Double flower formation induced by silencing of C-class MADS-box genes and its variation among petunia Cultivars

• Double flower formation by VIGS of C-class homeotic genes was examined in petunia.
• Cultivar-dependent new flower formation in pMDAS3/FBP6-VIGS flower was observed.
• Petaloid stamen size was mostly determined at the early developmental stage.
• A-class gene expression had little biological contribution to petaloid tissue development.

Double flowers induced by virus-induced gene silencing (VIGS) of two C-class MADS-box genes, pMADS3 and FBP6, were investigated in four cultivars of Petunia hybrida. In flowers induced by either pMADS3-VIGS or FBP6-VIGS, only small changes in commercial appearance were recognized regardless of cultivar, whereas in those induced by pMADS3/FBP6-VIGS, complete conversion of stamens into petaloid tissues and marked enlargement of upper limb-like tissues were observed, resulting in a decorative appearance in all the four cultivars. In whorl 4, cultivar-dependent conversion of carpels into new flowers was noted in pMADS3/FBP6-VIGS flowers. Of the four cultivars, only ‘Mambo Purple’ exhibited the development of new flowers instead of carpels and the emergence of ectopic new flowers from the axil of whorl 3 organs, which created an ornamental appearance with a high commercial value. Investigation of large and small petaloid stamens induced by pMADS3/FBP6-VIGS and pMADS3-VIGS, respectively, revealed only small differences in cell size compared to the large difference in total surface area. Quantitative RT-PCR analysis of SQUAMOSA/AP1/FRU type A-class genes, FBP29, PFG, and FBP26, showed no close relationship between the expression of those genes and petaloid stamen size. The suppressed C-class gene function at the late stage of flower development has little influence on the final size of petaloid tissue.