Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 99-106, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1113 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Harshita Dwivedi, Girjesh Kumar (2021) Colchicine induced manifes- tation of abnormal male meiosis and 2n pollen in Trachyspermum ammi (L.) Sprague (Apiaceae). Caryologia 74(3): 99-106. doi: 10.36253/caryologia-1113 Received: October 09, 2020 Accepted: September 24, 2021 Published: December 21, 2021 Copyright: © 2021 Harshita Dwivedi, Girjesh Kumar. 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. ORCID HD: 0000-0001-7197-3000 Colchicine induced manifestation of abnormal male meiosis and 2n pollen in Trachyspermum ammi (L.) Sprague (Apiaceae) Harshita Dwivedi*, Girjesh Kumar Plant Genetics Laboratory, Department of Botany, University of Allahabad, Prayagraj, 211002 India Corresponding author. E-mail: harshitadwivedi88@gmail.com Abstract. Unreduced gametes are the key source for the natural polyploidization in plants, but rate of its formation is very low in nature. Meiotic mutants are second source for the formation of 2n pollen. In this cytological investigation, the meiotic aberrations and its impact on post-meiotic products were analysed in autotetraploid Trachyspermum ammi (L.) Sprague (4n=36). The seedlings of T. ammi (L.) Sprague were treated with 3 different concentrations of colchicine (0.2, 0.4 and 0.5%, w/v) for 3 different durations. Six polyploid plants were induced which was confirmed on the basis of cytological analysis. Colchicine, an anti-microtubular drug induced different meiotic and post-meiotic abnormalities such as chromosomal bridges, lagging chro- mosomes, scattering, precocious, fragments, dyads, triads, and polyads. The formation of several abnormal sporads clearly signifies the meiotic restitution. The tendency of univalents to scattered in the cytoplasm at metaphase was identified as a peculiar aber- ration asynapsis. Pollen variability and fusion of pollen walls was reported and pollen fertility was calculated. The morphological analysis of the pollen allowed us to confirm the occurrence of 2n pollen. Keywords: unreduced gametes, 2n pollen, polyploidy, colchicine, meiotic aberrations, Trachyspermum ammi (L.) Sprague. INTRODUCTION Ploidy induction is an effective method to induce novel quantitative and qualitative traits in plants which make them superior to their diploid ances- tors. It is estimated that up to 70% of angiosperm species are polyploid, and this number is even higher if ancient polyploidization events are taken into account (Bretagnolle and Thompson 1995; Ramsey and Schemske 1998). Various polyploid plants have been reported in natural ecosystems; among them some are important crop species such as potato, coffee, banana, peanut, tobacco, wheat, oats, sugarcane, and many fruits (Bretagnolle and Thompson 1995; Stebbins 1950; Udall and Wendel 2006). Meiosis is a well-coordinated event which is vital for accomplishment of microsporogenesis. The product of male meiosis in plants is a tetrad of four 100 Harshita Dwivedi, Girjesh Kumar haploid microspores that are temporarily joined by a cal- losic wall (Brownfield and Kohler 2011). After release from the tetrad each microspore undergoes two mitotic divisions to produce a pollen grain containing the two sperm cells required for double fertilization (McCormick 2004). Whereas any disturbance to the meiotic event often has severe effects and leads to the abortion of the meiocytes or the developing gametophytes and thus ste- rility, a number of meiotic mutants that produce viable, unreduced gametes have been described in a range of plants (Bretagnolle and Thompson 1995; Ramanna and Jacobsen 2003). Ga metes w it h somatic chromosome numbers referred to as unreduced (2n) gametes. According to Bretagnolle and Thompson (1995), the production of unreduced gametes is considered the main cause of polyploid