Histochemical detection of H2O2 by tissue printing as a precocious marker of rhizogenesis in grapevine

For the histological detection and localisation of H2O2 as a precocious marker of rhizogenesis, a sensitive tissue-printing assay in freshly cut stem sections was used. With this technique, we could detect and localise H2O2 present in tissues during the adventitious rooting process of Vitis vinifera L. cuttings, induced in vitro by auxin. In non induced stems, H2O2 is preferentially associated to epidermis, pith parenchyma and vascular cambium, xylem and phloem appearing devoid of H2O2. Tissue-printing of auxin treated cuttings permitted the identification of a typical set of characteristics that allowed to define nine histological patterns during root induction and elongation. On the second day of auxin treatment, vascular cambium became irregular, with medullary rays losing their previous organisation; on day four, the vascular cambium was totally indistinct. After auxin removal, it was possible to identify the meristemoids initially as uncoloured spots and afterwards the adventitious root primordia; vascular cambium began to re-organise except in the zones of the root primordia. As root elongated, it was possible to identify the root vascular system as coloured cells, and the cortex cells as non coloured ones, contrasting with the stem pattern. Control material without auxin allowed the confirmation that the observed patterns were well associated to rhizogenesis.