Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 107-113, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-122 Citation: P. Mahditabar Bahnamiri, A. Mahmoudi Otaghvari, N. Ahmadian Chashmi, P. Azizi (2020) Electropho- retic study of seed storage proteins in the genus Hypericum L. in North of Iran. Caryologia 73(1): 107-113. doi: 10.13128/caryologia-122 Received: Januray 9, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 P. Mahditabar Bahnamiri, A. Mahmoudi Otaghvari, N. Ahmadian chashmi, P. Azizi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran Parisa Mahditabar Bahnamiri1, Arman Mahmoudi Otaghvari1,*, Najme Ahmadian Chashmi1, Pirouz Azizi2 1 Department of Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran 2 Department of Soil science, University of Guilan, Rasht, Iran *Corresponding author. E-mail: P.mahditabar@gmail.com, botany1347@gmail.com, najme.ahmadian@gmail.com Abstract. In this research we studied the electrophoretic of seed storage proteins in the genus Hypericum L. from Iran. The plant samples were collected from various phytoge- ographical regions of Iran to study the seed storage proteins. The study was performed to determine the boundary among different species of genus Hypericum using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All samples belong to three species of H. perforatum, H. tetrapterum and H. androsaemum. A total of 22 protein bands were observed in the studied species. The results show that H. perfora- tum, H. tetrapterum are closely related based on seed storage proteins. A closely rela- tionship and high protein similarity (J=0.66) were found between H. perforatum, H. tetrapterum. Electophoretic results compared with earlier molecular and morphological studies. The highest number of bands was observed in Kordkoy1 population (Pop12) and Gardane heyran population (Pop20) of H. perforatum and the lowest in Gorgan/ Naharkhoran population (Pop 25) of H. androsaemum. Our results showed the species of Hypericum were placed intermixed. The aim of this study to delimit species in the genus Hypericum and used these seeds storage protein for the correct identification. Keywords. Hypericum, North of Iran, Species relationships, SDS-PAGE. INTRODUCTION The genus Hypericum (Guttiferae, Hypericoideae) is perennial, belong- ing to the Hypericaceae family, having 484 species in forms of trees, shrubs, and herbs, distributed in 36 taxonomic sections (Crockett and Robson 2011). The species of the family are distributed worldwide in the temperate zones but are absent in extreme environmental conditions such as deserts and poles. Iranian species of this genus grow mainly in north, northwest and center of Iran and form floristic elements of Hyrcanian mountainous areas, Irano-Turanian, Mediterranean and Zagros elements. They generally prefer steep slopes of rocky and calcareous cliffs and margin of mountainous for- ests (Robson 1968; Azadi 1999). Robson (1968) introduced 21 species in the 108 Parisa Mahditabar Bahnamiri et al. area covered by Flora Iranica. Robson (1977) and Assa- di (1984) reported H. fursei N. Robson and H. dogon- badanicum Assadi as two endemics of North and South West of Iran. In Flora of Iran, Azadi (1999) identified 19 species, 4 subspecies arranged in 5 sections (compris- ing Campylosporus (Spach) R. Keller, Hypericum, Hir- tella Stef., Taeniocarpum Jaub. & Spach. and Drosanthe (Spach) Endl.), and two doubtful species including H. heterophyllum Vent. and H. olivieri (Spach) Boiss. Hypericum species are generally known locally in Iran with the names “Hofariqun” which Ebn Sina (or Bo Ali Sina) called it (Rechinger, 1986). Plants of the genus Hypericum have traditionally been used as medicinal plants in various parts of the world. Hypericum perfo- ratum L. is the source to one of the most manufactured and used herbal preparations in recent years, especially as a mild antidepressant, and thus is the most studied Hypericum species (Mozaffarian, 1998). According to Brutovská et al. (2000), H. perforatum is probably origi- nated from autopolyploidization of an ancestor closely related to diploid H. maculatum. The chemical composi- tion of H. perforatum oil has been the subject of many researcher in recent past (Cakir et al. 1997; Baser et al. 2002; Osinska 