NDT.net • June 2006 • Vol. 11 No.6

Structural Features of the Stony Cotton Seeds

V.A. Krakhmalev and A.A. Paiziev
Institute of Electronics, Uzbek Academy of Sciences, F.Khodjaeva str. 33, Tashkent 700125, Uzbekistan
Tel: 998-71-162-79-40 Fax 998-71-162-87-67 e-mail: adxam_payziev@rambler.ru


The roentgenograms of so-called "stony", non-swelling, hard seeds of Gossypium hirsutum L., G. barbadense L., G. herbaceum L. have been investigated. Abnormal morphological structures have been found without germination test. The original data on micro hardness of cotton seed peel and its structural layers are given. The obtained data form new approaches to a problem of seed hardness.

Key words: Gossypium, seed, stony seed, contact radiography, seed dormancy, micro hardness, light line of peel of seeds.


Hard or "stony" seeds with labored germination and dormancy are found for the majority of the plants with hard seed coat in particular, for Malvaceae family (Fryxell, 1969). Gossypium L. belongs to this family and it includes about 70 cotton variety ( Dariev and Abdullaev, 1985 ). Five of them are used for cultivation.

The problem of seed hardness is of biological and practical importance; many works are devoted to it (Walhood, 1956; Christiansen et. al., 1960; Konstantinov and Volkova, 1960; Curtis, 1963; Yatsu, 1965; Wareing, 1971; Poptsov, 1976. It was observed that seed hardness is strongly depended on moisture and temperature of the environment during seed maturation, as well as mechanical damage of seeds, micro flora, environmental gas composition, physical and chemical action of the seeds for seed hardness elimination, inorganic nutrition of plants, storage and structural features of the seed coat (Dariev and Abdullaev, 1985). Many researchers found that the seed hardness is related to the waterproofness and gasproofness seed coat, especially to its palisade, columnar layer.

For example, for cotton seeds, prismatic, thick-walled cells of this layer in the top of third part of its length very densely adjoin each other. When the cuts of the seed coat are examined, an original optical effect in the form of brightly luminous line is seen, which is called "light line" (Philimonov, 1961). The light line was found in all families, whose representatives have hard seeds (Krakhmalev and Sultanova, 1983).

It is known that among cotton cultivars the hardness of seeds is seldom observed (2-6%) (Konstantinov and Volkova, 1960). However, in cotton population there are the lines with mainly hard seeds (Poptsov, 1976), and their behavior is identical to the behavior of hard seeds of arboraceous-shrubby species of leguminous (Ganesan, 1952). Many of hard, stony seeds are found among natural cotton species (Fryxell, 1969; Dariev and Abdullaev, 1985; Konstantinov and Volkova, 1960; Christiansen et al., 1960).

The tests for germinating ability at 24°C were carried out on the seeds of three cotton species (Gossypium hirsutum, G. barbadense, G. thurberi) (Walhood, 1956). It has been shown that for G. hirsutum L. and G. barbadense L. the amount of hard seeds, ungerminated after five day of laboratory tests, was within the limits of 4-44% according with Walhood (1956). G. thurberi cotton seeds were non-germinated at all during this period (Walhood, 1956). They germinated only after thermal processing in hot and cold water after the fifth day by 82 % and by 84 % after drying and storage for two months. The rest non-germinated seeds 18 % of the investigated sample were absolutely hard.

In spite of seed hardness, which is characteristic for natural cotton species, they have dominant genes carrying extremely significant attributes, which are absent for the cultivated species. They are smaller susceptibility to verticilliose and fusarial wilt, a frost-resistance, a resistance to many diseases of bacterial character, etc. (Dariev and Abdullaev, 1985). As it is known, natural species involved into hybridization gives wide possibilities for genetic variability and allows obtaining the species with advanced properties. However, the inheritance of seed hardness also can take place. It has been found out, that one predetermine the presence of such 'stony' seeds in seed grain with sufficient accuracy without the use of standard methods of cotton seed germination under laboratory conditions. The radiographic investigations show that it is related to the features of morphostructure of a embryo. Our paper is devoted to the description of such features.

The objective of this paper is to investigate the X-Ray image analysis of embryos in hard seeds of Gossypium hirsutum L., G. barbadence L. and G. herbaceum L. and its comparison with microhardness of the seed coat.

