Interspecific Chromosomal and Genome Size Variations in In Vitro Propagated Willow Herb (Epilobium spp.) Medicinal Plant

Abstract

The genus Epilobium, belonging to the family Onagraceae, is an endemic Iranian medicinal plant that has illustrated a vast variety of pharmacologically important metabolites. The cytological characteristics of eight species collected from different regions of Iran were assessed for the first time. All species analyzed were diploid, six species (E. hirsutum, E. parviflorum, E. roseum, E. algidum, E. anatolicum, and E. confusum) had 2n=2x=36 chromosomes, except for two species (E. frigidum, E. lanceolatum) had 2n=2x=38 chromosomes with unequal in size. The mean chromosome length of all species was 0.53 µm, ranging from 0.46 to 0.64 µm. Propidium iodide (PI) and Solanum lycopersicum cv. Stupicke (2C DNA=1.96 pg) as an internal standard were used for flow cytometric survey of genome size and the levels of ploidy. The mean genome size (2Cx DNA) of all species was 0.86 pg, varied from 0.74 to 0.90 pg, confirming inter-specific variation and revealing considerable variation in genome size within each species. Valuable information on cytogenetics can be used in some research fields, including polygenetic analysis, taxonomic relationships, evolutionary characteristics, ecology, plant growth, and development of plant breeding.

Over the past few decades, plant cytogenetic studies have played a significant role in the advancement and expansion of genetic knowledge, plant breeding, evaluation of phylogenetic relatedness and taxonomic investigations as well as understanding the chromosomal and genetic structure of plant species (Hanrahan and Johnston 2011, Singh 2016) Specifically, karyotypes analysis, ploidy level, estimation of genome size, and monitoring the cell cycle using the flow cytometry (FCM) has a superior implication toward these goals. Chromosome counting can be both time-consuming and requires a certain expertise in cytological techniques, FCM has been devised to facilitate sample preparation and is very convenient to the determination of ploidy level and biosystematics analyses in different plant organs and tissues since the 1980s (Galbraith et al. 1983, Doležel and Bartoš 2005). Nowadays, due to the importance of medicinal species and the high demand for it, the use of FCM in medicinal plants can also provide relevant information about the improvement and production of bioactive compounds (Sliwinska 2018). One of the issues related to plant diversity is the systematic study of plant biological mechanisms that requires having sufficient knowledge and information about the genetic structure, morphological, ecological, and biological characteristics of chromosome complement and the studied plant species (Stebbin 1966, Gianfranco et al. 2008). The medicinal plant willow herb belonging to the genus Epilobium is the largest member of the family Onagraceae; comprising of 165 species (185 taxa) that most of these species are self-compatible and scattered worldwide (Rave 1967) and is divided into two subfamilies, Ludwigia and Onagroideae. Consists of ca. 20 species of the genus are previously found and reported in most provinces of Iran (Azizian 2005). Furthermore, these species grow in diverse habitats, including forests, mountainous regions, and rural areas (Dreger et al. 2016).

Previous cytogenetic assessments on this genus showed the difference between some of the Epilobium species in the base chromosome numbers as well as 2C DNA (Kurabayashi et al. 1962, Bennett et al. 1998, Kron and Husban 2009, Kubešová et al. 2010). Epilobium has been drawn considerable attention due to possessing important secondary metabolites such as flavonoids (kaempferol, quercetin, and myricetin), phenolic acids (ellagic acid, valoneic acid, gallic acid, protocatechuic acid), ellagitannins (oenothein A and B), fatty acids (linolenic, palmitic, linoleic, stearic), and vitamins (Lesuisse et al. 1996, Hiermann and Buca 1997, Velasco and Goffma 1999, Dreger et al. 2016, Kaškonienė et al. 2016). The aforementioned properties are chiefly involved in anti-inflammatory, antioxidant, anti-proliferative, and anti-microbial activities (Hiermann et al. 1986, Rauha et al. 2000, Vitalone et al. 2001, Steenkamp et al. 2006, Hevesi Tóth et al. 2009, Kiss et al. 2011). Some of Epilobium species are essentially well-known in folk medicine due to their beneficial attributes towards human health such as in the treatment of benign prostate hyperplasia (Granica et al. 2014). Epilobium is also used as a food resource. The young leaves of Epilobium could be eaten as salad vegetables or tea an excellent honey from their flowers (Bunney 1992). Despite these remarkable advantages, the required information addressing cytogenetic characteristics on Epilobium species is still elusive.

