Graduate Student Fort Valley State University Fort Valley, Georgia
Body of Abstract: The focus of this study was to establish an efficient protocol for somatic embryogenesis and plant regeneration in alfalfa, a perennial crop known for its nutritional and medicinal value as a forage legume. To initiate somatic embryogenesis from leaf explants of in vitro seed-derived plants, Gamborg's B5 basal (B5H) medium supplemented with 0.9 µM Kinetin, 4.5 µM 2,4-D, and different sugars (sucrose and maltose) was utilized. The objective was to optimize the protocol by evaluating the impact of different types and concentrations of carbohydrate sources on callus growth, the number of somatic embryos per leaf explant, and the percentage of globular embryos developing into cotyledonary embryos. Among the different sugars tested such as glucose, sucrose, maltose, mannitol, sorbitol, and fructose; maltose (3%) significantly enhanced somatic embryo production. After three weeks of culture on maltose medium, pro-embryonic cells were successfully initiated, whereas Calli obtained from sucrose-containing media were fragile/non-embryogenic and consisted predominantly of parenchymatous cells. Notably, the embryogenic calli cells on maltose medium exhibited rapid formation of globular embryos. Callus mass was found to increase with lower sugar concentrations (1% to 3%), while higher concentrations (up to 5%) led to alterations in callus color and reduced proliferation rate. The successful development of this regeneration system holds great promise for the large-scale production of somatic embryos, facilitating continuous cultivation of alfalfa and enabling genetic transformation studies.