Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(4): 15-22, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1955 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Rasekh, S.Z., & Karimza- deh, G. (2023). Chromosomal and genome size variations in Opium poppy (Papa- ver somniferum L.) from Afghanistan. Caryologia 76(4): 15-22. doi: 10.36253/ caryologia-1955 Received: December 25, 2022 Accepted: December 18, 2023 Published: March 14, 2024 Copyright: © 2023 Rasekh, S.Z., & Karimzadeh, G. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Chromosomal and genome size variations in Opium poppy (Papaver somniferum L.) from Afghanistan Sayed Zia Rasekh, Ghasem Karimzadeh* Department of Plant Genetics and Breeding, College of Agriculture, Tarbiat Modares Uni- versity, Tehran P. O. Box 14115-336, Iran *Corresponding author. E-mail: karimzadeh_g@modares.ac.ir Abstract. The genus Papaver classified in the Papaveraceae family, is a valuable, non- alternative medicinal plant which has illustrated a massive variety of pharmacologically important alkaloids. Chromosomal and monoploid genome size diversity of seven popu- lations collected from different districts of Balkh Province in northern parts of Afghani- stan were studied. All populations were diploid, six of which (P1-P6) had 22 chromo- somes, while P7 had 20 larger chromosomes. The mean chromosome length (CL) of P1-P6 populations was 1.32 μm (0.91-1.74 μm), but that of P7 population was 2.24 μm. The results of flow cytometric analysis showed that the mean monoploid 2Cx DNA of P1-P6 populations was 5.701 pg (5.574-5.901 pg), whereas that of P7 population was 5.795 pg, confirming intraspecific variation. This study is being reported for the first time from the northern part of Afghanistan’s opium cultivation area, and P7 population is also being reported for the first time in terms of chromosome number. Valuable information on Cytogenetics can be used in some research fields, including polygenetic analysis, taxo- nomic relationships, evolutionary characteristics, and plant breeding. Keywords: Papaver somniferum, chromosome, monoploid genome size, 2Cx DNA, flow cytometry, Balkh province, Afghanistan. 1. INTRODUCTION Opium poppy (Papaver somniferum L., 2n = 2x = 22) is one of the old- est cultivated medicinal plants that has been used for thousands of years (Askitopoulou et al., 2002; Vu et al., 2022). Its origin and domestication is not clear, but archeological findings and references prove that the Mediterra- nean is the origin of the poppy plant from the middle of the 6th millennium BC (Askitopoulou et al., 2002; Salavert et al., 2018; Vu et al., 2021; Jesus et al., 2021). Recently, poppy has been cultivated, both as a licit and illicit crop, in Asia, Europe, Oceania and South America as a main source of benzylisoqui- noline alkaloids (BIAs) (Askitopoulou et al., 2002; Beaudoin and Facchini, 2014; Guo et al., 2018; Vu et al., 2021). The word poppy has been used for many species of the Papaveraceae family, while opium word has been used for the air-dried latex extraction obtained from Papaver somniferum L. cap- http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-1955 https://doi.org/10.36253/caryologia-1955 https://doi.org/10.36253/caryologia-1955 http://www.fupress.com/caryologia mailto:karimzadeh_g@modares.ac.ir 16 Sayed Zia Rasekh, Ghasem Karimzadeh sules, one of the most useful plant species belonging to this family (Labanca et al., 2018). Opium poppy is one of the non-alternative sources of morphine, codeine, noscapine (Khan et al., 2011), and semisynthetic deriva- tives, including oxycodone and naltrexone (Carlin et al., 2020; Pei et al., 2021). Morphinan-based sedatives are obtained from opium poppy (Guo et al., 2018). There are about 600 species in the Papaveraceae. The majority of those are cultivated in gardens and their karyotypes are studied. In comparison, the meiotic chromosome numbers differ from 7 to 11(Sugiura, 1939). According to the latter report, the first karyological studies of Papav- eraceae were done by Nemec (1910) on Corydalis pumila and next Tahara on Papaver rhoeas, oriental, and som- niferum. Afterwards, Yasui (1921) and Ljungdahl (1922) reported that Papaver species are the chief studies in the Papaveraceae family. From which, meiotic karyotype studies of Papaver somniferum revealed that all popu- lations were diploid with 11 chromosomes (Kaul et al., 1979; Rezaei et al., 2014). Estimation of Papaver som- niferum genome size by flow cytometry is an easy and rapid technique that allows accurate value of nuclear DNA content (Kyrylenko et al., 2005). Some studies were carried out on the Papaveraceae family for genome size estimation, e.g. it was reported that the genome size of P. somniferum was 6.46 pg (Kyrylenko et al., 2005) and this amount for P. bracteatum was 6.15 pg (Tarkesh Esfaha- ni et al., 2016). Hence, the key objective of the current study was to investigate the chromosomal and genome size variations in seven populations of opium poppy (Papaver somniferum L.) medicinal plant. 