3.4. Wax response

For the SEM investigation, the needles were collected with tweezers and they were packed loosely to avoid compression. The samples were stored in a freezer until analysed. The middle parts of the needles were mounted on brass stubs and sputtered with gold-palladium on the next day (Polaron E5100).

In the Belgian experiment, the current-year needles (about 3 months old) were collected at the end of the experiment and micrographed (magnification 200x) (3/seedling) under a scanning electron microscope (JEOL JSM 6400) with a 12 kV power source. The wax structures of pine needles were scored using a classification method described by Turunen and Huttunen (1991). The epistomatal tubular waxes of spruce needles were classified by the method of Sauter et al. (1987). In the long-term Finnish greenhouse experiment, the current-year needles (about 5 months old) were collected at the end of the experiment and the wax tube distribution (WTD, %) of the pine and spruce needles (3 needles/seedling) was studied under a SEM with the help of a phase-analysing program of an EDS (LINK eXL) analyser. To increase the contrast between the areas covered by wax tubes and the eroded areas, processed (edge sharpen, erode and dilate) backscattered electron images (BEIs) were used. The WTD was scored as a percentage of the needle surface area from BEI micrographs of magnification 1300x (Manninen et al. 1996a,b, I).

Current-year needle samples (5/tree) from the long-term field experiment were taken twice in a growing season in 1996. In the other two growing seasons, needle samples (5/tree) were collected from the beginning of needle development to fully-grown needles three times/week in 1997 (15 samplings) and twice a month in 1998 (6 samplings). At the end of the growing season, all the previous-year needles which had developed during the experiment (c+1 and c+2 needles) were collected. The samples were analysed using the phase analysis method (Manninen et al. 1996a,b, I) or a slightly modified image analysis method (V, VI). The needle samples collected in 1997 were analysed with this new image analysis method and also with the wax classification method (Turunen & Huttunen 1991) (V).

In both greenhouse experiments, the quantity of chloroform-soluble waxes on Scots pine and Norway spruce needles (4–6 needles/seedling) was determined by the colorimetric method (Ebercon et al. 1977) with the exception that carbowax 3000 was used as a standard. The absorbances were measured at 590 nm by a spectrophotometer (Beckman DU‚-64). Needle dry weight and length were measured to express the results per needle dry weight (mg/g) and surface area (mg/cm²). Needle surface area was calculated according to Flower-Ellis and Olsson (1993) for Scots pine and according to Riederer et al. (1988) for Norway spruce. Needle waxes (5 needles/tree) from the long-term field experiment were analysed only in 1998 using the modified method of Schuck (1976). Current-year needles were sampled 6 times and in the last sampling previous-year needles (c+1 and c+2 needles) were also sampled. The wax amount was expressed as per needle surface area (mg/cm²).