Origin, timing, and gene expression profile of adventitious rooting in Arabidopsis hypocotyls and stems

Adventitious root (AR) formation is indispensable for vegetative propagation, but difficult to achieve in many crops. Understanding its molecular mechanisms is thus important for such species. Here we aimed at developing a rooting protocol for direct AR formation in stems, locating cellular AR origins in stems and exploring molecular differences underlying adventitious rooting in hypocotyls and stems.

In-vitro-grown hypocotyls or stems of wild-type and transgenic ecotype Columbia (Col-0) of Arabidopsis thaliana were rooted on rooting media. Anatomy of AR formation, qRT-PCR of some rooting-related genes and in situ GUS expression were carried out during rooting from hypocotyls and stems.

We developed a rooting protocol for AR formation in stems and traced back root origins in stems by anatomical and in situ expression studies. Unlike rooting in hypocotyls, rooting in stems was slower, and AR origins were mainly from lateral parenchyma of vascular bundles and neighboring starch sheath cells as well as, to a lesser extent, from phloem cap and xylem parenchyma. Transcript levels of GH3-3, LBD16, LBD29, and LRP1 in hypocotyls and stems were similar, but transcript accumulation was delayed in stems. In situ expression signals of DR5::GUS, LBD16::GUS, LBD29::GUS, and rolB::GUS reporters in stems mainly occurred at the root initiation sites, suggesting their involvement in AR formation.

We have developed an efficient rooting protocol using half-strength Lepoivre medium for studying AR formation in stems, traced back the cellular AR origins in stems, and correlated expression of rooting-related genes with root initiation sites.