Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(3): 53-61, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2768 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Morovati, Z., Karimzadeh, G., Naghavi, M.R. & Rashidi Mon- fared, S. (2024). Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia medicinal plant. Caryo- logia 77(3): 53-61. doi: 10.36253/caryolo- gia-2768 Received: May 17, 2024 Accepted: Oct 18, 2024 Published: March 25, 2025 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. 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. ORCID ZM: 0009-0000-7291-706X GK: 0000-0001-8209-3287 SRM: 0000-0001-5380-1387 Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia medicinal plant Zahra Morovati1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2, Sajad Rashidi Monfared3 1 Department of Plant Genetics and Breeding, College of Agriculture, Tarbiat Modares University, P. O. Box: 14115-336, Tehran, Iran 2 Department of Agronomy and Plant Breeding, College of Agriculture, University of Teh- ran, Karaj, Iran 3 Department of Agricultural Biotechnology, College of Agriculture, Tarbiat Modares Uni- versity, P. O. Box: 14115-336, Tehran, Iran *Corresponding author: E-mail: karimzadeh_g@modares.ac.ir Abstract. Datura stramonium and D. innoxia are among the important species of Datura genus. They have many uses in traditional and modern medicine. Since Iran is located in the origin area of Datura, it is expected that Iranian germplasms are factors of global genetic diversity of Datura. Ploidy level, chromosome number and length, and genome size estimation were studied on 15 populations of both Datura species mostly collected from different parts of Iran and a few from abroad. For chromosom- al preparations, root tip was squashed and stained with 1% (w/v) aceto-orcein. For genome size estimation, flow cytometric analysis was conducted on fresh developed leaves of Datura samples along with those of internal standard reference (Solanum lycopersicum cv. Stupick, 2C = 1.96 pg DNA), using PI fluorochrome. All the studied populations were diploids (2n = 2x = 24). The mean chromosome length in D. stra- monium and D. innoxia was determined as 1.97 µm and 2.39 µm, respectively; the lat- ter had 21% larger chromosomes. The mean monoploid genome size was determined as 3.80 pg in D. stramonium (ranged 3.65 pg to 3.93 pg) and 3.91 pg in D. innoxia (ranged 3.68 pg to 4.30 pg). The present study provides completely new information about cytogenetics in D. stramonium and D. innoxia populations from Iran for the first time, which is useful for whole genome sequencing and the construction of genetic and physical maps in the future. Keywords: chromosome, DNA C-value, monoploid genome size, Datura, Iran. INTRODUCTION Solanaceae is a large plant family that includes economically species and having still many members cytologically unexplored (Zhang et al., 2023). The genus Datura from the Solanaceae family produces various secondary metabolites, for example tropane alkaloids, terpenoids, and glycoalkaloids https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-2768 https://doi.org/10.36253/caryologia-2768 https://doi.org/10.36253/caryologia-2768 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0009-0000-7291-706X https://orcid.org/0000-0001-8209-3287 https://orcid.org/0000-0001-5380-1387 mailto:karimzadeh_g@modares.ac.ir 54 Zahra Morovati et al. to defense against natural enemies such as herbivorous insects, pathogenic agents (bacteria, fungi, viruses) and different abiotic stresses (De-la-Cruz et al., 2021). The classification of Datura species is organized into two primary groups. The first group, Ceratocauli, consists solely of the species D. ceratocaula. The second group encompasses a variety of other species and is further divided into two sections. The first section includes D. arenicola, D. discolor, D. ferox, D. kymatocarpa, D. leichhardtii, D. quercifolia, and D. stramonium, while the second section (termed polyphyletic) comprises D. innoxia, D. lanosa, D. metel, D. reburra, and D. wrightii (Bye and Sosa, 2013) which are native to North Amer- ica (De-la-Cruz et al., 2021), distributed in subtropical regions of the world (Hassan and Amer, 2019; Papagri- goriou et al., 2019). On the other hand, Karimi (2001) believed that the origin of D. stramonium are India and the western shores of the Caspian Sea. Hence, the two species D. stramonium and D. innoxia are found abun- dantly in Iran; D. innoxia is less distributed than D. stramonium in coastal areas, but it is more distributed in the outskirts of cities (Ghahraman, 1998; Muzafar- ian, 2000). Among the Iranian names of Datura weed, Tatore weed, Tatoleh, and Jozmash can be notified (Kirimi, 2001). In which, D. stramonium also known as the Thorn Apple, Jimson Weed, and Angel’s Trumpet (Disel et al., 2016). Both D. stramonium and D. innox- ia are important species of Datura genus (Batool et al., 2020; Al-Zharani et al., 2021), having several traditional and modern medicinal uses (Mohammed et al., 2021). Morovati et al. (2023) showed that the essential oil of the aerial parts of D. stramonium is rich in monoterpe- noid derivatives such as camphor and borneol, which are widely used as therapeutic agents against the prolif- eration of cancer cells for the treatment of neurological and antiviral disorders (Salakhutdinov et al., 2017). Genome size, chromosome number and structure changes play an important role in speciation events, adaptation and the development of new genetic networks during evolution (Pellestor and Gatinois, 2020; Winter- feld et al., 2020). Accordingly, analysis and chromosome observation and genome size estimates, elucidate phylo- genetic relationships, structure, function, organization, and evolution (Amosova et al., 2019). Such cytogenetic studies may be useful in establishing systematic and evo- lutionary relationships, resolving taxonomic ambiguities, and achieving a better understanding of the branching pattern of Datura genera (Dobigny et al., 2004; Knight et al., 2005; Bancheva and Greilhuber, 2006; Guerra, 2008; Bainard et al., 2013). Hence, for those reasons, many studies are conducted to genome size estimates and chromosomes studies (Burchardt et al., 2020). Variation of chromosome number in the Datura genus can indicate intra- and inter-specific differences in genomic DNA quantities and also, variation of intra/ interspecific genome size may ref lect karyotypic dif- ferences, such as differences in the case of