induction in nature. The unreduced (2n) gam- etes can be formed due to both pre- and post-meiotic genome duplication events, as well as meiotic restitution (Bretagnolle and Thompson 1995; Ramsey and Schemske 1998). The heterozygosity within 2n gametes depends on its cytological mechanism which is subdivided as first division restitution (FDR), second division restitu- tion (SDR) and indeterminate meiotic restitution (IMR) (Ramanna and Jacobsen, 2003). An easier diagrammatic representation of restitution mechanism was given by Ferris et al. (1992). A FDR 2n gamete comprises non- sister chromatids, whereas a SDR 2n gamete comprises two sister chromatids (Tang 2002). The third (IMR) type was observed in lily, carried characteristics similar to both SDR and FDR (Lim et al. 2001). In this intermedi- ate type, both univalents and bivalents are formed dur- ing metaphase I. Crossing 2n pollen with a normal female gamete has been shown to be one of the most effective methods to produce triploid individuals (Nilsson-Ehle 1936; Müntz- ing 1936). According to Dewitte et al. (2012), meiotic mutants are a second source of 2n gametes. Yet, it has been considered that the rate of occurrence of 2n pollen in nature is very low hence the production rate of poly- ploids was less than 0.1% (Liu et al. 2019). To increase the occurrence rate of 2n pollen, novel methods for inducing polyploid production in plants via the use of spindle inhibitors, such as dinitroanilines or colchicine, have been successfully explored and used in some spe- cies (Vaughn and Lehnen 1991; Hancock 1997; Zlesak et al. 2005; Dhooghe et al. 2009). Amongst several anti- microtubular drugs, colchicine is traditionally and pref- erably being used as doubling agent (Kumar and Dwive- di 2017). A widespread data has been accumulated over the last 60 years which specifies that the drug colchicine manifest defects in meiotic prophase. It reduces the fre- quency of chiasmata (Driscoll and Darvey 1970, Shepard et al. 1974) and impairs synaptonemal complex forma- tion (Tepperberg et al. 1997). The large number of world’s population rely either solely or largely on traditional herbal remedies for health care (Dwivedi 2016). One of these important medicinal plants is ajwain (Trachyspermum ammi (L.) Sprague, 2n = 2x = 18) which is a rich source of various neutra- ceutical components, due to which it occupies a signifi- cant economic position in pharmaceutical industries (Dwivedi and Kumar 2018). Many of the medicinal and aromatic plants do not have stable production in their growing areas and are usually gathered in accordance with conventional methods to meet demands (Dalkani et  al. 2012). Therefore to meet the growing pharmaceu- tical demands, attention need to be paid to medicinal plants with stable quality. 2n gametes are an effective and efficient way to transmit genetic diversity to the plants, including both valuable qualitative and quantita- tive traits (Peloquin et al. 1999). Recently, plant breeders have become interested in the practical use of 2n gam- etes in breeding program due to the new tools available for 2n gamete manipulation and insights into the genetic background of their formation (Dewitte et al. 2012). However artificial manipulation of ploidy in ajwain has been previously achieved by few researchers (Kumar and Dwivedi 2017 and Noori et al. 2017) yet the poten- tial role of unreduced gametes to create genetic variabil- ity in this crop is unmapped. Hence, through this study we had made an effort to understand the effect of in  vit- ro colchicine-induced disruption in meiotic products, mechanism of formation of 2n pollen and its repercus- sions on the crop fertility. MATERIAL AND METHODS Plant material Fresh and healthy seeds of T. ammi var. AA-1 were procured from National Research Centre for Seed Spic- es, Ajmer, Rajasthan, India. Seeds were sown in earthen pots in triplicates during