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A comparison of three methods used to estimate the stomatal density of …

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Materials and methods
- A comparison of three methods for determining the stomatal density of pine needles


P. taeda and P. ponderosa needles were sampled on 12 press (air)-dried herbarium sheets (six per species) collected throughout the eastern, south-eastern, and western United States. One needle per sheet was analysed to compare stomatal density estimates between the dissecting scope and SEM. A second needle was analysed to make similar comparisons between the dissecting scope and light microscopy (maceration). The abaxial and adaxial surfaces were counted separately resulting in two counts per needle.

Dissecting scope
Needle widths and stomatal counts were determined with a dissecting scope by placing each needle on its axis, perpendicular to the angle of view (Fig. 1Go). Needle width across the widest axis (W in Fig. 1Go) was measured to the nearest 0.05 mm with a 1 cm micrometer. Because the abaxial surfaces of the needles in a fascicle form a cylinder, the true abaxial and adaxial widths of three-needle pines are:


where W is the measured width of each needle, and r is the radius of the fascicle (Fig. 1Go). Because r cancels, equations 1 and 2 can be simplified to 1.209xW and 1.155xW, respectively. The length of the area measured was determined by placing a 7x7 mm (49 mm2) grid in the eyepiece of the microscope. The grid length projected through the eyepiece (L) was found by measuring its length against the 1 cm micrometer. All stomata within the eyepiece grid (SC) were counted, and the stomatal density calculated as:


Scanning electron microscopy
The needles were prepared for scanning electron microscopy by sectioning the same region that was analysed with the dissecting scope. Each sample was treated in chloroform for approximately 1 week to clear the surface of cuticular waxes. Needles were sputter-coated with a thin layer of gold and photographed under a scanning electron microscope (Hitachi S-570) at 50–70x magnification. Stomata were counted in four 44x44 mm transparent grids placed over each micrograph. Abaxial and adaxial needle widths were determined from the equations:


where Wm is the width of the needle measured from the micrograph and Mag is the magnification. The width is divided by two because each 44x44 mm grid represents only half the needle width on the micrograph. Because the geometric shape of pine needles remained intact throughout SEM preparation, equations 1 and 2 are applied to determine their true width. The stomatal density (no. of stomata mm-2) was determined as:

where 44 mm represents the length of each grid.

Light microscopy
In order to compare stomatal densities between the dissecting scope and the light microscope, the dissecting scope procedure described above was repeated. The needles were carefully sectioned to analyse the same area measured with the dissecting scope. After sectioning, the needles were placed in a wetting agent, Aerosol OT (Fisher Scientific), for 7–10 d (Wagner, 1981Go). Each sample was cleared in a concentrated 15% H2O2 solution and stained in safranin for approximately 24 h. Temporary slides were made by splitting each needle along its edge and spreading the abaxial or adaxial layer (depending on the sample) flat across a microscope slide. The slides were photographed with a Pentax 35 mm single lens reflex camera attached to a Zeiss compound microscope. Magnification was 50x and a micrometer was photographed with each roll of film to verify magnification after film processing. Because the needle width is greater than the width of the film plane at 50x, transparencies were generated from 8''x10'' enlargements and taped together as a continuation. A series of 126x68 mm grids were placed over the transparencies. The grids were separated into four quadrants and the stomata were counted in each quadrant. The abaxial and adaxial needle widths (Wlm) were determined as:


Because the needles were spread flat across the slide, the width did not need to be adjusted for the three-dimensional shape of the needle. The abaxial and adaxial stomatal densities were determined from the equation:

where 126 mm was the length of the grid.

Differences in needle widths between the dissecting scope and light microscope methods (from maceration) were corrected for by applying an expansion correction factor (EC) to equation 10. EC of individual needles was calculated as:

where Wdry is the width of the air-dried needles measured under the dissecting scope, and Wmac is the width of the macerated needle measured from photographs taken under the light microscope.

Thus, Wlm, corrected to the air-dried needle widths is:

and SDlm corrected for changes in leaf area is:

Changes in needle length will also lead to changes in leaf area. Changes in needle length between methods were estimated by sectioning each needle near the centre, and measuring the length of the section to the nearest 0.05 mm with a 1 cm micrometer. The lengths of the sections were re-measured after they were saturated in distilled H2O for 48 h.

Stomatal density estimates from fresh needles
To assess the relative change in stomatal densities when fresh, fully expanded needles are dried and re-wetted, fresh Pinus taeda and Pinus ponderosa needles were collected from Echols County, Georgia, USA, and Catron County, New Mexico, USA, respectively. Stomatal counts (SDFresh) were conducted on six needles per species with a binocular dissecting scope (method described above). Stomata were counted again on the same needles after they were oven-dried at 70 °C for 48 h (SDDry). A third set of stomata counts were conducted after the needles were re-wetted in distilled water for 48 h (SDWet).

A standard t-test was used to compare the stomatal densities among methods, and stomatal densities among treatments. JMP version 3.15 for Mackintosh (SAS Institute) was used for all statistical analysis.

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