Overexpression of miR319 in petunia (Petunia × hybrida) promotes de novo shoot organogenesis from leaf explants

Abstract

MicroRNA319 (miR319) plays an important role in leaf development. Although leaf discs are frequently used for plant transformation, little is known about the function of miR319 in shoot regeneration from leaf explants. To investigate the role of miR319 in de novo shoot organogenesis from leaf explants, the MIR319 gene was overexpressed in petunia (Petunia × hybrida), under the control of the 35S promoter. Petunia leaves overexpressing miR319 (miR319-OX) showed several morphological abnormalities, such as rounded, uneven, and curled leaf blade, larger leaf size, and smaller and rounded abaxial surface cells, which were differences compared to wild-type (WT) leaves. Additionally, miR319-OX leaf explants demonstrated enhanced shoot organogenesis on a growth medium containing 6-benzylaminopurine (BA) at shoot-inducing concentrations, and cytokinin-independent shoot regeneration on hormone-free medium compared with WT explants. Quantitative real-time PCR (qRT-PCR) analysis indicated that the expression of petunia NO APICAL MERISTEM (PhNAM) and SHOOT MERISTEMLESS (PhSTM) genes was markedly increased in miR319-OX explants compared with WT explants under shoot induction conditions, whereas the expression of petunia cytokinin-responsive type-A response regulator genes, PhRR2 and PhRR3, showed no differences in expression between WT and miR319-OX leaf explants. These results suggest that miR319 regulates the formation of shoot meristems from leaf explants in petunia during in vitro culture.