2. MATERIALS AND METHODS 2.1. Plant materials The ripped seeded capsules of seven populations of Papaver somniferum were collected from seven different districts of Balkh Province, Afghanistan in 2020. Seeds were provided from mature capsules and the seeds of each individual plant were collected separately and kept in small plastic bags. The characteristics of the local information of the collected sites and geographic distri- bution are presented in Table 1 and Figure 1. 2.2. Cytological preparation Seeds of seven Papaver somniferum L. populations were germinated on thick layers of petri paper in a glass petri dish at 23 ± 2 ºC (Kaul et al., 1979; Tarkesh Esfa- hani et al., 2020). Seeds were sterilized by immersing in 70% (v/v) ethanol three times and 30s each time, fol- lowed by sodium hypochlorite 5% (v/v) for 6 min, and rinsed by distilled water for 3 times. The sterilized seeds were then transferred to two layers of moisturized fil- ter paper in glass petri dishes and irrigated regularly by distilled water until germination (Tarkesh Esfahani et al., 2016). The seeds started germinating after 72 h in 16 h light and 8 h dark conditions at 23 ± 2 ºC. Since pretreatment is necessary (Rezaei et al., 2014), so the 2 cm-long roots were first cold pretreated for 1.5 h at 4 ºC, followed by chemical pretreatment in 0.05 M aque- ous colchicine solution for 2.5 h (Ahmadi-Roshan et al., 2016). They were then fixed in acetic acid: alcohol (1:3) for 1 h. The roots were hydrolyzed for 15 min in 1M HCl, followed by staining with aceto-orcein 2% (w/v) for 2 h (Chowa et al., 2020; Sayadi et al., 2021; Najafi et al., 2022). Squash method was applied at 45% HCl (v/v) for preparing slides. Photomicrographs were captured by a DP12 digital camera (Olympus Optical Corporation, Tokyo, Japan) appointed to a BX50 Olympus microscope (Olympus Optical Corporation, Ltd., Tokyo, Japan). Table 1. Collected sites locations of Papaver somniferum popula- tions of Northern part of Afghanistan in this study. Population codes Locality Latitude (N) Longitude (E) Altitude (m) P1 Chahar Kint, Balkh 36°20’33.43” 67°31’23.38” 1822 P2 Balkh, Balkh 36°45’54.62” 66°53’19.24” 341 P3 Kishindeh, Balkh 35°48’11.83” 67°05’38.92” 1862 P4 Chimtal, Balkh 36°28’27.42” 66°57’3.47” 553 P5 Khulm, Balkh 36°42’50.09” 66°57’7.92” 365 P6 Sholgara, Balkh 36°22’12.24” 66°53’29.67” 551 P7 Chahar Bolak, Balkh 36°48’5.64” 66°57’3.68” 335 Figure 1. Geographic distribution of sampled Papaver somniferum on the map of Afghanistan using ArcGIS. 17Chromosomal and genome size variations in Opium poppy (Papaver somniferum L.) from Afghanistan The chromosome length (CL) was measured using MicroMeasure software version 3.3. To estimate the genome size, the seeds were cul- tured in a plate culture, containing sterile perlite and coco-peat under room temperature conditions. After two months, the grown plants having four developed leaves were transferred to separate pots. One cm2 of young and well developed leaves of both Papaver somniferum plants and Solanum lycopersicum cv. Stupicke; 2C = 1.96 pg DNA (Doležel et al., 1998) as an internal reference stand- ard plant were chopped simultaneously by a sharp razor blade in a glass petri dish, containing one ml of Woody Plant Buffer (WPB) (Loureiro et al., 2007; Tarkesh Esfa- hani et al., 2020; Sayadi et al., 2022). The resultant nucle- ar suspension was filtered through a green Partec 30 μm-nylon mesh (Partec, Munster, Germany), followed by treating with 50 μg ml-1 RNase (Sigma-Aldrich Cor- poration, MO, USA) and 50 μg ml-1 Propidium Iodide (PI, Fluka) as DNA staining agent, and then incubated for 2 min at room temperature. To determine the nuclear monoploid 2Cx DNA, the nuclei suspension was ana- lyzed by a BD FACSCanto II flow cytometer (BD Bio- sciences, Bedford, MA, USA), using