Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 37-44, 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-269 Citation: S. Siddiqui, S. Al-Rumman (2020) Clethodim induced pollen steril- ity and meiotic abnormalities in vegeta- ble crop Pisum sativum L.. Caryologia 73(1): 37-44. doi: 10.13128/caryolo- gia-269 Received: May 25, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 S. Siddiqui, S. Al-Rumman. 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. Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. Sazada Siddiqui*, Sulaiman Al-Rumman Department of Biology, College of Science, King Khalid University, Abha 61413, Saudi Arabia *Corresponding author. E-mail: sasdeky@kku.edu.sa Abstract. Pesticides are highly noxious materials.Their poisonousness might not be unequivocally precise to the target entities but can unfavorably disturb various proce- dures in the non-target host plants. In the present study, the effect of application of clethodim on pollen sterility and meiotic anomalies of Pisum sativum L are studied. Pisum sativum L seeds are treated with different concentrations of clethodim varying from 0.01%, 0.02%, 0.03%, and 0.04% for exposure time of 1 hour and their effect on pollen sterility and chromosomal anomalies were investigated. The outcomes reveal that treatment of clethodim on Pisum sativum L seeds induces pollen sterility (PS) and chromosomal anomalies (CA) in a dose-dependent manner. Also in clethodim treated seeds, an elevation in the proportion of abnormal meiotic phases were observed which was time and concentration dependent. Secondary association (SeA), precociuos sepa- ration (PS), clumped nuclei (CNu) were reported in metaphase I & II, stickiness (Stc), bridges (Br) and laggards (Lg) in anaphase I & II. The results of the present study reveal that frequently used herbicide clethodim has a substantial cytotoxic effect on meiotic cells of Pisum sativum L. Keywords. Herbicide, Clethodim, Pollen sterility, Meiotic abnormality, Pisum sativum L. INTRODUCTION The application of herbicides has led to an alteration in the phytosocio- logical properties of weeds and to a choice of biotypes resilient to herbicides. In addition, it also causes effects in the health of humans and environment although they are categorized as an extremely efficient means in the regu- lation of weeds. According to He et al. (2012), all over the world, the most applied chemical materials are the herbicides. In the 90’s, the worldwide pes- ticide sales continued to stay moderately persistent, amongst 270 to 300 bil- lion american dollars, 79% of this amount is related to herbicides. Amongst the three foremost groups of pesticides i.e. herbicides, fungicides and insec- ticides; herbicides have taken the top slot since 2007 (Zhang et al. 2011). In order to upsurge agricultural output, the application of herbicides for regu- lating weeds is the most widely used practice in worldwide agriculture. Nev- ertheless, when these chemical substances are applied in an unrestrained 38 Sazada Siddiqui, Sulaiman Al-Rumman way, they can influence and disturb other organisms, particularly aquatic creatures (Nwani et al. 2011; Van Bruggen et al. 2018). Earlier reports have shown that much of the noxious effects of herbicides on plants and animals were inade- quately explored (Chevreuil et al. 1996; Kim and Feagley 1998; Abdel-Rahman et al. 1999). As a result of the absence of evidences about the effect of herbicides in the biotic environments, they can also epitomize a setback to health of human beings (Munger et al. 1997; Gorell et al. 1998). The influence of pesticide in the environments is determined by its noxiousness, concentration and dis- persion manner (Van der Werf 1996). The mutagenic effects of the herbicides can result from several reactions within the organism, as a direct action of the compound on the nuclear DNA; incorporation in the DNA during cell replication; interference in the activity of the mitotic or meiotic division, resulting in incorrect division of the cell (Timbrell 1999). A few herbicides affect directly the elongation, cell differentiation and cell division of plants. Herbicide clethodim falls into the cyclohexan- edione oxime class. From post-emergence, clethodim is active as an herbicide against various species of grass weeds mostly in sunflower, soybean, cotton and