Microsoft Word - 11451 NSB El Boukhari 2023.03.16.docx Received: 23 Jan 2023. Received in revised form: 03 Mar 2023. Accepted: 10 Mar 2023. Published online: 16 Mar 2023. From Volume 13, Issue 1, 2021, Notulae Scientia Biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. The journal will continue to appear quarterly, as before, with four annual numbers. SHSTSHSTSHSTSHST Horticulture and ForestryHorticulture and ForestryHorticulture and ForestryHorticulture and Forestry Society of TransylvaniaSociety of TransylvaniaSociety of TransylvaniaSociety of Transylvania El Boukhari A et al. (2023) Notulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia Biologicae Volume 15, Issue 1, Article number 11451 DOI:10.15835/nsb15111451 Research ArticleResearch ArticleResearch ArticleResearch Article.... NSBNSBNSBNSB Notulae Scientia Notulae Scientia Notulae Scientia Notulae Scientia BiologicaeBiologicaeBiologicaeBiologicae Flow cytometry and chromosome numbers variation in argan Flow cytometry and chromosome numbers variation in argan Flow cytometry and chromosome numbers variation in argan Flow cytometry and chromosome numbers variation in argan tree tree tree tree Argania spinosaArgania spinosaArgania spinosaArgania spinosa (L.) Skeels(L.) Skeels(L.) Skeels(L.) Skeels Ali EL BOUKHARI 1,2, Salma TABI1,4, Abdelhamid EL MOUSADIK2, Rachida EL BOULLANI2, Abdelghani TAHIRI1, Meriyem KOUFAN1, Hamid BENYAHIA3, Rachid BOUHARROUD1*, Naima AIT AABD1* 1Regional Center of Agricultural Research of Agadir, National Institute of Agricultural Research (INRA), Avenue Ennasr, B.P. 415 Rabat Principale, Rabat 10090, Morocco; abdelghani.tahiri@inra.ma; meriyem.koufan@inra.ma; rachid.bouharroud@inra.ma (*corresponding author); naima.aitaabd@inra.ma (*corresponding author) 2Ibn Zohr University, Faculty of Sciences, Laboratory of Biotechnology and Valorization of Natural Resources, PoB 8106, Agadir, Morocco; ali.elboukhari@edu.uiz.ac.ma; a.elmousadik@uiz.ac.ma; r.elboullani@uiz.ac.ma 3Regional Center of Agronomic Research of Kenitra, National Institute of Agronomic Research (INRA), Avenue Ennasr, B.P. 415 Rabat Principale, Rabat 10090, Morocco; hamid.benyahia@inra.ma 4Sultan My Sliman University, Faculty of Sciences and Technics, Laboratory of Natural Resources, Environment and Health, B.P 523, Beni Mellal, Morocco; salma.tabi@usms.ma AbstractAbstractAbstractAbstract Argania spinosa L. Skeels is an endemic species of west-central Morocco, which is characterized by a high diversity of morphological and genetic traits. It constitutes a natural resource for oleo-agro-sylvo-pastoral uses. All conservation and genetic breeding strategies aimed to domesticate argan require a good knowledge of the plant material. However, several studies focused on agronomical, morphological, phytochemical, and molecular characterization, while the cytogenetic aspects were less investigated. The objective of this work is to identify the chromosome number and ploidy level on the national argan collection at the Agadir Regional Agronomic Research Center, Morocco. The determination of the chromosome number was carried out on root tips of germinated seeds collected from five trees genotypes selected on various morphological aspects. As a result, chromosome count on active root tip cells showed variation in the number (2n = 20; 2n = 22; 2n = 24) with a stable ploidy level (2n = 2x) that is confirmed by flow cytometry. These results combine two previous findings (2n=20, 2n=24) and reveal a third existence of twenty-two chromosome. As a conclusion, A. spinosa has three chromosomal numbers which represent the genetic diversity of the chromosomal number that this species exhibits. More studies are required to explain this variation on chromosome numbers for future breeding programs and to avoid incompatibilities. Keywords:Keywords:Keywords:Keywords: Argane tree; chromosome number; cytogenetic; flow cytometry; genetic diversity https://www.notulaebiologicae.ro/index.php/nsb/index El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 2 IntroductionIntroductionIntroductionIntroduction Argania spinosa (L.) Skeels (Sapotaceae), the only representative of the family in Morocco, is an allogamous monoecious tree with entomophilic pollination (Nerd et al., 1998; Ajerrar et al., 2020). It is an endemic species of west-central Morocco; its forest area is estimated to 800,000 hectares (Msanda et al., 2021). The Arganereae offers several ecosystem services, namely the storage and sequestration of carbon, habitats of several endemic species, contributes to the conservation of biodiversity, improves soil fertility, prevents soil erosion, regulates and creates microclimates (Karmaoui, 2016). The Biosphere Reserve of Argania covers an area of 2,560,000 ha (Msanda et al., 2021), in which the local population (3 million people, including 2.2 million in rural areas) exploits this natural resource for energy purposes (wood and shell) as well as a fodder resource (leaves, and fruit pulp). Therefore, they are dependent on the exploitation of argan forests (Laaribya et al., 2017). The economic value of the argan tree is based mainly on its almonds, from which highly marketable oil is extracted (M'hirit, 1987). Argan oil is very known for its quality, with an interesting saponifiable composition, excellent effects, and several benefits (Berrougui et al., 2003; Drissi et al., 2006; Koufan et al., 2020a). Argane’s shells are used in combustion for heating and even as a bio-composite base. Biochar from argan shells enriches the soil by improving nutrient and water retention (Bouqbis et al., 2016). In addition, the pulp, bark, leaf, root, and even press cake have interesting medicinal uses (Moukal, 2004). Because of its importance and for its protection, UNESCO declared the Arganereae on December 8th, 1998, as the first Biosphere Reserve in Morocco. In 2021, UNESCO declared May 10th the international day of the argane tree. Well known for its high genetic diversity, efforts of scientific research achieved detection and characterization of specie’s genetic diversity on the morphological, agronomical, biochemical and molecular level and marked its exploitable genetic potential (El Mousadik et al., 1996; Ait Aabd et al., 2012; Ait Aabd et al., 2013; Ait Aabd et al., 2015; Mouhaddab et al., 2017; Koufan et al., 2020b). Unlike the cytogenetic approach, that is less studied. The breeding program from wild types seems to be the straighter method for genetic improvement and before conception of argane orchards. Genetic diversity can be found in genome sequence and the number of chromosomes (De Vicente et al., 2004). Polyploidy means increasing the number of chromosomes in an animal or plant cell (multiple of the number of haploids). It can be an auto-polyploidy (duplication without cytokinesis) or an allopolyploidy (fusion of two unreduced gametes). Also, the genetic variation can be at the origin of the anomalies of the chromosomal number like aneuploidy. It is defined as the presence of an abnormal number of chromosomes. In general, chromosome number variation can be a speciation factor. It plays a fundamental role in plants evolution, diversification, and ecological adaptation. Karyotype assessments by counting the number of metaphase chromosomes is time consuming and laborious. Highly skilled manipulators are needed to accomplish it, in addition, tissues containing few numbers of dividing cells, which may not be readily available. However, flow cytometry has arisen as a far more reliable methodology for the determination of genome size and ploidy level. It permits measurement of the fluorescence of large numbers of stained nuclei within seconds, more samples within minutes (Seker et al., 2003). In fact, with the enormous biodiversity at the biometric, phenological, agro-morphological, and molecular level, the allogamous pollination system, and pollen incompatibility (Ait Aabd et al., 2022), the existence of chromosomal variation remains to be checked in the argan tree species. Indeed, this study aims to investigate the chromosome number on a collection of genotypes morphologically and phenologically differentiated. El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 3 Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods Plant material The study was undertaken on five genotypes selected previously based on their morpho-biometry and phenology (tree shape, height, number of trunks, flowering period, fruit shape and fruit ripening duration). The trees were planted at Melk-Zhar experimental farm, Regional Agronomic Research Center, Agadir, Morocco. Chromosome counting The protocol of Majourhat et al. (2007) was adopted with minor modification. Mature fruits were collected from each tree and kept until drying. After pulping and crushing seeds, they were disinfected using Sodium Hypochlorite, rinsed with sterile water, and then germinated on Petri dishes with moist filter paper at 26 °C. Root tips were cut out when the radicles were about 2-3 cm long. They