October to November season to raise the seedlings. Agro-climatic conditions of experimental site The present experiment has been performed in the area of Roxburgh Botanical Garden, Department of Bot- any, University of Allahabad, Prayagraj, U.P., India, dur- ing the rabi season. The exact experimental location is 25°27”43.01’N, 81°51”10.42’E. Prayagraj is situated 98 m 101Colchicine induced manifestation of abnormal male meiosis and 2n pollen in Trachyspermum ammi Sprague above mean sea level. Prayagraj is in the sub-tropical cli- matic zone; the average rainfall is 1027 mm and relative humidity is 59%. Induction of autotetraploidy For the present work colchicine (C22H25NPO6) man- ufactured by Himedia Laboratory Pvt. Ltd., Mumbai, India was used. The treatment was given to the seed- lings within 2–3 days of germination (before third leaf emergence). Emerging shoot apices at cotyledonary stage of each of the 20 seedlings/pot were treated with aque- ous solution of 0.2, 0.4, and 0.6% concentration by cot- ton plug method for one, two and three consecutive days with an alternate recovery time period of 12 hours in each case. A set of control was also maintained of 20 plants/ pot. Meiotic preparation Small sized floral buds were fixed in Carnoy’s fixa- tive viz. glacial acetic acid: ethyl alcohol (1 : 3, v/v) and then transferred to 70% alcohol after 24 hours and stored at 4°C until use. Meiotic slides were prepared by using anther squash technique with 2% standard aceto- carmine stain, observed and microphotographed under Nikon Phase Contrast Research microscope (Nikon Eclipse, E200, Japan) at 40X magnification. Pollen viability assessment Pollen viability was estimated using a glyceroaceto- carmine i.e. stained cytoplasm with nucleus were con- sidered as fertile whereas unstained and shriveled pollen grains without nuclei were considered as sterile. Statistical analysis All the statistical analyses of morphological and cytological observations have been done by using SPSS 16.0 software to measure mean values of variables. A pair wise comparison of means was made using Dun- can’s Multiple Range Test (DMRT) at p≤0.05 signifi- cance level. RESULTS The diploid plant of Trachyspermum ammi (L.) Sprague of var. AA-1, used in the present study, had 2n=18 (n=9) as confirmed in mitotic as well as mei- otic studies (Figure 1.1). The meiotic behaviour of six induced polyploid plants of ajwain were analysed and found that the number of bivalents was more than 9 at early prophase (Figure 1.2). Moreover, the PMCs of col- chicine treated plants showed various chromosomal aberrations such as unorientation, laggard (Figure 1.9 and 1.11), bridge (Figure 1.9), scattering (Figure 1.4 and 1.10), precocious movement (Figure 1.7 and 1.8), frag- ment (Figure 1.3 and 1.10), etc. The range of these aber- rations was higher in metaphase I and II as compared Figure 1. 1. A PMC showing 9 bivalents at diakinesis (diploid), 2-12. Meiotic stages of autotetraploid plants (4n=36); 2. Normal PMC at early prophase, 3. Fragment at anaphase I, 4. Scattering at metaphase I, 5 and 6. Meiotic configurations of asynaptic plants, 5. 2IV+5II+10I, 6. 5II+26I, 7 and 8. Precocious movement at meta- phase I, 9. Bridge formation alongwith laggard at sticky anaphase I, 10. Scattering and fragment at anaphase I, 11. Laggard at anaphase I, 12. Fragment at anaphase II. Scale: 10µm 102 Harshita Dwivedi, Girjesh Kumar to both divisions of anaphase. The dividing phases of plants were less affected by lower doses of colchicine (24 and 36 hours treatment of 0.2% concentration) while it was higher in case of 0.4% and 0.6% concentrations. The highest total meiotic aberration (TMA) was observed at 0.6% concentration (10.51±1.22%) however it was range from 7.50±0.79% to 9.77±0.69% at 0.2% concentration (Table 1). The tendency of univalents to scattered in the cytoplasm at metaphase exhibited the peculiar phenom- enon