency. The rest of the treatments neither affect nor inhibit the embryonic response. A special treatment with sorbitol (219 mM)in the second stage of cultivation produced a slight increase in embryogenesis, but less than that obtained with proline.