were pre-cooled four hours in ice water. The samples are then fixed in glacial ethanol-acetic acid (3: 1) for 16 h at 4 °C, then transferred to 70% ethanol solution and kept at 4 °C. The root tips were hydrolysed in 5 N HCl for 10 min. Finally, the samples were stained in Orcein Acetic solution for two hours then squashed under coverslips. Slides were observed under different objectives to find well spread cells and show the best meta-phasic plates. Mitotic chromosomes were photographed under 1000X magnification using an optical microscope. Each photographed cell was drawn to count it accurately. To prevent experiment artefacts, more than 3 counts were made per each preparation and plant. Flow cytometry Cells were isolated from specimens collected directly from each genotype. Leaf samples (5 mm2) were chopped with razor blade in presence of 400 µL nuclei extraction buffer in Petri dishes. The samples were filtered directly into a sample tube of 50 µm and stained with 1,5 mL of 4’,6-diamidino-2-phenylindole buffer stain. Following 5 min incubation at room temperature, stained samples were run in flow cytometer. Analysis was repeated twice for each genotype. Results Results Results Results Chromosome numbers Chromosomal counting is carried out on well spread, drawn, and photographed plates. It was difficult to count the chromosomes given their small size (0.59 to 1.69 µm) (Majourhat et al., 2007). Despite, three chromosome numbers were counted: 20, 22 and 24 (Figure 1), which means three base numbers (n=10, n=11, n=12). The number of chromosomes varied between mother trees. Indeed, three genotypes with 22, one with 24, and one with 20 were observed. El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 4 Figure 1. Figure 1. Figure 1. Figure 1. Chromosomes count on A. spinosa: microscopic photography (A, B, C) and draw (a, b, c) of meta-phasic plate showing 22 chromosomes, 20 chromosomes and 24 chromosomes. Scale bars represent 5 µm. Flow cytometry profiles Figures 2, 3 and 4 show different raw flow cytometer profiles. First peak (around 50) is background noise and the second peak is cells fluorescence intensity emission. Profile 1 represent the maximal fluorescence intensity measured (around 230), profile 2 with intermediate intensity (around 200) while profile 3 represent the minimal fluorescence intensity detected (around 150). It was observed that fluorescence intensity of DAPI stain varied according to genotype. Cells intensity detected is correlated to relative nuclei DNA content. Figure 2. Figure 2. Figure 2. Figure 2. Argania spinosa flow cytometry profile 1, cells count represented on the Y axis while fluorescence intensity is represented on the X axis El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 5 Figure 3. Figure 3. Figure 3. Figure 3. Argania spinosa flow cytometry profile 2, cells count represented on the Y axis while fluorescence intensity is represented on the X axis Figure 4. Figure 4. Figure 4. Figure 4. Argania spinosa flow cytometry profile 3, cells count represented on the Y axis while fluorescence intensity is represented on the X axis DiscussionDiscussionDiscussionDiscussion Miège (1954) reported 20 chromosomes on A. spinosa somatic cells, while Humphries et al. (1978) discovered 24 chromosomes on pollen mother cells. The current study on A. spinosa showed a new finding as the counting of 2n = 22 chromosomes which has never been reported. The highest diversity observed on argan tree forest could give an explanation of this variability of chromosome numbers. Based on flow cytometry analysis, the 3 chromosomes numbers (20, 22 and 24) were confirmed and showed none variation in ploidy level. In fact, the intensity dissimilarity observed between genotypes cannot be a score as a variation in ploidy level. Nevertheless, this slight variation may be due to the genome size or genome composition El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 6 variations (DAPI binds specifically to AT nucleotides). Understanding the genetic diversity of the argan tree is an essential basis that requires more scientific effort to achieve the essential objective of the genetic improvement of this species. The basic data are chromosome numbers, but chromosome size, morphology, and staining characteristics may also be of important value (Stace et al., 2000). Systematic investigations are mainly based on molecular methods, while chromosome data provide essential and basic information, which may for example help in interpreting