chromo- some number and size (Bennett et al., 2008). Previously, Blakeslee (1921) reported various chromosome number in D. stramonium as 2n = 12, 25, 26, 36, and 48 in the USA, but in recent years Hassan and Amer (2019) stated that the commonly chromosome number in this spe- cies was 2n = 24. Confirming the latter report, Badr et al. (1997) verified the chromosome base number in D. innoxia and D. stramonium as n = x = 12. Moreover, recently, Sadeghian and Hatami (2022) clarified that D. innoxia is diploid with 2n = 24. Monoploid genome size (1Cx-value) as the amount of DNA of one basic chromo- some set (with chromosome base number x), regardless of the degree of generative polyploidy, aneuploidies, etc. (Greilhuber et al., 2005; Karimzadeh et al., 2011; Abedi et al., 2015). In previous study, the 2C DNA of D. stramonium was reported as 4.18 pg. (Kubešova et al., 2010). Also, in the report of Bennett and Smith (1976) who evaluated the absolute amounts of nuclear DNA for 753 species of angiosperms, using Feulgen microdensitometry. The 2C DNA of D. innoxia was reported as 4.60 pg (Bennett and Smith, 1976). Due to shortcomings in some of the used cytogenetic techniques and lack of access to detailed information on DNA C value, karyology, and ploidy lev- els of Datura genus and since Iran is located in the cent- er of the origin of diversity, so it is expected that Irani- an Datura germplasm indicates much of the worldwide genetic diversity of Datura. On the other hand, there is no reliable report regarding the number of chromosomes and genome size regarding Datura genus in Iran. Thus, reliable conclusions cannot be drawn on the actual range of chromosomal variation in Datura without consider- ing the Iranian germplasm. Hence, the current study, for the first time, was aimed to provide a detailed survey of chromosomal and genome size variation in the Iranian D. stramonium and D. innoxia by focusing on popula- tions that were not studied before. For this purpose, sev- eral Iranian populations of D. stramonium and D. innox- ia were investigated. MATERIALS AND METHODS Seed collection site The seeds of 13 Iranian endemic populations of Datura stramonium (9 populations) and D. innoxia (4 populations) were collected from different sites of Iran 55Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia during the October and November of 2021, also, two populations (P9, P10) of Datura stramonium species were prepared from Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany. The species code and geographical descriptions, including latitude, longi- tude, and altitude are shown in Table 1 and Figure 1. Plant material and growing conditions for genome size esti- mation For study the genome size, the collected seeds were planted in grow bags with 10 kg soil (sandy loam) and placed for three months in greenhouse of agricultural faculty of Tarbiat Modares University in Tehran. Under greenhouse conditions, average air temperature was 25 °C. Finally, the developed leaves at the four-leaf stage were collected to determine their genome size. Flow cytometric genome size estimation The 2C-value of each Datura species was determined by f low cytometric analyses. FCM (Flow cytometric) analysis was carried out by PI (Propidium Iodide) stain- ing technique and Solanum lycopersicum cv. Stupicke; 2C = 1.96 pg DNA (Doležel et al., 1998) as an internal ref- erence standard plant (Figure 2). About 2 cm2 of healthy fresh young leaves of Datura and internal reference standard were co-chopped with a sharp razor blade in a glass petri dish, containing one ml of ice-cold WPB buff- er (Woody Plant Buffer, Loureiro et al., 2007). The crude nuclei suspension was filtered through a 30 μm green nylon mesh (Partec, Münster, Germany). Then RNase (Sigma-Aldrich Corporation, MO, USA) and propidium iodide (PI; ach 50 μg ml−1) was added. For the resulting sample, the relative f luorescence intensity was calcu- lated. After incubation for two min at RT, to determine the amount of genomic 2C DNA, the nuclei suspension was examined by BD FACSCantoTM-KE flow cytometer (BD Biosciences, Bedford, MA, USA), equipped with an Table 1. Locality collection characteristics of D. stramonium and D. innoxia. Altitude (m) Latitude (N) Longitude (E) Local Collection locations Population codes 1723 35°43’57” 53°37’49” Semnan, Semnan, Iran S1P1 1612 32°36’12” 51°26’01” Isfahan, Isfahan, Iran S1P2 120 39°29’18” 48°07’49” Mughan plain, Ardabil, Iran S1P3 1500 36°42’15” 48°21’31” Zanjanrood, Zanjan, Iran S1P4 1362 36°26’17” 45°56’43” West Azerbaijan, Iran S1P5 65 38°06’44” 41°07’33” Saravan, Gilan, Iran S1P6 30 33°95’04” 41°55’89” Venous Rezvanshahr, Gilan, Iran S1P7 1505 29°34’80” 52°35’26” Shiraz, Fars, Iran S1P8 1880 11°16’46” 51°49’27” RuBland (RUS) S1P9 1880 11°16’46” 51°49’27” Brasitieh (BRA) S1P10 1269 35°44’17” 51°10’23” Tehran, Tehran, Iran S1P11 1800 37°12’11” 44°52’21” Turgor, Urmia, Iran S2P1 1914 34°27’00” 46°80’76” Mahidasht, Kermanshah, Iran S2P2 981 34°35’17” 50°49’02” Qom, Qom, Iran S2P3 838 29°49’17” 51°33’48” Kazerun, Fars, Iran S2P4 S1: Datura stramonium, S2: Datura innoxia. Figure 1. Location of the sampling sites of 13 Iranian endemic Datura populations on the map of Iran. 56 Zahra Morovati et al. argon ion laser (488 nm) via BD FACSDivaTM software. At least 5,000 nuclei were typically analyzed for each sample in three replications (Sayadi et al., 2022; Zarabi- zadeh et al., 2022). For create a histograms, the range of gating zone was calculated by using the Partec FloMax ver. 2.4e. (Partec, Münster, Germany). The measurements of relative fluorescence intensity of stained nuclei were performed on a linear scale. By calculating the values of the means of G1 peak, the absolute DNA amount of each sample was estimated (Doležel et al., 2003, 2007; Greilhu- ber et al., 2005; Karimzadeh et al., 2011) as follows: Sample 2Cx DNA (pg) = (Sample G1 peak mean/Stand- ard G1 peak mean) × Standard 2C DNA (pg). Value was calculated based on a conversion formula where 1 pg of DNA represents 978 Mbp (Doležel et al., 2003). Chromosome analysis Initially, the scraped seeds were placed in Petri dishes with sandpaper and germinated on moist filter paper at 20 - 25 °C under light conditions in a growth chamber. For the cytological preparations, each root tip (0.5 - 1 cm long) was removed and pretreated with 0.002 M 8- hydroxyquinoline at 25 °C for 2.5 h in the dark to induce cell cycle delay in metaphase. The roots were washed by distilled H2O in several times and