other broad-leaved plants (Edwards 2005). Earlier studies have emphasized the adverse effects of the herbicides on plant physiology and cytogenetics. Maleic hydrazide (MH) is one of the few herbicides which prevents the synthesis of proteins and nucleic acids (Siddiqui et al. 2008). Muta- genic action of glyphosate, alachor and maleic hydrazide has been reported by Siddiqui et al. (2012), diclofop- methyl and lindane has been stated by Anila and Ditika (2013) and anilofos has been described by Arzu et al. (2014) on root tip cells of plant. Pisum sativum L, (Fabaceae) is a plant useful in the manure production, ayurvedic medications and food (Davies et al. 1985). It is one of the most used legu- minous plant and is a source of protein. There are no studies available in the literature which have assessed the adverse effects of clethodim on Pisum sativum L, a potential multipurpose crop. In the current investiga- tion, we have assessed the noxious consequences of clethodim on meiotic cells of Pisum sativum L. MATERIALS AND METHODS Seeds and chemical Pisum sativum L seeds, variety ARKIL were obtained from the Indian Council of Agricultural Research - Bbhoj Krishi Vigyan Kendra, Near Village Naktara, P. O. Bankhedi, NH-86 Ext., Raisen Sagar Road, Bhopal, India. Clethodim herbicide was obtained from the Delta Chemical Company based in Riyadh, Saudi Arabia. Treatment of Pisum sativum L with clethodim Healthy and even sized seeds of Pisum sativum L were taken and submerged for 6 hours in double dis- tilled water of pH 6.7. In a glass beaker of 250 mL, seeds of Pisum sativum L were immersed for 1 hour in 150 mL clethodim solutions of various concentrations (0.01, 0.02, 0.03 and 0.04%). The seeds were shaken frequently for providing ample air to the seeds. For eliminating any remaining amount of clethodim, the treated seeds were properly cleaned with double distilled water. For control, a set of seeds were submerged in distilled water. In order to eradicate any remaining chemical sticking to the seed coat, the seeds were cleaned with tap water. Each set of seeds comprising of 10 seeds per pot were seeded in pots having a height of 24 cm and width of 17 cm. The entire experiment was repeated thrice under similar condi- tions. Pollen grain analysis For experiments related to pollen grains, f lower buds of similar age were collected from treated plants and control group. They were fixed in 70% alcohol. For identifying pollen sterility as well as fertility, pollens were stained with 1% propionocarmine. Pollens having uniform size and shape which were stained dark pur- ple in colour and filled with nuclei and cytoplasm were considered as fertile whereas pollen grains which were stained pale yellow colour or colourless, having irregular size and shape and without nuclei and cytoplasm were defined as sterile. Pollen sterility was calculated as the ratio of non viable pollen grains to the total number of pollens and expressed in percentage. No. of non viable pollen grains No. of sterile pollen grains = x 100 Total no. of pollen grains Meiotic analysis To assess the effect on meiotic cells, flower buds of similar size were fixed in 1:3 acetic acid saturated with iron and absolute alcohol for 24 hours and than passed to 70% alcohol. The meiotic cell preparations were prepared by traditional acetocarmine squash technique (Man- 39Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. ton 1950). By squashing the anther in an acetocarmine stain, slides were prepared. In normal butnyl alcohol (NBA) series, permanent slides were made and they were mounted in Canada balsam and than dried up at 45°C. Statistical analysis Statistical analysis was performed employing one way ANOVA test using GPIS software 1.13 (GRAPHPAD, California, USA) to detect the significance of differences of variables. All values are expressed as mean ± SE. RESULTS The occurrence of pollen sterility and meiotic chro- mosomal abnormalities were listed in (Figs 1, 2 and Table 1). It is clear from the results that clethodim was proficient in inducing pollen sterility and different types of meiotic chromosomal abnormalities such as second- ary association (SeA) (Fig. 2, A and B), precociuos sepa- ration (PS) (Fig. 2, C and D), and clumped nuclei (CNu) (Fig. 2, E and F) in metaphase I & II and stickiness (Stc) (Fig. 2, G and H), bridges (Br) (Fig. 