Analysis of indole-3-butyric acid-induced adventitious root formation on Arabidopsis stem segments

Root induction by auxins is still not well understood at the molecular level. In this study a system has been devised which distinguishes between the two active auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). IBA, but not IAA, efficiently induced adventitious rooting in Arabidopsis stem segments at a concentration of 10 microM. In wild-type plants, roots formed exclusively out of calli at the basal end of the segments. Root formation was inhibited by 10 microM 3,4,5-triiodobenzoic acid (TIBA), an inhibitor of polar auxin transport. At intermediate IBA concentrations (3-10 microM), root induction was less efficient in trp1, a tryptophan auxotroph of Arabidopsis with a bushy phenotype but no demonstrable reduction in IAA levels. By contrast, two mutants of Arabidopsis with measurably higher levels of IAA (trp2, amt1) show root induction characteristics very similar to the wild type. Using differential display, transcripts specific to the rooting process were identified by devising a protocol that distinguished between callus production only and callus production followed by root initiation. One fragment was identical to the sequence of a putative regulatory subunit B of protein phosphatase 2A. It is suggested that adventitious rooting in Arabidopsis stem segments is due to an interaction between endogenous IAA and exogenous IBA. In stem explants, residual endogenous IAA is transported to the basal end of each segment, thereby inducing root formation. In stem segments in which the polar auxin transport is inhibited by TIBA, root formation does not occur.