Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 133-144, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-169 Citation: S. Tarkesh Esfahani, G. Karimzadeh, M. Reza Naghavi (2020) In Vitro Polyploidy Induction in Per- sian Poppy (Papaver bracteatum Lindl.). Caryologia 73(1): 133-144. doi: 10.13128/caryologia-169 Received: February 15, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 S. Tarkesh Esfaha- ni, G. Karimzadeh, M. Reza Naghavi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) 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. In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Saeed Tarkesh Esfahani1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2 1 Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University (TMU), P. O. Box. 14115-336, Tehran, Iran 2 Department of Agronomy and Plant Breeding, College of Agricultural and Natural Resources, University of Tehran, Karaj, Iran *Corresponding author. E-mail: karimzadeh_g@modares.ac.ir Abstract. Papaver bracteatum Lindl. grows as a wild perennial medicinal plant in Northern Iran and is known mainly for its high amounts of the pharmaceutically valuable alkaloid of thebaine. In vitro production of tetraploid P. bracteatum through colchicine treatment of imbibed seeds is reported. Resulted tetraploid and mixoploid plants were effectively identified by chromosome counting and flow cytometry tech- nique. The chromosome number in diploid and successfully induced tetraploids were confirmed to be 2n=2x=14 and 2n=4x=28, where their calculated 2C DNA values were 6.15±0.03 and 11.95±0.07 pg, respectively. The highest induction efficiency was obtained by colchicine concentration of 0.05% and the treatment duration of 24 h. The effects of colchicine toxicity on plant survival and growth were proportional mainly to its concentration rather than duration of exposure to colchicine. Tetraploid plants possessed significantly larger and less frequent leaf stomata as well as a larger cell size. These attributes may serve as criteria for preliminary screening of P. bracteatum popu- lations for ploidy level. Keywords. Persian poppy, seed, tetraploid, colchicine, flow cytometry, 2C DNA value. INTRODUCTION Persian poppy (Papaver bracteatum Lindl.; 2n=2x=14) is a wild peren- nial medicinal plant belonging to the Papaveraceae family, section Oxytona that grows natively in the Alborz Mountains in the North of Iran in alti- tudes higher than 1800 m on the slopes facing the Caspian Sea (Sharghi and Lalezari 1967). It is mainly known for the high amounts of the valuable iso- quinoline alkaloid thebaine as the main secondary metabolite in different organs particularly in roots and capsules (Nyman and Bruhn 1979; Madam 2011) while some 20 other alkaloids are reported to be present in this spe- cies only in trace amounts (Wu and Dobberstein 1977). Persian poppy seeds contain 45-48% oil rich in nutritionally valuable unsaturated fatty acids, so the other important usage of the plant is in the food industry (Madam 2011). 134 Saeed Tarkesh Esfahani, Ghasem Karimzadeh, Mohammad Reza Naghavi Seddigh et al. (1982) reported mean seed yield and seed- oil yield of P. bracteatum to be 90 and 40 kg ha-1, respec- tively. Successful polyploidy induction has been reported in various medicinal and ornamental plants with the aim of producing plants with improved agronomical, phytochemical or economically important characteris- tics. Application of anti-mitotic agents such as colchicine (Chen and Gao 2007; Sakhanokho et al. 2009; Majdi et al. 2010; Omidbaigi et al. 2010a, b; Kaensaksiri et al. 2011; Wu et al. 2011; Marzougui et al. 2011; Tavan et al. 2015; Javadian et al. 2017; Sadat Noori et al. 2017), ory- zalin (Bouvier et al. 1994; Thao et al. 2003; Kermani et al. 2003; Lehrer et al. 2008; Sakhanokho et al. 2009), amiprophosmethyl (Rodrigues et al. 2011) and triflura- lin (Eeckhaut et al. 2002) has been reported as the most common procedure for in vitro polyploidy induction in plants with colchicine being the most commonly used anti-mitotic agent. Polyploid plants have been found to be valuable genetic resources due to possession of superior agro- nomic and phytochemical traits over their diploid pro- genitors. They have therefore attracted increasing atten- tion in breeding programs, agriculture and medicinal plants industries. Some of the more frequently reported advantages of induced polyploid plants include larger vegetative and reproductive organs such as leaves and flowers (Chen and Gao 2006; Majdi et al. 2010; Tang et al. 2010; Gantait et al. 2011; Miller et al. 2012), darker green leaves with a higher chlorophyll content and pho- tosynthesis capacity (Kulkarni and Borse 2010; Gan- tait et al. 2011), increased tolerance to environmental stresses (Natuli and Zobolo 2008), increased production of secondary metabolites (Dhawan and Lavania 1996; Kaensaksiri et al. 2011; Xu et al. 2013; Tavan et al. 2015; Javadian et al. 2017), increased expression of impor- tant genes and enzymes (Adams et al. 2003; Mishra et al. 2010; Miller et al. 2012; Xu et al. 2013) and delayed