Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(3): 47-64, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1666 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Durre Shahwar, Zeba Khan, Mohammad Yunus Khalil Ansari (2022). Cadmium induced genotoxicity and antioxidative defense system in len- til (Lens culinaris Medik.) genotype. Caryologia 75(3): 47-64. doi: 10.36253/ caryologia-1666 Received: May 22, 2022 Accepted: November 23, 2022 Published: April 5, 2023 Copyright: © 2022 Durre Shahwar, Zeba Khan, Mohammad Yunus Khalil Ansari. 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, 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. Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Durre Shahwar1,2,*, Zeba Khan3, Mohammad Yunus Khalil Ansari1 1 Cytogenetics and Molecular Biology laboratory, Department of Botany, Aligarh Muslim University, Aligarh, 202002, India 2 Plant Genomics and Molecular Biology laboratory, Department of Horticultural Biosci- ence, Pusan National University, Miryang 50463, Korea 3 Center for Agricultural Education, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, India *Corresponding author. E-mail: khanzeba02@gmail.com Abstract. Induced mutagenesis is considered a coherent mechanism in crop improve- ment programmes to produce novel plant varieties. Due to the insufficiency of desired genotypes, plant breeders are supposed to re-associate the gene of interest from the accessible gene pool of the related plant species through hybridization to develop new cultivars with desired traits. The present investigation was performed to evaluate cad- mium induced mutagenesis on growth performance, physio-biochemical traits and DNA damage studies in lentil. Growth and morphological parameters exhibited reduc- tion with increasing concentration of cadmium. Maximum devaluation was reported at the highest concentration. Physiological and biochemical traits were also affected by different cadmium concentrations and reduced as concentration increased. Lipid per- oxidation activity and antioxidant enzymes increased as mutagenic stress increased caused by cadmium. CAT and SOD concentration was found to increase initially and then decreased gradually at higher cadmium concentrations. SEM analysis of stomatal morphology revealed variation in stomatal shape and size in treated populations. There was a gradual enhancement in the percentage of DNA damage along with variation in morphological traits. The DNA damage was recorded as precocious movement, stray bivalent, laggard, stickiness, disorientation of chromosome, multi-bridge, disturbed polarity and micronuclei. It was concluded that at higher concentrations, cadmium cause DNA damage and these chromosomal alterations causes morpho-physiological and biochemical changes in lentil. Keywords: Abiotic stress, oxidative stress, antioxidant activity, DNA damage, Lens culinaris. ABBREVIATIONS Cd Cadmium CAT Catalase activity SOD Superoxide dismutase 48 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari ROS Reactive oxygen species EDTA Ethyl diamine tetra acetic acid INTRODUCTION Nowadays, world is posing a severe threat of mal- nutrition and food insecurity to human civilization. Scientists are involved in developing new and ingen- ious approaches to diminish hunger and malnutri- tion issues which are expanding day by day around the world. Pulses play a significant role in compensat- ing food insecurity, especially for low-income families (Kumar and Pandey 2020).India is one of world’s largest producer, importer and consumer of pulses, especially lentils, which have great potential to elucidate the global food crisis. Lentil is highly efficient inadjusting adverse climatic conditions, which resulted in a declaration by United Nations in 2016 as an International Year of Puls- es (IYP2016), with interdisciplinary research approaches towards the qualitative and quantitative improvement of pulses. Lentil is considered as essentially important nutri- tious crop rich in protein and minerals. Lens culinaris is self-pollinated, diploid (2n=14) crop with a genome size of 4063Mbp (Arumuganathan and Earle, 1991). Van Oss et al. (1997) suggested that the Lens genus has four wild species L.culinaris, L. lamottei, L. nigricans and L. ervoides, whereas (Ferguson et al. 2000) observed that Lens culinaris Medikus contain three wild sub- species: L. culinaris subsp. Orientalis and L. culinaris subsp. tomentosus and L. culinaris subsp. Odemen- sis of which L. culinaris subsp. orientalis is considered the ancestor of cultivated lentil. Full knowledge of len- til was given by Barulina (1930), who categorized Lens culinaris into two subspecies, of which one is named macrosperma (large seeds with 6-9 mm diameter) and the other microsperma (tiny seeds with 2-6mm diam- eter). Lentil is known to be a source of protein and high quality fiber among all pulses, because of this property, it is considered an economical food consumed all over the world. Lentil is an accomplished source of essential vitamins and minerals such as foliate vitamin B1, mag- nesium, phosphorus, potassium, copper complex carbo- hydrates and vegetable protein and a low amount of fat- free cholesterol (Tharanathan & Mahadevamma, 2003). Lentil contains macronutrients and also poses certain phytochemicals such as; flavonols, phenolic acids, phytic acid, soyasaponins and tannins (Xu & Chang, 2010). It can fix atmospheric nitrogen and increase soil fertility due to increased level of nitrogen in soil and by adding carbon and organic matter. Keeping all these attrib- utes in mind, it becomes necessary to ameliorate len- til variety to obtain genotype of good nutrient quality and yield-related traits. Induced mutagenesis is a help- ful technique in the plant-breeding programme for breeders or biological researchers with the embellish- ment in knowledge of technique for inducing mutation and mutation process itself to produce new cultivar of better quality by creating variability (Chaudhary et al. 2019).Mutagenesis has increased genetic variability for qualitative and quantitative traits and induces desir- able mutant alleles, which may not previously pre- sent in germplasm in a wide variety of species.Induced mutagenesis has played a significant role in overcom- ing food scarcity for world population and developed new mutant cultivars with increased nutritional values (Suprasanna et al. 2015). Cd is an anthropogenic genotoxic pollutant that is highly soluble in water (Jiang et al. 2001)and is readily absorbed by the plants. Cd toxicity reduces uptake and translocation of nutrients and water, increases oxida- tive damage, disrupts plant metabolism, and inhibits plant morphology and physiology (Haider et al. 2021). In plants, primary effect of metal toxicity is inhibition in root growth and cell division, protein denaturation, altered photosynthesis (Rathore et al. 2007; Akinci et al. 2010) and increases in the frequency of chromosomal aberrations as studied in different plants such as Allium by Liu et al., 1994, Allium sativum (Yi and Meng, 2003); Helianthus annuus (Kumar and Srivastava, 2006); Lathy- rus sativus (Kumar and Tripathi, 2007a) etc. Heavy met- al can induce reactive oxygen species (ROS) (Qian et al. 2009). Plants overcome the damage induced via metals stress by activating defense mechanisms which involve both -enzymatic components such as catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) to protect themselves from ROS (Ruley et al.2004) and non-enzymatic components such as glutathione–S-tran- ferase and glutathione reductase. An increase in ROS causes overproduction of MDA, therefore MDA in plant cell acts as a markerbetween production and scaveng- ing of free radicals. Production of ROS causes oxidative burst in biological macromolecules such as enzymes, proteins, membrane lipids, DNA, chloroplast and carot- enoids (Tripathy and Oelmüller 2012). Cadmium binds strongly to DNA and RNA, and alters the DNA tran- scription process so that DNA synthesis and mitotic activities are disturbed resulting in depolymerization, DNA strand breaks, generation of abnormal nitrog- enous bases, DNA – DNA cross-links and DNA – pro- tein cross-links. The present investigation examines cad- mium-induced mutagenicity and related stress in lentils by assessing the growth, yield, cytological, physiological and biochemical traits. 49Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype 2. MATERIALS AND METHODS 2.1 Seed procurement and treatments Dry, healthy, certified, uniform and equal size seeds of Lens culinaris variety L-4076 were obtained from Indi- an Agricultural Research Institute, New Delhi. Fresh, uniform and healthy seeds of lentil were presoaked in double-distilled water for 24 hours, and the mutagenic treatment of cadmium nitrate were given according to my previous study related to work (Shahwar et al. 2019). The comprehensive knowledge of induced mutagenesis and selection of mutant lines are described in detail in earlier study related to the work (Shahwar et al., 2022). Presoaked seeds were then subjected to different concen- trations (20,40,60,80 and 100ppm) of freshly prepared cadmium nitrate solution in double- distilled water at pH 7.0 for 12 hrs with intermittent shaking after an interval of 1 or 2 hours at room temperature of 25± 2°C. After treatment, the seeds were thoroughly washed with tap water to ensure the removal of adhered metal (Cd++) on the surface of the seed coat.Treated seeds of each con- centration were sown in replicates with their respective control in earthen pots having soil mixed with farmyard manure and irrigated regularly. 2.2 Growth and morphological study The experiment was carried out to demonstrate the cadmium stress on the growth and morphology of Lens culinaris. Root and shoot length were measured from randomly selected seedlings of each replicate for 30 days. Agronomical parameters such as plant height, number of branches per plant, yield and yield related traits were recorded during the development. 2.3Determination of physiological and biochemical param- eters 2.3.1 Estimation of chlorophyll and carotenoid content The photosynthetic pigments (chlorophyll a, b and carotenoid) were determined by acetone method (Arnon 1949) following pigment extraction. For the purpose,1 g fresh leaves were ground with 80% acetone and the extract was diluted with double distilled water and the final volume was made 10mL. The optical density (O.D) of photosynthetic pigments were measured at wave- lengths of 663 and 645nm (Smith and Benitez, 1955) using UV-VISspectrophotometers. Photosynthetic pig- ment of the sample was calculated using the following formula: chlorophyll a = 12.7 (O.D.) 663-2.69(O.D.) 645×v/ w×1000 chlorophyll b = 22.9 (O.D.) 645-4.68(O.D.) 663×v/ w×1000 Total chlorophyll = 20.2 (O.D.) 645+8.02 (O.D.) 663×v/ w×1000 carotenoids = 46.95 (O.D. 440.5-0.268× chlorophyll (a+b) Where W=fresh weight of extracted tissue in grams V= total volume of extract 2.3.2 Analysis of stomatal morphology and mineral ele- ments Stomatal morphology was studied using scanning electron microscopy (JEOL, JSM-6510LV, JAPAN). Scan- ning electron microscopy andenergy dispersive X-ray microanalysis(EDX) of leaf sample were performed fol- lowing the protocol proposed by Daudet al. (2009) with minor changes. The leaf samples were fixed in 2.5 % glutaraldehyde and 2% paraformaldehyde in 0.1M phos- phate buffer (pH 7.0) for 4 hrs and washed for 15 min with phosphate buffer thrice at each step. Leaf sam- ples were then re-fixed for 1 hour with OsO4 (osmium tetraoxide) in 0.1 M of potassium phosphate buffer (pH 7.0) and were again washed for 15 min with the same phosphate buffer thrice at each step. The dehydration was done after fixation using ethanol series (30%, 50%, 70%, 90%, and 100%) for 15-20 min thrice for each cycle and transferred in the mixture of alcohol and isoa- myl acetate (1:1) for half an hour and in pure isoamyl acetate for one hour. Dehydration of specimens were done by Zeiss Evo 60 (Carl Zeiss SEM, Germany) criti- cal point dryer using liquid carbon dioxide, the samples were coated with a thin layer of Palladium and observed under SEM at 15 kv with x1500 magnifications. Pre- pared leaf samples were analyzed through EDX for min- eral element analysis. 