The objective of this study was to investigate the phenotypic and genetic effects of long-term (12 years) in vitro culturing of Astragalus chrysochlorus callus tissue. Murashige and Skoog medium supplemented with 0.5mg/l 2,4-dichlorophenoxyacetic acid was used for the production of young callus (2 months old) from hypocotyl explants. Morphological analysis showed that long-term cultures were well adapted, with greener and more friable calli, and a higher growth index (2.11 +/- 0.39) was compared with that in short-term cultures (1.2 +/- 0.73). Genetic variation between the two types of calli was investigated by randomly amplified polymorphic DNA analysis, which showed different amplification patterns with a polymorphism level of about 45%. We furthermore measured RNA transcript levels for two key enzymes of the important stress-related phenylpropanoid pathway, PAL and C4H, via quantitative polymerase chain reaction. The gene expression levels of C4H and PAL were 0.205- and 0.584-fold lower in old cultures than in young cultures. Metabolic analysis by high performance thin layer chromatography clearly showed differences in flavonoid type metabolic content. Together, these data show that long-term culturing of callus tissue leads to genetic heterogeneity and variations in secondary metabolite content.