Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago truncatula cv. Jemalong

摘要

Plants were successfully regenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertn. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30d in vitro-grown plants. Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 mu M 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 mu M kinetin (Kin) and subcultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 mu M 2,4-D and 0.91 mu M zeatin (Zea), during 1, 2, and 3 wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of I and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3 wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5 +/- 1.6, 52.5 +/- 18.5, and 41.6 +/- 8.4% for 0, 1, and 2wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.