fixed in 3:1 (v/v) of ethanol and glacial acetic acid (Carnoy solution) at 4 °C for 17 h. The fixed roots were washed in distilled H2O, hydrolyzed in 1 M HC1 at 60 °C (11 min for D. stramonium and 13 min for D. innoxia) in a water bath, and washed in water, then stained by aceto-orcein 1% (w/v) at 25 °C (50 min for D. stramonium and 60 min for D. innoxia) in darkness (Reference). Finally, for micro- scopic studies, the five root tips from different individu- als were squashed in a drop of 45% (v/v) acetic acid and analyzed per Datura populations. Slides were examined and High-resolution microscopic digital photographs (Super High Quality; SHQ; Tiff format images) were acquired, using an Olympus BX50 (Olympus Optical Co., Ltd., Tokyo, Japan) microscope equipped with an Olympus DP12 digital camera. It is reminded that each replicate is a cell from the meristem of the plant and five slides from the terminal meristem of five different plants were prepared from each population. Statistical analysis The karyotypic and flow cytometric data was ana- lyzed according to analysis of variance based on a com- pletely randomized design with five and three replica- tions, using SAS Statistical Package Program version 9.0 and SPSS software version 20. The PROC UNIVARIATE within SAS was used to test the assumptions of ANOVA, and residuals were normally distributed. The means were compared through the least significant difference (LSD) posthoc test at the 5% probability level. Moreover, the standard errors of the means were calculated. RESULTS Chromosome counts and length and ploidy level Figures 3 show the somatic complement karyotypes in the 15 D. stramonium and D. innoxia populations. All cells studied of the examined Datura populations consistently had ploidy levels and chromosome number of 2n =2x= 24 were with small chromosomes. Based on ANOVA results, among populations D. stramonium for Chromosome length (CL), were significant differences (P < 0.05; Table 2). The mean chromosome length (CL) was determined as 1.966 μm, varied from 1.627 μm (S1P2) to 2.286 μm (S1P8, Table 3). On the other hand, the ANO- VA results, among populations D. innoxia verified sig- nificant differences (P < 0.01; Table 2) in Chromosome length (CL). The highest and the least values of Chromo- some length (CL) in S2P1 (2.819 μm) and S2P2 (1.967 Figure 2. Two species of Datura in grow bags in greenhouse (a). Datura innoxia, (b) D. stramonium (c). Solanum lycopersicum cv. Stu- picke (2C = 1.96 pg DNA) the internal reference standard plant (d). 57Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia μm), respectively and the mean Chromosome length in this species was 2.388 μm (Table 3). Flow cytometric analysis of monoploid genome size The nuclear DNA values of 15 populations of two species of Datura genus were estimated by flow cytom- etry. In the process of estimating the DNA content of the nuclei in the leaf tissue, two peaks were observed in the obtained histograms. In all populations under study, the left peak corresponds to the Solanum lycopersicum cv. Stupicke (2C value = 1.96 pg DNA) internal reference standard plant, and the right peaks refer to the Datura populations (Figures 4). Based on the ANOVA results (Table 4), no significant difference in the comparison Figure 3. Somatic chromosomes (2n = 2x = 24) of 11 Datura stramonium populations and four D. innoxia populations. Scale bar = 5 μm. 58 Zahra Morovati et al. of genome size among populations D. stramonium was observed. However, the mean monoploid genome size was determined as 3.8 pg, varied from 3.650 pg (S1P5) to 3.934 pg (S1P4). Also, the ANOVA results, between four populations D. innoxia verified significant differences in genome size (Table 4). The mean genome size (Table 5) was determined as 3.91 pg, varied from 3.682 pg (S2P2) to 4.305 pg (S2P1). DISCUSSION Fifteen Datura populations we studied, among which nine populations of Datura stramonium and four populations of D. innoxia were of Iranian endemic ori- gin. The results of the current study, which were used to examine karyotype diversity and estimate genome size from the new and unworked populations of Datura plant, are being reported for the first time in the world. Our results provide basic cytogenetic information for these two species, which are helpful for the whole- genome sequencing and the construction of genetic and physical maps in the future. Cytogenetic investigations carried out on the populations of Datura stramonium and D. innoxia showed that all the studied populations were diploid with chromosome number of 24, which was completely consistent with the results of previous reports (Badr et al., 1997; Hassan and Amer, 2019; Sadeghian and Hatami, 2022). Because of short chromosomes’ lengths, the locations of the centromeres could not be identified clearly, hence chromosome length (CL) param- eter was measured, as reported for different species by researchers (e.g. Morales Valverde, 1986; Karimza- deh et al., 2010; Abbasi-Karin et al., 2022; Rasekh and Karimzadeh, 2023, Yari et al., 2024). According to the results of the current study, the mean chromosome length (CL) in D. stramonium and D. innoxia popula- tions was 1.97 µm and 2.39 µm, respectively. In other words, D. innoxia populations had 21% larger chromo- somes. Moreover, in the present study, the leaf materi- als were used for the estimation of genome size, using Table 2. ANOVA of chromosome length (µm) of Datura stramoni- um and D. innoxia populations. S.O.V. D. stramonium D. innoxia df MS df MS Population 10 0.17039* 3 0.6176** Error 44 0.06506 8 0.1081 Total 54 11 CV% 12.97 13.77 *Significant (P<0.05); **Significant (P<0.01). Table 3. Means (±SE) and the range comparisons of chromosome length (µm) of Datura stramonium and D. innoxia populations. Population codes CL (µm) D. stramonium Population codes CL (µm) D. innoxia S1P1 2.032 ± 0.212abc S2P1 2.819 ± 0.076a S1P2 1.627 ± 0.071d S2P2 1.967 ± 0.142b S1P3 2.133 ± 0.132ab S2P3 2.323 ± 0.119ab S1P4 1.757 ± 0.061cd S2P4 2.441 ± 0.216ab S1P5 1.883 ± 0.086bcd --- --- S1P6 2.006 ± 0.100abc --- --- S1P7 2.005 ± 0.092abc --- --- S1P8 2.286 ± 0.119a --- --- S1P9 1.985 ± 0.068abc --- --- S1P10 2.092 ± 0.160ab --- --- S1P11 1.820 ± 0.040bcd --- --- Mean 1.966 2.3876 Range 1.627-2.286 1.967-2.819 LSD5% 0.325 0.607 CL: chromosome length (µm), S1: Datura stramonium, S2: D. innoxia. Table 4. ANOVA of monoploid genome size (2Cx DNA, pg) of Datura stramonium and D. innoxia populations. S.O.V. D. stramonium D. innoxia df MS df MS Population 10 0.03099ns 3 0.25142* Error 44 0.05217 8 0.04042 Total 54 11 CV% 6.0 5.12 ns non-significant (P>0.05); * Significant (P<0.05).. Table 5. Means (±SE) and the range monoploid genome size (DNA 2Cx value, pg) of D. innoxia populations. Population codes 2Cx genome size (pg) 1Cx genome size (pg) 1Cx genome size (Mbp) S2P1 4.305 ± 0.032a 2.152 2104.66 S2P2 3.682 ± 0.122b 1.841 1800.50 S2P3 3.708 ± 0.119b 1.854 1813.21 S2P4 3.963 ± 0.155ab 1.982 1938.40 Mean 3.914 1.957 1914.19 Range 3.682-4.305 1.841-2.152 1800.50-2104.66 LSD5% 0.377 59Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia N um be r o f n uc le i Relative nuclear DNA content (Arbitrary units) !"