Plants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of
Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of
30 d
in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented
with 2.3 μ
M 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μ
M kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45
μ
M 2,4-D and 0.91 μ
M zeatin (Zea), during 1,2, and 3wk 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 1 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 3wk 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 2 wk induction treatments, respectively.
These plants were successfully transferred to the greenhouse where they matured and produced seeds.
Key words embryogenic competence - cell suspension culture -
Medicago truncatula
- line M9-10a