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Betanin belongs to a large family of betacyanin pigments, betalains, and in food industry it is established as a powerful antioxidant and natural colorant. The main source of betanin is a red beet (Beta vulgaris L.), whose yield depends on abiotic and biotic factors in the field. Sugar beet cells, transformed by a wild strain B6S3 of Agrobacterium tumefaciens, overproduce betanin and could be its alternative and stable production source. With the aim to enhance its production, cell suspensions were initiated. Biomass accumulation, betanin production and yield were monitored during 21 days in relation to changes of sucrose content, modifications of minerals in nutrient medium, and usage of elicitors. Results showed that elevating sucrose levels from 3 to 4, 5, or 6% (w/v) in original medium strongly induced biomass accumulation, resulting in increase in betanin production and yield up to 250%. Modification of minerals in the medium additionally increased it up to 20%. The highest production at 4.1 mg L-1 day-1 and yield at 41 mg L-1 was recorded at day 7 of subculture in modified medium with 6% of sucrose. Calcium and yeast extract were used as elicitors in early exponential phase of subculture (day 7). Calcium ions (at 10 fold higher concentration than in control) failed to increase betanin yield as well as yeast extract at 0.25% (w/v). Yeast extract at 1.25% provoked excretion of betanin at day 4, and cell necrosis at day 7 after elicitation. Taken together, sucrose was stronger stimulant of betanin production than medium modifications or elicitors. Yeast extract could be used for reverse-sequestration of betanin where the cells can be used over an extended period.