of asynapsis which was witnessed at 0.4% concen- tration of colchicine (figure 1.5 and 1.6). Owing to the outcomes of these meiotic aber- rations, abnormal post-meiotic products have been reported among which triads showed predominance over the dyads and polyads. An increasing trend for polyads formation was recorded with respect to colchi- cine i.e. 4.19±0.42% to 8.20±0.51% in a dose depend- ent manner (Table 2). The arrangement of these spo- rads was quite different from the normal tetrad. Figure 2.3 showed dyad with one small microcyte unlike the dyad shown in figure 2.4. In some instances, the poly- ads were recorded in which the sporads were not joined by callosic wall (figure 2.8 and 2.9) unlike the normal tetrad stage. Such sporads can produce gametes of dif- ferent ploidy levels. As a consequence of these abnormal sporads, meiotic index (MI) was decreased along with increasing the concentrations of colchicine. The MI was Table 1. Effect of colchicine on different meiotic phases of Trachy- spermum ammi (L.) Sprague. Concentra- tion (%) Duration (hours) Plant No. Meiotic Abnormalities (Mean±S.E.) Meta I/II Ana I/II TMA Control - P1-P10 - - - 0.2 24 P-1 4.22±0.58a 3.28±0.26ab 7.50±0.79a 36 P-2 5.07±0.80ab 4.26±0.26ab 9.33±1.68ab 36 P-3 5.50±0.39ab 4.27±0.38ab 9.77±0.69ab 36 P-4 6.68±0.34b 2.97±0.29a 9.65±0.41ab 0.4 24 P-5 5.47±0.17ab 4.50±0.14bc 9.97±0.28ab 0.6 12 P-6 5.72±0.47ab 4.79±0.76c 10.51±1.22b *Mean±S.E., Values followed by the superscript differ at p<0.05 between treatments by the DMRT. Table 2. Effect of colchicine on post-meiotic products, meiotic index and Pollen fertility of Trachyspermum ammi (L.) Sprague. Concentration (%) Duration (hours) Plant No. Post-meiotic Abnormalities (Mean±S.E.) MI (%) PF (%)Dyads Triads Polyads Control - P1-P10 - 1.75±0.24a - 98.72±0.33f 98.99±0.22f 0.2 24 P-1 7.80±0.43b 21.35±0.09b 4.19±0.42b 64.86±0.30e 59.30±0.20e 36 P-2 8.16±0.36b 21.49±0.33b 5.69±0.43c 62.11±0.15c 58.80±0.17d 36 P-3 8.20±0.64b 21.92±0.42bc 6.43±0.47c 64.05±0.07d 58.32±0.22cd 36 P-4 8.70±0.42bc 22.96±0.47cd 6.15±0.41c 63.40±0.22d 58.29±0.25c 0.4 24 P-5 8.80±0.40bc 23.64±0.26d 6.52±0.60c 57.20±0.19b 41.20±0.14b 0.6 12 P-6 9.75±0.43c 24.03±0.52d 8.20±0.51d 55.10±0.20a 39.10±0.09a TMA- Total Meiotic Abnormalities, MI-Meiotic Index, PF- Pollen Fertility. *Mean±S.E., Values followed by the superscript differ at p<0.05 between treatments by the DMRT. Figure 2. 1. Normal tetrad of diploid plant, 2. Normal tetrad of autotetraploid plant, 3. Dyad with a microcyte, 4. Dyad, 5. Triad, 6. Pentad, 7. Polyads, 8. A polyad with fused microspore in two groups, 9. a polyad completely fused microspore. Scale: 10µm 103Colchicine induced manifestation of abnormal male meiosis and 2n pollen in Trachyspermum ammi Sprague recorded as 64.86±0.30% at 24 hour duration of 0.2% concentration whereas 55.10±0.20% at 0.6% concentra- tion of colchicine (Table 2). On account of these aberrant post meiotic products, the process of microsporogenesis is significantly affected and consequently resulted variability in pollen grains (Figure 3). The pollen grains exhibited variability in terms of their shape and size. The shape was observed to be remarkably transformed which is represented in figure 3. The fertility of pollen grains were gradually reduced (Table 2) and ranged from 59.30±0.20% (at 0.2% concen- tration) to 39.10±0.09% (at 0.6% concentration). Figure 4.1 and 4.2 represents the pollen cytomixis in which the chromatin material was transferred through both wide and narrow channels to the proximate pollens. Onset of passing event was evident by the assemblies of pol- lens where the dissolution of walls was observed (Figure 4.2). Direct fusion of pollen grains is represented in fig- ure 4.1. The size of pollen grain of diploids was registered as 4.42 0.04 μm × 2.67 ± 0.12 μm while 7.09±0.17 μm × 5.31±0.20 μm in case of autotetraploids. The pollens of polyploids