2002; Schwob et al. 2002; Mockute et al. 2003; Smelcerovic et al. 2004). The methanolic extract from the aerial parts of Hypericum plants typically con- tain hypericins, hyperforins and phenolic compounds (Osinska 2002). Proteins and enzymes, characterized as primary gene products, are important parameters in biochemical taxonomy. Storage proteins separated by electrophoretic methods are thought to undergo the process of evolu- tion with relative slowness due to their “non-essential nature’’ (Margoliash and Fitch 1968), while enzymes are thought to be extremely sensitive to selection pres- sures in evolution and thus to survival of the organism (McDaniel 1970). Analysis of proteins and isozymes is a tool for supplementing the evidence obtained by com- parative morphology, breeding experiments and cytolog- ical analysis. Seed protein electrophoresis for the study of phylogenetic relationship in Capsicum L. was per- formed by Panda et al. (1986). Although phenotypic traits are important for diver- sity studies, they need to be supported by molecu- lar markers to give robust genetic diversity estimates (Esfandani-Bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). Genetic diversity studies in Capsicum using morpho- logical, cytological and biochemical marker systems (Kaur and Kapoor 2001; Gopinath et al. 2006) are also conducted. The data on agronomic, morphological and physiological plant traits are generally used to estimate the magnitude of genetic diversity present in the germ- plasm. However, such data may not provide an accurate indication of genetic diversity because of environmental influences upon the expression of observed traits and also the time consuming and laborious field evaluation procedures. The introduction of biochemical techniques like Sodium dodecyl sulphate polyacrylamide gel elec- trophoresis (SDS-PAGE), isozyme markers has been particularly helpful in deducing systematic relationships between groups where morphological and cytological data were not corollary. SDS-PAGE is an economical, simple and extensively used technique for describing the seed protein diversity of crop germplasm (Fufa et al. 2005; Iqbal et al. 2005). Furthermore, seed proteins, used as genetic markers convey greater precision to measures of genetic diversity because they are the pri- mary products of structural genes (Srivalli et al. 1999). Seed protein electrophoresis for the study of phylogenet- ic relationship in Capsicum annuum was performed by Panda et al. (1986) and of diploids and tetraploid hybrids of Capsicum was initiated by Srivalli et al. (1999). There is no report of SDS-PAGE in Hypericum species in Iran. The present study was conducted on the genetic diversity of Hypericum genotypes from different loca- tions which will be useful for breeding programmes and also for conservation of germplasm. To use genetic resources adequately, it is necessary to understand the extent and pattern of genetic diversity. Therefore, an attempt with the present investigation was undertaken to evaluate the extent of variability existing in 29 geo- graphical populations belonging to three species of Hypericum of North region of Iran through seed protein analysis to provide a scientific basis for future selection and crop improvement program. The objective of this study was to assess the level of seed electrophoretic pat- terns of Hypericum taxa in Iran and used it for the cor- rect taxonomy of the genus. MATERIAL AND METHODS Plant Material Extensive field visits and collections were under- taken during 2016-2017 throughout the north of Iran. In present study 63 plant samples from 29 geographi- cal populations belonging to three species of Hyperi- cum in Iran were collected from field: H. perforatum L., H. tetrapterum Fries. and H. androsaemum L. Different references were used for the correct identification of spe- cies (Rechinger, 1999 , Azadi, 1999). The details of the voucher specimens and their localities are given in Table 1 and Fig 1. All materials were examined with a stere- omicroscope (NIKON-SMZ1) and all voucher specimens 109Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran are deposited at the University of Mazandaran Herbari- um (HUMZ). Protein extraction & Electrophoresis An amount of 0.1 g of