#A%CD#C(CF*C+,-./01"2/CP4RSTC89CCCCCC4.-,"W#A+%C;<<*T?"/A%C@@C+ > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> !DAT=CCC W0 21 ,+ > "> #>> #"> $>> $"> > $( "% '# #>' #)" DDAT=CCC W0 21 ,+ > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> 4A-A4TAB;T;T=CCC W0 21 ,+ > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> !DAT=CCC DD AT =C CC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> DDAT=CCC 4A -A 4T AB ;T ;T =C CC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> 4A-A4TAB;T;T=CCC 4A -A 4T AB ;T ;T C CCC G+,-./M# > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> !DAT=CCC W0 21 ,+ G+,-./M# > "> #>> #"> $>> $"> > $( "% '# #>' #)" DDAT=CCC W0 21 ,+ G+,-./M# > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> 4A-A4TAB;T;T=CCC W0 21 ,+ G+,-./M# > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> !DAT=CCC DD AT =C CC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> DDAT=CCC 4A -A 4T AB ;T ;T =C CC 89 G+,-./M# > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> 4A-A4TAB;T;T=CCC 4A -A 4T AB ;T ;T C CCC a.-,AWC4=D M-N234 G+,- R4N+,-6 73U4, 73U4,V:; ? =-+4>? 7@>?< G=4>A =-+4>A 7@>A< M# BC34-a #ECC//// #ECC//// >//// )EF)$ "CF(%/// "EFE#/// )#F>) C(F(%/// C(FE)/// 'F#' S1P1 !"#A%CD#C(CF*C+,-./01"2/CP4RSCTC89CCCCCC4.-,"W#A+%C;<<*T?"/A%C@RC+ > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> !DAT=CCC W0 21 ,+ > "> #>> #"> $>> $"> > ## $$ (( %% "" DDAT=CCC W0 21 ,+ > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> 4A-A4TAB;T;T=CCC W0 21 ,+ > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> !DAT=CCC DD AT =C CC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> DDAT=CCC 4A -A 4T AB ;T ;T =C CC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> 4A-A4TAB;T;T=CCC 4A -A 4T AB ;T ;T C CCC )G+,-./# > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> !DAT=CCC W0 21 ,+ )G+,-./# > "> #>> #"> $>> $"> > ## $$ (( %% "" DDAT=CCC W0 21 ,+ )G+,-./# > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> 4A-A4TAB;T;T=CCC W0 21 ,+ )G+,-./# > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> !DAT=CCC DD AT =C CC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> DDAT=CCC 4A -A 4T AB ;T ;T =C CC 89 )G+,-./# > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> 4A-A4TAB;T;T=CCC 4A -A 4T AB ;T ;T C CCC a.-,AWC4=D /,MN23 )G+, 43MG+,R 6273+ 6273+UV: ;)G+,R )<3=> <,G3=> 6?=>; )<3=@ <,G3=@ 6?=@; /# AB23,C #%#a.... #%#a.... =.... $'E(' #>"E'(.. ###EC%.. (>EC" #>FE>'.. #>aE(".. $#EC" S1P2 !"#A%CD#C(CF*C+,-./01"2/CP4RSTC89CCCCCC4.-,"W#A+%C;<<*T?"/A%C@9C+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DAT=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! ' #C $% ($ %! DDAT=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-A4TAB;T;T=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DAT=CCC DD A T= CCC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDAT=CCC 4A -A 4T A B; T; T= CCC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-A4TAB;T;T=CCC 4A -A 4T A B; T; TC CCC )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DAT=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! ' #C $% ($ %! DDAT=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-A4TAB;T;T=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DAT=CCC DD A T= CCC )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDAT=CCC 4A -A 4T A B; T; T= CCC 89 8a )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-A4TAB;T;T=CCC 4A -A 4T A B; T; TC CCC E.-,AWC4=D M,O34R )G+, 6ROG+,7 U4VR+ U4VR+:;< =)G+,7 )>R?@ >,GR?@ UA?@= )>R?B >,GR?B UA?B= M# )# $"C..... $"C..... ?.... $Ca!E '$aC"... '$a'%... CaCC #!"aC".. #!CaE".. #"a"C M$ )# C$'..... C$'..... ?.... C(aE# #""aC%.. #""aE#.. "aEC ECaE!... E'a'$... #%a$( S1P3 !"#A%CD#C(F*C+,-./01"2/CP4RSTC89CCCCCC4.-,"W#A+%C;<<*T?"/A%C9<@C+ > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> !DAT=CCC W0 21 ,+ > "> #>> #"> $>> $"> > #C ($ %' C% '> DDAT=CCC W0 21 ,+ > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> 4A-A4TAB;T;T=CCC W0 21 ,+ > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> !DAT=CCC DD A T= CCC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> DDAT=CCC 4A -A 4T A B; T; T= CCC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> 4A-A4TAB;T;T=CCC 4A -A 4T A B; T; TC CCC )G+,-./# > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> !DAT=CCC W0 21 ,+ )G+,-./# > "> #>> #"> $>> $"> > #C ($ %' C% '> DDAT=CCC W0 21 ,+ )G+,-./# > "> #>> #"> $>> $"> > $>> %>> C>> '>> #>>> 4A-A4TAB;T;T=CCC W0 21 ,+ )G+,-./# > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> !DAT=CCC DD A T= CCC > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> DDAT=CCC 4A -A 4T A B; T; T= CCC 89 )G+,-./# > "> #>> #"> $>> $"> > "> #>> #"> $>> $"> 4A-A4TAB;T;T=CCC 4A -A 4T A B; T; TC CCC a.-,AWC4=D /,MN23 )G+, 43MG+,R 6273+ 6273+UV: ;)G+,R )<3=> <,G3=> 6?=>; )<3=@ <,G3=@ 6?=@; /# AB23,C #>#(.... #>#(.... =.... $>a$C "Ca(>... "Ea'>... (Ca%$ '$a'F... '%a'(... $(a'C S1P4 !"#A%CD#C(CF*C+,-./01"2/CP4RCST89CCCCCC4.-,"W#A+%C;<<*T?"/A%CR@C+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DAT=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $! %! C! '! #!! DDAT=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-A4TAB;T;T=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DAT=CCC DD AT =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDAT=CCC 4A -A 4T AB ;T ;T =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-A4TAB;T;T=CCC 4A -A 4T AB ;T ;T C CCC ()G+,-(#./#-M/-/$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DAT=CCC W0 21 ,+ ()G+,-(#./#-M/-/$ ! "! #!! #"! $!! $"! ! $! %! C! '! #!! DDAT=CCC W0 21 ,+ ()G+,-(#./#-M/-/$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-A4TAB;T;T=CCC W0 21 ,+ ()G+,-(#./#-M/-/$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DAT=CCC DD AT =C CC ()G+,-(#./#-M/-/$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDAT=CCC 4A -A 4T AB ;T ;T =C CC 89 8a ()G+,-(#./#-M/-/$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-A4TAB;T;T=CCC 4A -A 4T AB ;T ;T C CCC E.