Abstract
Betalains are natural pigments with diverse biological properties found in Celosia argentea var. plumosa, a member of the Amaranthaceae family. This study established an optimized cell suspension culture system for enhanced betalain production from C. argentea var. plumosa through combined elicitor treatment. Three elicitors were evaluated: 6-benzylaminopurine (BAP), methyl jasmonate (MeJA), and copper sulfate (CuSO(4)), using response surface methodology (RSM) based on central composite design (CCD). Under standard conditions, maximum total betalain content (TBC) and dry weight reached 35.61 mg/L (1.95 mg/g DW) and 19.90 g/L, respectively, on day 15. The optimal formulation consisted of 2.28 µM BAP, 49.97 µM MeJA, and 6.71 µM CuSO₄, applied during the exponential growth phase on day 9. These optimized conditions achieved a 3.9-fold increase in betalain production on day 15, reaching a maximum TBC of 139.99 mg/L (7.54 mg/g DW) with biomass of 16.90 g/L. Additionally, betalain extracts from cells cultured under optimal conditions demonstrated higher antioxidant capacity than unoptimized culture extracts. These findings suggest that using statistical experimental design with combined elicitors provides an optimized platform for scalable betalain production with enhanced bioactive properties. This information will be valuable for food, pharmaceutical, and cosmetic industries requiring natural colorants with functional benefits.