An easy method for cutting and fluorescent staining of thin roots

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. 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. 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. 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.