mature seeds were selected from each population and crushed in liquid nitrogen at low temperature. After obtaining a fine powder, proteins were extracted under cool conditions with 3 ml of Tris- Glycin buffer (pH 8). The resulting samples were centri- fuged twice for 5 min at 11000 g. The protein electro- phoresis was based on Laemmlis procedure (1970), using a discontinuous vertical slab gel. The separating gel com- prises 12 ml of 30 % acrylamid stock solution, 2.5 ml Tris-HCl 1.5 M (pH 8.8), 100 μl SDS 10 %, 2.3 ml water, 7 μl TEMED, and 60 μl APS (Ammonium per Sulfate). After polymerization of the separating gel, the stacking gel with 530 μl of 30 % acrylamide stock solution, l ml Tris 0.5 M (pH 6.8), 40 ml SDS 10 %, 2.37 ml water, 5μl TEMED, and 40 μl APS was polymerized on the separating gel. The electrophoresis was carried out at a constant voltage of 100V for 7-15h. Gels were stained in Coomasie Brilliant Blue for 1-2 h and overnight distained with ace- tic acid and methanol (Laemmli 1970). We used Jaccard similarity coefficient. Standard pro- teins (b-galactosisase, Ovalbumin, Lactate dehydroge- nase, lactoglobulin-b, Lysozyme and Bovine serum albu- min) were used to evaluate the molecular weight of the unknown proteins. The protein density was determined by Bradford Protocol(Bradford, 1976). Protein banding profile analysis Number and location of each protein band were identified and their RF (relative factor) and molecular weight were estimated. In statistical analysis, each pro- tein band was considered as a qualitative character and coded as 1 (presence) versus 0 (absence). For grouping of the plant specimens,Ward (minimum spherical char- acters) were used (Podani 2000). PCA (principal compo- nents analysis) biplot was used to identify the most vari- able characters among the studied populations (Podani 2000). PAST version 2.17 (Hammer et al. 2012) RESULTS A total of 22 protein bands were observed for these taxa. (Fig. 2 and Table 2). All studied taxa had bands 76.12 KD and 45 KD except for Gorgan/Naharkhoran population (Pop 25) of H. androsaemum. The highest number of bands was observed in Kordkoy1 population (Pop12) and Gardane heyran population (Pop20) of H. perforatum and the lowest in Gorgan/Naharkhoran pop- ulation (Pop 25) of H. androsaemum (Fig. 2 and Table 2). In order to find out the most variable protein bands in the studied taxa, a Principal Component Analysis was implemented. Primitive analysis showed that three fac- tors were responsible for 62.37 % of total studied varia- tion in the taxa. In the first factor, with almost 37.81 % of the total variation, bands 6.12, 9.87, 34.87, 51.12 KD had the highest correlation. In the second factor, with about 14.63 % of the observed variation, bands 27.37, 30.19, 76.77 and 81.67 KD had the highest positive correlation. In the third factor, with 9.92 % of the total variation, bands 21.54, 78.14, 93.16 KD had the highest correlation. Both clustering and PCA analyses of the Hypericum species studied produced similar groupings and there- fore only WARD clustering characters are presented here (Fig. 3). Two major clusters were formed in WARD clus- tering (Fig.3). WARD clustering, of the studied popula- tions did not entirely delimit the studied species and revealed that plants in these species are intermixed. In WARD dendrogram, a higher degree of intermixture occurred between H. perforatum, H. tetrapterum and H. androsaemum. Also WARD dendrogram revealed that although population of the species H. perforatum is more distinct than the other two species, but it showed a high degree of intraspecific genetic variability as they are positioned in different places of the dendrogram. Figure 1. Distribution map of Hypericum populations studied. 