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Background  Phenotypic and genotypic variations, collectively called somaclonal variations, are induced during tissue culture.    Results  We studied the phenotypic variation in pollen viability of regenerants of torenia after subculturing for one to nine generations. We found that pollen viability of regenerants continuously decreased with increasing subculture time. High concentrations of plant growth regulators applied to the Murashige and Skoog (MS) medium also resulted in diminished pollen viability. Furthermore, antibiotic application during gene transformation also decreased pollen viability of the transformants. However, the process of long-term culture did not significantly change pollen viability. The mean methylation level of regenerants showed a 0.28% to 3.95% decrease in seedlings subcultured in vitro for nine generations. Moreover, when the ninth subcultured regenerants with reduced pollen vibility were recovered in soil to get seeds, the pollen viability of seed-derive plants was similar to that of the wild type.    Conclusions  The results show that plant growth regulators, antibiotics, and the number of subculture generations influence somaclonal variations in torenia. The somaclonal variations in torenia may results from epigenetic changes.    Electronic supplementary material  The online version of this article (doi:10.1186/1999-3110-54-36) contains supplementary material, which is available to authorized users.