2, I and J) and lag- gards (Lg) (Fig. 2, K and L) in anaphase I & II. Effect of clethodim on pollen sterility of Pisum sativum L. The observation reveals that the percentage of pollen mother cells (PMCS) showing pollen sterility increased with increase in concentrations of clethodim at different exposure times and was null in control (Fig. 1). A low- er percentage of pollen sterility were observed at 0.01% concentration which was 60% at one hour and it was highly significant (p>0.001) when compared to control. In addition, at highest concentration high increase in pollen sterility were reported which reached 83% at one hour in clethodim treated PMCS. Pollen sterility is a sign of disturbance in reproduc- tive process which was observed in clethodim treated pollen mother cells of Pisum sativum L at 1 hour (Fig. 1). The increase in the ratio of sterile pollen grains in the clethodim treated groups as the dose increases might be due to their toxic effects on pollen grains. Effect of clethodim treatment on chromosomal aberration (CA) of Pisum sativum L Cytological analysis also documented numerous anomalies in the PMCS of clethodim treated plants at one hour, as shown in Table 1. Different types of CA such as SeA, PS, and CNu were noticed in metaphase I & II and Stc, Br and Lg were observed in anaphase I & Figure 1. Pollen sterilty of Pisum sativum L PMCS exposed to differ- ent concentrations of clethodim for 1 hour. ¥ p≤0.001 compared to control. Data are mean of three replicates ± SE; 0.0 = Control group. Table 1. Percentage of meiotic abnormality in metaphase I & II and anaphase I & II plate of Pisum sativum L in PMCS exposed to different concentrations of clethodim for 1 hour. (%) Concentration time Treatment PMC cells Total Metaphase I/II (Mean ±S.E) Anaphase I/II (Mean ±S.E) Sec. asso. Pre.sep. Clumped nuclei Stickiness Bridges Laggards Total anomalies 1 h 0.00 110 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 00.0 ± 0.0 0.01 125 1.5 ± 0.34 2.6 ± 0.29 3.5 ± 0.2 1.8 ± 0.3 2.4 ± 0.4 1.4 ± 0.2 13.2 ± 1.73 0.02 115 2.8 ± 1.20 3.8 ± 0.93 4.6 ± 0.9 2.2 ± 0.7 3.1 ± 0.7 2.8 ± 0.6 19.3 ± 5.03 0.03 128 3.2 ± 1.70 4.2 ± 1.04 6.4$ ± 1.3 3.5 ± 1.2 3.8 ± 0.3 3.4 ± 1.2 24.5 ± 7.34 0.04 130 6.5# ± 2.80 6.1$ ± 2.1 7.4$ ± 2.4 6.7¥# ± 2.0 6.5# ± 2.7 6.2$# ± 2.1 39.4$ ± 14.1 p≤ 0.05; $ p≤0.01; ¥ p≤0.001 compared to control. Data are mean of three replicates ± SE; 0.0 = Control group. 40 Sazada Siddiqui, Sulaiman Al-Rumman II (Table 1 and Fig. 2). Cytological investigations clearly revealed that the level of CAs gradually increased along with increasing concentrations of clethodim at one hour. Studies of different stages of meiotic division shows that almost all the stages of division were affected to a cer- tain extent. The percentage of formation of SeA, PS, and CNu in metaphase I & II were reported maximum in 0.04% (6.5% - significant p≤0.05, 6.1% - very significant p≤0.01 and 7.4% - very significant p≤0.01) as compared to con- trol, at one hour and reported minimum in 0.01% (1.5%, 2.6% and 3.5%) at one hour of clethodim treated plants. The percentage of formation of Stc, Br, and Lg in anaphase I & II were reported maximum in 0.04% (6.7% - very significant p≤0.01 and significant p≤0.05, 6.5% - significant p≤0.05 and 6.2% - very significant p≤0.01) as compared to control at one hour and reported minimum in 0.01% (1.8%, 2.4% and 1.4%) at one hour of clethodim treated plants. Increased incidence of CAs were in the following order for 1 hour clethodim treatment: CNu > Stc > BR = SeA > Lg > PS. DISCUSSION In the present study, the occurrence of laggards and bridges in anaphase I & II were strictly correlated with sterility. Similar finding were reported by Liang et al. (1967) on sorghum by Atrazine treatment which affected meiotic stability. Çalli (2008) reported that the Equation Pro (22.5% Famoxadone+30% Cymoxanil) fungicide instigates abnormalities in the meiosis of pollen grains and thus resulting in pollen sterility. Dubey et al. (1977) reported a decrease in viability of about 60% in the pol- len of eggplants, indicated by the joint effect of a dinitro herbicide and two organophosphate insecticides. Fungi- cide propiconazole produced damaging effects on pollen tube development and pollen germination of Tradescan- tia virginiana (He et al. 1995). Rana and Swaminathan (1964) and Ramanna (1974) found that any deviance in cytokinesis or karyokineses could generate non-viable microspores. Sinha and Godward (1969;1972) found that translocations were liable for reduced pollen fertil- ity. In H.orientalis, tube growth and in vitro pollen ger- mination was disturbed and affected by the herbicide A E DCB JI HGF K L Figure 2. (A-L) Meiotic aberrations induced by clethodim in Pisum sativum L PMCS. (A-B) Secondary association; (C-D) Precocious sepa- ration; (E-F) Clumped nuclei at Metaphase I & II, (G-H) Sticky chromosome; (I-J) Bridges; (K-L) Laggards at Anaphase I & II in PMCS in Pisum sativum L. 41Clethodim induced pollen sterility and meiotic abnormalities in vegetable crop Pisum sativum L. Quizalofop-p-ethyl (QPE) treatments. Especially, the maximum QPE concentration instigated alterations in the morphological attributes of H. orientalis. Pollen ger- mination is decreased by three times by utmost vigorous QPE application. Within the pollen tube morphological anomalies were also detected (Deveci et al. 2017). Pisum sativum L and Allium cepa L are generally used in order to check the genotoxicity and cytotoxicity of several chemicals (Bonciu et al. 2018 a and b; Siddiqui 2018). Previous works have revealed that herbicides have mutagenic effect on Pisum sativum L and Allium cepa L (Siddiqui et al. 2008; Rosculet et al. 2019). Cytologi- cal abnormalities in plants can be a useful monitoring method for the recognition of chemicals present in the environment that might cause a genetic threat. In the current study, we found numerous kinds of chromosom- al irregularities such as SeA, PS, CNu in metaphase I & II, Lg, Br, and Stc in anaphase I & II in Pisum sativum L after treatments with clethodim of one hour. The prevalence of secondary association has been found in treated plants. Incidence of secondary associa- tion is a regular attribute occurring primarily because of the existence of more than two homologous chro- mosomes. In the course of secondary pairing, there is a debate on the participation of homologous chromosomes amongst the researchers. Hirayoshi (1957) differed with the postulation that contemplates the participation of homologous chromosome in secondary association and after investigating thorough outcomes in Zizanieae and Oryzeae offered the inference that secondary associa- tion might be an incidence effective under physico and bio-chemical effects and has got no relation to the exact homology of chromosomes. Secondary pairing within bivalents is well thought to be a sign of polyploid attribute of a species as found in Kidney bean (Girjesh and Nitu, 2014), Ocimum (Mukherjee and Datta 2005), Uraria picta (Bhattacha- rya and Datta 2010). As per Stebbins (1950), secondary association may be contemplated to be a phenomenon that illustrates the polyploid attribute of a species but detailed phylogenetic estimations may not be framed from this since secondary pairing within bivalents is substantially altered by further chromosomal altera- tions. In the present study, the occurrence of precocious movement also appeared enhanced with rising cletho- dim concentations. Precocious separations were formed due to unstable spindle process or inactivation of spin- dle formation and interrupted homology for pairing of chromosomes that might lead to the precocious motion of chromosomes (Umar and Singh 2003). It might also be attributed due to the occurrence of breakage of chro- mosomes or because of the heavy metals breaching the protein moiety of the nucleoprotein backbone. In the current study, the prevalence of clumped nuclei also get boosted with rising clethodim concen- tations. Because of the turbulences at the cytochemi- cal level, clumped nuclei were created. Clumping of the chromosomes can occur with regular arrangement at metaphase and separation may result in the creation of bridges. Some types of gene mutations that cause incor- rect coding of some non-histone proteins involved in chromosomal organization can lead to chromosomal clumping. There is a possibility that the mutagen reacts with the available histone proteins and results in an alteration in the surface property of the chromosomes because of incorrect folding of DNA and hence causing the chromosomes to stick or clump (Grant 1978). Stickiness was also reported with increase in the clet hodim concentrations. Stick