Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(1): 15-21, 2019 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/cayologia-247 Citation: Ramanpreet, R. Chand Gupta (2019) Meiotic studies in genus Withania Pauquy, from Indian Thar Desert. Caryologia 72(1): 15-21. doi: 10.13128/cayologia-247 Received: 19th July 2018 Accepted: 20th December 2018 Published: 10th May 2019 Copyright: © 2019 Ramanpreet, R. Chand Gupta. 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 Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Meiotic studies in genus Withania Pauquy, from Indian Thar Desert Ramanpreet*, Raghbir Chand Gupta Department of Botany Punjabi University, Patiala, 147002, Punjab, India * Corresponding author: ramanbrar247@gmail.com Abstract. Detailed male meiosis has been made in two species of genus Withania col- lected from desert regions of Rajasthan, India. The study revealed 2n=48 for both the species. The meiotic analysis of W. somnifera is reported to be normal whereas, W. coagulans showed abnormal meiosis with the presence of abnormalities like spindle irregularities, chromatin transfer, laggards and irregular microsporogensis with the presence of monads, dyads, triads, polyads and tetrads with micronuclei which further lowered the pollen fertility and giving rise to varying size pollen grains. Both the spe- cies are widely used for various medicinal purposes by local/tribal people of the state. Keywords. Withania, Medicinal plant, Abnormal meiotic behavior, Indian Thar desert. INTRODUCTION The genus Withania belongs to the family Solanaceae. The family com- prises of 100 genera and about 2500 species (Hunziker 2001, Olmstead et al. 2008). The genus is distributed throughout the tropical and sub-tropical regions of the world with 26 species (Ahmad 2014). W. somnifera is well dis- tributed in India with and is growing well in dry parts of tropical and sub- tropical regions extending to the elevation of 1500 m. But W. coagulans is rare and endangered plant species found only in few localities in Rajasthan. Both the species are well distributed in East of Mediterranean regions extending to South Asia. Regarding the medicinal properties of the species, a composite Ayurve- dic medicine “Liv 52” commonly used as intestinal infections is a hepatopro- tective herbal preparation and contains extracts from W. coagulans and W. somnifera (Mishra et al. 2013). W. coagulans is commonly known as “Indian cheese maker” since fruits and leaves of the plants are used as coagulant, as it contain an enzyme called Withanin, having milk coagulating activity (Naz et. al. 2010). In some parts of India and Pakistan, both the species are used as blood purifier, for cleaning teeth and plant smoke is inhaled for relief in toothache (Dymock et al. 1972, Krishnamurthi 1969). The plant possesses 16 Ramanpreet, Raghbir Chand Gupta antitumor, antimicrobial and anti-inflammatory prop- erties. Since the plant is toxic in nature so it should be used with caution (Purohit and Yyas 2004). Flow- ers of its species are used to treat nervous exhaustion, insomnia and impotence. The meiotic course of the spe- cies reveals the presence of 2n=48 from West Pakistan (Baquar 1967) as well as outside India. W. somnifera commonly known as “Ashwangan- dha” or “Indian ginseng” is extensively studied from India by Bir et al. (1978) Bir and Sidhu (1979 and 1980) from Punjab plains, Koul et al. (1976) from Jammu and Kashmir, Madhavadian (1968) from Tamil Nadu, Bha- duri (1933), Datta et al. (2005), Iqbal and Datta (2007) from West Bengal. It is also extensively worked out from Pakistan by Baquar (1967) and Khatton and Ali (1982) and from Saudi Arabia by Al-Turki et al. (2000). Earlier meiotic studies reveals the presence of intraspecific dip- loid cytotype (2n=24), tetraploid cytotype (2n=48) and hexaploid cytotypes (2n=72). The karyotype analysis of the species shows seven groups of the chromosomes with occurrence of metacentric and sub-metacentric types (Samaddar et al. 2012). The species also shows polyso- matomy (2n=12, 18, 24, 36, 48, 72) with predominance of 2n=48.Whereas cytologically, W. coagulans is not as much explored. There is no cytological report of it from the studied area. Present research work is undertaken by keeping in view the existence of cytological diversity. MATERIALS AND METHODS Floral buds were fixed in freshly prepared Carnoy’s fixative (6 parts of absolute alcohol: 3 parts of chloro- form: 1 part of glacial acetic acid) for 24-48 hours. After- wards, these