florescence time (Gu et al. 2005). On the other hand, decreased fertility, increased level of mitotic disruptions and pollen sterility (Liu et al. 2012) and facilitated bio- logical invasion (Beest et al. 2012) are reported as the most important unfavorable consequences of induced polyploidy. In vitro polyploidy induction in several Papa- veraceae plants has been previously studied mainly with the aim of obtaining an increased content of medici- nal alkaloids. Mishra et al. (2010) successfully induced tetraploidy in Papaver somniferum L. and reported a significant enhancement in the morphine content and increased expression level of important genes involved in alkaloid biosynthesis pathway in tetraploid plants. Milo et al. (1987) reported the induction of tetraploidy in P. bracteatum through colchicine treatment of apical meristems and subsequent production of triploid plants by crossing induced tetraploids to diploid plants. They suggested the ploidy breeding and tetraploidy induc- tion as the most promising approach for development of thebaine-rich poppy lines. In their studies, they selected the plants with different ploidy levels through chromo- some counting and cytological techniques. So an effec- tive, easy and clearly described method for in vitro poly- ploidy induction in P. bracteatum and effective discrimi- nation of tetraploid and mixoploid results based on flow cytometric (FCM) technique and 2C DNA value is not reported yet. Consequently, we report for the first time the in vitro production of autotetraploid P. bracteatum by colchicine treatment of imbibed seeds followed by FCM identification of polyploidy. The differences in the DNA C-value, anatomical and morphological traits between diploid and induced tetraploid plants were also measured and their capability for being employed as reliable indicators of ploidy level in the plant populations was described. MATERIAL AND METHODS Plant material Seeds of mature Persian endemic Papaver bractea- tum plants were collected in Polour region (Latitude 35° 52’ 16.99” N, Longitude 52° 04’ 38.62” E, Altitude 2489 ± 50 m) from hillsides of Alborz Mountains in north- ern Iran. The seeds from each individual plant were col- lected separately and kept in small plastic bags. Since P. bracteatum is a self-incompatible totally cross-polli- nating plant (Nyman and Bruhn 1979), the seeds which originated from each individual plant were considered as progenitors for future colchicine-treated plants. The seeds of each maternal plant were collected separately so that the eventual comparisons between different ploidy levels could be conducted between two half-sib plants rather than two plants with completely different genetic backgrounds. For this purpose, all treated seeds in poly- ploidy induction assay were selected from the seeds of an individual maternal plant. Polyploidy induction Seeds obtained from one capsule from an individual P. bracteatum plant were sterilized by immersing in eth- anol 70% (v/v) for three 30 s times, followed by sodium hypochlorite 5% (v/v) for 7 min. The seeds were then rinsed with distilled water for 5 min and transferred on 135In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) two layers of moistened filter papers in glass petri dish- es and irrigated regularly with distilled water to allow water imbibition. Germination process progressed up to the radicle emergence. After 8-10 days, the imbibed seeds (swollen seeds without a well-defined radicle apex) were transferred to tubular penicillin vials containing 1000 µl of colchicine solutions (Sigma-Aldrich Corpora- tion, MO, USA) with different concentrations compris- ing 0.00, 0.025, 0.050, 0.075, 0.10 and 0.20% (w/v). The vials were placed on a shaker with a rotation speed of 95 rpm and shaken for predetermined durations, including 4, 8, 12, 24, 36, 48, 72, and 168 h. At the end of treat- ment duration, the treated seeds were washed thorough- ly with distilled water for 3 × 3-min and transferred to a 250-ml glass baby food jars containing 40 ml of ½ Murashige and Skoog (1962) medium with 1 g l-1 char- coal. The latter was used in order to prevent the seed phenolic compounds from interfering with the emer- gence and growth of new seedlings. Seven treated seeds were cultured in each jar (representing one replication for a given treatment in data analysis). The seedlings were re-cultured once a month on a new medium with the above-mentioned composition and conditions. After three months, the plants with 6-7 true developed leaves were transferred to 500 g pots containing sand, com- mercial potting soil and vermiculite as the main compo- nents mixed with a ratio of 2:2:1 orderly. Flow cytometry analysis One cm2 of young, healthy and fully green devel- oped leaf material from each examined Persian poppy plant together with about 1/3-1/2 in area of leaf mate- rial from Pisum sativum cv. ‘Citrad’ (2C DNA = 9.09 pg; Doležel et al. 1998) as an internal reference standard, were chopped into small pieces by a sharp razor blade in a 100 mm glass petri dish, containing one ml of Woody Plant Buffer (WPB; Loureiro et al. 2007). The result- ant nuclear suspension was filtered through a Partec (Partec, Münster, Germany) 30 µm-nylon mesh, 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 two min at room temperature. To determine nuclear 2C DNA amount, the nuclei suspension was analysed by a BD FACSCanto II flow cytometer (BD Biosciences, Bedford, MA, USA), using BD FACSDivaTM Software. Output data were then transferred to a Flowing Soft- ware version 2.5.0 to be editable in Partec FloMax ver. 2.4e (Partec, Münster, Germany). The measurements of relative fluorescence intensity of stained nuclei were per- formed on a linear scale, analysing 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. 