were near about two times larger as compared to diploids thus it was considered as unreduced pollen (2n) (figure 3.2 and and 4.1-4.4). DISCUSSION The repercussions of colchicine induced meiotic aberrations resulted to genetically unstable polyploid plants. The proper spindle formation and its precise ongoing activity during whole meiotic event are essen- tial for the accomplishment of fertile progeny. However, Levan (1939) stated that colchicine causes a temporary inactivation of the spindle mechanism without damag- ing any other life processes of the chromosomes. Thus, testing the stability of induced polyploids is critical and should be studied at various stages as the plant material matures (Harbard et al. 2012). Meiotic prophase I is characterized by chromosome cohesion, pairing, and recombination (Ma, 2006). These bivalents are the physical sites of crossover between homologous chromosomes and are only established if pairing and recombination occur normally. The muta- tions concerning to meiotic prophase I that often result in univalents (paired sister chromatids) rather than bivalents (Ross et al. 1997; Bai et al. 1999; Bhatt et al. 1999; Couteau et al. 1999; Caryl et al. 2000; Grelon et al. 2001; De Muyt et al. 2009). The unequal segrega- tion of univalents in meiosis I followed by an equal second division, results in the formation of aneuploid cells which abort during development, however in some mutants, a few functional gametes are produced (Cou- Figure 4. 1. Fusion of wall between two pollen grains, 2. Fusion of walls in a group of pollen grains, 3 and 4. Pollen variability. Scale: 10µm. Figure 3. 1. A diploid pollen (2n), 2-4. Different autotetraploid pollen grains (4n), 2. a fertile tetraploid pollen 3. a sterile budding pollen grain, 4. A fertile pollen showing variability in shape. Scale: 10µm. 104 Harshita Dwivedi, Girjesh Kumar teau et al. 1999; Azumi et al. 2002; Ravi et al. 2008). In both meiosis I and II, bridge formation accompanied with the fragment/s was exhibited at the higher concen- tration of colchicine which attributed to the presence of paracentric inversion (Sybenga, 1996). Chromosome bridges have often been studied by observing cancer cells containing chromosomes damaged by spontaneous telomere loss (Fouladi et al. 2000; Lo et al. 2002; Aci- lan et al. 2007). These damaged chromosomes enter the breakage–fusion–bridge cycle (Zheng et al. 1999). Bajer (1964) mentioned that chromatin bridges are responsible for retardation of kinetochore movement, or, frequent bridges formation may result in nuclear restitution. In the present study, the phenomenon of asynapsis has been observed which showed the inability of univalents to assemble at the equatorial plate and widely scattered in cytoplasm at metaphase stage suggests lack of pairing at early prophase. According to Gottschalk and Kaul (1980 a, b), the absence or failure of synapsis is termed as asynapsis. The asynaptic mutant induced by colchicine was previously described in soybean by Kumar and Rai (2007); proposed that this mutation might have affected specific genes for chromosome pairing. The sporads are highly specialized cells which are able to produce four haploid cells after a series of geneti- cally controlled steps (Caetano-Pereira et al. 1999). The elimination or addition of one or more chromosomes (as a consequence of laggard, precocious, bridge, fragments, etc.) is responsible for the formation of anomalous post- meiotic products such as monads, dyads, triads and polyads resulted to the unreduced gametes (Golubovs- kaya 1989) or sterile (Bosco et al. 1999) pollen grains. According to Kiihl et al. (2011), the meiotic phases that precede cytoplasm cleavage might have caused failure in the cytokinesis process resulting into monads, dyads, triads and polyads. There are three main types of abnor- mal spindles orientation i.e. parallel, fused and tripolar that have been