2.3.3 Estimation of proline content Leaf sample was homogenized in 10 mL of 3% aque- ous sulfosalicylic acid and centrifuged at 9000 rpm for 10 min. 2ml glacial acetic acid was added to 2 mL of supernatant; further 2ml ninhydrin solution in 30ml acetic acid and 20mL of 6M H3PO4were added. The solution was incubated at 100oC for 1 hour and OD was recorded at 520 nm using toluene as blank. Proline con- tent in test sample was calculated using a standard curve (Bates et al. 1973). 50 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari 2.3.4 Determination of lipid peroxidation/MDA content Malondialdehyde (MDA) content was measured fol- lowing the protocol proposed by Hodges et al. (1999) and expressed as µ moles g -1. 2.3.5 Antioxidant enzyme activity assay Antioxidant enzyme assay was done by the method proposed by Sinha et al. (2018) with slight modification. Fresh leaves tissues were grinded in 1 ml extraction buff- er having 80 mM sodium phosphate buffer, 1mM EDTA, 1 m Mphenylsulfonylfluride (PMSF), 1% polyvinyl pyr- rolidone (PVP), and 0.5% (v/v) Triton X-100 and centri- fuged at 11000 rpm for 25 min at 4°C. The supernatant kept at -20°C was used to determine antioxidant enzyme activities such as catalase (CAT) following protocol pro- posed by Yu and Rengel (1999), superoxide dismutase (SOD), Gallego et al. (1996) and peroxidase (POX) Kar and Mishra (1976). 2.3.6 Estimation of protein content Dry seeds (0.5g) were ground in 10ml water and 1ml of 10%trichloroaceticacid was added to the extract. The sample was kept in an ice bath for 10 min. and the supernatant was collected and centrifuged at 5000 rpm for 10 min at 4oC. 20 ml sodium hydroxide (0.1N) was added to dissolve the protein and the total volume was made the nearest whole number. Seed protein content of the extract was determined by Lowry’s method (1951) using BSA (Bovine serum albumin) as standard and absorbance were measured at 650 nm. 2.4 DNA damage Studies For chromosomal studies, young and small-sized f lower buds were collected from treated and control plants, fixed in freshly prepared Carnoy’s fluid (1:3:6 ratio of glacial acetic acid, chloroform and alcohol) and were preserved in 70% alcohol. For DNA dam- age studies,anthers of appropriate size were squashed in 0.5% propionocarmine stain, dehydrated in normal butyl alcohol series and mounted on Canada balsam to prepare permanent slides. Microphotographs of chro- mosomal lesion or DNA damage were taken from tem- porary and permanent slides by “Olympus” microphoto- graphic unit. 2.5 Statistical interpretation The results were analyzed and interpreted statisti- cally using software SPSS version 20 for windows 10 using one-way ANOVA. For determinationof least sig- nificant difference (LSD) at 5% and 10% probability (p < 0.05, 0.01), data analysis of variance, one-way ANOVA was done using Duncan’s Multiple Range Test (DMRT) (Duncan, 1955) 3. RESULTS 3.1 Effect of heavy metal stress on growth and morphologi- cal parameter 3.1.1 Germination, survival and pollen fertility Effects of cadmium stress on seedling growth were investigated on 15 days old seedling. It was observed that plant germination, survival and pollen fertility decreased linearly in dose-dependent manner. The inhibitory effect on germination and related parameters were evident at the highest concentration of heavy metal. Fig. 1A depicts a gradual decrease in these characters as concentration increases. The highest concentration (100 ppm) of mutagen exhibited a maximum reduction in all these parameters. 3.1.2 Effect of Cd heavy metal on root and shoot length (cm) A more pronounced impact of cadmium stress on root and shoot lengths were observed in treated plants. Fresh weight of the seedlings decreased significantly with increase in cadmium concentration. The decrease was significantat 80 and 100 ppm for root length and in 40-100 Cd(NO3)2 for shoot length.Inhibitory effect on the seedling growth was higher in the root than in the aerial segment. (Fig. 1B). 