2003, 2007; Doležel and Bartoš 2005; Mahdavi and Karimzadeh 2010; Karimzadeh et al. 2010, 2011; Abedi et al. 2015) as follows: Sample 2C DNA (pg) = (Sample G1 peak mean/Standard G1 peak mean) × Standard 2C DNA (pg) The analysed samples were classified based on the FCM results into diploid (2x), tetraploid (4x) and mixop- loid (2x and 4x) samples. Chromosome counting The ploidy status of the induction results were addi- tionally confirmed by microscopic chromosome, count- ing in 10 randomly sampled plants from each class of ploidy level. Root tips from confirmed diploid and tetra- ploid plants were pretreated with α-bromonaphthalene for 1 h at 24 °C, followed by rinsing with distilled water for 3 × 3 min. The pretreated roots were then fixed in Carnoy solution (3:1 ethanol:glacial acetic acid) and stored at 4 °C followed by washing in distilled water, hydrolyzing with 1 N HCl for 8 min at 65 °C and stain- ing with 1% (w/v) aceto-orcein for 1 h. Treated 1-2 mm long root tips were excised and squashed on slide glass, with a drop of 45% (v/v) acetic acid, and protected with a cover slip. Chromosome counts were analysed by observation under a BX51 Olympus light microscope (Olympus Optical Co., Tokyo, Japan), equipped with an Olympus DP12 digital camera (Olympus Optical Co., Tokyo, Japan) using WH10X (FN22) eyepiece and 100x objective lens. Anatomical and morphological analysis To assess any possible relations between anatomical traits and ploidy level in P. bracteatum, leaf cell size as well as the dimensions and the frequency of leaf stomata were measured on the lower epidermis of 20 fully devel- oped leaves each taken from an individual plant in each confirmed class of ploidy. Data on stomata dimensions were measured on 60 stomata from leaves of the plants in each class of ploidy level. The stomata were visual- ized by the impression method (Majdi et al. 2010; Tavan et al. 2015). The density of the stomata was counted at 200x and the area of stomata were measured at 1000x magnification, using high resolution microscopic digital photographs taken with an Olympus DP12 digital cam- era (Olympus Optical Co., Tokyo, Japan) interfaced to 136 Saeed Tarkesh Esfahani, Ghasem Karimzadeh, Mohammad Reza Naghavi an Olympus BX50 (Olympus Optical Co., Tokyo, Japan) microscope. The stomata and the cells in three random microscopic fields per each leaf were counted and meas- ured. The measurements of stomata dimensions, includ- ing area and large diameter (length) as well as the cell area were determined, using the captured images and the UTHSCSA ImageTool program (University of Texas Health Science Center at San Antonio, Texas, USA). Data analysis The data regarding the effect of time and concen- tration of the colchicine treatment on survival rate and polyploidy induction were analysed through a Complete- ly Randomized Design (CRD) with three replications. Mean comparison was carried out by using Least Signifi- cant Difference (LSD) test at P<0.05. Seeds germination percentage was calculated as GP = (number of germinat- ed seeds / total planted seeds) * 100. Tetraploidy induc- tion efficiency was assessed using the method reported by Bouvier et al. (1994) and Majdi et al. (2010) as follows: Induction efficiency = % Seedling survival × % Tetra- ploidy induction For induction efficiency calculation, a seedling was considered as survived if it persisted for three months after being treated with colchicine and had enough green leaf area to be analysed by FCM. The resultant data were first tested for normality with the Kolmogo- rov-Smirnov test. The logarithmic transformation was then used for both stomata area and stomata frequency data. Mean comparisons between two different ploidy levels for anatomical traits were conducted, using Stu- dent’s t test. All statistical analysis were conducted, using SPSS 18 (Chicago: SPSS Inc., 2009). RESULTS Survival and the growth of colchicine-treated seeds The toxic effects of colchicine on the treated Persian poppy (Papaver bracteatum Lindl.) plants were different in terms of their survival and consecutive growth. Some treated seeds could no longer survive colchicine treat- ment, as they remained as dark necrotic non-emerging seeds without any root appearance. Whereas some oth- ers could survive in the colchicine and remained alive initially at the time of transferring to the medium, but their