results from molecular studies (Baltisberger and Widmer, 2006). It is worth noting that the high genetic diversity revealed by molecular tools can be explained at chromosome’s scale. Possession of more or fewer chromosome numbers influences the presence/absence of alleles on the whole genome. There is no report of intraspecific cytological variation in Sapotaceae until now. Johnson (1991) evaluated the number of chromosomes of 95 Sapotaceae species. All specimens showed n = 10, 11, 12, 13, or 14 and almost at the diploid level. The highest numbers (n = 13, 14) predominate in the tribes Chrysophylleae and Omphalocarpeae, whereas the numbers in Mimusopeae, Isonandreae and Sideroxyleae are almost on n = 10 or 11. Intraspecific cytological variation is frequent in plants, especially polyploidy. However, intraspecific variation in chromosome number is rare (Severns et al., 2008). Among species, genome size variation can occur as different ploidy levels (Terlević et al., 2022) or chromosome number variation. It can vary or not at the same ploidy level (Bagheri et al., 2022). Moreover, genome size variation can be correlated with morphological traits (Hoang et al., 2019). In general, descending dysploidy and polyploidy played crucial roles in chromosome number evolution in angiosperm (Carta et al., 2020) and may be the origin of this observed variation. In addition, abnormal meiocytes may be observed as a variation on chromosome numbers such as cytomixis, which is defined as the migration of chromatin between adjacent cells through cytoplasmic connection channels (Bellucci et al., 2003). This phenomenon detected in the meiocytes of several plants can cause variation in the chromosome number. However, this variation can be prevailing in populations of wild-type (Kaur and Singhal, 2015), having the form of different types of aneuploidy (trisomy, tetrasomy, double trisomy) or polyploidy (diploid, triploid, tetraploid, octaploid). Tetrasomy can be the origin that explains this variability (2n=20: no tetrasomy, 2n=22: one chromosome tetrasomy, 2n=24: two tetrasomy chromosomes). The union of two n+1 gametes can increase the number of chromosomes and restore euploidy in the offspring (2n+2) (Mayrose and Lysak, 2021). In addition, chromosome drive may be responsible for this diversity in chromosomal numbers (Camacho et al., 2000). Organisms can use a greater number of genes with an increase in the number of allelic variants. This is considered as interesting to plants in terms of synthesis rate and or the variability of the metabolic compounds produced (Pan et al., 2009). Duplicated genes can be maintained in copies (duplication divergence) which often undergo neo-functionalization or under-functionalization (Comai, 2005). A cytomorphologic study by counting chromosomes from the somatic cells (buds) on more genotypes is desirable to confirm these findings and achieve a genotype characterization associated with phenotypes. In addition, deepening in the study of the nature of chromosomes that makes the difference, their segregation, and their sequences is crucial. Furthermore, an analysis of genome size by a non-specific staining (like Iodide Propidium) to set standard genome size is important to facilitate the detection of cytotypes for rapid breeding program then avoid incompatibility during cross pollination process. ConclusionsConclusionsConclusionsConclusions A. spinosa has three possible chromosome numbers representing the highest genetic diversity observed. This diversity reflects another side of the adaptability of this species to mitigate extreme climate conditions. These findings will make possible determination of high diversity and ecotypes identification belonging to this species based on chromosomal numbers. El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 7 Authors’ ContributionsAuthors’ ContributionsAuthors’ ContributionsAuthors’ Contributions NAA Conceptualization; AEB, NAA, ST, HB, AT, RB, methodology; NAA, AEM, REB, MK, validation; AEB, NAA, HB, MK investigation; AEB, NAA, AEM, REB, RB, writing—original draft preparation; AEB, NAA, AT, RB writing—review and editing; NAA, AEM, REB, MK supervision; NAA, MK, RB funding acquisition. All authors read and approved the final manuscript. Ethical approvalEthical approvalEthical approvalEthical approval (for researches involving animals or humans) Not applicable. AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements This work was supported and funded by National Institute of