Materials and methods

Mature seeds of following cotton varieties were investigated: Tashkent-1, 108-F, C-4727 (Gossypium hirsutum L.), C-6030, C-6524 (G. barbadense L.), widely cultivated in the Republic of Uzbekistan, and also coarse-fibered form Turfan Guza ( G. harbaceum L.). After hand ginning the seeds with dark brown and brown coloration in a quantity not less than 1000 seeds were placed into sandy germinators and couched under laboratory conditions in thermostats during 10 day according to a procedure established by Rakhimov and Rudenko (1976). Non-germinated seeds with undamaged seed coat were cleaned from the rests of sand and were dried at room temperature; then they were set on special holder in the "Electronics-25" device for radiographic analysis. Special frames allowed to place cotton seeds directly on a negative film with the size of 13x18 cm. After exposure to soft x-rays the films were developed and explain roentgenograms of "stony" seeds were obtained. The operating modes were the following: tube voltage - 18 kV, tube current - 40 mkA, exposure time - 2 min (Krakhmalev and Alimova, 1981; Krakhmalev, 1991). Contact roentgenograms of the seeds were placed into photographic enlarger, giving on photographic paper the increased patterns of fine structure of stony seed embrio, available for observation. It is necessary to note that in addition to stony seeds, the empty seeds also did not germinate during laboratory tests and the rest of endosperm was seen inside them. In the given case the use of radiography is caused by its high resolving power and by the possibility to study the seed germs without its breaking (Smirnova, 1978).

Results and discussion

When the hairs removed, mature cotton seeds have dark or dark brown color of seed coat and high microhardness almost of all constituent structural layers (Krakhmalev and Gershman, 1990). For example, if in process of maturing, the microhardness of external epidermis of seeds for 108-P G. hirsutum L. cotton sort changes within the values of 18-28 kg/mm2, the microhardness columnar (palisade) layer (at a level of middle of cell cavities) increases from 20 to 30 kg/mm2. The greatest jump of microhardness during maturation is observed in a thin interlayer of seed coat, passing through top ends of columnar cells, where under microscopic observations the so called "light line" is found. The microhardness of this layer of seeds during their maturation increases from 45 to 80 kg/mm2 (Krakhmalev and Sultanova, 1983).

Comparison between microhardness of various peel layers (Krakhmalev and Gershman, 1990) and microhardness of known metals has shown, that for most mature cotton seeds, as, for example, for 108-F cotton sort, the external epidermis, the external pigmental and crystalliferous layers have approximately the same hardness, as annealed aluminum, and the columnar layer at a level of a light line - as the annealed copper. Such feature of microhardness distribution among structural layers is also characteristic for the seeds of other cotton sorts (in sum, 30 types have been investigated), and it, apparently, have an essential influence on seed hardness or stoniness of the seeds. In roentgenographic investigations it has been found for selected mature seeds of the investigated cotton species with the laboratory germinating ability of 98%, that the shadow x-ray images of the germs (roentgenograms) of such seeds (or germs) had uniform blackening on positive images. Only narrow parts of germ edges are slightly clarified as they have oval form. The germs occupy the seed cavities almost completely (Krakhmalev, 1991) .

Follow facts are very important for the technology of the seed treatment under hulling and pelleting. Every mature seed with the moisture of 8-10% and lower, always has narrow and hollow gap between seed coat and embryo. Its size is 0.05-0.1 mm (Krakhmalev, 1991; Krakhmalev and Gershman, 1990). For immature seeds these gaps are significantly larger, though their external sizes can be close to external sizes of more mature seeds. According to Rakhimov and Rudenko, (1976) dormancy cotton seeds quantity varies from 2.0 to 4-8 %. We have show that dormancy seeds have morphological features of embryo (Fig. 1, b).

Fig.1. X-Ray image of cotton seeds (positive image):
a- Perfect seeds (uniform distribution of germ tissue density). Sort Tashkent-1 G.hirsutum;
b- Stone seeds. We can see irregularity distribution of germ tissue density. Sort Tashkent-1 G.hirsutum.

Their positive images in soft x-ray beams are characterized by various brighter strips. The decoded roentgenograms have shown that brightness of background is, apparently, related to internal structure of a embryo, to original arrangement of cotyledons and seed roots. The shadow images of the germs of hard seeds for different cotton sorts are characterized by their individual patterns of brightened shadow backgrounds of the germs (Fig. 2). But in general case, the most character feature of the roentgenograms of hard, stony seeds is brightness around a rootlet of a embryo (Fig. 2, d).

Fig.2. X-Ray image of stone cotton seeds (positive image):
a- Sort 108-F G.hirsutum;
b- Sort C-4727 G.hirsutum
c- Sort C-6030 G.barbadense L.;
d- Sort Turfan guza G. herbaceum L.

Taking into account a nature X-Ray image formation of a embryo we can say that such locally brightened background on the roentgenograms is connected with smaller absorption of X-Ray beams in the given areas of embryo material, or is related to existence of free spaces-gaps between structural elements of a embryo. First assumption is related to the fact that the absorption of x-ray beams, transmitting through complex objects, is determined, in addition to their energy, by atomic composition, density and thickness of a sample (Krakhmalev and Sultanova, 1983). The heavier chemical elements entering into the object and the higher their density and thickness, the more intensive x-ray absorption takes place. As a result, a distinctive shadow image arises on photoemulsion after its appropriate processing (Burger , 1981). On the prints the images get opposite visibility; dark parts of a negative become bright on the photos. Thus, the roentgenogram of investigated object differs in substance from its optical image obtained with optical microscope (Burger, 1981). The patterns are similar only by their contour borders. For second assumption on occurred brightened parts on the photographic images it is necessary to recognize the presence of continuity breaks within the volume of germ body.

To explain the occurrence of contrast inhomogeneity on X-Ray images of stony cotton seeds, longitudinal and cross cuts of these seeds have been investigated. Among 65 stony seeds of five cotton kinds (Tashkent-1, 108-F, C-4727 Gossypium hirsutum L., C-6030, C-6524) 50 seeds were randomly selected for the embryo cut preparation. The distinctive features of the seed germ structures, which are characteristic for stony seeds have been found out. They are the gaps of various sizes inside a body of a embryo, which separate the rootlet off the seed-lobes. In this respect the cross cuts of the seed germs were the most informative (Fig. 3). The gaps between a tissue of the cotyledons and the rootlets (cuts of the seed-roots in Fig. 3 were denoted by r) for some stony seeds were significant and well observable (Fig. 3a). For example, in Fig. 3a the size of large gap has the cross size of 1.5 mm by 1.0 mm; and the size of smaller gap 1.0 mm by 0.3 mm. The gaps of very small sizes were also found with microscope (Fig.3d, arrows). Their sizes were about 0,1x0,1 mm. Thus, the inhomogenity of X-Ray image structure on roentgenograms of cotton seeds is caused first of all by anomalous arrangement of the germ parts.

Fig.3. Structure of the cross section surface of the stone cotton seeds germ (optical microscope):
a- Sort Tashkent-1 G.hirsutum;
b- Sort C-4727 G.hirsutum
c- Sort C-6030 G.barbadense L.;
d- Sort Turfan guza G. herbaceum L.
Cross sections of germ roots is designated as "r". Arrows indicate to gaps small size between seed-lobes tissue and rootlet.

Longitudinal cuts of stony seeds also show the presence of longitudinal slot-like hollows in a body of germs, which are often oriented along a conditional axis of a germ. The powerfully developed rootlet elastically supports the micropilar part of a peel, compressing the laid cotyledons in halasal part of a seed cavity. On longitudinal and cross cuts of stony seeds the spherical particles of gossypol are also seen. Using a technique of photography of the cut surfaces according to (Krakhmalev et al., 1979), it is possible to determine the content of gossypol in separately taken cotton seeds.

The structural attribute of cotton seed hardness is clearly evident. But it is impossible to exclude the influence on this phenomenon of such fact, as occurrence of chemical heterogeneity in the structural elements of the germs and seed peel. The physiologists, dealing with cotton, know that the concentrations of the elements of inorganic nutrition are non-uniformly distributed: calcium and magnesium are accumulated in the leaf, in the folds of bolls, in seed peel; potassium prevails in a fibre; zinc, copper, phosphorus prevail in the seed nuclei. By the end of cotton vegetation more than 60 % of total quantity of nitrogen, phosphorus and potassium, removed from the soil, pass into the seeds. Such non-uniformity in the element composition of cotton materials is easily found using the radioisotope methods of analysis in cotton industry (Grober, 1966). It is naturally to assume, that the increased concentration of various chemical elements in structural formations of the seeds results, for example, in abnormal jump of microhardness in the zone of a so-called light line (Krakhmalev and Sultanova,1983). Probably, this fact and also the presence of inhibiting substances in cotton seeds (which are similar to the substances found in the seeds of Astragalus tennesseans (Baskin and Quarterman, 1969) and cause the obvious dormancy epicotyl's in a plant germ) give rise to the seeds become stony, though the germ is well developed and structurally differentiated.

Human eye have resolution about 0.125 mm and we can see details of morphostructure of dormancy cotton seeds on the roentgenograms But now modern high-speed computers let us considerably to short time of measurement and increase its resolution (Krakhmalev et al., 1986). Therefore it is possible to develop such X-Ray device, which will allow to process 15-20 and more seeds per second. A technique of cotton seed radiography is very informative. In addition to determination of the seed hardness it is possible to use the roentgenograms in selection works for forecasting an opportunity of formation of such important seed defect in new cotton sorts, as peel with a fibre at primary processing of a raw cotton (Krakhmalev and Alimova, 1984). The radiography also is only one possible simple method allowing quality estimation of pelleted and sterilized seed grain of cotton (Krakhmalev and Gershman, 1986; Krakhmalev and Gershman, 1990).


We would like to thank H. Abdullaev for excellent technical assistance during sampling. The research described in this paper was supported, in part, by a grant from the Center Science and Technology Republic of Uzbekistan (Contract P-17.64).


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