3.1.3 Plant height At maturity plant height was found to be maximum in control 43.26±1.52 and decreased significantly from 39.53±3.24 to 31.80±3.31 in 20 to100 ppm both at 5% (p < 0.05) and 1 % level (p < 0.01) (Table 1). 3.1.4 Number of branches per plant Mean for number of branches per plant was found to be 3.86±0.49 in control and decreased significantly at 51Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Figure 1. Effect of Cd(NO3)2 on germination, survival, and pollen fertility, root and shoot length (cm), photosynthetic pigments and proline content (µmoles/g dry wt) in Lens culinaris. Medik L. (M1 generation). Data means within columns followed by the same letter is not differ- ent at the 5% level of significance, based on the Duncan Multiple Range Test. 52 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari 1% (p < 0.01) from lower to higher concentration. Coef- ficient of variation increased with the increasing concen- tration of mutagens (Table 1). 3.1.5 Yield attributing traits Number of pods per plant, number of seeds per pod, total number of seeds per plant, 100 seed weight and total yield per plant are the yield related traits. All these parameters were found to reduce significantly at 5% (p < 0.05) and 1% level (p < 0.01) when compared with their respective control (Table-1). Number of pods per plant decreased significantly at 1% level (p < 0.01) from 34.46±3.36 to 31.66±4.09 (60-100 ppm) concentra- tion and the number of seeds per pod decreased at 1% level in 60-100 ppm Cd(NO3)2 (Table-1). Total number of seeds per plant, 100 seed weight and total yield per plant significantly decreased minimally from lower to higher doses of cadmium nitrate. Coefficient of variation increased with increasing concentration of cadmium which means the coefficient of variation is directly pro- portional to the concentration of mutagen. 3.2 Physiological and biochemical study 3.2.1. Photosynthetic pigment Estimation of photosynthetic pigments revealed some significant variations in control and treated plants (Fig. 1C-E). Photosynthetic pigments reduced as Cd con- centration increased. Chlorophyll ‘a’, ‘b’ and carotenoid significantly decreased from 40-100 ppm and the maxi- mum reduction was recorded at highest concentrations- with minimum chlorophyll contents. 3.2.2 Proline content Proline content increased remarkably by Cd expo- sure. Lowest concentration of proline was observed at 20 and 40 ppm, i.e. 2.12 and 2.35 µ moles/g fw, respective- ly compared to the other treatments, (Fig. 1F) while its production enhanced insignificantly with the increasing concentrations. Maximum significant increase in pro- line concentration (3.24 µ moles/g fw) was recorded at 100 ppm. Increased proline concentrations are common symptoms of metal stress and served as a non-specific index of Cd-toxicity. 3.2.3 Lipid peroxidation assay Estimation of lipid peroxidation was done by deter- mining the malondialdehyde content in control and cadmium stressed plants. The MDA content enhanced significantly in all concentrations over the control. The maximum increase of MDA content was 1.10 µ M g-1 at 100 ppm of Cd(NO3)2 (Fig. 2A). Table 1. Growth and Yield Studies in Cd(NO3)2treated Lens culinaris Medik. Conc. ppm Cd(NO3)2 Plant Height (cm) Mean±SD CV No. of Branches/Plant Mean±SD CV No. of Pods/Plant Mean±SD CV Length/pod (cm) Mean±SD CV No. of Seeds/ pod Mean±SD CV Total no. of Seeds/Plant Mean±SD CV 100-Seeds Weight (g) Mean±SD CV Total Yield/ plant(g) Mean±SD CV Control 43.26±1.52 3.51 3.86±0.49 12.69 38.53±1.25 3.24 1.06±0.16 15.09 2.0±0.36 18.0 77.06±2.08 2.69 3.10±0.20 6.45 2.38±0.45 18.90 20 39.53*±3.24 8.25 2.93**±0.57 19.45 37.26±2.48 6.65 1.00±0.23 23.00 1.66±0.44 26.50 61.85**±4.42 7.14 2.94±0.36 12.24 1.81±0.69 38.12 40 38.93*±3.31 8.50 2.73**±0.67 24.54 36.13±2.67 7.38 0.96±0.24 25.00 1.46*±0.48 32.87 52.74**±4.64 8.79 2.88±0.39 13.54 1.51*±0.78 51.65 60 35.00**±4.22 12.05 2.66**±0.73 27.44 34.46**±3.36 9.75 0.92±0.25 27.17 1.33**±0.49 36.84 45.83**±5.15 11.23 2.80±0.41 14.64 1.28**±0.81 63.28 80 32.46**±4.68 14.41 2.40**±0.80 33.33 32.53**±3.79 11.65 0.87 ±0.27 31.03 1.26**±0.49 38.88 40.98**±5.81 14.17 2.72*±0.46 16.91 1.11**±0.75 67.56 100 31.80**±4.96 15.59 2.26**±0.78 34.51 31.66**±4.09 12.91 0.84*±0.28 33.33 1.20**±0.48 40.00 37.99**±6.09 16.03 2.68*±0.50 18.65 1.01**±0.70 69.30 LSD at 5% (*) 3.37 0.60 2.70 0.20 0.41 4.28 0.34 0.64 LSD at 1% (**) 4.72 0.84 3.78 0.29 0.59 5.99 0.48 0.90 SD= Standard Deviation, CV= Coefficient of Variations, LSD= Least Significant Difference. 53Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype 3.2.4 Antioxidant enzyme activities Antioxidant activity (CAT, SOD) in leaves were found disturbed under cadmium stress. The anti- oxidant enzyme activity of lentil was found to be increased initially and then fall at higher doses. The catalase activity increases insignificantly over control in 20 and 40 ppm cadmium whereas it increased sig- nificantly in 60-100 ppm (Fig. 2B). On the other hand, SOD activity was significantly enhanced at 40-80 ppm cadmium respectively and thereby decreases (0.71 mg-1 protein) with their respective control (0.87 mg-1 pro- tein) at 100 ppm (Fig. 2C). 3.2.5 Estimation of protein content Result of estimation of protein content in Lens culi- naris is depicted in (Fig. 2D). Protein content decreased as cadmium concentration increased. Highest concen- tration (100 ppm) showed lower percentage of protein (23.0%) over control. An inverse relationship between cadmium concentration and protein content was observed. Statistical analysis shows a significant dif- ference in each treatment except 20 ppm of Cd at (p < 0.05). Figure 2. Effect of different concentrations of Cd(NO3)2 on lipid peroxidation (MDA content µmoles/g FW), catalase activity (CAT) (µmoles min-1g-1) and superoxide dismutase (SOD) (U mg-1 Protein) and protein content (%) in Lens culinaris Medik. Data means within columns followed by the same letter is not different at the 5% level of significance, based on the Duncan Multiple Range Test. 54 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari 3.2.6 Stomatal behavior and mineral element analysis Variation in structure of guard cells in treated populations was determined through scanning electron microscopy (SEM). Th e SEM image showed variation in shape, length and width of guard cells in treated popula- tions. Cadmium treatment induced partially closed sto- mata. Stomatal opening slightly increases over control in lower doses while it reduced in higher doses with their respective control (Fig. 3; a-f). EDX profi ling of leaf was Figure 3. Scanning electron micrographs exhibiting morphology of stomata in control (A) and diff erent shape and size of stomata in vari- ous concentrations of cadmium nitrate (20-100 ppm) (B-F). 55Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype (a) (b) (c) Element Weight % Atomic % C K 47.80 60.92 O K 37.58 35.96 Mg K 0.89 0.56 K K 1.98 0.78 Ca K 2.44 0.93 Zn K 0.77 0.18 Au M 8.53 0.66 Element Weight % Atomic % C K 44.04 58.09 O K 38.97 38.58 Mg K 0.95 0.62 K K 2.40 0.97 Ca K 1.99 0.79 Zn K 0.12 0.03 Au M 11.54 0.93 Element Weight % Atomic % C K 33.56 46.67 O K 46.44 48.48 Mg K 0.93 0.64 Cl K 1.20 0.57 K K 4.35 1.86 Ca K 1.58 0.66 Fe K 0.53 0.16 Au M 11.41 0.97 Figure 4. EDX profiling of mineral content of leaf (a) control; (b) 40 ppm Cd; (c) 80 ppm Cd. 56 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari (a) (b) (c) Figure 5. Graphical representation of EDX profiling of mineral content of treated plant of lentil along with control plant. 57Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype also done via energy dispersive X-ray analyser (EDX)to estimate mineral element of control as well as treated plants. Treated populations exhibited a slight reduction and enhancement in mineral elements as compared to control when expressed in percentage content (Fig. 4 and 5a-c) 3.3 DNA damage Meiotic studies in pollen mother cells treated with different concentrations of Cd are shown in Fig. 6. The aberrant cells increased as heavy metal concentrations increased. Untreated plants exhibited normal meiotic cells at metaphase I (control) (Fig. 6a).Various chromo- a b c d e f g h i Figure 6. a: Metaphase I (control), b: Metaphase I (precocious movement of chromosome), c: Anaphase I (unequal division with two lag- gards), d: Telophase I (sticky chromosomes), e: Metaphase II (stray chromosomes), f: Anaphase II (disturbed polarity with multi bridge for- mation) g, h: Anaphase II (disturbed polarity), i: Telophase II (two micronuclei). 