-,AWC4=D /+1O34 ()G+ R41)G+6 73U4G 73U4GV:; <()G+6 (=4>? =+)4>? 7@>?< (=4>A =+)4>A 7@>A< /# (# #B%----- #B%----- >---- ##C!% %$C#$--- %$C$"--- aCE$ a#CC$--- a#CEB--- ECCC /$ (# #!'!---- #!'!---- >---- ''CEC '#C%C--- '#CC!--- "C'% a#C$E--- a#C%"--- aC!% S1P5 !"#A%CD#C(CF*C+,-./01"2/CP4RRSTC8RCCCCCC4.-,"9#A+%CW;;;CCC<9=*T>"/A%C?@C+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DATR?@ >,GR?@ UA?@= )>R?B >,GR?B UA?B= M# )# $(C..... $(C..... ?.... $%C%( aaCC"... aaC'"... aC!E #!CC!!.. #!CCE#.. #(C$! M$ )# a(!..... a(!..... ?.... a"C"a #%#C(%.. #%#CC$.. CC(( EaC"(... E'C($... #$CEE S1P6 !"#A%CD#C(CF*C+,-./01"2/CP4RST8C9RCCCCCC4.-,"W#A+%C;<<*8?"/A%CSSC+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !D@8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! ( ) ## #" #G DD@8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-@48@A;8;8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !D@8=CCC DD @8 =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DD@8=CCC 4A -@ 48 @A ;8 ;8 =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-@48@A;8;8=CCC 4A -@ 48 @A ;8 ;8 B CCC +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !D@8=CCC W0 21 ,+ +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! ( ) ## #" #G DD@8=CCC W0 21 ,+ +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-@48@A;8;8=CCC W0 21 ,+ +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !D@8=CCC DD @8 =C CC +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DD@8=CCC 4A -@ 48 @A ;8 ;8 =C CC 9R 9C +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-@48@A;8;8=CCC 4A -@ 48 @A ;8 ;8 B CCC a.-,AWC4=D O.4R67 +,-. U74,-.V :6;7- :6;7-<=> ?+,-.V +@7AB @.,7AB :CAB? +@7Aa @.,7Aa :CAa? O# +# $(CMMMMM $(CMMMMM AMMMM C(EC# "(E""MMM "%E#(MMM #%E'! )CEG!MMM ))E(!MMM #!E%G O$ +# #("MMMMM #("MMMMM AMMMM (CE(G ##!EC"MM ##!EG)MM )EC) )CEG'MMM ))E#%MMM CE)$ S1P7 !"#A%CD#C(CF*C+,-./01"2/CP4RST8C9RCCCCCC4.-,"W#A+%C;<<*8?"/A%C@AC+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DB8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! ( #' $) GC %" DDB8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-B48BC;8;8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DB8=CCC DD B8 =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDB8=CCC 4A -B 48 BC ;8 ;8 =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-B48BC;8;8=CCC 4A -B 48 BC ;8 ;8 a CCC +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DB8=CCC W0 21 ,+ +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! ( #' $) GC %" DDB8=CCC W0 21 ,+ +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-B48BC;8;8=CCC W0 21 ,+ +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DB8=CCC DD B8 =C CC +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDB8=CCC 4A -B 48 BC ;8 ;8 =C CC 9R 9E +,-./M+#1O#M3OMO$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-B48BC;8;8=CCC 4A -B 48 BC ;8 ;8 a CCC c.-,AWC4=D O.4R67 +,-. U74,-.V :6;7- :6;7-<=> ?+,-.V +@7AB @.,7AB :CAB? +@7Aa @.,7Aa :CAa? O# +# G"$MMMMM G"$MMMMM AMMMM G)E$" %%E$%MMM %%E%'MMM #!E"" C(E#(MMM C(E%$MMM 'EC! O$ +# "(GMMMMM "(GMMMMM AMMMM C$E)" 'CEG#MMM 'CE"'MMM )E(C C(E$$MMM C(E$(MMM %E)$ S1P8 !"#A%CD#C(CF*C+,-./01"2/CP4RST89RCCCCCC4.-,"W#A+%C;<<*8?"/A%C@AC+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DB8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! #! $! (! %! "! DDB8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-B48BC;8;8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DB8=CCC DD B 8= CCC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDB8=CCC 4A -B 48 B C; 8; 8= CCC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-B48BC;8;8=CCC 4A -B 48 B C; 8; 8a CCC )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DB8=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! #! $! (! %! "! DDB8=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-B48BC;8;8=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DB8=CCC DD B 8= CCC )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDB8=CCC 4A -B 48 B C; 8; 8= CCC 9R 9E )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-B48BC;8;8=CCC 4A -B 48 B C; 8; 8a CCC c.-,AWC4=D M,O34R )G+, 6ROG+,7 U4VR+ U4VR+:;< =)G+,7 )>R?@ >,GR?@ UA?@= )>R?B >,GR?B UA?B= M# )# (%!..... (%!..... ?.... (!C(( "!C'"... "!Ca(... "CEE CCC(a... CCC%"... %C(( M$ )# E'#..... E'#..... ?.... CaCCE aaCC$... aaC'!... "Ca# C'C(E... C'C%C... "C!% S1P9 !"#A%CD#C(CF*C+,-./01"2/CP4RST8C9WCCCCCC4.-,";#A+%C*8?"/A%C@G+,-U G?6@A ?-+6@A VB@A> G?6@C ?-+6@C VB@C> 1# G# C"////// C"////// @//// #%aE% "'a"%/// "'a")/// %a#! E#a%C/// E#a'(/// #!a%) 1$ G# (EC///// (EC///// @//// '"a$C )(aC"/// )(a'C/// CaE$ C)aCE/// C)aEE/// "a"$ S1P10 !"#A%CD#C(CF*C+,-./01"2/CP4RST8C9SCCCCCC4.-,"W#A+%C;<<*8?"/A%C@RC+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DA8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! ' #C $% ($ %! DDA8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-A48AB;8;8=CCC W0 21 ,+ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DA8=CCC DD A8 =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDA8=CCC 4A -A 48 AB ;8 ;8 =C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-A48AB;8;8=CCC 4A -A 48 AB ;8 ;8 C CCC )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DA8=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! ' #C $% ($ %! DDA8=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! 4A-A48AB;8;8=CCC W0 21 ,+ )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DA8=CCC DD A8 =C CC )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDA8=CCC 4A -A 48 AB ;8 ;8 =C CC 9S 9R )G+,-.)#/M#.1M.M$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 4A-A48AB;8;8=CCC 4A -A 48 AB ;8 ;8 C CCC a.-,AWC4=D M,O34R )G+, 6ROG+,7 U4VR+ U4VR+:;< =)G+,7 )>R?@ >,GR?@ UA?@= )>R?B >,GR?B UA?B= M# )# #$$..... #$$..... ?.... #'C$a %EC#(... %EC(a... #!CC' CaCC"... CaC'"... ECC" M$ )# "%"..... "%"..... ?.... '#CE# a$C#'... a$C%'... 'C!! E$C%E... E$CC"... EC#$ S1P11 !"#A%CD#C(CF*C"++,-"KC/01OPC41CRSC/DA#AT8A9OCCCCCC0KW8"T#A;%C<===CCC>T?*P@"AA%C1BCC; ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DaP>CCC T, E+ 8; ! "! #!! #"! $!! $"! ! #( $C () "$ C" DDaP>CCC T, E+ 8; ! "! #!! #"! $!! $"! ! (! C! )! #$! #"! 0AWa0PacCCC T, E+ 8; ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DaP>CCC DD aP >C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDaP>CCC 0A Wa 0P ac

C CC ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 0AWa0PacCCC 0A Wa 0P ac

