2.4. Fluorescence and bright field microscopy
For microstructural characterization, four potatoes of
each variety (Asterix andBi ntje) were steam cooked to a
temperature of 80C in the center, or usedraw. Samples
from the selectedtissue zones were extractedwith a cork
borer andcut into 5mm cubes. The cubes were fixedin
glutaraldehyde, 1 g/100 ml 0.1M phosphate buffer (pH
7), and dehydrated in a graded ethanol series prior to
infiltration andpolyme rization using a Historesin
Embedding Kit (Jung, Germany). Thin sections, 4 mm,
were cut (Leica 2055 Autocut Rotary Microtome) and
stained. For fluorescence microscopy Calcofluor White
(0.01 g/100 ml) andAcid Fuchsin (0.1 g/100 ml), staining
cell walls blue andpro tein orange, respectively, were
used. For bright field microscopy, samples were stained
with Lugol (I2/KI) solution andLight Green (0.01 g/
100 ml), staining starch blue andprotei n green. An
Olympus BX-50 microscope (Olympus, Japan) was used
together with AnalySIS Pro 3.1 software (Soft Imaging
System GmbH, Germany). Isolatedstarch was studied
under the microscope with polarized light with no
embedding or pretreatment. The granule size distribution
in isolatedstarch from different tissue zones was determined by measuring the starch granule diameter of
approximately 600 starch granules.
2.5. Statistical analysis
For analysis of variance (ANOVA) calculations
STATGRAPHICS Plus Version 5.0 (Manugistics Inc.,
Rockville, MD, USA) were used. Means were compared
by least significant difference (LSD). Numbers given for
results in the text are significant at the 95.0% confidence
level ðPo0:05Þ: