Abstract: The present investigation is carried out in order to gain insight into the genetic variability which correlates with seed polymorphism in Calendula micrantha Tineo et Guss. based on evidence obtained from karyotype analysis and electrophoretic patterns of seed proteins. Six distinct morphs of achene heteromorphism have been reported in C. micrantha collected from the Mediterranean desert of Egypt. These morphs are balloon-shaped lacking spines, balloon shaped with spines, insect leg, winged, ring-like and curved achenes. All of these morphs were observed on every infructscence. All achene forms of C. micrantha has a diploid chromosome number of 2n = 44. Cytological analyses include nucleic acid content DNA and RNA (mg g-1), total chromosome length (TCL), mean chromosome length (MCL), total chromosome volume (TCV), mean arm ratio (Mr value), intra chromosomal index (A1), inter chromosomal index (A2), total form percent (TF%) and karyotype formulae carried out for all the studied samples. Karyotype characteristics are given here for the first time. The karyotype formulae revealed that all of them had symmetrical karyotype except in insect leg form. The electrophoretic analysis of total seed protein extracts using discontinuous SDS-PAGE gel for the studied samples are recorded. The total number of recorded bands were 26. Such bands are not always expressed in all samples. Each sample exhibited a distinctive electrophoretic pattern. From this study, it could be suggested that the genetic differentiation in C. micrantha might be the consequence of an adaptive process.
Introduction
The existence of heteromorphic fruits has been well described in a number of families including Asteraceae, Brassicaceae, Poaceae and Chenopodiaceae (Tanowitz et al., 1987). The production of heteromorphic seeds has been shown for many plant species inhabiting unpredictable environments, such as frequently disturbed habitats (Harper, 1977) and arid and semi-arid environments (Venable and Lawlor 1980; Ellner and Shmida, 1984).
Achene polymorphism in Asteraceae most likely spreads germination out in space and time and thereby increases the number of safe sites an individual percent can exploit in disseminating off springs (McEvoy, 1984). Seed heteromorphism has been assumed to increase adaptation in highly variable environments because different seed types have been shown to function differentially in dispersal (e.g. Gymnarrhena micrantha, koller and Roth 1964; Picris echioides, Sorensen 1978; Crepis sancta, Imbert et al., 1996), within-or among year time of germination (e.g. Plantago coronopus, Schat 1981; Senecio jacoboea, Mc Evoy 1984; Heterotheca latifolia, Venable and Levin 1984; Hemizonia increscens, Tanowitz et al., 1987 and Cakile edentula, Zhang, 1993).
Heteromorphic achenes have been reported in Calendula micrantha (Asteraceae) (Zohary 1950, Täckholm 1974 Syn: C. arvensis L., C. aegyptiaca Pers., Bolous 1995). The environment of the Egyptian Mediterranean desert is highly unpredictable in time and space (El-Keblawy et al., 1997). It seems that C. micrantha may survive such conditions by producing different achenes capable of success in different habitats and different times.
El-Keblawy (1999) described six distinct morphs for C. micrantha collected from the Mediterranean desert of Egypt. The different morphs of C. micrantha differ in size, shape, dispersal and total germination percentage. He found that there are positive relationship between achene size and germination. Also, the variation in achene shape and colour was associated with variation in achene mass. Calendula arvensis (Syn. C. micrantha) is important for its economic value as it used as anti-inflammatory agents for skin disease (Graf, 2000), calendula cream for grazes and scalds (Kaplan, 1994). Also, it is used as antiviral agent (Bogdanova et al., 1970). Also, Elias et al. 1990 and Chemli et al. 1990 reported that the species contains saponins, as well as calendic acid (Chisholm and Hopkins, 1967 and Fritsche et al., 1999) which are used in the treatment of skin diseases.
However, no study, to my knowledge, examined the genetic variability which correlates with seed polymorphism in C. micrantha. This investigation is carried out in order to gain insight into its genetic variation based on evidence obtained from karyotype analysis and electrophoretic patterns of seed proteins.