BD FACSDivaTM Software. Output data were then transferred to FloMax Software for Partec Flow Cytometer 2.4.1. The measure- ments of relative fluorescence intensity of stained nuclei were performed on a linear scale, analyzing at least 5,000 nuclei for each sample. The absolute DNA amount of a sample was calculated based on the values of the G1 peak means (Doležel et al., 1998; Bennett et al., 2000; Brown and Wittwer, 2000; Loureiro et al., 2007; Abedi et al., 2015; Tarkesh Esfahani et al., 2020; Abbasi-Karin et al., 2022; Sayadi et al., 2022) as follows: Sample 2Cx DNA (pg) = (Sample G1 peak mean/Stand- ard G1 peak mean) × Standard 2C DNA (pg) 2.3. Statistical analyses The normality test was first applied to chromosome length (CL) and genome size data, followed by ANOVA, using a completely randomized design (CRD) with five and three replications, respectively. The least significant difference (LSD) mean comparisons were carried out, using the general linear model (GLM) procedure in SAS 9.1 software (SAS Institute Inc 2009). 3. RESULTS Karyotypic study results show that all of the exam- ined seven opium poppy (Papaver somniferum L.) popu- lations of Balkh Province, Afghanistan were diploids; six among which possess 2n = 2x = 22 chromosomes, while the other one had 2n = 2x = 20 chromosome (Figure 3, Table 3). This study has being reported for the first time 1 a b c Figure 2. Growing stage of seven population of Papaver somniferum in plate culture after 70 d (a). Transfer of every single population in separate pots in age 95 d (b). Growing stage of Solanum lycopersicum cv. Stupicke (2C = 1.96 pg DNA) the reference standard plant (c). Table 2. ANOVA of chromosome length (CL) and monoploid genome size (2Cx DNA; pg) of Papaver somniferum Populations. S.O.V. Df MS CL Df MS 2C DNA (pg) Population 6 36.215** 6 0.04174ns Error 373 0.426 14 0.02597 Total 379 20 CV% 21.7 8.93 ns Non significant difference (P > 0.05); ** significant difference (P < 0.01) 18 Sayed Zia Rasekh, Ghasem Karimzadeh on opium cultivation area in Balkh Province, the north- ern part of Afghanistan. ANOVA results indicate sig- nificant differences (P < 0.01) for CL between the studied populations, showing intraspecific diversity (Table 2). The mean CL of all populations are shown in (Table 3). The mean CL of the first six populations (P1-P6) with 2n = 2x = 22 chromosomes was 1.32 μm, ranging from 0.91 μm (P1) to 1.74 μm (P6), but that of the P7 population was 2.24 μm (Table 3). The monoploid nuclear DNA contents of seven studied populations are shown in figure 4. The mean 2Cx DNA amount of the P1-P6 populations with 22 chromosomes was 5.701 pg, ranging from 5.574 pg (P5) to 5.901 pg (P1), while that of P7 population was 5.795 pg (Table 3). The coefficients of variation for G0/G1 peaks of all species were less than 5%. The ANOVA of genome size indicates non-significant differences (P > 0.05). Hence, to make sure of any possible difference, furthermore, LSD mean comparisons were carried out at 0.05 probability level, indicating significant differences between P1 and P3 and P5 (Table 3). The results of histogram analysis are complementary and confirm the karyotypic studies, indi- cating the diploid nature of the examined populations. 1 P1 P2 P3 P4 P5 P6 P7 Figure 3. Karyotypes of somatic chromosomes of Papaver somniferum populations. Scale bars = 5 μm. 19Chromosomal and genome size variations in Opium poppy (Papaver somniferum L.) from Afghanistan Table 3. Means (± SE) comparisons of chromosome length (CL) and monoploid genome size (2Cx DNA; pg) of Papaver somniferum popu- lations from Balkh-Afghanistan Population Locality 2n CL 2Cx DNA (pg) Mean ± Se 1Cx genome size (pg) 1Cx genome size (Mbp) P1 Chahar Kint 22 0.91f ± 0.030 5.901a ± 0.11 2.95 2885.10 P2 Balkh 22 1.50c ± 0.047 5.779ab ± 0.04 2.89 2826.42 P3 Kishindeh 22 1.08e ± 0.039 5.613b ± 0.03 2.81 2748.18 P4 Chimtal 22 1.41cd ± 0.045 5.716ab ± 0.08 2.86 2797.08 P5 Khulm 22 1.27d ± 0.033 5.574b ± 0.18 2.79 2728.62 P6 Sholgara 22 1.74b ± 0.062 5.625ab ± 0.08 2.81 2748.18 P7 Chahar Bolak 20 2.24a ± 0.074 5.795ab ± 0.06 2.89 2826.42 Means P1-P6 1.32 5.701 -- -- P7 2.24 5.795 LSD1% 0.32 0.28 -- -- Means followed by the same letter within (CL) and “2Cx DNA (pg)” columns indicate they are not significantly different at (P > 0.01) and (P > 0.05), respectively, using LSD test. Figure 4. Flow cytometric histograms of 2Cx DNA content of seven Papaver somniferum populations. The left peaks refer to G1 of the Solanum lycopersicum cv. Stupicke; 2C = 1.96 pg DNA internal reference standard and the right peak is G1 of the sample (Papaver somniferum L.). 