seedlings could not keep normal growing and turned to dark brown-colored necrotic tissues during later three weeks after being transferred. The degree of mortality caused by different colchicine either concentra- tions or durations were variable. The toxic effects of col- chicine treatment on the survival and on the growth of the seeds were therefore assessed 30 days after treatment induction. The general colchicine-induced mortality was divided and expressed as two different criteria includ- ing seed mortality and seedling mortality (Table 1). Seed mortality was calculated based on non-emerging seeds which could not survive in the colchicine treatment, while seedling mortality was reflected by the emerged seedlings with inhibited successive growth. The results showed that increased colchicine concentration signifi- cantly increased the level of lethal effects (Table 1, Fig. 1), so that only 4.76±2.38% seed survival but no subsequent growth was observed in the seeds treated with the 0.2% (w/v) colchicine solution. While no seedling growth was identified in the 0.2% colchicine treatment, the survival and the growth in the seeds treated with other concen- Table 1. Results of the analysis of variance (ANOVA) for the effect of colchicine concentration and treatment duration on the mortality of Papaver bracteatum seeds. Source of Variation Mean Squares Seed mortality Seedling mortality Colchicine concentration (C) 13097.26** 13173.03** Treatment duration (D) 126.22n.s. 162.97* C*D interaction 116.51* 70.15n.s. **, * and n.s. indicate significant at 1%, 5% level and and not sig- nificant, respectively. Figure 1. Effect of colchicine concentration on seed survival and seedling growth of treated Papaver bracteatum explants. All values are in percentage. Mean values specified by the same latter are not significantly different at P<0.05 by LSD test. Letters with a prime symbol designate mean differences in seedling growth. 137In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Table 2. Effect of colchicine concentration and treatment duration on the percentage (mean ± standard error) of seed mortality, seedling mortality, diploid (2x), mixoploid (2x+4x), and tetraploid (4x) explants in Papaver bracteatum. Colchicine concentration (%) Exposure duration (h) Seed mortality (%) Seedling mortality (%) 2x (%) 2x + 4x (%) 4x (%) 0.000 4 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 8 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 12 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 24 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 36 0.00 ± 0.00 9.52 ± 9.52 90.48 ± 9.52 0.00 ± 0.00 0.00 ± 0.00 48 0.00 ± 0.00 0.00 ± 0.00 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 72 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 168 0.00 ± 0.00 14.29 ± 8.25 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 0.025 4 4.76 ± 4.76 4.76 ± 4.76 90.48 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 8 14.29 ± 8.25 0.00 ± 0.00 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 12 0.00 ± 0.00 9.52 ± 9.52 90.48 ± 9.52 0.00 ± 0.00 0.00 ± 0.00 24 14.29 ± 8.25 4.76 ± 4.76 80.95 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 36 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 48 0.00 ± 0.00 14.29 ± 8.25 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 72 0.00 ± 0.00 4.76 ± 4.76 95.24 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 168 4.76 ± 4.76 9.52 ± 4.76 85.71 ± 8.25 0.00 ± 0.00 0.00 ± 0.00 0.050 4 9.52 ± 4.76 14.29 ± 8.25 33.33 ± 17.17 19.05 ± 4.76 23.81 ± 12.60 8 28.57 ± 8.25 4.76 ± 4.76 19.05 ± 4.76 23.81 ± 12.60 23.81 ± 12.60 12 28.57 ± 14.29 9.52 ± 4.76 0.00 ± 0.00 23.81 ± 4.76 38.10 ± 9.52 24 4.76 ± 4.76 4.76 ± 4.76 23.81 ± 17.17 33.33 ± 4.76 33.33 ± 17.17 36 23.81 ± 9.52 9.52 ± 9.52 19.05 ± 12.60 19.05 ± 4.76 28.57 ± 16.50 48 19.05 ± 9.52 19.05 ± 12.60 38.10 ± 12.60 14.29 ± 0.00 9.52 ± 9.52 72 0.00 ± 0.00 19.05 ± 4.76 57.14 ± 0.00 23.81 ± 4.76 0.00 ± 0.00 168 28.57 ± 8.25 19.05 ± 4.76 28.57 ± 16.50 14.29 ± 0.00 9.52 ± 9.52 0.075 4 28.57 ± 14.29 14.29 ± 0.00 9.52 ± 9.52 33.33 ± 4.76 14.29 ± 8.25 8 9.52 ± 4.76 19.05 ± 4.76 14.29 ± 8.25 23.81 ± 4.76 33.33 ± 4.76 12 42.86 ± 8.25 4.76 ± 4.76 14.29 ± 0.00 28.57 ± 8.25 9.52 ± 9.52 24 9.52 ± 4.76 9.52 ± 4.76 23.81 ± 4.76 23.81 ± 4.76 33.33 ± 9.52 36 38.10 ± 4.76 9.52 ± 4.76 4.76 ± 4.76 23.81 ± 4.76 23.81 ± 12.60 48 33.33 ± 4.76 14.29 ± 8.25 9.52 ± 4.76 19.05 ± 4.76 23.81 ± 4.76 72 38.10 ± 4.76 14.29 ± 8.25 19.05 ± 9.52 19.05 ± 4.76 9.52 ± 4.76 168 33.33 ± 9.52 14.29 ± 0.00 19.05 ± 12.60 9.52 ± 4.76 23.81 ± 4.76 0.100 4 33.33 ± 9.52 19.05 ± 4.76 0.00 ± 0.00 23.81 ± 9.52 23.81 ± 4.76 8 33.33 ± 4.76 19.05 ± 12.60 9.52 ± 4.76 14.29 ± 0.00 23.81 ± 9.52 12 47.62 ± 4.73 14.29 ± 4.25 0.00 ± 0.00 14.29 ± 0.00 23.81 ± 4.76 24 23.81 ± 9.52 28.57 ± 8.25 4.76 ± 4.76 19.05 ± 4.76 23.81 ± 4.76 36 28.57 ± 8.25 33.33 ± 12.60 9.52 ± 9.52 14.29 ± 0.00 14.29 ± 8.25 48 28.57 ± 14.29 28.57 ± 8.25 14.29 ± 8.25 14.29 ±0.00 14.29 ± 8.25 72 38.10 ± 9.52 28.57 ± 8.25 4.76 ± 4.76 9.52 ± 4.76 19.05 ± 4.76 168 47.62 ± 9.52 28.57 ± 0.00 9.52 ± 4.76 4.76 ± 4.76 9.52 ± 4.76 0.200 4 76.19 ± 12.60 23.81 ± 12.60 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 8 90.48 ± 9.52 9.52 ± 9.52 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 12 95.24 ± 4.76 4.76 ± 4.76 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 24 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 36 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 48 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 72 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 168 100.