Karyological study of the medicinal plant Papaver rhoeas from northwest of Iran

Karyotype and morphology of mitotic chromosomes in six populations of the medicinal plant Papaver rhoeas collected from various geographical locations of the northwest of Iran were studied. Chromosome characteristics were measured from 10 complete metaphase cells using micromeasure software. The results showed that P. rhoeas is a diploid species with 2n=2x=14 chromosomes. The karyotype consisted of seven pairs of submetacentric chromosomes with one pairs of SAT chromosome (chromosome 2) having a secondary constriction at the end of its short arm. Karyological characteristics of all the materials studied were similar to each other; however, there were some variations on chromosome arm ratios and relative lengths among the different populations. All of the populations are placed on 4A class of Stebbin’s asymmetry categories.

Genus Papaver has the highest level of botanical and phytochemical variability, embracing many species with numerous subspecies and varieties yielding approximately 170 alkaloids from 13 alkaloid groups.Different authors have divided Papaver genus into five to eleven sections.The latest revision was made by Kadereit (1990) who divided the 11 sections of the genus into four groups based on morphological traits, primarily on the characteristics of the capsules.Chromosome numbers in the family are mainly multiples of x = 6, 7, 8, 9, 10, 11 *Corresponding author.E-mail: rrasghari@yahoo.com.Tel: 98-451-5510140 or 98-914-354-1783.(Mihalik, 1998).
In conjunction with data from other approaches, chromosome information and numerical analysis based on karyotype data continues to be useful in assessing generic and tribal relationships in large and complicated families of plants.P. rhoeas (2n= 2x= 14) belong to the section Rhoeadium in Papaver genus.There are a few reports on the chromosome number of Papaver rhoeas (Lavania and Srivastava, 1999;Vorniceanu et al., 2004).In this study, an attempt was made to develop detailed karyotype of this species.

MATERIALS AND METHODS
Seeds of six populations of P. rhoeas, collected from various geographical locations from north and northwest of Iran (Table 1), were soaked for germination in Petri plates on filtered paper moistened with distilled water in darkness at room temperature (22 to 24°C).The root tips were pretreated in 0.05% solution of colchicine for 2.5 h at room temperature before they were fixed in chromic acid -formalin (1:1 v:v) at 4°C for 25 h.Staining with hematoxylin was done as described earlier by Asghari-Zakaria et al. (2002).
Chromosome measurements including long arm, short arm and chromosome lengths, arm ratio index and relative chromosome length were made from 10 enlarged well-spread metaphase cells for each population, using micromeasure software developed by the  Biology department of Colorado State University, available on internet at http://www.colostate.edu.Depts.Biology.The nomenclature of chromosomes followed Levan et al. (1964) and chromosomes were named as 1 to 7 in descending order of length.Karyotype asymmetry was estimated using the ratio of the shortest/longest pair (R), the total form percent (TF), difference between relative length of the longest and the shortest chromosomes (DRL), centromeric gradient (CG), coefficient of variation for chromosome length (CV), dispersion index (DI) (Lavania and Srivastava, 1992), Stebbins (1971) asymmetry category, the intra-chromosomal (A1) and the inter-chromosomal asymmetry index (A2) indices (Romero Zarco, 1986).

RESULTS AND DISCUSSION
Mitotic chromosomes of the six P. rhoeas populations are shown in Figure 1 and karyotypic characters of the seven mitotic chromosomes are shown in Table 2.The data on  karyotype formulae, difference between relative length of the longest and the shortest chromosomes, total form percentage, ratio of the longest to the shortest chromosome, intra-chromosomal and inter-chromosomal asymmetry indices for each population are also present-ed in Table 3.
The results showed that P. rhoeas is a diploid species with 2n= 2x= 14 chromosomes.This is in agreement with the finding of Lavania and Srivastava (1999) and Vorniceanu et al. (2004).Arm ratio index of chromosomes ranged from 2.26 in chromosome 7 to 2.76 in chromosomes 1 and 3. Karyological characteristics of all the materials studied were similar to each other; however, there were some variations among populations on chromosome arm ratios and relative lengths.For example, chromosome 3 in the population G2 and chromosomes 1, 3, 4 and 6 in population G4 had arm ratio values greater than 3 and then according to Levan et al. (1964) were acrocentric chromosomes (Table 2).However, other chromosomes had arm ratio values smaller than 3.00 in all of the populations.
According to the relative length of chromosomes, there were some variations among populations.Chromosome 1 was the longest chromosome in all of the populations except for G2 and G3.However, chromosome 7 was the smallest one in all of the populations (Table 2).One of the chromosomes (chromosome 2) had a secondary constriction on the end of short arm.The SAT chromosome had major variation in size among cells and populations, probably due to differences in chromosome contraction.
In general, the karyotype of this species consisted of seven pairs of submetacentric chromosomes.Vorniceanu et al. (2004) showed that the chromosomes of P. rhoeas Table 3. Karyotype formulae (KF), difference between relative length of the longest and the shortest chromosomes (DRL), total form percentage (TF), ratio of the longest to the shortest chromosome (R), centromeric gradient (CG), coefficient of variation for chromosome length (CV), dispersion index (DI), the intra-chromosomal asymmetry index (A1) and the interchromosomal asymmetry index (A2) and Stebbin's asymmetry category (ST) in six populations (Pop.) of Papaver rhoeas.consisted of six pairs of submetacentric and one pairs of metacentric chromosomes and the arm ratio values was between 1.69 and 2.78.In addition, Lavania and Srivastava (1999) reported four pairs of submetacentric and three pairs of metacentric chromosomes in the karyotype of this species with the arm ratio values between 1.29 and 2.60.However, in this study, all of the chromosomes had arm ratio values greater than 2 and then were submetacentic (acrocentric in some populations).

Pop
According to asymmetry indices, the category of all the populations was the same and placed on 4A class of Stebbins (1971) asymmetry categories with minor variations among the populations (Table 3).This was dissimilar with previous reports (Lavania andSrivastava, 1992, 1999) in which they placed it on 2A category.Vorniceanu et al. (2004) also showed that the karyotype of this species is symmetric and less evolved.This considerable increase in asymmetry indicates that these populations experienced structural changes of the chromosomes or inter species mating.Hybridization often occurs among P. rhoeas and Papaver carmeli and spontaneous hybrids P. rhoeas× Papaver dubium occur quite often (Kadereit, 1990).

Table 1 .
Origin of P. rhoeas populations used in this study.

Table 2 .
The karyotype characteristics of seven mitotic chromosomes in six populations of Papaver rhoeas.

m Long arm ( m Length of chr (µm) Relative length (%) Arm ratio index
Chr, Chromosome; pop, population.Data are mean ± standard error.