iness could occur because of incomplete detachment of the nuclear pro- teins and changes in their design of association or because of incomplete detachment of the nucleoproteins and amendment in their design of association or because of depolymerization of nucleic acids instigated by cletho- dim treatment. Stickiness might result from disorders in cytochemical balance reaction (Dewitte et al. 2010; Ros- culet et al. 2019). Depolymerisation of nucleic acids due to herbicidal treatment or by incomplete detachment of nucleoproteins (Kaufman et al. 1955) or by the limited separation of nucleoprotein variation in their design of organization (Evans 1962) may cause stickness. Numerous bridges were formed in anaphase I & II in the treated plants. Bridges were probably created by breaking and combination of chromosomes bridges which augmented with the treatment of clethodim. For- mation of chromosomal bridges could occur because of the chromosomal stickiness and consequent failure of free anaphase division or because of inversion of chro- mosome fragments or irregular translocation (Gomur- gen 2000). Rosculet et al. (2019) reported that the origin of bridges were mainly because of the merger amongst broken chromosomes. The laggards noticed within the current investi- gation could be due to the failure of the movement of chromosomes or because of the deferred termination of stickiness of the ends of chromosomes. Lagging of chro- mosomes might occur because of the interruption of the motion of bivalents towards equatorial plate at meta- phase I. The utmost regular occurence was the lagging of single univalent (Zeyad et al. 2019). Barthelmess (1957) reported that more than one lagging chromosomes in meiosis might occur because of the interruption of the prometaphase motion of chromosomes supplemented by 42 Sazada Siddiqui, Sulaiman Al-Rumman union of the centromeric to the adjoining inner surface of plasma. Bridges and laggards might have been formed because of deferred terminalisation of stickiness of the ends of chromosomes (Kaur and Grover 1985). Crea- tion of micronuclei at telophase I is caused by laggards. Generation of micronuclei at telophase II is caused by laggards or acentric fragments and thus it results in the alteration in numeral and size of pollen grains ensuing from mother cells. As reported in previous findings, our outcomes suggest the possibility of these chemicals to instigate meiotic anomalies (Namrata and Alka 2014; Das et al. 2018). The chromosomal abnormalities caused by these herbicides might be because of their impeding effect on spindle proteins and their capability to instigate swap over of sister chromatids (Tartar et al. 2006; Siddiqui et al. 2009). Previous investigations have revealed that free radicals might be the reason for genomic uncertainty in cells. Damages in DNA, disarray in the cytoskeleton and disproportion in energy metabolism might result in chromosomal anomalies as reactive oxygen species are extremely unstable (Siddiqui et al. 2012; Siddiqui 2015). Due to genetic and physiological disarrays, numer- ous types of meiotic abnormalities might have been cre- ated. It has become clear from the above findings that clethodim used in the current work is probably capable of instigating several types of chromosomal anomalies. CONCLUSION Within the experimental settings applied in the cur- rent work, clethodim displayed a sturdy genotoxic effect on Pisum sativum L. It is obligatory to do additional research work on the attribute of the harvests resulting from seeds and plants subjected to these chemicals in relation to their nutritive worth and their vulnerability to acclimatize with diseases. Additionally, it is signifi- cant to note that the pesticide preparations encompass- ing these active constituents may or may not have a par- allel effect on the plant cells. Regarding this, supplemen- tary researches are essential to evaluate their outcome on biological procedures. ACKNOWLEDGMENTS We are grateful to Deanship of Scientific Research at King Khalid University, Abha, Kingdom of Saudi Arabia for providing technical and administrative support. DECLARATION The authors declare no conflict of interest. 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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.) 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