Materials and Methods
Calendula micrantha is an annual monoecious plant, flowering from early winter (Nov., or Dec.) to spring (April). It grows in a variety of habitats ranging from sand to clay and loamy compact soils. It is observed to be insect pollinated.
Mature fruits were collected from about 40 plants from the Mediterranean desert near El-Arish, North Sinai, Egypt. Achenes were separated into six morphs described as follows: balloon-shaped achenes lacking spines at the back of the pericarp, balloon-shaped with spines, insect leg, large achenes with big wide wings, ring-like and curved achenes (Fig. 1). All of these morphs were observed in every infructscence.
Seeds of the studied samples were germinated. Actively growing root tips were pretreated for 2-4 h in 0.002 M 8-hydroxyquinoline (Tjio and Levan, 1950), fixed in 3:1 (absolute ethanol: acetic acid), hydrolysed for 5 min. in 1N HCl at 60°C and stained by using modified carbol fuchsin (for 2 h.) (Koa, 1975 aandb) followed by aceto orcein stain. Well spread metaphase plates were selected and photographed. Determination of karyotype parameters was carried out using image and working with photomicrographs. Mean descriptive values for karyotypes were calculated from a minimum five scattered metaphase plates measured in each sample. Karyograms were drawn, lengths of long arm (L) and short arm (S) were measured for karyotype analysis. The nomenclature used for the description of chromosome morphology is that proposed by Abraham and Prasad (1983). Mean chromosome length (MCL) in μ and mean arm ratio (Mr value) in μ were determined. The total chromosome volume (TCV) was estimated using the formula: TCV=2 (II x r2 x TCL) where r is the average radius of the chromatid and TCL the total chromosome length. To estimate karyotype asymmetry, two numerical parameters were used according to Romero Zarco (1986): A1 (intra chromosomal asymmetry index) and A2 (inter chromosomal asymmetry index). Also, the total form percent (TF%) i.e. the average degree of symmetry over the whole karyotype was calculated according to Huziwara (1962). The values of analysed criteria were expressed by calculating the standard error (SE) of these parameter.
For nucleic acids extraction, the method based on that of Shibko et al. (1967). DNA was estimated by diphenylamine colour reaction described by Burton (1968). RNA was determined followed the method of Dische (1962).
For protein electrophoretic analysis, the method for discontinuous SDS-PAGE techniques was based on that of Laemmli (1970). For the determination of the molecular weight a mixture of the marker proteins are used. The banding profile in gel was photographed. The number of bands for each sample was scored. The analysis percentage of the bands were carried out using Gel Doc 2000, Bio Rad Densitometer scanner.
Results
All the samples under study were diploid. In the analyzed samples 44 chromosomes were observed in somatic cells (Fig. 2).
Cytological analyses include nucleic acid content DNA and RNA (mg g-1), total chromosome length (TCL), mean chromosome length (MCL), total chromosome volume (TCV), mean chromosome arm ratio (Mr value), intra chromosomal index (A1), inter chromosomal index (A2), total form percent (TF%) and karyotype formula were carried out for all the studied samples, data are summarized (Table 1). Karyograms of the studied samples illustrated in (Fig. 3).
The electrophoretic analysis of total seed protein extracts using discontinuous SDS-PAGE gel for the studied samples are recorded in Fig. 4. The scanning of SDS-PAGE gel of the different samples, their molecular weight (MW), relative front (RF), number of bands and average optical density (OD) are shown in Fig. 5 and Table 2, 3.
Discussion
Seed polymorphism is a common phenomenon associated with discrete and (or) continuous morphological or physiological variation among individual seeds produced by a plant or population (Venable, 1985; Silvertown 1984). Nevo et al. (1998) indicated that micro climatic conditions generate both protein and DNA patterns of polymorphism that parallel macroscale environmental pattern. They also emphasized that natural selection appears to be a major differentiating and orienting force of regional evolutionary change, maintaining genetic polymorphism under conditions of environmental heterogeneity and stress. Fruit heteromorphism in C. micrantha, as in most species of Asteraceae, represents a form of bet-hedging in the face of environments that may vary significantly in time (Venable, 1985), or space (Schoen and Lloyed, 1984).