Therefore, the main objective of this study was to investigate the cytogenetic interspecific variations of eight species of Iranian Epilobium include E. hirsutum, E. parviflorum, E. roseum, E. algidum, E. anatolicum, and E. confusum. Accordingly, chromosome number and chromosome detail, ploidy level, and genome size of the collected Epilobium species were determined. The Cx-values and karyological parameters for this genus were also calculated, and the association of genome size and chromosome length with geographical parameters was discussed.

Materials and methods

Plant materials

The seeds and whole plant of five Epilobium species were collected from natural habitats of Iran in the maturity growth stage of seed in Jun–July 2014 (Table 1). Seeds of eight Epilobium species were provided from the Iranian Biological Resource Center (Table 1). The distribution of the studied is in the areas near to Caspian Sea (Khazar Lake), and characteristics of the local information of the collection sites and geographic distribution are presented in Table 1 and Fig. 1.

Table 1. Locations of collection sites and endemic Epilobium spp. of Iran in this study.

Species name	IBRC No	Species codes	Local collection locations	Longitude (E) Latitude (N)	Altitude (m)
E. hirsutum	IBRC P1012152	S1	Chalus, Mazandaran, Iran	36°38′, 51°24′	80
E. parviflorum	IBRC P1012148	S2	Urmia, West Azerbaijan, Iran	37°10′, 45°7′	1,719
E. roseum	IBRC P1012149	S3	Liqvan, East Azerbaijan, Iran	37°49′, 46°23′	2,516
E. algidum	IBRC P1012150	S4	Hovir, Tehran, Iran	35°41′, 52°24′	2,221
E. anatolicum	IBRC P1012141	S5	Sorkheh Dizaj, zanjan, Iran	36°35′, 48°51′	1,978
E. confusum	IBRC P1012143	S6	Shahrud, Semnan, Iran	36°32′, 54°49′	2,154
E. frigidum	IBRC P1012139	S7	Tange Vashi, Tehran, Iran	35°54′, 52°43′	2,591
E. lanceolatum	IBRC P1012138	S8	Evan lake, Qazvin, Iran	36°29′, 50°27′	1,857

Fig. 1. Geographic distribution of sampled Epilobium species on the map of Iran using ArcGIS.

Cytological preparation and microscopy

To evaluate the ploidy level and mitotic chromosome number, the seeds were antisepticised by 2% (v/v) sodium hypochlorite for 8 min in clean Petri dishes on a wet filter paper at 25°C. Then, 0.5 cm of root tips were cut multiple times from in vivo plants and pretreated with 2 mM 8-hydroxyquinoline at 4°C for 5 h. The samples were then fixed in ethanol–glacial acetic acid (3 : 1, v/v) for 24 h. The fixed roots were rinsed with deionized water (dH₂O), the seedlings were hydrolyzed in 1 M HCl solution at 55°C for 4 min, then washed twice (each time for 2 min) in dH₂O and stained with 4% (w/v) hematoxylin at 60°C for 6 h in a water bath (Kurabayashi et al. 1962). The stained meristems were thereafter squashed on a glass slide. Photomicrographs were captured by a DP12 digital camera (Olympus Optical Corporation, Tokyo, Japan) appointed to a BX50 Olympus microscope (Olympus Optical Corporation, Ltd., Tokyo, Japan). Then, the chromosome length (CL) was measured with MicroMeasure (version 3.3) software.

In vitro micropropagation

To evaluate the genome size, the seeds were firstly cultured in an MS medium (Murashige and Skoog 1962). For this purpose, the seeds were rinsed in running double-distilled water for 5 min to pre-sterilized their surface, and soaked in 70% (v/v) ethanol (20 s); 2% (v/v) sodium hypochlorite (10 min); afterward, washed three times with sterile distilled water (Turker et al. 2008). The sterilized seeds were placed in an MS medium containing 8% (w/v) sucrose, agar at pH 5.8, and incubated at room temperature with 16/8-h light/darkness conditions (Fig. 2). To acclimatization, the in vitro-grown plants were transferred to plastic pots containing sterile perlite and cocopeat under growth chamber conditions.

Fig. 2. In vitro regeneration of E. parviflorum (a) Seedlings grown on hormone-free MS medium, (b) acclimatization of a seedling under in vitro conditions.

