Ivan Zelko

An easy method for cutting and fluorescent staining of thin roots

BACKGROUND AND AIMS Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here. METHODS To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light. KEY RESULTS Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots. CONCLUSIONS This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.

Growth and anatomical parameters of adventitious roots formed on mung bean hypocotyls are correlated with galactoglucomannan oligosaccharides structure.

The effect of galactoglucomannan oligosaccharides (GGMOs) compared with chemically modified oligosaccharides, GGMOs-g (with reduced number of D-galactose side chains) and GGMOs-r (with reduced reducing ends) on mung bean (Vigna radiata (L.) Wilczek) adventitious roots formation, elongation, and anatomical structure have been studied. All types of oligosaccharides influenced adventitious root formation in the same way: stimulation in the absence of exogenous auxin and inhibition in the presence of exogenous auxin. Both reactions are probably related with the presence/content of endogenous auxin in plant cuttings. However, the adventitious root length was inhibited by GGMOs both in the absence as well as in the presence of auxin (IBA or NAA), while GGMOs-g inhibition was significantly weaker compared with GGMOs. GGMOs-r were without significant difference on both processes, compared with GGMOs. GGMOs affected not only the adventitious root length but also their anatomy in dependence on the combination with certain type of auxin. The oligosaccharides influenced cortical cells division, which was reflected in the cortex area and in the root diameter. All processes followed were dependent on oligosaccharides chemical structure. The results suggest also that GGM-derived oligosaccharides may play an important role in adventitious roots elongation but not in their formation.