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 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 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subcultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM 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 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 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 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.
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1 May 2006
EFFICIENT SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM LONG-TERM CELL SUSPENSION CULTURES OF MEDICAGO TRUNCATULA CV. JEMALONG
ANA SOFIA DUQUE,
ANA SOFIA PIRES,
DULCE METELO DOS SANTOS,
PEDRO FEVEREIRO
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In Vitro Cellular and Developmental Biology - Plant
Vol. 42 • No. 3
May 2006
Vol. 42 • No. 3
May 2006
cell suspension culture
embryogenic competence
line M9-10a
Medicago truncatula