CCC T, E+ 8; G+,-./G#M1#/O1/1$ ! "! #!! #"! $!! $"! ! #( $C () "$ C" DDaP>CCC T, E+ 8; G+,-./G#M1#/O1/1$ ! "! #!! #"! $!! $"! ! (! C! )! #$! #"! 0AWa0PacCCC T, E+ 8; G+,-./G#M1#/O1/1$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! !DaP>CCC DD aP >C CC G+,-./G#M1#/O1/1$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! DDaP>CCC 0A Wa 0P ac

C CC 41 4B G+,-./G#M1#/O1/1$ ! "! #!! #"! $!! $"! ! "! #!! #"! $!! $"! 0AWa0PacCCC 0A Wa 0P ac

D 1-34R6 G+,- 763+,-U VR:6, VR:6,;<= >G+,-U G?6@A ?-+6@A VB@A> G?6@C ?-+6@C VB@C> 1# G# %a#///// %a#///// @//// "!E%( %"EC'/// %"E'C/// 'Ea% a'E(C/// a'ECa/// )E!" 1$ G# %C(///// %C(///// @//// %)E"a #!(E(!// #!(E('// (E)" '#E!a/// '#E(#/// aE)# S2P1 !"#A%CD#C(CF*C"++,-".C/012P4CR1CCCCCC0.ST"8#A9%CW;;;CCC<8=*4>"?A%C2WC9 ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !D@4R?@ >,GR?@ UA?@= )>R?B >,GR?B UA?B= M# )# "!C..... "!C..... ?.... %'C$' "'C$!... "'C($... CC%a E'C("... E'C%"... "C## M$ )# "%$..... "%$..... ?.... "#CE$ #!CCEE.. #!CCaE.. CC#C ECC'a... ECCaa... "C$' S2P2 !"#A%CD#C(CF*C"++,-".C/012P4CR1CCCCCC0.ST"8#A9%CW;;;CCC<8=*4>"?A%C@1C9 ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DA4G+,-U G?6@A ?-+6@A VB@A> G?6@C ?-+6@C VB@C> 1# G# #a(///// #a(///// @//// (!E)' CaE$a/// CaEC"/// #!E(# '$E#)/// '$E%(/// )Ea) 1$ G# %(%///// %(%///// @//// CaE$$ ##aE"!// ##aEa$// 'E%C )'E$%/// )'E()/// "E)" S2P3 !"#A%CD#C(CF*C"++,-".C/012P4CRSCCCCCC0.T8"9#AW%C;222CCC<9=*4>"?A%C@ACW ! "! #!! #"! $!! $"! ! $!! %!! C!! '!! #!!! !DB4G+,-U G?6@A ?-+6@A VB@A> G?6@C ?-+6@C VB@C> 1# G# $$'///// $$'///// @//// CCaCE C"a!)/// C"a$(/// 'a(% '%a$%/// '%a%'/// EaCE 1$ G# ##%///// ##%///// @//// ))a)) #)Ea#"// #)Ea$(// %a"# '#a))/// '#a%)/// %a(# S2P4 File: Sl + D. stramonium (P2)- R1 Particles: 5000 Acq.-Time: 44 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 27 54 81 108 135 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: R1 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: R1 0 50 100 150 200 250 0 27 54 81 108 135 SSC-A co un ts Gate: R1 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: R1 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 Gate: R1 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 1966 1966 - 39.32 56.74 59.91 31.03 67.74 67.93 8.18 File: Sl + D. stramonium (P3) - R1 Particles: 5000 Acq.-Time: 83 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 11 22 33 44 55 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: R1 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: R1 0 50 100 150 200 250 0 11 22 33 44 55 SSC-A co un ts Gate: R1 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: R1 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 Gate: R1 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 1419 1419 - 28.38 105.83 111.64 30.65 107.08 109.35 21.65 File: Sl + D. stramonium (P4)- R1 Particles: 5000 Acq.-Time: 81 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 8 16 24 32 40 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 8 16 24 32 40 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 257 257 - 26.09 82.65 82.84 6.77 105.75 106.95 15.57 R2 G1 728 728 - 73.91 155.64 155.91 5.97 97.90 98.82 14.23 File: Sl +D. stramonium (P7)- R1 Particles: 5000 Acq.-Time: 109 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 16 32 48 64 80 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: R1 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: R1 0 50 100 150 200 250 0 16 32 48 64 80 SSC-A co un ts Gate: R1 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: R1 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 Gate: R1 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 1013 1013 - 20.26 56.30 59.80 36.42 82.87 84.83 23.86 File: Sl + D. stramonium (P8 )-R1 Particles: 5000 Acq.-Time: 89 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 20 40 60 80 100 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 20 40 60 80 100 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 134 134 - 11.04 42.12 42.25 7.92 71.62 71.93 9.66 R2 G1 1080 1080 - 88.96 81.46 81.60 5.84 71.29 71.45 7.04 File: Sl + D. stramonium (P11)- R1 Particles: 5000 Acq.-Time: 86 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 8 16 24 32 40 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 8 16 24 32 40 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 236 236 - 24.43 77.65 77.85 7.09 106.00 106.91 13.20 R2 G1 730 730 - 75.57 141.34 141.62 6.33 97.53 98.32 12.99 File: Sl + D. stramonium (P14)- R1 Particles: 500 Acq.-Time: 44 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 3 7 11 15 19 SSC-A co 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 3 7 11 15 19 SSC-A co Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 236 236 - 63.61 53.55 54.13 14.80 76.90 77.30 10.49 R2 G1 135 135 - 36.39 110.65 110.97 7.67 76.98 77.14 6.72 File: Sl + D. stramonium (P17)- R1 Particles: 5000 Acq.-Time: 86 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 9 18 27 36 45 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 9 18 27 36 45 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 352 352 - 37.25 44.24 44.48 10.55 69.19 69.42 8.60 R2 G1 593 593 - 62.75 86.31 86.58 7.96 69.22 69.29 4.72 File: Sl + D. stramonium (P18)-R1 Particles: 5000 Acq.-Time: 43 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 10 20 30 40 50 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 10 20 30 40 50 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A SS C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A Pe rC P- C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A Pe rC P- C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 340 340 - 30.33 50.85 50.93 5.77 66.39 66.45 4.33 R2 G1 781 781 - 69.67 99.62 99.80 5.91 68.37 68.46 5.04 File: Sl + D. stramonium (P20)- R1 Particles: 5000 Acq.-Time: 85 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 6 13 19 26 33 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 6 13 19 26 33 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 65 65 - 14.74 58.54 58.59 4.10 71.46 71.83 10.49 R2 G1 376 376 - 85.26 93.65 93.86 6.72 69.67 69.77 5.52 File: Sl + D. stramonium (P21)- R1 Particles: 5000 Acq.-Time: 62 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 8 16 24 32 40 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 8 16 24 32 40 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 122 122 - 18.29 47.13 47.39 10.68 69.65 69.85 7.65 R2 G1 545 545 - 81.71 92.18 92.48 8.00 72.47 72.65 7.12 File: Sl + D. innoxia (P1)- R1 OK (Selected) Particles: 5000 Acq.