Carbon sources play a critical role in plant tissue culture. This study evaluates the effects of different carbon sources—including monosaccharide hexoses (fructose, glucose, and galactose), disaccharides (maltose, sucrose, and lactose), and sugar alcohols (mannitol, sorbitol, and glycerol)—at concentrations ranging from 1% - 5% on in vitro plant regeneration in alfalfa (Medicago sativa L.). All leaf explants successfully induced callus formation when cultured on Gamborg’s B5 (B5h) medium supplemented with either monosaccharides or disaccharides in darkness for three weeks. Similarly, somatic embryo induction and maturation were enhanced in media containing monosaccharides (85.4% - 100%) and disaccharides (97.5% - 100%). Among all treatments, maltose at 3% exhibited rapid progression to the globular stage, yielding a 96.9% embryo-to-shoot conversion rate and the lowest incidence of shoot loss. Regenerated plantlets were successfully acclimatized and transferred to soil, achieving survival rates ranging from 50% - 100%, depending on the carbon source. These findings underscore the importance of carbon source selection for optimizing somatic embryogenesis protocols and advancing large-scale micropropagation of alfalfa and related legumes.
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