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 138 Saeed Tarkesh Esfahani, Ghasem Karimzadeh, Mohammad Reza Naghavi trations varied widely, where the mean germination per- centage and the mean percentage of developed seedlings in the 0.1% treatment were 64.88±3.22 and 39.88±2.72, respectively (Table 2). These values were 95.24 ± 1.86 and 88.69 ± 2.27 in the 0.025% treatment, respectively (Fig. 1). Flow cytometry analysis of ploidy level The ploidy level of colchicine-treated plants was determined by FCM analysis. All treated plants with apparently normal growth and development were clas- sified into three main classes including diploids (2x), mixoploids (2x+4x) and tetraploids (4x). As shown in related histograms (Fig. 2), diploid and tetraploid plants revealed a peak at the position of channels 50 and 95 of the relative fluorescent intensity respectively, while mix- oploids revealed two peaks with variable heights at both channels 50 and 95. The 2C DNA contents of the diploid and induced tetraploid plants were estimated as 6.15±0.03 (Fig. 2a) and 11.95±0.07 pg (Fig. 2c), respectively. The ploidy sta- tus of the resultant events was additionally confirmed by microscopic chromosome counting in plants with different ploidy levels. It was indicated that all diploid plants had a chromosome number of 2n=2x=14 (Fig. 3a), whereas all tetraploids had 2n=4x=28 (Fig. 3b). In the present study, significant differences between induction treatments were seen based on the induction efficiency data. The results showed that both higher concentrations of colchicine and longer durations of exposure to colchi- cine resulted in a significantly larger percentage of tetra- ploid plants (Table 3). The highest induction efficiency (31.29) was yielded by 0.05% (w/v) colchicine concen- tration at exposure duration of 24 h. The next two most efficient treatments were 0.75% (w/v)-24 h and 0.05% (w/v)-12 h with induction efficiency values of 27.89 and 25.85 respectively (Figs. 4, 5). Anatomical and morphological characteristics Stomata data measured on the leaves of plants in each class of ploidy showed that the stomata size in Figure 2. Flow cytometric histogram of the relative fluorescence intensity of nuclei isolated from Papaver bracteatum plants. Histograms show the nuclei isolated form diploid (a), mixoploid (b), and induced tetraploid (c) plants. The S in each histogram indicates the peak resulted by the cells of the Pisum sativum cv. ‘Citrad’ (2C DNA=9.09 pg) used as the internal standard. Figure 3. Chromosome numbers of surveyed Papaver bracteatum plants. Number of chromosomes in diploid (2n=2x=14) (a) and artificially induced tetraploid (2n=4x=28) (b) plants are compared. Table 3. Results of the analysis of variance (ANOVA) for the effect of colchicine concentration and treatment duration on the poly- ploidy induction rate in Papaver bracteatum. Source of Variation Mean Squares Colchicine concentration (C) 16.98** Treatment duration (D) 2.57* C*D interaction 1.11n.s. **, * and n.s. indicate significant at 1%, 5% level and and not sig- nificant, respectively. (a) (a) (b) (b) (c) 139In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) tetraploid plants was larger than that in diploids (P<0.01; Figs. 6a, 6b). The average stomata area for the dip- loid and tetraploid (Figs. 7a, b, respectively) plants was 531.44±24.02 and 868.98±55.66 µm2 respectively indi- cating a 63.51% increase in stomata area of tetraploid plants. It was also included that the stomata length in tetraploid plants (40.46±1.33 µm) was 30.06% larger than that in diploids (31.10±0.89 µm). In addition, a significant difference was identified between diploid and polyploid plants in the stomatal density (P<0.01), where the average number of stomata per square millimetre in the leaves of diploid and tetra- ploid (Figs. 7c, d, respectively) plants was 236.18±10.54 and 157.14±3.78, respectively (Fig. 6c). In other words, tetraploidy induction caused a 50.3% decrease in sto- Figure 6. Comparison of anatomical traits between diploid and tetraploid plants of Papaver bracteatum at the cellular level. The stomata length (a), stomata area (b), stomata frequency (c), and stomata cell area (d) are significantly changed in induced tetraploid plants. Mean values specified by different letters are significantly different at P<0.05 by Students’s t test. Bars show standard errors. Figure 4. Effect of colchicine concentration and treatment dura- tion on tetraploidy induction efficiency in Papaver bracteatum. Bars show standard errors. Figure 5. Effect of colchicine concentration