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Cytogenetic Studies in Four Species of Flax (Linum spp.)



Afsanah Samadi, Ahmad Mahmodzadeh, Abdollah Hasanzadeh Gorttapeh and Mohammad Reza Torkamani
 
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ABSTRACT

This research a karyological investigation which was carried out in three species of Flax, which is a multipurpose and valuable plant. Plants and achenes specimens were collected and identified from different ecological regions in West-Azerbaijan and Markazi Provinces, Iran. Root tip meristems obtained from germinated of achenes were pretreated with saturated solution of α-Brumonaphtalene and fixed in Levitsky solution and stained in Aceto-Iron-Hematoxylin. Stained slides were photographed by camera equipped microscopes and five appropriate metaphase plates were used for analyzing karyotypeparameters. A number of statistical parameters were also estimated for all the species to investigate karyotype asymmetry. According to the results of this study L. austeriacum species is diploid that its karyotype consists of 18 chromosomes (2n = 2x = 18) whose size are between 2.246-3.44 micron and chromosome No. 1 includes satellite. All of the chromosomes were metacenteric type. L. nervosum species, as this study explains, is diploid and its karyotype includes of 18 chromosomes (2n = 2x = 18). With size of 2.95-4.291 micron. Also according to the results of this research, L. usitatissimum species is diploied, with 30 chromosomes (2n = 2x = 30), that their size are between 1.292-2.968 micron. Type of all the chromosomes are metacentric except chromosome No. 4 that is submetacentric and its arms ratio is 1.8. The karyotype Linum spp. species consists 30 + 1 chromosome and is Anioploied. That the sizes of the chromosomes are between 1.224- 1.992 micron. Mostly chromosomes of this species are metacentric type except chromosomes No. 1 and 2. that are submetacentric. Based on investigating asymmetry, L. austeriacum is being estimated to be the most developed one among them. It needs more researches to be proved.

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Afsanah Samadi, Ahmad Mahmodzadeh, Abdollah Hasanzadeh Gorttapeh and Mohammad Reza Torkamani, 2007. Cytogenetic Studies in Four Species of Flax (Linum spp.). Journal of Applied Sciences, 7: 2832-2839.

DOI: 10.3923/jas.2007.2832.2839

URL: https://scialert.net/abstract/?doi=jas.2007.2832.2839

INTRODUCTION

The genus of Linum of Linaceae comprises 200 distributed throughout the world, 15 of which occur in Iran (Sharifnia and Assad, 2001). Flax is making its mark in the world food supply as a functional food. Functional foods deliver a boost beyond what might be expected from their traditional nutrient content (Hasler, 2002).

Muravenko et al. (2001a), were studied C-banding patterns of the karyotypes of two closely related wild flax species, Linum austeriacum L. and Linum grandiflorum DESF. According results of this study the karyotype of both species were similar in the chromosome morphology and size. In each species, metacentric and acrocentric chromosomes (1.7-4.3 μm) and satellite chromosome were observed (Muravenko et al., 2001a).

Muravenko et al. (2004), fluorescence in situ hybridization was for the first time, used to study the chromosomal location of the 45S (18S-5. 8S-26S) and 5S ribosomal genes in the genomes of five flax species of the section linum. In L. usitatissimum L., L. angustifolium Huds and L. bienne Mill, a major hybridization site of 45s rDNA was observed in the pericentric region of a large metacentric chromosome. Sites of 5s rDNA were colocalized with those of 45s rDNA, but direct correlation between signal intensities from the 45s and 5s rDNA sites was observed only in some cases (Murvenko et al., 2004).

The C-banding technique was used to study flax chromosomes. Hetrochromatin was mainly locate in pericentromeric regions of chromosomes (Muravenko et al., 2001b).

Muravenko et al. (2003), chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimum L., 2n = 30; L. angustifolium Huds., 2n = 30 and L. bienne Mill., 2n = 30). In each case, the karyotype included metacentrics, submetacentrics and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1-3 micron) and C-banding pattern and slightly differed in size of hetrochromatic regions (Muravenko et al., 2003).

Karyotypic studies are used to compare the existing differences among individuals of a group, to clarify the developmental process of changes in forming chromosomes of genome. And plays an important role in determining the affinities of species. It can be the first step in phylogenic analysis and the development of relative groups. One of the effects of the development process in plants is the changes that it makes in the absolute length of chromosomes, number and situation of satellites and chromosomal unit number. This information is used in plant classification too (Stebbins, 1971). Flax, for being autogamy, has a very low genetic diversity in the natural colonies. Therefore, syngamy will cause Flax to have got more genetic diversity and recombination virtue. The intra specific inoculations, performed between some of these species, are necessary to bring the desirable virtues in to a genotype; these species, karyotypic and chromosomal properties requires to be suitable. As a matter of fact in order to achieve a more and better recognition of flax, three species of this valuable plant will be karyologically studied in this research.