FCM analysis

The genome size of eight species Epilobium was determined by using FCM. To prepare nuclear suspensions, young and well-developed leaves were used, and S. lycopericum cv. Stupicke, with a known DNA content of 2C=1.96 pg used as an internal reference standard (Doležel et al. 1992). To this end, the leaves of Epilobium and internal standard were finely chopped using a sharp and clean razor blade in 1 mL Woody Plant Buffer (Loureiro et al. 2007) and filtrated through a 30-µm green nylon mesh (Partec, Münster, Germany). Afterward, isolated nuclei were added by 50 µL of PI and 50 µL of RNase with a concentration range of 1 µg mL⁻¹. Estimation of the 2Cx DNA value of the obtained nuclei suspension was carried out by a BD FACSCanto II flow cytometer (BD Biosciences, Bedford, MA, USA), and the data were analyzed by BD FACSDivaTM software. Histograms were gated using Partec (Partec, Münster, Germany) Flomax ver. 2.4e. Relative fluorescence intensity of the stained nuclei were also measured on a linear scale. The genome size of the studied samples were calculated based on the G1 peak means values (Doležel et al. 2003, 2007, Doležel and Barto 2005, Karimzadeh et al. 2010, 2011, Mahdavi and Karimzadeh 2010, Hamidi et al. 2018, Tarkesh Esfahani et al. 2021):

  

The histograms used for estimating genome size illustrated two peaks, the first peak refers to the G1 of Epilobium species samples and the second peak represents the G1 of the standard sample (2C DNA=1.96 pg).

Statistical analysis

The initially karyotypic and nuclear DNA content data were tested for normality and homogeneity of variances. Moreover, analysis of variance was carried out using an unbalanced completely randomized design (CRD), and mean comparisons were performed by the least significant differences (LSD) method at 0.01 probability levels using the general linear model’s procedure (Proc GLM), with SAS (version 9.2) software. Linear regression analysis was also conducted to examine any relationships among 2Cx DNA and CL and some geographical parameters (latitude, longitude, and altitude) using SPSS software (Version 22, SPSS Inc, Chicago, IL, USA).

Results

The result of chromosomal study indicates that all of the examined species were diploid; six among eight species (E. hirsutum, E. parviflorum, E. roseum, E. algidum, E. anatolicum, and E. confusum) had 2n=2x=36 chromosomes, while the other two species (E. frigidum and E. lanceolatum) had 2n=2x=38 (Fig. 3, Table 3) and this chromosome number has been reported for the first time. The base chromosome number of x=18 and diminutive chromosomes with conspicuously different in chromosome length has been previously reported for Epilobium species (Lewis and Raven 1961, Mosquin 1966, Husband and Schemsk 1998, Wagner et al. 2007). The ANOVA results showed significant differences (p＜1%) for CL among the studied plants, indicating interspecies diversity (Table 2). The mean CL of all species was 0.53 µm, ranging from 0.46 (S4) to 0.64 (S1) µm. Based on the various karyotypic symmetrical indices, Epilobium species represented different symmetrical features (Table 3).

Fig. 3. Somatic chromosomes of eight Epilobium spp. Scale bar=5 µm.

Table 2. ANOVA of chromosome length (CL) and monoploid genome size (2Cx DNA; pg) of Epilobium spp.

S.O.V.	Df	MS	Df	MS
		CL		2Cx DNA (pg)
Species	7	0.0379**	7	0.0283**
Error	48	0.0030	58	0.001
Total	55		65
CV%		9.88%		4.22%

**Significant difference (p＜0.01).

　

Table 3. Means (±SE) comparisons of chromosome length (CL), and karyotypic. Parameters of Iranian endemic Epilobium spp.

Species	2n	CL	S%	CVCL%	DRL%	Asymmetry index (A2) (Zarco 1986)
S1	36	0.647±0.026a	52.27	17.52	3.47	0.17
S2	36	0.529±0.028bc	54.16	19.14	3.29	0.19
S3	36	0.645±0.046a	50.00	18.85	3.72	0.18
S4	36	0.441±0.044c	5.35	21.72	3.36	0.21
S5	36	0.563±0.032ab	58.21	18.59	3.72	0.18
S6	36	0.519±0.036bc	58.46	16.30	3.18	0.16
S7	38	0.522±0.040bc	52.94	16.96	3.17	0.16
S8	38	0.469±0.043c	47.76	19.32	3.79	0.19
LSD1%		0.048

Means followed by the same letter within “CL” column indicate they are not significantly different (p＞0.01), using LSD test. S: Species

Flow cytometric histograms of nuclear DNA amounts of eight studied species are displayed in Fig. 4. The mean 2Cx DNA of all species was 0.86 pg, ranging from 0.74 to 0.90 pg (Table 4), and E. algidum and E. lanceolatum had the lowest and largest genome size among other species, respectively. The coefficients of variation for G₀/G₁ peaks of all species were 5%, while those of the internal reference standard mean were 4.92%. The statistical analysis of genome size showed significant differences (p＜1%) for interspecific variation (Table 3). The results of histograms analysis are complementary and confirming to the karyotypic studies, indicating the diploid nature of the eight Epilobium species. It should also be noticed that the C-value of Epilobium species in the current study has not been reported yet, except for E. hirsutum (0.83 pg) (Stehlik et al. 2007, Kron and Husband 2009).