and treatment duration on the contribution (%) of seed mortality, seedling mortality, dip- loid, mixoploid, and tetraploid explants produced during polyploidy induction in Papaver bracteatum. Figure 7. Impressions illustrating the size and density of the stomata in the leaf lower epidermis of Papaver bracteatum plants. The smaller stomata size in diploid (a) than in induced tetraploid (b) plants and the lower stomata density in diploid (c) than in tetraploid (d) plants are illustrated. (a) (a) (b) (b) (c) (c) (d) (d) 140 Saeed Tarkesh Esfahani, Ghasem Karimzadeh, Mohammad Reza Naghavi mata density in studied P. bracteatum plants. Data regarding the cell size showed that the average leaf cell area in diploid and tetraploid P. bracteatum plants was 1187.20±73.93 and 1515.12±118.31, respectively indicat- ing a 27.62% increase in epidermal cell area of tetraploid plants (P < 0.05; Fig. 6d). Unlike in the stomatal mor- phology, tetraploid plants exhibited no remarkable dif- ferences in visible morphological traits such as plant height, shoot or leaf thickness and growth rate com- pared to their diploid counterparts (Fig. 8). DISCUSSION The results regarding the colchicine effects on sur- vival of seeds and seedlings are in agreement with those reported in various plant species e.g. Berberis thunbergii (Lehrer et al. 2008), Tanacetum parthenium (Majdi et al. 2010) and Thymus persicus (Tavan et al. 2015), indicat- ing that the toxic effects of colchicine as an anti-mitotic chemical is largely influenced by the colchicine concen- tration (Table 1). The toxic effects of colchicine on the treated Persian poppy were expressed as two different criteria including seed mortality and seedling mortality. The results showed that increased colchicine concentra- tion significantly increased the level of both lethal effects and decreases the ability of treated plants to survive and grow (Table 2). The C-value index defined as the DNA content of an unreplicated haploid chromosome complement (i.e. a gamete) is highly useful in systematics, genome size estimation and many other biological fields related to eukaryotic organisms (Doležel and Bartoš 2005). Dur- ing normal mitosis two daughter cells each with 2C DNA content is formed (Doležel et al. 2003). Therefore, the 2C DNA content of a tetraploid cell which is resulted by mitotic arrest is expected to be about two times that of the progenitor diploid cells. In the present study, the calculated 2C DNA contents of the diploid and tetra- ploid plants were estimated as 6.15±0.03 (Fig. 3a) and 11.95±0.07 pg (Fig. 3b), respectively, indicating the suc- cessful induction of tetraploidy. Furthermore, these results suggest the effectiveness of FCM-based analy- sis as a rapid and reliable strategy for discriminating P. bracteatum from other identified or unidentified Papaver species with similar morphological traits and different 2C DNA values. Tetraploid induction efficiency has frequently been used in previous studies (Bouvier et al. 1994; Lehrer et al. 2008; Majdi et al. 2010) as a measure for identify- ing the most effective treatments capable of inducing complete and stable polyploidy. It is known as reliable index because it takes into account not only the rate of conversion of diploidy tetraploidy, but the survival rate of successful tetraploid events (Lehrer et al. 2008). Our results showed that both higher concentrations of col- chicine and longer durations of exposure to colchicine resulted in a significantly larger percentage of tetraploid seedlings, but their interaction effect was non-significant (Table 3). There are several reports about the evaluation of the effects by the concentration of the anti-miotic agent and treatment duration as the two main determin- ing factors in polyploidy induction efficiency in different plant types. For instance, Stanys et al. (2006) working on Chaenomeles japonica reported that with both colchicine and oryzalin as the anti-mitotic agents, the efficiency of ploidy induction was mainly dependent on the concen- tration of anti-mitotic agent rather than its exposure duration. On the other hand, several authors suggested that the polyploidy induction efficiency is associated with both optimum concentration and the duration of anti-miotic (Gu et al. 2005; Tang et al. 2010; Tavan et al. 2015). The results of FCM analysis showed that mixop- loid plants could be expected to form a large contribu- tion of polyploids sometimes as high as 33% of induc- Figure 8. Comparison of visible morphological traits at the whole plant level. The illustrated diploid (a) and tetraploid (b) Papaver bractea- tum plants are all at the same age (195 d) and acclimated under similar environmental condition. (a) (b) 141In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) tion results obtained by in vitro polyploidy induction in P. bracteatum (Table 2; Fig. 5). In polyploidy induction works, high percentage of mixoploid results are gener- ally known as a drawback of the procedure because their unstable polyploidy state often reverts partially or total- ly to the