Agronomic Research of Agadir, Morocco (Grant id. PRMT 2020-24). Conflict of InterestsConflict of InterestsConflict of InterestsConflict of Interests The authors declare that there are no conflicts of interest related to this article. ReferencesReferencesReferencesReferences Ait Aabd N, El Asbahani A, El Alem Y, El Finti A, Msanda F, El Mousadik A (2013). Variation in oil content and fatty acid composition in preselected argan trees with morphological characters and geographical localization. Mediterranean Journal of Nutrition and Metabolism 6(3):217-225. https://doi.org/10.3233/s12349-013-0134-2 Ait Aabd N, Msanda F, El Mousadik A (2012). Univariate and multivariate analysis of agronomical traits of preselected argan trees. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 40:308-316. https://doi.org/10.15835/nbha4028209 Ait Aabd N, Msanda F, El Mousadik A (2015). Genetic diversity of the endangered argan tree (Argania spinosa L.) (Sapotaceae) revealed by ISSR analysis. Basic Research Journal of Agriculture Science and Review 4:176-186. Ait Aabd N, Tahiri A, Qessaoui R, Mimouni A, Bouharroud R (2022). Self- and cross-pollination in argane tree and their implications on breeding programs. Cells 11(5):828. https://doi.org/10.3390/cells11050828 Ajerrar A, Akroud H, Ait Aabd N, Qessaoui R, Amarraque A, Lahmyd H, … Bouharroud R (2020). Pollination system and effective pollinators of Argania spinosa (L. Skeels). Journal of the Saudi Society of Agricultural Sciences 19:375-382. https://doi.org/10.1016/j.jssas.2020.04.002 Bagheri A, Akhavan Roofigar A, Nemati Z, Blattner F. R (2022). Genome Size and Chromosome Number Evaluation of Astragalus L. sect. Hymenostegis Bunge (Fabaceae). Plants 11(3):435. https://doi.org/10.3390/plants11030435 Baltisberger M, Widmer A (2006). Chromosome numbers of plant species from the Canary Islands. Botanica Helvetica 116:9-30. https://doi.org/10.1007/s00035-006-0739-x Bellucci M, Roscini C, Mariani A (2003). Cytomixis in pollen mother cells of Medicago sativa L. Journal of Heredity 94:512-516. https://doi.org/10.1093/jhered/esg096 Berrougui H, Ettaib A, Gonzalez MH, De Sotomayor MA, Bennani-kabchi N, Hmamouchi M (2003). Hypolipidemic and hypocholesterolemic effect of argan oil (Argania spinosa L.) in Merionesshawi rats. Journal of Ethnopharmacology 89:15-18. https://doi.org/10.1016/S0378-8741(03)00176-4 El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 8 Bouqbis L, Daoud S, Koyro HW, Kammann CI, Ainlhout LFZ, Harrouni MC (2016). Biochar from argan shells: production and characterization. International Journal of Recycling of Organic Waste in Agriculture 5(4):361- 365. https://doi.org/10.1007/s40093-016-0146-2 Camacho JPM, Sharbel T F, Beukeboom L (2000). W. B-chromosome evolution. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 355:163-178. https://doi.org/10.1098/rstb.2000.0556 Carta A, Bedini G, Peruzzi L (2020). A deep dive into the ancestral chromosome number and genome size of flowering plants. New Phytologist 228(3):1097-1106. https://doi.org/10.1111/nph.16668 Comai L (2005). The advantages and disadvantages of being polyploid. Nature Reviews Genetics 6:836-846. https://doi.org/10.1038/nrg1711 De Vicente MC, Lopez C, Fulton T (2004). Genetic diversity analysis with molecular marker data: learning module. International Plant Genetic Resources Institute. Drissi A, Bennani H, Giton F, Charrouf Z, Fiet J, Adlouni A (2006). Tocopherols and saponins derived from Argania spinosa exert, an antiproliferative effect on human prostate cancer. Cancer Investigation 24:588-592. https://doi.org/10.1080/07357900600894815 El Mousadik A, Petit R J (1996). High level of genetic differentiation for allelic richness among populations of the argan tree [Argania spinosa (L.) Skeels] endemic to Morocco. Theoretical and Applied Genetics 92:832-839. https://doi.org/10.1007/BF00221895 Hoang PT, Schubert V, Meister A, Fuchs J, Schubert I (2019). Variation in genome size, cell and nucleus volume, chromosome number and rDNA loci among duckweeds. Scientific Reports 9(1):3234. https://doi.org/10.1038/s41598-019-39332-w Humphries C J, Murray BG, Bocquet G, Vasudevan K (1978). Chromosome numbers of phanerogams from Morocco and Algeria. Botaniska Notiser 131:391-406. Johnson M (1991). The Genera of Sapotaceae. Cytology. Kew Publishing, Surrey, Gran Bretaña 15-22. Karmaoui A (2016). Ecosystem Services of the Argan Forest, the Current State and Trends. Advances in Research 1-13. https://doi.org/10.9734/AIR/2016/21353 Kaur M, Singhal