110 Parisa Mahditabar Bahnamiri et al. Table 1. Voucher details of Hypericum species examined in this study from Iran. Population No. Species Population cod Locality / Voucher number 1 H. perforatum L. Hp1 Mazandaran , Ramsar, 1723 HUMZ 2 H. perforatum L. Hp2 Mazandaran , Ramsar/Javaherde1,1724 HUMZ 3 H. perforatum L. Hp3 Mazandaran , Ramsar/Javaherde2/daryache ghoo,1725 HUMZ 4 H. perforatum L. Hp4 Mazandaran , Savadkoh/Alasht,1726 HUMZ 5 H. perforatum L. Hp5 Mazandaran , Babolkenar1,1727 HUMZ 6 H. perforatum L. Hp 6 Mazandaran , Babolkenar2,1728 HUMZ 7 H. perforatum L. Hp 7 Mazandaran , Galogah1,1729 HUMZ 8 H. perforatum L. Hp 8 Mazandaran , Galogah2,1730 HUMZ 9 H. perforatum L. Hp9 Mazandaran , Aliabad katool,1731 HUMZ 10 H. perforatum L. Hp10 Golestan , Gorgan,1732 HUMZ 11 H. perforatum L. Hp 11 Golestan , Ziarat,1733 HUMZ 12 H. perforatum L. Hp 12 Mazandaran ,Kordkoy1,1734 HUMZ 13 H. perforatum L. Hp13 Mazandaran , Kordkoy2,1736 HUMZ 14 H. perforatum L. Hp14 Mazandaran , Kelachay,1737 HUMZ 15 H. perforatum L. Hp15 Guilan , Langrood,1738 HUMZ 16 H. perforatum L. Hp16 Guilan , Lahijan/Bam Lahijan,1739 HUMZ 17 H. perforatum L. Hp17 Guilan , Somesara,1740 HUMZ 18 H. perforatum L. Hp18 Guilan , Asalem,1741 HUMZ 19 H. perforatum L. Hp19 Guilan , Heyran,1742 HUMZ 20 H. perforatum L. Hp20 Guilan , Gardane heyran,1743 HUMZ 21 H. perforatum L. Hp21 Mazandaran , Nowshahr/Sisangan,1744 HUMZ 22 H. perforatum L. Ht22 Guilan , Astara,1745 HUMZ 23 H. tetrapterum Fries. Ht23 Mazandaran , Savadkoh/Alasht,1746 HUMZ 24 H. tetrapterum Fries. Ha24 Guilan , Asalem,1747 HUMZ 25 H. androsaemum L. Ha25 Golestan , Gorgan/Naharkhoran,1748 HUMZ 26 H .androsaemum L. Ha26 Guilan , Astara,1759 HUMZ 27 H. androsaemum L. Ha27 Mazandaran, Ramsar/Bam Ramsar,1750 HUMZ 28 H. androsaemum L. Ha28 Mazandaran , Aliabad Katool/ Kabodval,1751 HUMZ 29 H. androsaemum L. Ha29 Mazandaran , Amol/Sangchal,1752 HUMZ Figure 2. SDS-PAGE electrophoresis profiles of the studied population of Hypericum. Note: Populations abbreviations are according to Table 1. 111Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran DISCUSSION In the present study, 29 geographical populations belonging to three species of Hypericum of North region of Iran was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Elec- trophoretic data of the seed storage proteins presented in this study have shown that the dendrogram obtained from the studied species is not capable of the species recognition. We found findings the seed storage was often incongruent with the result of Faghir, et al. (2018) and Mahmoudi Otaghvari & al, (2015) and Bayat & al., (2015) for pollen data. In previous studies, the micromorphology of pollen grains was performed in several species and their impor- tance in plant taxonomy was emphasized (Faghir, et al. 2018; Mahmoudi Otaghvari & al,2015; Bayat & al., 2015). Faghir, et al. (2018) pollen grains of ten species and two subspecies of the genus Hypericum in Iran belong- ing to four sections were studied using light and scan- ning electron microscopy. Palynological analysis of selected species of the genus Hypericum revealed impor- tant pollen morphological characters, especially pollen outline, numbers and types of apertures, colpus length; presence and absence of operculum; exine sculpturing type, pore shape, size and arrangements. These traits can be used for infrageneric classification, especially at sec- tional and species levels (Faghir, et al. 2018). Different techniques including morphological, bio- chemical and especially molecular markers let scien- Table 2. Band number and molecular weight for each studied population of Hypericum. (1- band is present in the seed sample, 0- band is absent in the seed sample). Band No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 MW(kd) 99 95 94 92 85 82 76 68 65 62 59 55 51 45 40 37 34 27 22 18 12 8 Pop 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 4 1 0 0 0 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 7 0 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 0 0 1 1 1 8 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 0 0 1 1 1 9 0 1 1 1 1 1 1 0 0 0 0 1 1 1 0 1 1 0 0 1 1 1 10 0 0 0 0 1 1 1 0 0 0 0 1 1 1 0 0 1 0 0 1 0 0 11 1 1 1 0 1 1 1 0 0 0 0 1 1 1 0 0 1 0 0 1 0 0 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 14 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 0 1 1 15 1 1 1 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 17 0 0 0 0 1 1 1 0 0 0 0 0 1 1 1 0 1 1 1 0 0 0 18 0 0 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 0 0 19 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 0 0 0 1 1 1 1 1 0 0 0 1 1 1 1 0 1 0 1 1 0 0 22 1 0 1 1 1 1 1 1 0 0 0 1 1 1 1 0 1 0 1 1 1 1 23 0 0 0 1 1 1 1 1 0 0 0 1 1 1 1 0 0 0 0 1 1 1 24 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 