20 Sayed Zia Rasekh, Ghasem Karimzadeh 4. DISCUSSION Many pharmaceutical components and medical ben- efits have been reported for Opium poppy (Khan et al., 2011; Heydari et al., 2013; Labanca et al., 2018). Opium poppy is an important drug plant used in the manufac- ture of benzylisoquinoline and phenanthrene groups of alkaloids (Gümüşçü et al., 2008). For using the potential applicability, this plant still requires more research on its genetic characteristics as well as developing breeding methods. In the current study, we studied seven popula- tions of opium poppy (Papaver somniferum L.) in terms of the chromosomal and genome size variations. Somatic chromosome morphology of Papaver somniferum shows that their chromosomes are numbered from 1 to 11 in order of differentiation in chromosome length (Kaul et al., 1979). The karyotype of cultured poppy plant root tips showed 22 chromosomes in all well spread root tip and shoot tip cells with more variation in length and centromere positions (Wakhlu and Bajwa, 1987). The karyotypic study in the present research showed that all populations were diploid and, in terms of chromosome numbers, P1-P6 had 22 and P7 had 20 chromosomes. The 22-chromosome number is in agreement with the studies conducted by Kaul et al. (1979) and Wakhlu and Bajwa, (1987), but not for the 20-chromosome P7 popu- lation. Considerable variation in somatic chromosome numbers of many plants, especially in the root tips, has been reported (Winterfeld., 2020; Mehravi et al., 2022). The first karyological studies of the Papaveraceae were done by Tahara on Papaver somniferum, P. orientale, and P. rhoeas which reported 2n = 22 for poppy (Papa- ver somniferum) species (Sugiura, 1940). That is in exact conformity with P1-P6 populations in the current study, but differed from those in P7. On the other hand, in recent studies, the chromosome number of Iranian pop- py (Papaver bracteatum L.) in diploid and in induced tetraploids showed 14 and 28, respectively (Tarkesh Esfahani et al., 2020), showing massive difference with that in the present study. Based on the obtained results of the current study, the P1-P6 populations were diploid with the base chromosome number of x = 11, the same base chromosome number of 11 was reported by previ- ous studies (Sugiura, 1940; Kaul et al., 1979; Wakhlu and Bajwa, 1987; Tetenyi, 1994; Rezaei et al., 2014). Flow cytometry describes the use of this technique for the estimation of genomic DNA amount in cell nuclei (Doležel and Bartoš, 2005). In a research, the average 2C DNA content of all Persian poppy plants (Papaver brac- teatum) was estimated as 6.15 ± 0.03 pg (Tarkesh Esfa- hani et al., 2016, 2020), indicating differences with that in the present study on P. somniferum populations. It can be noted that the 2Cx DNA content of P. somnifer- um species, having less variation was previously report- ed by researchers (Kyrylenko et al., 2005; Rezaei et al., 2014; Tarkesh Esfahani et al., 2016; Vu et al., 2021; Pei et al., 2021). The genome size of the first six populations (P1-P6) with 22 chromosomes was 5.701 pg (5.574-5.901 pg), which is similar to the previous report of Kyrylenko et al. (2005) in poppy species (P. somniferum) in terms of chromosome numbers possess 6.46 pg genomes size, showing a difference of 0.76 pg (13% reduction than that in the present study). By division of genome size by the number of chromosomes pg/chr, which was previously done in Mahdavi and Karimzadeh (2010) study on Thy- mus species (Lamiaceae), was also conducted in the cur- rent research. Hence, the genome size of the P1-P6 pop- ulations on the chromosome was equal to 0.259 pg/chr. Such statistics was calculated in the previous report by Kyrylenko et al. (2005) for P. somniferum with the same chromosome number to be 0.294 pg/chr, revealing 13% more than that in our six populations in the current study. More interestingly, 0.439 pg/chr was calculated in the Iranian poppy species (P. bracteatum) in the study of (Tarkesh Esfahani et al., 2020) which showed about 1.69 and 1.52 times increases compared to that in the first six populations and in the 7th population in the present study, respectively. 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