Preparation of the Plant Material

For wood anatomical studies and for age determinations the most important plant part is the transition zone between root and stem, (root collar). For rhizomes the oldest end of the rhizome system has been used. For collection the plant was usually excavated.

Plant material was conserved in the field in 40% ethanol or any commercial alcohol, and stored and transported in thick-walled plastic bags which were collectively stored in plastic boxes.

Each sample was labeled with a sticker inscribed with a soft pencil (not alcohol soluble) containing the Latin plant name, the identification of the plant part (root, rhizome etc.), the plant life form according to Raunikiaer, plant height, phenology and obvious stem deformations, site conditions, altitude, location and sampling date.

The sample preparation was described in detail by Chaffey (2002) and Schweingruber et al. (2006).

All stems were cut as fresh material (not embedded in paraffin) into 1 cm3 sections. Large stems were sectioned in pieces near the pith (juvenile wood) and near the cambium (adult wood). Thin stems were clamped in cork (Quercussuber). Sections were cut with a Reichert microtome or with the GSL-sliding microtom. The knives of the Reichert microtome were sharpened with the Leica knife sharpening machine. The GSL-microtome uses disposable paper knife blades.

The thin sections are placed on a glass object holder (slide) and covered with glycerol. Staining liquids are dropped in excess to run off into a container. The sections were stained with Astrab-lue (0.3 g in 100ml aqua dest. with 2 ml acetic or tartaric acid) and Safranin (0.4 g in 100 ml aqua dest.) mixed in a 1:1 ratio. A drop of the solution is placed on the section every 3 minutes.

The stained sample is washed with 95% alcohol and dehydrated with absolute alcohol. The absolute alcohol is replaced again by 95% alcohol mixed with 2,2-Dimethoxypropanaceton-di-methyacetat (Fluka). Finally, a drop of xylol tests for the presence of any water. Dehydration is incomplete, and requires more washing with absolute alcohol, if the xylol turns milky. A drop of Canada Balsam is placed on the dehydrated section with a cover glass pressed on top. To avoid buckling of the sample, two PVC-plastic stripes are placed above and below the slide and pressed together using two small magnets while drying in an oven at 60°C for 12 hours.

Specimens containing slimes (mucilage), starch or dark-stain-ingsubstances (phenols) were initially soaked in a drop of calcium hypochloride (Bleach, Javelle water) for 5-10 minutes. The section is then rinsed with water until any chloride smell disappears.

Sections were microscopically inspected using magnifications of 20-1000 (Olympus BX51 with camera Olympus C5050).

Polarized light is an extremely useful technique for the differentiation of the cell wall construction. Cell walls with a net-like, unordered fibril orientation disappear in polarized light. Cells with more ordered fibrils exhibit birefringency when illuminated with polarized light. Birefringence, or double refraction, is the decomposition of a ray of light into two rays when it passes through certain types of material. Therefore primary and tertiary walls (S1 and S3) and parenchyma cells with a non-crystalline fibril construction appear black and all cells with secondary walls (S2) and with parallel ordered fibrils appear lighter. The practical value of Astrablue/Safranin staining and the use of polarized light is demonstrated in the figure on the next page.

stained with Astrablue / Safranin unpolarized light polarized light

Apollonias barbujana tree with dense wood

lignified unlignified unlignified unlignified lignified r pa v f lignified unlignified unlignified unlignified lignified with secondary walls (S2)

only with primary walls (S1)

all cells with S2

with secondary walls (S2)

only with primary walls (S1)

all cells with S2

unlignified lignified unlignified r

Erysimum crepidifolium dwarf shrub with soft stem chamaephyte unlignified lignified unlignified only with S1

with S2 only with S1

only with S1

with S2 only with S1

unlignified

—slightly lignified lignified unlignified

Cerastium semidecandrum annual, fragile herb unlignified lignified with S2

unlignified

Be only with S1

ph xy unlignified

—slightly lignified lignified unlignified with S2

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