0 0 1 1 1 0 25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 26 0 0 0 0 1 1 1 0 0 0 1 0 1 1 1 0 1 0 1 1 1 0 27 0 1 1 1 0 0 1 0 1 1 1 0 1 1 1 0 1 1 1 1 1 0 28 1 1 1 1 0 0 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 0 29 0 0 0 1 0 0 1 0 0 0 0 0 0 1 1 0 1 1 0 1 1 0 112 Parisa Mahditabar Bahnamiri et al. tists to study genetic variability of plants. As molecular markers present reproducible results regardless of envi- ronmental conditions, they have gained nowadays con- siderable attention for studies relating to the genetic diversity (Farooq and Azam 2002). According to Morshedloo et al. (2015) genetic vari- ability among ten wild populations of H. perforatum growing in different climatic regions of Iran via ISSR markers. They observed the studied populations were classified into four main groups which was, to the some extent, in accordance with their geographical origins. Also they recovered, ISSR markers revealed relatively a high level of genetic variability among Iranian H. perfo- ratum populations suggesting that the ISSR technique is efficient and powerful for assessment of genetic diversity at the intraspecific level. The present study also provide the way for use of molecular systematics within genus Hypericum. The taxa are not clearly separated on the basis of electrophoretic data of seed storage proteins. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 1 - 2020 Firenze University Press Karyotypic investigation concerning five Bromus Species from several populations in Iran Sara Sadeghian, Ahmad Hatami, Mehrnaz Riasat High genetic diversity and presence of genetic structure characterise the endemics Ruta corsica and Ruta lamarmorae (Rutaceae) Marilena Meloni1, Caterina Angela Dettori2, Andrea Reid3, Gianluigi Bacchetta2,4,*, Laetitia Hugot5, Elena Conti1 Cytogenetic effects of C6H4 (CH3)2 (xylene) on meristematic cells of root tips of Vicia faba L. and mathematical analysis Cihangir Alaca1, Ali Özdemir1, Bahattın Bozdağ2, Canan Özdemir2,* Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. Sazada Siddiqui*, Sulaiman Al-Rumman Temporal Analysis of Al-Induced Programmed Cell Death in Barley (Hordeum vulgare L.) Roots Büşra Huri Gölge, Filiz Vardar* Genetic diversity, population structure and chromosome numbers in medicinal plant species Stellaria media L. VILL. Shahram Mehri*, Hassan Shirafkanajirlou, Iman Kolbadi A new diploid cytotype of Agrimonia pilosa (Rosaceae) Elizaveta Mitrenina1, Mikhail Skaptsov2, Maksim Kutsev2, Alexander Kuznetsov1, Hiroshi Ikeda3, Andrey Erst1,4,* Study regarding the cytotoxic potential of cadmium and zinc in meristematic tissues of basil (Ocimum basilicum L.) Irina Petrescu1, Ioan Sarac1, Elena Bonciu2, Emilian Madosa1, Catalin Aurelian Rosculete2,*, Monica Butnariu1 Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts Nicoleta Anca Şuţan1,*, Andreea Natalia Matei1, Eliza Oprea2, Victorița Tecuceanu3, Lavinia Diana Tătaru1, Sorin Georgian Moga1, Denisa Ştefania Manolescu1, Carmen Mihaela Topală1 Phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm Bombyx mori induced by microsporidian infection Hungund P. Shambhavi1, Pooja Makwana2, Basavaraju Surendranath3, Kangayam M Ponnuvel1, Rakesh K Mishra1, Appukuttan Nair R Pradeep1,* Electrophoretic study of seed storage proteins in the genus Hypericum L. in North of Iran Parisa Mahditabar Bahnamiri1, Arman Mahmoudi Otaghvari1,*, Najme Ahmadian chashmi1, Pirouz Azizi2 Melissa officinalis: A potent herb against EMS induced mutagenicity in mice Hilal Ahmad Ganaie1,2,*, Md. Niamat Ali1, Bashir A Ganai2 Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers Rayan Partovi1, Alireza Iaranbakhsh1,*, Masoud Sheidai2, Mostafa Ebadi3 In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Saeed Tarkesh Esfahani1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2 Long-term Effect Different Concentrations of Zn (NO3)2 on the Development of Male and Female Gametophytes of Capsicum annuum L. var California Wonder Helal Nemat Farahzadi, Sedigheh Arbabian*, Ahamd Majd, Golnaz Tajadod A karyological study of some endemic Trigonella species (Fabaceae) in Iran Hamidreza Sharghi1,2, Majid Azizi1,*, Hamid Moazzeni2 Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Kishan Saha*, Rabindra Kumar Sinha, Sangram Sinha