were transferred to 70% ethyl alcohol and stored in refrigerator at 4˚C until use. For chromosomal preparations, anthers were crushed and tapped to prepare a smear of pollen mother cells (PMCs) in 1% acetocar- mine (Belling 1921). A number of PMCs were observed and chromosome counts were confirmed. In case of spe- cies with meiotic abnormalities, large numbers of PMCs are observed to confirm frequency of various abnormali- ties. Pollen fertility was observed by mounting the pol- len grains in 50% glycerol-aceto carmine (1:1) solution (Marks 1954). Pollen grains with stained nuclei were taken as fertile and viable, whereas, unstained pollen grains marked as sterile ones. Pollen grain size was meas- ured using an occulometer. The photomicrographs of the PMCs and pollen grains were taken from the temporary slides by using, Nikon 80i digital imaging system. The species are collected from different localities of district Churu and Jodhpur. W. somnifera is more fre- quent in its appearance as compared to W. coagulans, which is a rare medicinal plant and is collected only from single locality of the studied area. The plant speci- mens were identified with the help of various floras such as Flora of North East Rajasthan (Sharma and Tiagi 1979), Flora of the Indian Desert (Bhandari 1978), Flora of Rajasthan (Shetty and Singh 1993) and by comparing the plant specimens with the samples lying in of Herbar- ia of Department of Botany, Punjabi University,  Patiala (PUP) and Botanical Survey of India, Jodhpur (BSI). The identified voucher specimens of plants have been depos- ited in the Herbarium, Department of Botany, Punjabi University Patiala (PUN), India. RESULTS Detailed cytological investigation is carried out in two species of genus Withania i.e., W. somnifera and W. coagulans. Meiotic studies of W. somnifera reveals the presence of 24 bivalents at metaphase-I (Fig. 1). PMCs also show the presence of equal segregation of 24:24 chromosomes at anaphase-I (Fig. 2). The meiotic course is normal with high pollen fertility (97.66%). The meio- sis of W. coagulans depicts the presence 24 bivalents at metaphase-I (Fig. 3). These chromosomes are unable to segregate on the spindle and are scattered all over the cytoplasm (Fig. 4). A very few the cells also shows equal segregation of 24:24 chromosomes at anaphase-I (Fig. 5). The meiotic course reveals spindle irregularities in the PMCs as the chromosomes are unable to move towards the poles (Figs. 6, 7) and several chromosomes remain in the cytoplasm to the form of laggards (Fig. 8) at ana- phase-I. The spindle abnormalities (Figs. 9, 10) and lag- gard formation is also observed at anaphase-II stages of meiosis (Fig. 10). Further the formation of bridges (Fig. 11) is also observed at telophase-II. All these abnormali- ties lead to the formation of multipolarity (Fig. 12) as chromosomes are not able to move towards their respec- tive poles and remain in the cytoplasm. Microsporogen- esis is highly abnormal with the presence of monad (Fig. 13), dyad (Fig. 14) and triad (Fig. 15). Chromatin trans- fer within tetrads (Fig. 16), polyad (Fig. 17) and polyads with micronuclei (Fig. 18) is also observed. Pollen grains with unequal size (Fig. 19) and fertile and sterile pollen are also observed (Fig. 20) which leads to low pollen fer- tility. The majority of the PMCs depicted abnormal spin- dle formation which resulted in irregular arrangement of bivalents at the spindle plate during metaphase-I and segregation of chromosomes during anaphase-I/teolo- phase-I and anaphase-II/telophase-II. Chromosomes also lack the ability of congregation at a single pole and 17Meiotic behavior of a tetraploid cytotype of Brazilian nightshade Figs. 1-20. Withania somnifera 1) PMC at metaphase-I showing 24 bivalents; 2) PMC at anaphase-I showing 24:24 chromosomes; W. coag- ulans 3-4) PMC at metaphase-I showing 24 bivalents; 5) PMC at anaphase-I showing 24:24 chromosomes; 6-7) PMC showing spindle irreg- ularities with scattered chromosomes (scattered chromosomes arrowed); 8) PMC at anaphase-I showing laggards (laggards arrowed); 9, 10) PMCs at anaphase-II showing spindle irregularities in form of laggards (laggards arrowed); 11) PMC at telophase-II showing laggards and bridges (bridge arrowed); 12) PMC showing multipolarity; 13) Monad; 14) Dyad; 15) Triad; 16) Chromatin transfer within tetrad (chro- matin transfer arrowed); 17) Chromatin transfer within polyad (chromatin transfer arrowed); 18) Polyad with micronuclei (micronuclei arrowed); 19) Unequal sized pollen grains; 20) Sterile and fertile pollen grains. Scale bar=10µm. 18 Ramanpreet, Raghbir Chand Gupta remained scattered in the cytoplasm or in small groups. Chromosomes in these PMCs failed to reach their respective poles and constitute micronuclei during late telophase stages and sporad formation. Irregular spin- dles in these plants are also depicted in the meiocytes, which showed multipolar presence of chromosomes. DISCUSSION AND CONCLUSIONS Meiosis is most sensitive stage in the life cycle for all sexual species and has direct relevance to natural selec- tion; it leads to the formation of gametes, contributes to genome stability and generates genetic diversity. The process of meiosis depends upon interrelated events of homologous chromosome recognition, intimate associa- tion, synapsis and recombination (Hamant et al. 2006, de Muyt et al. 2009). In plants, it is affected by various genetic and environmental factors (Ahmad et al. 1984, Viccini and Carvalho, 2002, Sun et al. 2004, Bajpai and Singh 2006, Rezaei et al. 2010). There are various meiotic abnormalities which hinder the path of normal meio- sis and are the cause of changes in the morphology and genetic constitutions of the plant. The evolution of vas- cular plants is dependent upon the variation in chro- mosome numbers which may be caused due to genomic mutations especially polyploidy (auto or allopolyploidy) (Soltis et al. 2009, Bedini et al. 2012). There are num- ber of research papers on the phenomena of polyploidy, emphasizing its origin, impact and role in speciation (Stebbins 1985, Ramsey and Schemske 1998, Otto and Whitton 2000, Cifuentes et al. 2010, Jiao et al. 2011). The autotetraploids are generally characterized by the presence of quadrivalents due to homology of 4 sets of chromosomes, whereas, in allopolyploids there is nor- mal pairing because of existence of two separate sets of chromosomes. On the other hand in segmental allo- tetraploids due to the partial homology of two genomes there is low frequency of quadrivalent formation. In the present study W. somnifera shows normal bivalent formation in all the PMCs, without any quadrivalent formation which indicates its allotetraploid behavior. However, the absence of quadrivalents does not con- firm that it is an allotetraploid because there are many artificially produced autotetraploids where there is only bivalent formation because the formation of quadriva- lents depends upon many other factors such as localiza- tion of chiasmata, small size of chromosomes, and pres- ence of some suppressor genes etc., which does not allow the pairing between the homeologous chromosomes (Morrison and Rajhathy 1960, Gottschlk 1978). On the other hand in W. coagulans the meiosis is highly abnor- 19Meiotic behavior of a tetraploid cytotype of Brazilian nightshade mal with the presence of spindle abnormalities which indicates the absence of multivalents and also indicates that it might be hybrid or more probably due the pres- ence of specific genes which interfere in the pairing and functioning of spindle (Baum et al.1992, Risso-Pascotto et al. 2003; Kumar and Singhal 2008; Singhal and Kaur 2009). The basic function of the spindle is to attach at kinetochore and separate the chromosome or chroma- tids at anaphases (Wadsworth et al. 2011), these attach to the centromeres (Qu and Vorsa 1999) and rearrange the chromosomes on the equatorial plate and bring them together at metaphase-I (Qu and Vorsa 1999). But, if due to some factors (genetic or enviornmental) the spindle activity fails then chromosomes are unable to line up in the equator and then separate at Anaphases of the mei- osis, which leads to abnormal meiotic course. Earlier, a number of plants have been reported with abnormalities like irregular spindle activity, cytomixis and chromatin stickiness leading to abnormal microsporogenesis (Baum et al. 1992, Caetano-Pereira and Pagliarinini 2001, Kumar and Singhal 2008, Rai and Kumar 2010, Sin- ghal and Kaur 2009). Spindle irregularities are generally divided into 4 categories-multipolar, monopolar, radial and apolar. Multipolar spindles are those in which 3 or more poles are formed, in monopolar only one spindle formation takes place, in radial spindles ends are located at the periphery and near the equator (Shamina et al. 2000a) and apolar spindles have randomly oriented set of fibers (Shamina et al. 2000b, Seriukova et al. 2003). 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