reported to produce 2n pollen (Mok and Peloquin 1975; Veilleux 1985; Bretagnolle and Thomp- son 1995; Ramsey and Schemske 1998; Zhang et al. 2009; Zhang and Kang 2010; Silva et al. 2011). The paral- lel spindles resulted in two FDR 2n pollens while fused spindles are accountable to form a dyad and then two FDR 2n pollens. The tripolar spindles develop a mother cell to produce one FDR 2n pollen and two 1n pollen (Zhang and Kang 2013). However, Ramanna and Jacobson (2003) stated that FDR is typical in synaptic mutants or distant hybrids, in which homologous chromosome pairing (bivalent formation) is completely absent, although other mecha- nisms, such as cytokinesis failure or spindle abnormali- ties during metaphase II, can also lead to an equiva- lent of FDR. FDR gametes contain an equal number of parental chromosomes due to the equatorial division of sister chromatids. Therefore, FDR pollen are key enti- ties in producing heterozygous hybrids, because of the highly heterozygous 2n gametes formed (Bretagnolle and Thompson 1995). On the contrary, sister chromosomes move to the same daughter cell in case of SDR gametes, thus due to the absence of crossing-over, it exhibit maxi- mum homozygosity (Hermsen 1984; Veilleux 1985; Pelo- quin et al. 1999). Owing to the abnormal spindle activ- ity, the unreduced pollen of T. ammi genetically repre- sents the FDR mechanism. Naturally, the establishment of new polyploid geno- types is infeasible without the existence of 2n gametes. Thus its role has great significance in evolution, as the cytological mechanisms of 2n gamete formation dem- onstrate that it might be the source of variable genetic combinations. Since, our investigation was performed only to determine the cytological behavior of unreduced gametes; further researches are required to understand the extent of heterozygosity of 2n gametes and its influ- ential role to create variability in ajwain crop. ACKNOWLEDGMENTS Authors are thankful to NRCSS, Ajmer, Rajasthan, India for supplying seeds of T. ammi var. AA-1. One of the authors (Harshita Dwivedi) thanks the University Grant Commission (UGC) for financial assistance and the Head of the Department of Botany, University of Alla- habad, Prayagraj, for providing the necessary facilities. Sincere thanks are also due to all the members of Plant Genetics Laboratory for their encouragement and support. FUNDING This work was supported by the University Grants Commission. REFERENCES Acilan C, Potter DM, Saunders WS (2007). DNA repair pathways involved in anaphase bridge formation. Gen Chrom Cancer. 46:522–531. Azumi Y, Liu D, Zhao D, Li W, Wang G, Hu Y, Ma H. 2002. Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein. EMBO Journal. 21:3081–3095. 105Colchicine induced manifestation of abnormal male meiosis and 2n pollen in Trachyspermum ammi Sprague Bai X, Peirson BN, Dong F, Xue C, Makaroff CA. 1999. 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Tek*, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım Development of a protocol for genetic transformation of Malus spp Federico Martinelli1,*, Anna Perrone2, Abhaya M. Dandekar3 Cytogenetic and cytological analysis of Colombian cape gooseberry genetic material for breeding purposes Viviana Franco-Florez, Sara Alejandra Liberato Guío, Erika Sánchez-Betancourt, Francy Liliana García-Arias, Víctor Manuel Núñez Zarantes* Palynological analysis of genus Geranium (Geraniaceae) and its systematic implications using scanning electron microscopy Jun Wang1,2,*, Qiang Ye1, Chu Wang2, Tong Zhang2, Xusheng Shi2, Majid Khayatnezhad3, Abdul Shakoor4,5 Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats Yuri A. Kirillov1, Maria A. Kozlova1, Lyudmila A. Makartseva1, Igor A. Chernov2, Evgeniya V. Shtemplevskaya1, David A. Areshidze1,* The morphological, karyological and phylogenetic analyses of three Artemisia L. 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