Scanning Electron Microscopy

Sefa-Dedeh and Stanley (1979) found that anatomical structures could be used to explain functional properties such as water absorption. Their results indicated that the factors affecting water absorption contributed unequally during the soaking process.

During the initial stage the external structure of the seed was expected to be important.

The porous cotyledon structure and the thin, amorphous seed coat explained the rapid absorption of water. In the later stages the protein content became increasingly important.

Muzilla et al. (1991) indicated from SEM examinations that the more open and less dense ultrastructure of soybean hulls facilitate the movement of water into the hull.

Tang and Sokhansanj (1993) developed a physical model of laird lentil, based on the SEM examinations of lentil microstructure.

Neményi and Szabó (1987) and Neményi (1983, 1988 and 1993) studied extensively the pericarp of maize grains with optical microscope. They found connections between thickness and structures of pericarps and the permeability of different hybrids.

Although the optical microscope gives very expressive pictures of two-dimensional excisions, like pericarp, it is not able to make three-dimensional images, if e.g. the air spaces among starch granules in endosperm are needed to study.

Pomeranz (1972) studied the endosperm of malted barley with SEM. He found that the central starchy endosperm showed starch granules embedded in a protein matrix. It was found from the SEM investigations of the buckwheat kernel that the starch granules in the center of the endosperm of buckwheat kernel filled the contents of cells surrounded by relatively thin cell walls (Pomeranz and Sachs, 1972). Higher magnifications (1,310) of the center of the endosperm indicated that the starch granules were not free but surrounded by a matrix, presumably proteinaceous, which strengthened the structural unity of the cell contents.

Palmer (1972) studied the morphology of starch granules in cereal grains with SEM and light microscopes. It was observed that there were fewer small granules at the distal (non-embryo) end of the barley endosperm. In the dry endosperm the small starch granules were normally associated with protein material forming clumps. It was found that the starch granules of the mealy (opaque) area were loosely associated with paper sheets of protein material. There was a noticeable absence of small starch granules.

Conversely, the steely periphery was tightly packed with a rigid starch-protein matrix. The starch granules were seen to be angular and were intimately associated.

Smaller starch granules were present to a greater extent than in the mealy region and, when dislodged, they left indentations on the surface of the large granules.

Stenvert and Kingswood (1976) studied the moisture penetration during tempering of wheat grains. They found that the endosperm structure had been shown to be of primary importance in water penetration and its more ordered structure - vitreous grains - strongly retarded the rate of moisture penetration. The more closely the protein

matrix occluded the starch granules, the harder the endosperm became and the rate of moisture penetration was slower.

Gunesekaran et al. (1985) studied the stress cracks in maize kernels by SEM. Stress cracks were observed to originate at the inner core of floury endosperm and propagate radially outward along the boundary of starch granules. Robutti et al. (1974) examined the structure of normal and modified opaque-2 corn endosperms with SEM. They found loosely packed, nearly round starch granules associated with thin sheets of protein and many intergranular air spaces in the soft endosperm. The hard endosperm had tightly packed, polygonal starch granules associated with a continuous protein matrix and no intercellular spaces. They gave an explanation as to why the starch granules differed in shape.

The microstructure of opaque mutants in contrast with normal maize (W64A) were studied by SEM (Dombrink-Kurtzman, 1994). The starch granules of all mutants were free and adherent as well the protein bodies, which were smaller than in the normal line.

Wolf et al. (1969) studied eight different varieties of maize. They found that all varieties of corn showed that the outer endosperm had a higher portion concentration than in the inner endosperm. Other general trends apparent from the photomicrographs were an increase in size of both cells and starch granules from the aleuron layer inward to the centre of the kernel. They also found that the endosperm protein fills the space between starch granules.

Nass and Crane (1970) stated that drying rate of maize seeds in the field is regulated in part by colloids in the endosperm.

Wang et al. (1993) studied the morphology of starch granules of 17 maize mutant genotypes. They found that genetic background played a major rule in determining the fine structure of starch components. They have also concluded that the combination of different genes (wx, h wx, sh2 wx, wx du1, ae etc.) creates additional variations in the structure and shape of starch granules. Hence, the SEM examinations of endosperm structures in connections with artificial drying characteristics surely play a role in plant breeding research too.

4.3. MATERIALS AND METHODS

The scanning electron microscopic studies were carried out at the KU Leuven, Belgium in 1995/96. Single kernels were studied each time with SEM. Prior to the examinations kernels were cut into two halves with a razor blade and glued to aluminum stubs. The surface of specimen was sputter-coated with gold (200Å). A JEOL JXA-733 Electron Probe, Japan SEM was used for viewing and photographing the microstructure of the half kernels with 600 and 1500 magnifications.

The images were photographed directly on Polaroid films and the pictures were evaluated visually. Figure 22 shows a typical image of SEM. The distinguishing particles were the starch granules (size, homogeneity of size, shape), the protein matrix surrounding the starch granules (presence, continuance) and air spaces between the constituents.

