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
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
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).
||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.
||Total Form Percentage; ratio of short arms of one taxa chromosomes/length
of all chromosomes of that taxa
||The difference between minimum and maximum relative Length of chromosomes
||Relative Length of the shortest chromosome
||Total Length; Total length of a group of chromosomes by micron
||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).
||A) Mitotic metaphase plate of L. austeriacum, B) Karyogram,
constructed from chromosomes of this plate (2n = 2x = 18), Bar = 5 μm
||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.
||A) Mitotic metaphase plate of L. usitatissimum, B)
Karyogram, constructed from chromosomes of this plate (2n = 2x = 30), Bar
= 5 μm
||A) Mitotic metaphase plate of Linum spp., B) Kayogram,
constructed from chromosomes of this plate (2n = 2x = 30+1), Bar = 5 μm
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.
||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
||Numerical data concerning the karyotype of L. nervosum
(N = 5)
|Chromosomal formula is 2n = 2x = 18 = 16m+2M
||Numerical data concerning the karyotype of L. usitatissimum
(N = 5)
|Chromosomal formula is 2n = 2x = 30 = 26 m +2 sm +2M, sm:
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:
||Numerical data concerning the karyotype of Linum spp.
(N = 5)
|Chromosomal formula is 2n = 30+1= 4 sm + 24 m + 3M
||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.
||A) Mitotic metaphase plate of Linum spp., B) Kayogram,
constructed from chromosomes of this plate (2n = 2x = 30), Bar = 5 μm
||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.
||Idiogram of haploid chromosomes of L. nervosum L. 2n
= 18, Bar = 5 μm
||Idiogram of haploid chromosomes of L. usitatissimium
L. 2n = 30, Bar = 5 μm
||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
||The study performed on this species/population showed (that), it had high
seed yield in comparison with cultivated genotypes.
We thank Mr. Seyyedzadeh for their valuable cooperation and guidance for carrying out this project.