In vitro optimization of gallic acid biosynthesis from Camellia sinensis callus culture using ethanol extraction and high-performance liquid chromatography characterization

Gallic acid is a phenolic compound of high pharmacological and industrial relevance, commonly found in Camellia sinensis leaves. Conventional extraction from field-grown tea plants faces challenges of environmental dependence and inconsistent yields. Plant tissue culture provides a sustainable platform for secondary metabolite production under controlled conditions. This study aimed to optimize gallic acid biosynthesis through in vitro callus culture of C. sinensis leaves. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). Morphological changes and biomass accumulation were monitored over 35 days. Callus extracts were prepared using 70% ethanol and analyzed by high-performance liquid chromatography (HPLC) with a C18 column at 280 nm to identify gallic acid based on retention time comparison with authentic standards. Explants exhibited progressive dedifferentiation and callus formation, reaching compact bluish-green morphology by day 35. Biomass increased steadily throughout culture, peaking at 1.83 ± 0.07 g per explant. HPLC chromatograms confirmed gallic acid presence at a retention time of 4.36 ± 0.03 min, matching the standard peak. The findings demonstrate that C. sinensis callus retains biosynthetic capacity for phenolic compounds via the shikimate pathway. The established in vitro culture system effectively produced gallic acid, validating tea callus as a renewable biofactory for phenolic metabolites. Further optimization through hormonal regulation and elicitor application could enhance yield, supporting scalable and sustainable biotechnological production of natural antioxidants.