VK (2015). Cytomorphological diversity in some members of family Asteraceae from the ecologically disturbed habitats of Solang valley, Kullu district, Himachal Pradesh. Cytologia 80:203-222. https://doi.org/10.1508/cytologia.80.203 Koufan M, Belkoura I, Mazri MA, Amarraque A, Essatte A, Elhorri H, ... Alaoui T (2020a). Determination of antioxidant activity, total phenolics and fatty acids in essential oils and other extracts from callus culture, seeds and leaves of Argania spinosa (L.) Skeels. Plant Cell, Tissue and Organ Culture (PCTOC) 141:217-227. https://doi.org/10.1007/s11240-020-01782-w Laaribya S, Alaoui A, Gmira N (2017). The Moroccan Forest and sustainable development case of the argan tree Argania spinosa L. Skeels in Morocco. Biyolojik Çeşitlilikve Koruma 10:1-7. Majourhat K, Jabbar Y, Araneda L, Zeinalabedin M, Hafidi A, Martínez-gómez P (2007). Karyotype characterization of Argania spinosa (L.) Skeel (Sapotaceae). South African Journal of Botany 73:661-663. https://doi.org/10.1016/j.sajb.2007.06.007 Mayrose I, Lysak MA (2021). The evolution of chromosome numbers: mechanistic models and experimental approaches. Genome Biology and Evolution 13(2):evaa220. https://doi.org/10.1093/gbe/evaa220 M'hirit O, Benzyane M, Benchekroun F, El Yousfu SM, Bendaanoun M (1987). L’arganier, une espece fruitiere, forestiere a usages multiples des zones arides méditerranéens. Saragosse, Espana. Miège J (1954). Nombres chromosomiques et répartition géographique de quelques plantes tropicales et équatoriales. Revue de Cytologie et de Biologie Végétales 15:312-348. Mouhaddab J, Msanda F, Filali-Maltouf A, Belkadi B, Ferradouss A, El Modafar C, … El Mousadik A (2017). Using microsatellite markers to map genetic diversity and population structure of an endangered Moroccan endemic tree (Argania spinosa L. Skeels) and development of a core collection. Plant Gene 10:51-59. https://doi.org/10.1016/j.plgene.2017.05.008 Moukal A (2004). L’arganier, Argania spinosa L.(skeels), usage thérapeutique, cosmétique et alimentaire. Phytothérapie 2:135-141. https://doi.org/10.1007/s10298-004-0041-2 El Boukhari A et al. (2023). Not Sci Biol 15(1):11451 9 Msanda F, Mayad EH, Furze JN (2021). Floristic biodiversity, biogeographical significance, and importance of Morocco’s Arganeraie Biosphere Reserve. Environmental Science and Pollution Research 1-10. https://doi.org/10.1007/s11356-020-11936-0 Nerd A, Irijimovich V, Mizrahi Y (1998). Phenology, breeding system and fruit development of Argan [Argania spinosa, Sapotaceae] cultivated in Israel. Economic botany 52:161-167. Pan YZ, Gao W, Yu AM (2009). MicroRNAs regulate CYP3A4 expression via direct and indirect targeting. Drug Metabolism and Disposition 37:2112-2117. https://doi.org/10.1124/dmd.109.027680 Seker M, Tuzcu O, Ollitrault P (2003). Comparison of nuclear DNA content of citrus rootstock populations by flow cytometry analysis. Plant Breeding 122:169-172. https://doi.org/10.1046/j.1439-0523.2003.00821.x Severns PM, Liston A (2008). Intraspecific chromosome number variation: a neglected threat to the conservation of rare plants. Conservation Biology 22:1641-1647. https://doi.org/10.1111/j.1523-1739.2008.01058.x Stace CA (2000). Cytology and cytogenetics as a fundamental taxonomic resource for the 20th and 21st centuries. Taxon 49:451-477. https://doi.org/10.2307/1224344 Terlević A, Bogdanović S, Frajman B, Rešetnik I (2022). Genome size variation in Dianthus sylvestris Wulfensensulato (Caryophyllaceae). Plants 11(11):1481. https://doi.org/10.3390/plants11111481 The journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. Users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. License License License License ---- Articles published in Notulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia Biologicae are Open-Access, distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) License. © Articles by the authors; Licensee SMTCT, Cluj-Napoca, Romania. The journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. Notes:Notes:Notes:Notes:  Material disclaimer: The authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  Maps and affiliations: The publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  Responsibilities: The editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. The statements and opinions published represent the views of the authors or persons to whom they are credited. Publication of research information does not constitute a recommendation or endorsement of products involved.