-Time: 123 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 13 26 39 52 65 SSC-A co un ts 0 50 100 150 200 250 0 30 60 90 120 150 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 13 26 39 52 65 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 30 60 90 120 150 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 471 471 - 50.43 45.68 45.86 8.74 78.36 78.67 9.05 R2 G1 463 463 - 49.57 103.30 103.38 3.95 81.07 81.31 7.91 File: Sl + D. innoxia (P17)- R1 Particles: 5000 Acq.-Time: 75 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 12 24 36 48 60 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 12 24 36 48 60 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 506 506 - 48.28 58.20 58.32 6.49 78.35 78.45 5.11 R2 G1 542 542 - 51.72 106.77 106.97 6.16 76.89 76.99 5.28 File: Sl + D. innoxia (P18)- R1 Particles: 5000 Acq.-Time: 31 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 4 8 13 17 22 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 4 8 13 17 22 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 193 193 - 30.78 69.29 69.65 10.31 82.17 82.43 7.97 R2 G1 434 434 - 69.22 119.50 119.92 8.46 78.24 78.37 5.75 File: Sl + D. innoxia (P20)- R1 Particles: 5000 Acq.-Time: 79 s 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts 0 50 100 150 200 250 0 4 9 13 18 23 SSC-A co un ts 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 FSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 4 9 13 18 23 SSC-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 200 400 600 800 1000 PerCP-Cy5-5-A co un ts Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 FSC-A S S C -A Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 SSC-A P er C P -C y5 -5 -A R1 R2 Gate: G1=R1 OR R2 0 50 100 150 200 250 0 50 100 150 200 250 PerCP-Cy5-5-A P er C P -C y5 -5 -W partec PAS Region Gate Ungated Count Count/ml %Gated GMn-x Mean-x CV-x% GMn-y Mean-y CV-y% R1 G1 228 228 - 66.67 65.03 65.29 8.94 84.24 84.48 7.67 R2 G1 114 114 - 33.33 137.15 137.29 4.51 81.33 81.43 4.91 S2P4S2P3S2P2 S2P1S1P11S1P10 S1P9S1P8S1P7 S1P6S1P5S1P4 S1P3S1P2S1P1 Relative nuclear DNA content (Arbitrary units) ucle un ts un ts Figure 4. Histograms of monoploid genome size (2Cx DNA content) of Datura stramonium and D. innoxia populations. The left peaks refer to the G1 peaks of Solanum lycopersicum cv. Stupicke (2C = 1.96 pg DNA) as an internal reference standard plant and the right peaks refer to the G1 peaks of the samples. 60 Zahra Morovati et al. flow cytometric analysis (Mohammadpour et al., 2022; Rasekh and Karimzadeh, 2023; Yari et al., 2024). The mean monoploid genome size in D. stramonium and D. innoxia populations was 3.799 pg and 3.914 pg, respec- tively. The cytogenetic information obtained from this research is more than the mean chromosome length and the mean genome size reported in the previous studies (Bennett and Smith et al., 1976; Badr et al., 1997). The reason for this is unknown, but this difference could be related to the cell cycle, the rate of cell division, ecologi- cal behavior in plant communities and life forms, and differences between the methods of nuclear DNA con- tent analysis (Bennett et al., 2000). On the other hand, previous studies have only been conducted on one popu- lation. In general, it can be concluded that the average chromosome length and average monoploid genome size in D. innoxia species are 0.40 and 0.11 times higher than those in D. stramonium species, respectively. ACKNOWLEDGMENT Authors acknowledge the Tarbiat Modares Universi- ty (TMU) and Iran National Science Foundation (Grant Number: 4014886) for financial supporting of this research work. The authors would also like to acknowl- edge the financial support of Modares Science and Tech- nology Park for this project. REFERENCES Abbasi-Karin Sh, Karimzadeh G, and Mohammadi- Bazargani M. 2022. Interspecific chromosomal and genome size variations in in vitro propagated wil- low herb (Epilobium spp.) medicinal plant. Cytologia 87(2): 129-135. Abedi R, Babaei A, and Karimzadeh G. 2015. Karyologi- cal and flow cytometric studies of Tulipa (Liliaceae) species from Iran. Plant Syst. Evol. l301: 1473-1484. Al-Zharani M, Nasr FA, Alqahtani AS, Cordero MAW, Alotaibi AA, Bepari A, Alarifi S, Daoud A, Barnawi IO, and Daradka HM. 2021. In vitro cytotoxic evalua- tion and apoptotic effects of Datura innoxia grown in Saudi Arabia and phytochemical analysis. Appl. Sci. 11(6): 2864. Amosova AV, Zoshchuk SA, Rodionov AV, Ghukasyan L, Samatadze TE, Punina EO, Loskutov IG, Yurkevich OY, and Muravenko OV. 2019. Molecular cytoge- netics of valuable Arctic and sub-Arctic pasture grass species from the Aveneae/Poeae tribe complex (Poaceae). BMC Genetic 20(1): 1-16. Badr A, Khalifa SF, Aboel-Atta AI, and Abou-ElEnain MM. 1997: Chromosomal criteria and taxonomic relationships in the Solanaceae. Cytologia 62(2): 103- 113. Bainard JD, Forrest LL, Goffinet B, and Newmaster SG. 2013. Nuclear DNA content variation and evolution in liverworts. Mol. Phylogenet. Evol. 68: 619-627. Blakeslee AF. 1921. Types of mutations and their possible significance in evolution. Am Nat. 55: 254-267. Bancheva S and Greilhuber J. 2006. Genome size in Bul- garian Centaurea s.l. (Asteraceae). Plant Syst. Evol. 257: 95-117. Batool A, Batool Z, Qureshi R, and Raja NI. 2020. Phy- tochemicals, pharmacological properties and biotech- nological aspects of a highly medicinal plant: Datura stramonium. J. Plant Sci., 8(2): 29-40. Bennett MD, Bhandol P, and Leitch I J. 2000. Nuclear DNA amounts in angiosperms and their modern uses- 807 new estimates. Ann. Bot. 86: 859-909. Bennett MD and Smith JB. 1976. Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society B-Biological Sciences 274: Issue 933. Bennett MD, Price HJ, and Johnston JS. 2008. Anthocya- nin inhibits propidium iodide DNA fluorescence in Euphorbia pulcherrima: implications for genome size variation and flow cytometry. Ann. Bot. 101: 777-790. Burchardt P, Buddenhagen CE, Gaeta ML, Souza MD, Marques A, and Vanzela ALL. 2020. Holocentric karyotype evolution in Rhynchospora is marked by intense numerical, structural, and genome size changes. Front. Plant Sci. 11: 1390. Bye R, and Sosa V. 2013. Molecular phylogeny of the jim- sonweed genus Datura (Solanaceae). Systematic Bot- any, 38(3): 818-829. De-la-Cruz IM, Hallab A, Olivares-Pinto U, Tapia-López R, Velázquez-Márquez S, Piñero D, Oyama K, Usadel B, and Núñez-Farfán J. 2021. Genomic signatures of the evolution of defense against its natural enemies in the poisonous and medicinal plant Datura stramo- nium (Solanaceae). Sci. Rep. 11(1): 1-19. Disel NR, Yılmaz M, Kekec Z, and Karanlık M. 2016. Poisoned after diner: Dolma with Datura stramoni- um. Turkish Journal of Emergency Medicine, 15(1): 51-55. Dobigny G, Ducroz JF, Robinson TJ, and Volobouev V. 2004. Cytogenetics and cladistics. Syst. Biol., 53: 470- 484. Doležel J, Bartos J, Voglmayr H, and Greilhuber J. 2003. Nuclear DNA content and genome size of trout and human. Cytometry 51: 127-128. Doležel J, Greilhuber J, Lucretti S, Meister A, Lysak M. A, Nardi L, and Obermayer R. 1998. Plant genome size 61Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia estimation by flow cytometry: Inter-laboratory com- parison. Ann. Bot., 82(Suppl. A): 17-26. Doležel J, Greilhuber J, Suda J. 2007. Estimation of nucle- ar DNA content in plants using flow cytometry. Nat. Protoc., 2: 2233-2244. Ghahraman A. 1998. Flora of Iran. Research Institute of Forests Rangelands. Tehran, Iran, pp. 17. (In Persian) Greilhuber J, Doležel J, Lysák MA, and Bennett MD. 2005. The origin, evolution and proposed stabilization of the terms ‘genome size’and ‘C-value’to describe nucle- ar DNA contents. Ann. Bot. 95: 255-260. Guerra M. 2008. Chromosome numbers in plant cyto- taxonomy: concepts and implications. Cytogenet. Genome. Res. 120: 339-350. Hassan RA and Amer WM. 2019. Biosystematic study of the Egyptian Datura stramonium (Solanaceae). Phy- totaxa 408(3): 178-194. Karimi H. 2008. Weeds of Iran. Iran University Press, Tehran, Iran, 419 p. (In Persian). Karimzadeh G, Danesh-Gilevaei M, and Aghaalikhani M. 2011. Karyotypic and nuclear DNA variations in Lathyrus sativus (Fabaceae). Caryologia 64: 42-54. Karimzadeh G, Mousavi SH., Jafarkhani-Kermani M, and Jalali-Javaran M. 2010. Karyological and nucle- ar DNA variation in Iranian endemic muskmelon (Cucumis melo var. Inodorus). Cytologia 75: 451-461. Knight CA, Molinari NA, and Petrov DA. 2005. The large genome constraint hypothesis: evolution, ecology and phenotype. Ann. Bot. 95: 177-190. Kubešova M, Moravcova L, Suda J, Jarošik V, and Pyšek P. 2010. Naturalized plants have smaller genomes than their non-invading relatives: a flow cytometric analy- sis of the Czech alien flora. Preslia 82(1): 81-96. Loureiro J, Rodriguez E, Doležel J, and Santos C. 2007. Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann. Bot. 100: 875-888. Mohammadpour S, Karimzadeh G, and Ghaffari SM. 2022. Karyomorphology, genome size, and variation of antioxidant in twelve berry species from Iran. Car- yologia 75(4): 133-148. Mohammed FS, Kına E, Sevindik M, Dogan M, and Pehlivan M. 2021. Datura stramonium (Solanaceae): Antioxidant and antimicrobial potentials. Turk. J. Agric. Food Sci. Technol. 9(4): 818-821. Morales Valverde R. 1986. Taxonomía De Los Géneros Thymus (Excluida De La Sección Serpyllum) Y Thymbra En La Península Ibérica). CSIC - Real Jardín Botánico (RJB), Ruizia. Monografias del Jardín Botánico 3: 324 p. Muzafarian V. 2000. Plant Classification. Amirkabir Pub- lications, Tehran, Iran, pp. 393 (In Persian). Papagrigoriou G, Papazoglou D, Lazari D, Zorić L, and Tsialtas J. 2019. Hybridization effects on seed traits of annual Datura accessions focusing on oil concentra- tion and composition. Ind. Crops Prod. 132: 69-75. Pellestor F and Gatinois V. 2020. Chromoanagenesis: A piece of the macroevolution scenario. Mol. Cytogen- et. 13: 3. Rasekh SZ and Karimzadeh G. 2023. Chromosomal and genome size variations in opium poppy (Papaver somniferum L.) from Afghanistan. Caryologia 76(4): 15-22. Sadeghian S and Hatami A. 2022. Chromosome number reports and karyotype analysis of seven species from the flora of Iran. Iran. J. Bot. 28(2): 165-169. Salakhutdinov NF, Volcho KP, and Yarovaya OI. 2017. Monoterpenes as a renewable source of biologically active compounds. Pure. Appl. Chem. 89(8): 1105- 1117. Sayadi V, Karimzadeh G, Naghavi MR, and Rashidi Mon- fared S. 2022. Interspecific genome size variation of Iranian endemic Allium species (Amaryllidaceae). Cytologia 87(4): 335-338. Winterfeld G, Ley A, Hoffmann MH, Paule J, and Röser M. 2020. Dysploidy and polyploidy trigger strong variation of chromosome numbers in the prayer- plant family (Marantaceae). Plant Syst. Evol. 306: 36. Zarabizadeh H, Karimzadeh G, Rashidi Monfared S, and Tarkesh Esfahani S. 2022. Karyomorphology, ploidy analysis, and flow cytometric genome size estimation of Medicago monantha populations. Turk. J. Bot. 46: 50-61. Zhang Y, Guo W, Yuan Z, Song Z, Wang Z, Gao J, Fu W, and Zhang G. 2023. Chromosome-level genome assembly and annotation of the prickly nightshade Solanum rostratum Dunal. Sci. Data 10: 341. Yari A, Karimzadeh G, Rashidi Monfared S, and Sayadi S. 2024. Mixed-ploidy in Iranian endemic Cymbopogon olivieri (Boiss.) Bor: A chromosomal and holoploid genome size study. Cytologia 89(2): 1-5. The genome of the southern short-horned tree dragon Acanthosaura meridiona Trivalairat, Sumontha, Kunya & Chaingkul, 2022 (Squamata, Draconinae) was analyzed using classical and molecular techniques to identify and study its chromosomal and repetitive ele Praween Supanuama, Sittisak Jantaratb*, Thaintip Kraipromb, Somsak Buathipb, Sarun Jumrusthanasanc, Sarawut Kaewsric, Nattasuda Donbunditd, Phichaya Buasriyote, Weera Thongnetrf, Sumalee Phimphang, and Alongklod Tanomtongd Amelioration strategy of saline stress in wheat with salicylic acid: a review Syeda Afia Fairoj1,†, Uttam Kumar Ghosh1,†, Md. Moshiul Islam1,*, Khurshida Jahan1, Anamika1, Sazada Siddiqui2, Mohammed O. Alshaharani2, Ayesha Siddiqua3, Habab Merghani Yassin2 Genomic in situ hybridization (GISH) and performance analysis in intergeneric hybrids from five consecutive generations of Erianthus x Saccharum V. P. Sobhakumari*, K. Mohanraj Giemsa-based chromosome staining and comparative fluorescent banding pattern in five valuable Indian plant species Timir Baran Jha1,*, Mihir Halder2, Biplab Kumar Bhowmick3 Karyomorphology and microsatellites characterization of Limnonectes gyldenstolpei: first report from Thailand Sumalee Phimphan1,*, Suracheat Aiumsumang1, Kan Khoomsab2, Itsara Tangsuwan3, Alongklod Tanomtong4 Chromosome, ploidy analysis, and flow cytometric genome size estimation of Datura stramonium and D. innoxia medicinal plant Zahra Morovati1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2, and Sajad Rashidi Monfared3 Report of genomic doubling in Cyamopsis tetragonoloba (L.) Taub. (Fabaceae): salient features and effects Shefali Singh1,*, Girjesh Kumar2