Fig. 4. PI fluorescence histograms of nuclei isolated from in vitro derived leaves of eight Epilobium species. The left peaks refer to G1 of the Epilobium samples and the right peaks to G1 of the S. lycopericum cv. Stupicke (2C DNA=1.96 pg) reference standard.

Table 4. Means (±SE) comparisons of genome size (2Cx DNA; pg) of Epilobium spp.

Species	2n	2Cx DNA (pg) Mean±SE	1Cx DNA (pg)	1Cx DNA (Mbp)
S1	36	0.90±0.006a	0.450	440.10
S2	36	0.87±0.009ab	0.435	425.43
S3	36	0.82±0.006c	0.410	400.98
S4	36	0.74±0.008d	0.370	361.86
S5	36	0.83±0.020bc	0.415	405.87
S6	36	0.76±0.008d	0.460	449.88
S7	38	0.75±0.018d	0.375	366.75
S8	38	0.92±0.003a	0.380	371.64

Means followed by the same letter within “2Cx DNA (pg)” column indicate they are not significantly different (p＞0.01), using LSD test.

Discussion

Although the chromosome size varies within the Onagraceae members, there are obvious similarities within each genera, with an exception in the Clarkia. According to the earlier karyotypic studies, Epilobium has the smallest chromosome size; on the other hand, the genus Lopezia has the largest chromosome in this family (Håkansson 1943). The chromosomes of Epilobium were smaller than Boisduvalia and Ludwigia (Kurabayashi et al. 1962). In addition, Epilobieae like Jussiaeae had diminutive chromosomes with variety in size (Kurabayashi et al. 1962). The results of chromosome count for Epilobium species demonstrated a variety of base chromosome numbers for species, which are in consists with previous reports in some of the genera of Onagraceae such as Clarkia and Boisduvalia (Kumar et al. 2012, Rani et al. 2012). This variation in Eukaryotes is probably stemmed from several potential reasons: duplication or deletion of chromosomes, polyploidy, displacement, inversion, fusion, and/or the translocation heterozygosity of the chromosomes. One of the main reasons underlying the evolution of translocation is the change in the number of chromosome sets leading to the origin of diversified karyotypes between species; also the presence of translocation in the Onagraceae proving for the first time and found in the Clarkia, Oenothera, Gayophytum, Gaura, and Onagreae and in populations of Clarkia leading to the reduced number of chromosome (Håkansson 1943, Gardé and Gardé 1950, Lewis and Rave 1961, Greilhuber 1995, Luceño and Guerr 1996, Coghlan et al. 2005).

Stebbins (1966) and Raven (1976) considered the diverse base chromosome numbers including of x=9, 10, 12, 13, 15, 16, and 18 had reported in tribe Epilobieae (Skvarla et al. 1975), and proposed that the species of Boisduvalia (now a section of Epilobium). Recognition of the karyotypic features and ploidy level is an early necessity for a breeding program; for instance, to produce fertile hybrids. As far as the previously performed cytogenetic studies on the Onagraceae family are concerned, the mean genome size of the Epilobium is 786 Mbp, and other genera such as Ludwigia, Oenothera, and Clarkia have the mean DNA contents of 2127.15, 2378.95, and 3614.53 Mbp, respectively. Herein, we revealed that the Epilobium has the least content of genome within its family (Bennett et al. 1998, Talluri and Murra 2009). Correlation analysis also showed that there was no significant correlation between genome size, CL, and the geographical parameters, i.e., longitude, latitude, and altitude.

The result of Talluri and Murray (2009) on Fuchsia shows no association between the genome size and geographical factors, albeit having variation in genome size across geographically different regions. Likewise, the results of this study can be profitable to augment pharmaceuticals by improving breeding efficiency in the species of Epilobium. This research provides new information about genome size diversity, chromosome number, and ploidy screening in Epilobium species for the first time, which open up new avenues towards reaching genetically improved lines.

Acknowledgments

The authors gratefully acknowledge the support provided for this research project by the Tarbiat Modares University and by the Iranian Research Organization for Science and Technology.

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