The legend on the bottom of the pictures contains (from left to right) the following:

kilo-voltage used by the probe; magnification: first two digits (decimal number) multiple by ten over the third digit, (1500 = 15·10²  152). The third label is the number of the picture; and the last one is a vertical bar stands for as many micrometers as written above (in Figure 22 it is 10 m).

Figure 22. Scanning electron micrograph of the floury endosperm of a maize kernel (W153R) showing the basic constituents.

4.4. RESULTS

The examined hybrids were classified in two categories: slow drying (Dea, Helga and Stira) and fast drying types (Janna, Florencia and MTC 344), according to the results of the thin layer drying experiments (chapter 3.4.2. Artificial Drying Characteristics of Hybrids and Local Strains).

Floury and horny endosperms of each kernel were studied with SEM. There were no significant differences among the micrographs of horny (vitreous) endospems. The starch granules were very tightly packed surrounded by continuous, unbroken protein matrix as shown in Figure 23. Small differences occurred only in the shape of starch grains (spherical vs. polymorphous).

Figure 23. Scanning electron photomicrograph (600) of the horny endosperm (Marietta).

Structural differences were found only in floury endosperms. Figure 24 shows an image of a floury endosperm of a slow drying type hybrid. The starch granules were packed with few air spaces. The diameter of all granules was very similar. The shape of the starch bodies was not totally spherical but rather amorphous.

Figure 24. Scanning electron microscopic image (1500) of the floury endosperm of the hybrid Dea (slow drying type).

In spite of this, in Figure 25, where a fast drying type can be seen, the starch granules are more spherical and differ very much its sizes. The proteinaceous matrix is less present. The three above mentioned hybrids were harvested at an experimental field of Pioneer Co in Belgium.

Figure 25. Scanning electron micrograph of the fast drying type hybrid Janna (1500).

Floury endosperms of two artificially slow drying hybrids (Helga and Stira) from the Hungarian field experiments were studied with SEM (Figure 26-29). The characteristics of particles were the same (like by the hybrid Dea): dense structure of non-spherical starch granules with very similar sizes; protein-matrix were well visible, but not always uninterrupted. However, the hybrid Stira showed slower drying rate (Figures 26 and 28) in this case Helga showed a bit denser structure (Figures 27 and 29) which could refer higher resistance to water removal.

Figure 26. SEM image (1500) of the floury endosperm of Stira (slow drying type).

Figure 27. SEM image (1500) of the floury endosperm of Helga (slow drying type).

Figure 28. Scanning electron micrograph of the slow drying type hybrid, Stira (600).

Figure 29. Scanning electron micrograph of the slow drying type hybrid, Helga (600).

Floury endosperms of two fast drying hybrids (Florencia and MTC 344) were examined, too (Figure 30 … 33). Their starch granules are much smoother and more spherical (Figures 30 and 32). In smaller magnification (600) the whole structure of soft endosperms seem to be more disconnected with lots of air space (Figures 31 and 33).

The protein matrix is still present and sometimes shows not distinguishable differences with the slow drying ones (Figure 27 and 32). The major dissimilarity is the size of starch granules. While it was nearly the same in slow drying hybrids, here the divergence is apparent.

One can find granules five-ten times bigger than its neighbours (Figure 33). This inhomogenity shows irregular starch development, which does not allow the granules to be put into symmetrical order.

The morphological inhomogenity can cause open structure of endosperms which facilitates quick moisture loss, but it can be also attributed to have different chemical composition, chain lengths of amylose-amylopectin etc. (Hoover et al., 1996). Hence it is not questionable that the drying characteristics is influenced both morphological and structural composition, which points out the future research.

Figure 30. SEM image of the soft endosperm of good drying hybrid, MTC 344 (1500).

Figure 31. Scannig electron picture of the same (MTC 344) hybrid (600).

Figure 32. SEM image of the soft endosperm of good drying hybrid, Florencia (1500).

Figure 33. Scannig electron picture of the same (Florencia) hybrid (600).

4.5. CONCLUSIONS

Scanning electron microscopic (SEM) images of opaque endosperm showed distinctly different structure according to the drying characteristics. The technique seemed to be able to find an explanation why one hybrid dries faster than the other.

Fast drying types showed loose structure, lots of open airspace derived the unequally developed starch granules, and meanwhile slow drying types had dense, compact structure with similar sized starch grains.

The results explain one of the reasons of differences in artificial drying characteristics of maize. Moreover this can be very useful information for plant breeders because genes which affect the quality and quantity of carbohydrate (basic part of endosperm) are well known (Marx, 1981). Investigations, improved techniques (without sputter coating etc.) and studies of more repetition from each variety could result more detailed consequences. In addition these experiments ought to associate with chemical analysis.