DNA localized in cell nuclei codes most of the genetic information of an organism (Dolezel et al., 1998).
| Table 1: | Cytological data [ Nucleic acid content DNAandRNA (mg g-1), Total chromosome length (TCL), Mean chromosome length ± SE (MCL), Total chromosome volume (TCV), Mean chromosome arm ratio ± SE (Mr value), Intra chromosomal index (A1), Inter chromosomal index (A2) Total form percent (TF%) and karyotype formula of the different achene morphs of Calendula micrantha. |
| M: Metacentric, SM: SubMetacentric, nm: nearly metacentric,
nsm(+): nearly submetacentric (+), nsm(-): nearly submetacentric (-), nst (-): nearly subtelocentric (-) |
|
| Table 2: | Protein electrophoretic analysis of different achene morphs of Calendula micrantha: curved (1), Balloon-like (2), Balloon-like with spines (3), winged (4), insect leg (5), and ring like (6) |
From Table 1 it is clear that, the nuclear DNA contents ranges from 0.094 mg g-1 to 3.333 mg g-1. The maximum value of 3.333 mg g-1 was recorded in curved achene and the minimum value of 0.094 mg g-1 was recorded in winged achene. The RNA content ranges from 0.619 mg g-1 to 7.721 mg g-1. The highest content value was found in ring-like achene of 7.721 mg g-1, followed by curved form the RNA content found (3.529 mg g-1) while the lowest calculated content was found in winged form (0.619 mg g-1).
Cytological features used to determine the degree of karyotype variation of the studied samples are summarized in Table 1. All achene forms of C. micrantha has a diploid chromosome number of 2n= 44 (Fig. 2). This diploid chromosome number confirmed the previous reports by Hazra (1970), Strother (1972), Nordenstam (1972), Powell et al. (1974), Heyn et al. (1974), Van Loon (1974) and Borgen (1975).
| Table 3: | Comparative analysis of relative concentrations and molecular weights (M.Wt) of extracted proteins of different achene morphs of Calendula micrantha separated using SDS-PAGE technique |
The karyotype characteristics are given here for the first time (Table 1, Fig. 3).
The TCL (total chromosome length) at mitotic metaphase varies between 39.71 and 71.14 μm. The TCV (total chromosome volume) shows differences among the studied achene forms, being lower in ring-like (24.42 μm3) and curved form (24.74 μm3) than in insect leg form (130.17 μm3) (Table 1).
Karyotypic differences were observed between different achene forms with regard to chromosomal morphology and chromosome length.
| Fig. 1: | Shapes and relative size of different achene morphs of Calendula micrantha: Baloon-like (1), Baloon like with spines (2), Insect-leg (3), Winged (4), ring-like (5) and Curved (6) |
| Fig. 2: | Somatic chromosomes of different achene morphs of Calendula micrantha: Balloon-like (A), Balloon like with spines (B), Insect-leg (C), Winged (D), ring-like (E) and Curved (F) (X=1000) |
The chromosomes of insect leg form falls in 6 groups (Metacentric, nearly metacentric, nearly submetacentric (-), Sub Metacentric, nearly subme tacentric (+) and nearly subtelocentric (-) types) where balloon-like achene had 3 groups of chromosomes (Metacentric, nearly metacentric and nearly submetacentric (-) (Table 1).