MATERIALS AND METHODS

Plant samples and seeds, were collected from the different eco-geographical regions of West-Azerbaijan and Markazi Provinces (Fig. 1). The experiments were conducted in Karaj, institute of national plant Gene-Bank, Iran in 2005 year. In order cytogenetical studies seeds were sterilized in Sodium Hypochlorite solution for 30 min first. Then were germinated on damp filter paper in petri dish in 20-25°C and fresh root tips were collected for karyotypic study.

Different pretreatments were used and best result was obtained from saturated solution of α- Brumonaphtalene for four hours in 4°C (Agayev, 1998). And then were fixed in Levetsky solution for 48 h.

Next stage roots tip were hydrolyzed in normal one NaOH for 10 min, in 60°C. And were stained by Aceto-Iron-Hematoxilin for 16 h in 30-32 degree (Agayev, 1998). Finally in order to microscopic studies, Cytaze enzyme was used for destroy cell walls. Then were squashed in one drop of 45% Acetic acid and supplied microscopic samples and prepared samples were examined by camera equipped microscopes (Axiophot, Zeiss) with 10, 20, 40 degree binoculars and the best metaphase cells were selected. Then were re-examined by 100 degree binoculars and 5 metaphase plats were used for the analysis of karyotype parameters such as: (short arm length, long arm length, ratio of long arm length to short one, total length of chromosomes and relative length of chromosomes and Centromeric index).

Fig. 1: Dispersion area the species of Linum in Iran

Chromosomes were identified according to Levan method Levan et al. (1964) and karyograme of chromosomes was prepared after analyzing data and idiogrames were designed using Excel software.

Parameters of symmetrical evaluation: Several parameters have been presented and applied for karyotypic symmetrical evolution; the following parameters were used in this research.

TF%: Total Form Percentage; ratio of short arms of one taxa chromosomes/length of all chromosomes of that taxa
DRL: The difference between minimum and maximum relative Length of chromosomes
S%: Relative Length of the shortest chromosome
TL: Total Length; Total length of a group of chromosomes by micron
S/L: The ratio of the smallest chromosome to the longest one

RESULTS AND DISCUSSION

Cytogenetic evolution of L. austeriacum species:According to the results of this study number of somatic chromosomes of L. austeriacum species is 18 chromosomes (2n = 2x = 18) and diploid, which is similar to the previous reports (Muravenko et al., 2003).

There is a pair of satellites on the short arm of one of the chromosomes. These satellites can be applied in other chromosomes as a chromosomal remark. Types of the chromosomes were determined by Levan method after the karyograme was prepared by five metaphase plates.

Results suggest that in this species the length of the longest chromosome (No. 1) and the shortest chromosome (No. 9) were (3.444±0.103) with satellite and (2.246±0.178) micron, respectively. All of the chromosomes were metacentric type. Average length of the chromosome was 2.56±0.067 micron and the length of the total genome was 23.126 micron.

Karyotypic detail of this species are presented in Table 1 and Fig. 2A shows the metaphasic plate of this species and Fig. 2B shows karyograme prepared of this metaphase. Figure 7 shows the idiograme of this species with 9 haploid chromosomes. Average of ratio arms is 1.122±0.020 is defined and chromosomal formula of this species is as link 1.

Link 1 &rrar; 2n = 2x = 18 = 12 m + 2sat + 4M

Cytogenetic evolution of L.nervosum species: According to the results obtained of chromosomic images (Fig. 3), the number of somatic chromosomes is 18 and diploid (2n = 2x = 18).

Fig. 2: A) Mitotic metaphase plate of L. austeriacum, B) Karyogram, constructed from chromosomes of this plate (2n = 2x = 18), Bar = 5 μm

Fig. 3: A) Mitotic metaphase plate of L. nervosum, B) Karyogram, constructed from chromosomes of this plate (2n = 2x = 18), Bar = 5 μ

Whilst widespread attempts have been done to find the resources of this species, yet no definite resource has been found. It seems that no study has been done in this case.