diploid condition after successive cell division cycles. It occurs mainly because the remaining diploid cells proliferate at higher rates than the tetraploid ones (Mergen and Lester 1971). During artificial polyploidy induction, colchicine influences actively dividing cells in the treated tissues and polyploidization therefore occurs unequally among explant cells, leading to the occurrence of mixoploids and chimeras (Wan et al. 1989). Accord- ingly, a low growth rate and intrinsically stunted devel- opment in treated plants are expected to aggregate these effects particularly in short exposure durations lead- ing to a higher proportion of mixoploid events among polyploidy induction results. Chakraborti et al. (1998) suggested that in in vitro induction methods, the occur- rence of mixoploids may have been minimized by grow- ing the treated plants under more favorable conditions. They stated that the uniformity of environmental factors like temperature and photoperiod may favor the syn- chronous division of meristematic cells and help mini- mize the mixoploid events leading to a high tetraploidy rate (Chakraborti et al. 1998). The ratio of tetraploid to diploid cells based on the analysed FCM data can be calculated as a measure of the contribution of tetraploid cells within a mixoploid event. The values higher than one indicate a higher percentage of tetraploid cells than the diploids. In the present study, the obtained values indicated a high degree of variabil- ity in polyploidy induction capability of colchicine in P. bracteatum (detailed data not shown because of the large number of hits and lack of a significant interaction). In addition, it was interestingly observed that varying val- ues of the ratio of tetraploid to diploid cells might be obtained by the same concentration-duration combina- tion. For example, both values of 0.19 and 1.75 which indicate a low and a high contribution of tetraploid cells respectively, were seen in the mixoploid events resulted by the 0.05%- 24h treatment combination. So despite of favorable and controlled environmental factors within in vitro induction and growth environments, large dif- ferences between induction capabilities of certain con- centration-duration combinations were revealed by the variable degrees of mixoploidy obtained by the same treatment combination. These results indicate that the effectiveness of anti-mitotic agent in polyploidy induc- tion in P. bracteatum can be determined mainly by the explant and cell-related factors rather than those related to the induction environment. However, the rather high percentage of mixoploid events yielded by in vitro col- chicine treating of P. bracteatum explants as well as the wide range of the ratio of tetraploid to diploid cells with- in mixoploid events can be explained by the incomplete effects of colchicine on meristematic cells in the treated explants. These incomplete effects could be aggravated by the intrinsically stunted growth of P. bracteatum. Indeed, short exposure times and lower concentrations of the anti-mitotic agent during polyploidy induction may in turn decrease the chance for complementation of polyploidization process leading to a higher degree of mixoploidy (Wan et al. 1989). A recent study by Tavan et al. (2015) on Thymus per- sicus has showed that the mixoploid events which were produced during polyploidy induction in Thymus persi- cus had a considerable capability for producing pharma- ceutically important compounds. They reported signifi- cantly increased production of medicinal triterpenoids in both tetraploid and mixoploid results as compared to their diploid progenitors, where mixoploid plants inter- estingly yielded significantly higher contents of Betulinic acid, Oleanolic acid and Ursolic acid even than success- fully induced tetraploids (Tavan et al. 2015). Addition- ally, successful generation of tetraploid plants from mixoploid progenitors using tetraploid cells of leaf cal- lus through callus-based techniques and tissue culture strategies has been previously reported in various plant species such as Humulus lupulus L. (Roy et al. 2001), Astragalus membranaceus (Chen and Gao 2007) and Echinacea purpurea L. (Dahanayake et al. 2010). Like- wise Shao et al. (2003) reported that further subculture of mixoploid events resulted by in vitro colchicine treat- ment of shoots in Punica granatum L. resulted in their separation to tetraploid and diploid progenies. This strat- egy has been recommended to be employed when an anti-mitotic agent generates a high degree of mixoploid events during tetraploidy induction. In general, the mix- oploids can therefore be considered as valuable sources of genetic material in ploidy breeding programs. They particularly can be employed for certain plant species which are expected to produce high numbers of mixop- loid events during polyploidy induction works. Applica- tion of tissue culture techniques in P. bracteatum has been well established, where successful in vitro regenera- tion pf this species using callus derived from seedlings (Ilahi and Ghauri 1994), roots, seeds and cotyledons (Rostampour et al. 2010) as well as through cell sus- pension culture (Farjaminezhad et al. 2013) and hairy roots (Sharafi et al. 2013) have previously been reported. Therefore, it allows combination polyploidy induction strategies and tissue culture techniques to achieve higher goals in P. bracteatum breeding programs. 