The karyotype formulae of the studied samples revealed that all of them had symmetrical karyotype except in insect leg form where nearly subtelocentric (-) chromosomes appeared and thus gave the asymmetric affinity. The total form percentages (TF%) of all the studied samples ranged from 33.530 to 43.039%. Taxa with asymmetric karyotype tend to have low TF% (Huziwara, 1962), therefore, insect leg (TF% = 34.639%) and winged (TF% = 33.530%) achenes are presumed to have more asymmetrical karyotype than other forms.
| Fig. 3: | Karyograms of different achene morphs of Calendula micrantha: Balloon-like (A), Balloon like with spines (B), Insect-leg (C), Winged (D), ring-like (E) and Curved (F) |
The symmetry of the chromosomes within the samples is expressed by interchromosomal index (A2) for each of the studied samples. The lower A2 value (0.190) was recorded in balloon like form and the higher value (0.283) in winged form.
Karyotypic variations exist not only between different species but also within the same species. This karyologic differences within different cytotypes of the same species have been observed previously in some species such as Amaryllis balladonna (Guha, 1979) and Astragalus (Ashraf and Gohil, 1988).
| Fig. 4: | Polyacrylamid gel illustrating protein bands of different achene morphs of Calendula micrantha: Curved (1), Balloon-like (2), Balloon like with spines (3), Winged (4), Insect-leg (5) and Ring-like (6) |
| Fig. 5: | The densitometer scanning pattern of extracted protein of different achene morphs of Calendula micrantha: Curved (A), Balloon-like (B), Balloon-like with spines (C) Winged (D), Insect-leg (E) and Ring-like (F) |
The DNA content is generally known to increase as the number of chromosomes increase (Ramachandran and Narayan, 1985), but there are many complicated factors affecting the amount of the DNA content, such as total chromosome length (TCL), total chromosome volume (TCV) (Furuta and Nishikawa, 1991), chromosome folds and V-shaped chromosomes Nishikawa et al. (1979).
Nishikawa, et al. (1979) observed the greater number of V-shaped chromosomes in the complement of Lycoris, is accompanied by the greater DNA content. He assumed that, this may be due to the presence of V-shaped chromosomes carried an extra segment on both sides of centromere. This may be applied to both curved and balloon like with spines achene characterized by greater number of nearly submetacentric (-) (22) as well as the highest DNA content.
The use of seed protein electrophoresis has provided valid evidence concerning the genetic variability which could help in understanding the mechanisms controlling the biosynthesis of the different protein fractions and the source of information for genetic and systematic relationships of several plant groups (Stegemann, 1984; Evans 1985, Sammour, 1987). It was applied to Trifolium (Badr, 1995), Gossypium barbadense L. cultivars in Egypt (Sammour, 1990) and Khalifa et al. 1998 on Solanaceae. Jones and Luchsinger (1987) reported that proteins represent direct products of DNA code. Seed proteins are usually considered to be more stable than many other characters used in classification (Larsen, 1967 and Larsen and Coldwell, 1968). So using the electrophoretic techniques provide the information about genetic relationships among plant species.
Electrophoretic analysis of seed protein showed variations among the studied samples of C. micrantha. Each sample exhibited a distinctive electrophoretic pattern. The observed changes were both qualitative and quantitative and may be illustrated by the disappearance of some bands, changes in band intensity, changes in band relative mobility or changes in some fractionation of some bands (Fig. 3). From, Table 2 and 3 it is obvious that the total number of recorded bands were 26. Such bands are not always expressed in all samples. Thus, the total number of bands in the studied samples ranges between 3 (in balloon-like and insect leg form) to 6 (in curved and ring form) bands. There is no common bands in all samples. However, there are a manifested band at approximate M wt 141.611 KDa recorded in all samples, except in balloon like with spines and insect leg form. Protein variation in natural populations Hordeum spontaneum was observed by Nevo (1997) who indicated that the pattern of protein diversity was often associated with ecological factors. El-Monayeri et al. (2001) revealed that ecological factors (climatic, edaphic and vegetational) play the major roles in the genetic diversity in Zilla spinosa natural populations.
From this study, it could be suggested that the genetic differentiation in C. micrantha might be the consequence of an adaptive process.