Fig. 4: A) Mitotic metaphase plate of L. usitatissimum, B) Karyogram, constructed from chromosomes of this plate (2n = 2x = 30), Bar = 5 μm

Fig. 5: A) Mitotic metaphase plate of Linum spp., B) Kayogram, constructed from chromosomes of this plate (2n = 2x = 30+1), Bar = 5 μm

The length of the longest chromosome (No. 1) and the shortest chromosome (No. 9) were (4.291±0.401) and (2.95±0.168) micron, respectively. All of them were metacentric type. Average length of the chromosomes was 3.661±0.096 micron and the length of the total genome was 32.603 micron.

Karyotypic details of this species are shown in Table 2 and Fig. 3A shows the metaphase plate of this species and Fig. 3B shows karyogramme prepared of this metaphase.

Figure 8 shows the idiograme of this species with 9 haploid chromosomes. Average ratio of arms is 1.32±0.063 and its chromosomal formula is defined as link 2:

Link 2 &rrar; 2n = 2x = 18 = 16 m + 2M

Cytogenetic evolution of L. usitatissimum L. species: Gained results of the studies based upon chromosomal images of the metaphases of this species (Fig. 3) show that the number of the somatic chromosomes is 18 and diploid (2n = 2x = 18) that is similar to Muravenko et al. (2003), reports in Russia. against to what these researchers said, there is no satellite on its chromosomes; this result which refers to the genotype of the plant, location of geographical plant and its growth place. In this species the length of the longest chromosome (No. 1) and the shortest chromosome (No. 15) were (2.968±0.133) and (1.292±0.110) micron, respectively and all of the chromosomes were metacentric type except number 4 which is a submetacentric chromosome and its arms ratio is 1.8. Average length of the chromosomes was 1.758±0.074 micron and total genome was 26.342 micron.

Karyotypic details of this species are shown in Table 3 and Fig. 4A shows the metaphase plate of this species and Fig. 4B shows karyograme prepared of this metaphase.

Figure 9 shows the idiogramme of this species with 15 haploid chromosomes. Average ratio of arms is 1.323±0.049, the chromosomal formula of this species is defined as link 3:

Link 3 &rrar; 2n = 2x = 30 = 26 m + 2 sm + 2M

Cytogenetic evolution of L. spp. species: According to the fact that this species has morphologic and karyotypic specialties different from cultivated species, such as that the petals are white and yellowish anthers and the peduncle are longer than those of cultivated ones and consists white seeds, it is probably a different species or is going to make new one. Having been studied, it is clarified that the number of somatic chromosomes is 30+1 chromosomes and aneuploid (2n = 30 + 1) (Fig. 5) for the reason that monosomic type is fataller case except in the some polyploids, then so aneupoidy of this plant is from trisomic type and gametes of this plant should be n+1 and n also additional chromosome should be isochromosome.

Table 1: Numerical data concerning the karyotype of L. austeriacum (N = 5)
Chromosomal formula is 2n = 2x = 18 = 12m + 2m sat + 4M, L: Length of the long arm of the chromosome (μm), S: Length of the short arm of the chromosome (μm), CI: Centromere index, (m: Centromer in middle region of chromosome), (M: Centromer in center of chromosome), Centromere type according to Levan et al. (1964), sat: satellite, N = Number of mitotic metaphases used to determine the karyotype, L% and S%: Indices that express the contribution of each arm of each chromosome to the total length of karyotype

Table 2: Numerical data concerning the karyotype of L. nervosum (N = 5)
Chromosomal formula is 2n = 2x = 18 = 16m+2M

Table 3: Numerical data concerning the karyotype of L. usitatissimum (N = 5)
Chromosomal formula is 2n = 2x = 30 = 26 m +2 sm +2M, sm: submetacentric

The longest chromosome (No. 1) and the shortest chromosome (No. 15) were (1.992±0.065) and (1.224±0.045) micron. Most of the chromosomes are of metacentric type except chromosomes number 1 and 2 which are of submetacentric type. Average ratio of arms in this species is 1.81 and 1.84. Average length of the chromosomes is 1.545±0.039 micron and the length of the total genome is 24.727 micron.

Karyotypic details of this species are shown in Table 4 and Fig. 6A shows the metaphase plate of this species and Fig. 6B shows karyograme prepared of this metaphase.

