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Background: The origin of somaclonal variation has not been questioned previously, i.e., ''pre-existing mutations'' in explants and ''newly induced mutations'' arising from the tissue culture process have not been distinguished. This is primarily because there has been no reliable molecular method for estimating or quantifying variation. Methodology/Principal Findings: We adopted a petal-variegated cultivar of Saintpaulia 'Thamires' (Saintpaulia sp.) as the model plant. Based on the difference between the pre- and post-transposon excision sequence of the promoter region of flavonoid 39, 59-hydoroxylase (F3959H), we estimated mutated (transposon-excised) cell percentages using a quantitative realtime PCR. Mutated cell percentages in leaf laminae used as explants was 4.6 and 2.4% in highly or low variegation flower plants, respectively, although the occurrences of blue color mutants in their regenerants were more than 40%. Preexisting mutated cell percentages in cultured explants were considerably lower than the mutated plant percentage among total regenerants via tissue culture. Conclusions/Significance: The estimation of mutated cell percentages became possible using the quantitative real-time PCR. The origins of mutations were successfully distinguished; it was confirmed that somaclonal variations are mainly caused by newly generated mutations arising from tissue culture process [ABSTRACT FROM AUTHOR]