(800-31) Influence of activated charcoal, cefotaxime, and amino acids on the production and development of somatic embryos and plant regeneration in alfalfa (Medicago sativa)
Post doctoral researcher Fort Valley State University Fort Valley, Georgia
Body of Abstract: Alfalfa (Medicago sativa) is a widely cultivated perennial crop known for its significance as a forage legume and its nutritional and medicinal value. This study aimed to investigate the effects of activated charcoal, cefotaxime, and amino acids on callus induction, somatic embryogenesis, and subsequent plant regeneration in alfalfa leaf explants. The initiation of embryogenic callus (97.5%) was achieved from alfalfa leaf explants on Gamborg's B5 basal (B5H) medium supplemented with 4.5 μM 2,4-D, 0.9 μM kinetin, 6.65 g Glutamine, 0.8 g Serine, 0.004 g Adenine, and 0.08 g L-glutathione after a three-week incubation period in the dark. Various concentrations of activated charcoal (0, 0.25, 0.5, 0.75, 1, 2, 4%) (w/v) were tested in B5H medium with and without 4.5 µM 2,4-D. The highest percentage (85.2%) of somatic embryos was obtained with a medium containing 4.5 µM 2,4-D. Different concentrations of cefotaxime (0, 200, 300, 400, 500, 600, and 700 mg/L) were examined, with the highest percentage (93.8%) of globular structured somatic embryo induction and maturation observed on a medium containing 500 mg/L cefotaxime after three to four weeks of incubation in the dark. Higher concentrations of cefotaxime hindered somatic embryo formation. Somatic embryos cultured on a medium supplemented with 1.0% activated charcoal and 500 mg/L cefotaxime successfully regenerated into normal plantlets, with a slightly lower regeneration rate of 65.4% compared to 79.8% on MS basal medium, respectively. Concentrations of activated charcoal exceeding 2.0% and cefotaxime beyond 500 mg/L resulted in the formation of abnormal somatic embryos, which subsequently produced abnormal plantlets. Each healthy plantlet was transferred to an acclimatization medium containing half-strength MS medium with 3% sucrose and 0.03% GELRITE®. The in vitro regenerated plantlets were carefully transplanted into soil cups, initially maintained in a growth chamber for one week, and later hardened and grown to maturity in the field.