Critical steps and troubleshooting in sample preparation for wood and phloem formation: from sampling to microscopic observation

Abstract

We present a technical note that supplements published procedures on optimal sample preparation for performing wood and phloem formation analyses. Before beginning sampling, it is important to learn about the characteristics of the tree or shrub species to be investigated. Some tips are given how to use the Trephor tool in the best way, how to remove the outer hard bark (periderm), how microcores should be handled after removal from the tree, and how they should be oriented for embedding in paraffin, and cutting thin sections for microscopy. Possible defects that may result from improper handling are illustrated and discussed. We also present optimal images to accurately identify different cell development stages in phloem and xylem, which is particularly challenging in hardwoods and Mediterranean tree and shrub species.

Anatomy of xylem and phloem in stems and roots of Populus sibirica and Ulmus pumila from semi-arid steppe in Mongolia

“…Therefore, tissues must be efficiently fixed and appropriately stored before section preparation can begin. Cutting is usually complicated by the fact that plant tissue is generally heterogeneous and fragile (Balzano et al, 2022). These limitations can be exacerbated when we sample species for which there are few or no studies of wood and bark anatomy, and when we work in difficult, remote environments, as in our case in semi-arid areas in the Mongolian steppe, far from the laboratory.…”

“…The microcores were initially stored in FAA (mixture of formalin, acetic acid and ethanol) and then dehydrated in gradient ethanol series (70, 90, 95 and 100%), infiltrated with bio-clear (D-limonene) and embedded in paraffin blocks using a Leica TP1020-1 tissue processor (Nussloch, Germany). Using a semi-automatic rotary microtome (RM 2245, Leica, Nussloch), cross-sections (9 µm thick) were obtained, and subsequently stained with safranin (0.04%) and astra blue (0.15%) water solution (Prislan et al, 2013a;Balzano et al, 2022;Prislan et al, 2022). The samples were mounted in Euparal (Bioquip Rancho Domingez, CA, USA) and observed under a light microscope -transmitted light mode Zeiss Axio Imager A.2 light microscope (Carl Zeiss Microscopy, White Plains, NY, USA), while images were acquired with a Zeiss Axiocam 712 color (Carl Zeiss Microscopy GmbH, Jena, Germany).…”

“…In the present context, the term 'cambium' is used to describe the cambial zone, which consists of several cell layers, including actively dividing cambial initials with phloem and xylem mother cells (Catesson et al, 1994;Larson, 1994;Lachaud et al, 1999;Gričar & Čufar, 2007). The study of cambial activity, and its productivity, usually involves collecting tissues from a living tree, fixation and preparation of tissues, cutting and staining of thin sections, observation under a microscope, image analysis and quantification (counting and measuring) of cambial cells in radial rows (e.g., Prislan et al, 2009;Balzano et al, 2022;Prislan et al, 2022). Studies can also include an analysis of cambial cell morphology, cell content and the developmental stages of their derivatives.…”

“…When studies focus on the characteristics and dynamics of the cambial activity, xylem and phloem formation in response to climatic and environmental changes, it is important to prepare an adequate sampling design, along with an appropriate technique for tissue collection and sample preparation, as described by Prislan et al (2014a;2014b; and Balzano et al (2022). It is also important to have a thorough knowledge of the anatomy of the secondary tissues of the species in question.…”