A simple and reliable method was established for the maintenance of a permanent stock of several Medicago truncatula genotypes selected from a general seed stock by their in vitro culture amenability and embryogenic capacity. In the first step, multiple shoots were induced from the cotyledon axillary meristem meristem of pre-germinated seeds in Murashige and Skoog medium supplemented with cytokinins (9.3 M zeatin, 22.2 M benzylaminopurine or 4.5 M thidiazuron). In the second step, the induced shoots were allowed to develop in growth-regulator-free medium. Benzylaminopurine at 22.2 M supported the best combination of shoot quality and number of shoots produced. Rooting of microshoots depended on the cytokinin used for shoot induction and was faster for zeatin-treated shoots. In this work a propagation system was devised where the addition of growth regulators was restricted to the induction phase therefore reducing the risks of epigenetic and somaclonal variation.