Figure 10 shows the idiograme of this species with 15 + 1 haploid chromosomes. Average ratio of arms is 1.262±0.033 and chromosomal formula of this species is described as link 4:

Table 4: Numerical data concerning the karyotype of Linum spp. (N = 5)
Chromosomal formula is 2n = 30+1= 4 sm + 24 m + 3M

Table 5: Karyotypic symmetrical parameters
TF%: Total form percentage, DRL: The difference between minimum and maximum, relative Length of chromosomes, S%: Relative length of the shortest chromosome, TL: Total Length; Total length of a group of chromosomes by micron, S/L: The ratio of the smallest chromosome to the longest one. sat: satellite

Fig. 6: A) Mitotic metaphase plate of Linum spp., B) Kayogram, constructed from chromosomes of this plate (2n = 2x = 30), Bar = 5 μm

Fig. 7: Idiogram of haploid chromosomes of L. austeriacum L. 2n = 18, Bar =5 μm

Link 4 &rrar; 2 n = 30 + 1 = 4 sm + 24 m + 3 M

Flax has different species but L. usitatissimum L. species from the point of commercial view is more valuable than the others as this species is of the oil seeds and fiber plants. Also medically and industrially it is valuable. Little cytological and limited chromosomal counting studies related to these species have been made all of which similar to our research (Muravenko et al., 2001a; Muravenko et al., 2003). The differences between absolute size of the chromosomes being manifested in species probably show the differences in one-by-one expansion of genes or the acceleration of polytenychromonemata.

Fig. 8: Idiogram of haploid chromosomes of L. nervosum L. 2n = 18, Bar = 5 μm

Fig. 9: Idiogram of haploid chromosomes of L. usitatissimium L. 2n = 30, Bar = 5 μm

Fig. 10: Idiogram of haploid chromosomes of Linum spp., L. 2n = 30, Bar = 5 μm

And the differences in number and position of satellite show the existing differences in place and size of nucleus areas. This phenomenon is followed by the differences in hetrochromatination (Agayev, 1998). As observed from Table 1-4 absolute size of the chromosomes is different in these four species. It may be because of that the developmental process has effected the changes in absolute size of the chromosomes. Karyotype asymmetry parameters are presented in Table 5. According to these karyotypic parameters symmetry is different in these three species as in TF% view the most is for L. austeriacum species and the least for L. nevosum species. It should be mentioned that TF% parameter has an opposite relation with karyotype symmetry. So L. nevosum has the most symmetrical karyotype and L. austeriacum has the non-symmetrical karyotype. And from DRL parametrical view the less amount shows the less symmetry. In vegetative series, symmetrical karyotypes are primary and the tension is from symmetrical to non-symmetrical side. So based on this statement, it seems that L. austeriacum type has the most non-symmetric karyotype ratio to the others among which have been studied. And probably, it is more developed than two other types that proving this theses needs more examinations to be performed. There are spreaded karyotypical diversity in ploid level, the number of the base chromosomes and karyotypical asymmetry parameters within (our) four studied cases. Which could not only make genetical diversity, but also prevent of performing successful intra specific inosculation that this research was being performed in order to show the possibility of this.

For doing intra-interspesific inosculation, choosing of base parents should be performed on the basis of high similarity at karyotypes of course in respect of these parameters, could prevent of successfully doing intra specific inosculation; subsequently lead to inabequancy in reproduction and also producing fules seeds as results. In other words, it is possible the results of this inosculation to be somewhat barren. High autogamy and lack of karyotypical asymmetry in flax species have made the act of breeding so difficult. As previously mentioned, Mouravenko et al. (2001a), conducted the study on the (L. austeriacum L. and L. grandiflorum), so the results of this study are not similar to our results both in size and morphology of chromosomes.

There are two reasons of those dissimilarities: first, the material and methods and the facilities of lab used, by them, were different by those that we-applied. Socond, these species are dispersed in different climates throughout the world, so that the diversity of karyotype and in the number of base chromosomes have been brought into different populations of the species.

Finally, the most important result taken of this study is Linum spp. species/population that has shown the different morphologic and karyotypic diversity in comparison with other species. There are several interesting cases about this species that we would make to point to some items:

This species/population was wild plant and collected from Markazi province=s regions.
This species/population was not showing similarity with any present species in Iran.
The study performed on this species/population showed (that), it had high seed yield in comparison with cultivated genotypes.

ACKNOWLEDGMENT

We thank Mr. Seyyedzadeh for their valuable cooperation and guidance for carrying out this project.

REFERENCES
Agayev, Y.M., 1998. Advanced squash methods for investigation of plant chromosomes. Key-note papers. Proceedings of the 4th Iranian Congress in Crop Production and Breeding Science, August 25-28, 1998, Esfahan University of Technology, Esfahan, Iran, pp: 57-58.

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Murvenko, O.V., A.V. Amosova and T.E. Samatadze, 2004. Chromosome localization of 5s and 45s ribosomal DNA in the genomes of Linum L. species of the section Linum (syn.proto Linum and Aden linum). Russ. J. Gene, 40: 193-196.

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