142 Saeed Tarkesh Esfahani, Ghasem Karimzadeh, Mohammad Reza Naghavi These obtained results indicating increased stomata size (Fig. 6a) and decreased stomata frequency (Fig. 6c) in tetraploid plants as compared to their diploid progenitors are in agreement with those of several previous reports in different plant types. Furthermore, differences in stomata size and density are frequently reported to successfully discriminate plants with different ploidy levels, where polyploid plants are often known to have, on average, a lower stomata number per leaf area unit and increased size of the stomata and guard cells (de Carvalho 2005; Tang et al. 2010; Gantait et al. 2011; Aina et al. 2012) as well as an increased number of chloroplasts in stomatal guard cells (Ewald et al. 2009). Gantait et al. (2011) sug- gested that the lower density of stomata in polyploid plants was due to the larger stomata and epidermal cell size, as well as reduced stomata differentiation. The cell size is also reported to be related to poly- ploidy level and to be significantly different between induced tetraploid plants and their diploid progenitors (Melaragno et al. 1993). It is suggested as a potential anatomical indicator of ploidy level being capable of dis- criminating plants in a mixed population of tetraploid and diploids (Zeng et al. 2006). The results regarding the cell size in the plants with different ploidy levels showed that the tetraploid P. bracteatum plants had significantly larger leaf epidermal cells than diploid plants (Figs. 6b, 6c). These results confirm previous reports where larger cell dimensions were reported for tetraploid plants as compared to their diploid progenitors in various plant species such as Fortunella crassifolia, Citrus sinensis (Zeng et al. 2006), Tanacetum parthenium (Majdi et al. 2010) and Thymus persicus (Tavan et al. 2015). The results obtained in this study indicated that certain anatomical traits such as leaf epidermal cell and stomata size and frequency may serve as reliable crite- ria for screening of P. bracteatum plants for ploidy level. However, other routinely suggested anatomical, morpho- logical or physiological charachteristics such as chloro- phyll florescence, flower size and morphology, pollen grain size, chloroplast density, enzymatic activity, etc. have to be evaluated precisely before being employed as potential indicators of ploidy level in quick evaluation and successful screening of large numbers of P. bractea- tum plants. Because in the present study, unlike in the stomatal morphology, the colchicine-treated plants did not exhibit any remarkable differences in visible mor- phological traits such as plant height, shoot or leaf thick- ness and growth rate (Fig. 8). These observations were in agreement with those reported by Milo et al. (1987) who stated that there were no significant differences between P. bracteatum plants with different ploidy levels in the morphological traits such as plant height, flower size or in the height of the flowering stem. However, the arti- ficially induced tetraploid plants of P. bracteatum were reported to flower later than diploid plants. Their cap- sules also matured significantly later than their diploid plants (Milo et al. 1987). The lack of visible morphological distinctions between diploid and tetraploid P. bracteatum plants might be attributed to the high morphological variation observed in this species particularly in its natural habitat. Wild P. bracteatum plants naturally exhibit high variation in plant size, growth rate and other visible characteristics. So, the new polyploid variants are likely to still fall with- in the wide variation range that already exists in wild P. bracteatum populations. Consequently, like their diploid progenitors, tetraploid plants are expected to reveal a wide range of morphological variation. Hence, ploidy statuses of the induction results need to be confirmed by quick, easy and reliable criteria such as flow cytometry tech- niques rather than morphological measures. In conclusion, polyploidy was successfully induced in diploid Persian poppy (Papaver bracteatum Lindl.) through colchicine treatment of newly germinated seeds. Tetraploid and mixoploid progenies were quickly and effectively recognized by FCM technique and the 2C DNA content. Both colchicine concentration and expo- sure duration were known as determining factors in success of in vitro tetraploidy induction. Morphologi- cal traits like stomata size and frequency and cell size were significantly associated with ploidy level in Persian poppy and were known as reliable criteria for prelimi- nary screening of mixed populations based on ploidy level. 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