For determination of the endogenous and exogenous causes of somaclonal variation in in vitro culture, a bioassay system was developed using the variegated Saintpaulia (African violet) ‘Thamires’ (Saintpaulia sp.), having pink petals with blue splotches caused by transposon VGs1 (Variation Generator of Saintpaulia 1) deletion in the promoter region of flavonoid 3',5'-hydroxylase. Not only true-to-type but also many solid blue and chimeric plants regenerate in vitro-cultured explants of this cultivar. Using multiplex PCR that enables the determination of these variations, we attempted to evaluate the effects of four candidate triggers of mutation: pre-existing mutated cells, shooting conditions in vitro or ex vitro, cutting treatment of explants, and addition of plant growth regulators (PGRs) to the medium. The percentages of somaclonal variations among total shoots regenerated from leaf segments and stamens were 46.6 and 56.5, which were higher than the percentages expected from pre-existing mutated cells (3.6 and 1.4, respectively). These results indicate that pre-existing mutated cells are not a main cause of somaclonal variations. The percentage of somaclonal variation was independent of culture conditions for mother plants; the mutation percentages of adventitious shoots regenerated from ex vitro- and in vitro-grown leaves were 9.2% and 8.5%, respectively. In addition, the percentage of somaclonal variations of adventitious shoots regenerated under in vitro conditions from the in vitro grown mother plants was also low, at 4.9%. This indicates that the in vitro condition itself is not a main cause of somaclonal variation. However, when adventitious shoots were regenerated from 10 × 5-mm cut-leaf laminas on a PGR-free medium, the percentage of somaclonal variation was 26.4%. In addition, the percentage of somaclonal variations dramatically increased when PGRs were added to the medium for both leaves and leaf segments (39.9 and 46.6, respectively). The bioassay system using Saintpaulia ‘Thamires’ will enable the screening of many environmental factors because of its rapidity and ease of use and will facilitate the development of a new tissue culture technology for avoiding mutation.