Plant Regeneration via Direct Organogenesis in Three Alfalfa (Medicago sativa L.) Cultivars Using Stem Nodal Explant

In this study, direct shoot regeneration of three alfalfa cultivars was investigated. Stem nodal explants isolated from in vitro germinated seedlings were cultured on Murashige and Skoog (MS) basal medium with Gamborg’s B5 vitamins supplemented with different concentrations and factorial combinations of BAP/NAA or TDZ/AgNO3. The analysis of variance revealed highly significant (P< 0.01) differences among the cultivars and among the different levels of growth regulators in view of shoot buds induction and regeneration. The maximum average number of induced shoot-bud (5.80 buds per explant, the average of three cultivars) and regenerated shoot (5.73 shoots per explant, the average of three cultivars) was achieved with the higher level of BAP (1 mgl-1) and NAA (0.2 mgl-1), while the maximum values (5.77 and 5.69 shoot buds and regenerated shoots, respectively) were obtained with the lowest level of TDZ (0.01 mgl-1) along with 3 mgl-1 AgNO3. Cultivars Ghareyonjeh, Hamedani and Ordobad were ranked, respectively, from point of view of shoot development and regeneration potentials. Regenerated plantlets were remarkably rooted on half-strength MS medium supplemented with 1.5 mgl-1 NAA and 1 mgl-1 IBA and rooted plants were acclimatized successfully in the greenhouse with a survival rate of 100%. The simple, rapid and efficient regeneration protocol presented in this study can be used in breeding programs as well as for genetic engineering of a wide range of Medicago species.