58 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari somal anomalies in pollen mother cells of treated popu- lations were observed, such as precocious movement of two univalents at metaphase I (Fig. 6b), unequal division with two laggards at anaphase I (Fig. 6c), stickiness at telophase I (Fig. 6d), stray chromosomes at metaphase II (Fig. 6e), disturb polarity with multi bridge formation at anaphase II (Fig. 6f), disturbed polarity at anaphase II (Fig. 6g), laggards at telophase II (Fig. 6h), two micro- nuclei at telophase II (Fig. 6i). In the present investiga- tion, chromosomal aberrations and frequency of meiotic abnormalities at each concentration were calculated in percentage (Table 2). Maximum frequencies of chro- mosomal aberrations were observed at 100 ppm. The total percentage of abnormal PMCs ranged from 8.88 to 38.34% (Table 2, Fig. 7) 4. DISCUSSION As reported earlier by many researchers, Cd is a non-essential element that is readily taken by plants and inhibits plant physiological processes such as water absorption, photosynthesis, stunted foliage, withering of leaf and alters normal meiotic division (Patra et al. 2004).The present study showed that exposure of lentil genotypes to different doses of heavy metal (Cd) exhib- ited substantial alterations in the phenotypic and geno- typic makeup of the plant. During growth and develop- Ta bl e 2. F re qu en cy o f c hr om os om al a no m al ie s i nd uc ed b y C d( N O 3) 2 in L en s c ul in ar is M ed ik . ( M 1G en er at io n) . Pr op ha se -I (D ia ki ne sis ) M et ap ha se -I /I I A na ph as e- I/ II Te lo ph as e- I/ II Conc .of mutagen (ppm) Total no. of PMCs observed Univalents Multivalents % of Abn. PMCs (A) Univalents Multivalents Precocious Mov. of chromosomes Stray chromosomes Stickiness % of Abn. PMCs (B) Laggards Disturbed polarity Unequal Sep. of chromosomes % of Abn. PMCs (C) Laggards Bridges Unequal Sep. of chromosomes Micro nucleate cells Multi nucleate cells Disturbed polarity Cytomixis % of Abn. PMCs (D) Total No. of Abnoral PMCs observed Total % of Abnormal PMCs observed A+B+C+D C on tr ol 28 9 - - - - - - - - - - - - - - - - - - - - - - - C d( N O 3) 2 ( pp m ) 20 27 0 2 2 1. 4 1 1 2 1 2 2. 59 2 1 2 1. 85 2 - - 2 1 2 1 2. 96 24 8. 88 40 26 5 3 2 1. 8 3 2 4 3 3 5. 66 3 2 4 3. 39 3 1 1 3 2 3 2 5. 66 44 16 .5 9 60 26 1 2 3 1. 9 3 3 5 4 5 7. 66 5 3 4 4. 59 4 2 2 3 3 4 3 8. 04 58 22 .2 0 80 25 0 3 4 2. 8 4 4 6 5 7 10 .4 6 4 6 6. 40 5 3 3 4 4 5 5 11 .6 0 78 31 .2 0 10 0 24 5 4 5 3. 6 5 6 8 6 8 13 .4 6 7 4 7 7. 34 6 3 4 5 5 6 5 13 .8 7 94 38 .3 4 Figure 7. Effect of Cd(NO3)2 on percentage of total chromosomal aberrations in Lens culinaris Medik. 59Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype mental stages, morpho-physiological parameters were examined as well as biochemical parameters, antioxidant enzymes activity, DNA damage, SEM and EDX analysis of leaf were also performed to evaluate the overall effect of Cd on plant ecology. 4.1. Growth and morphology 4.1.1 Seed Germination, Survival and pollen fertility Germination percentage, survival and pollen fertil- ity were found to decrease as cadmium doses increased in the present investigation. Similar observations were reported by (Choudharyet al. 2012) in Trigonella, (Shah- war et al. 2016) in Vicia faba and (Shahwar et al. 2018, Sharma et al. 2022) lentil, (Petrescu et al. 2020) in Oci- mum. Inhibition in germination and root development was due to Cd (Pandit and Prasannakumar 1999) low water uptake, reduction in cell division and metabolic activity and enlargement of the embryo. It was reported by (Moreno et al. 1999) Cd disrupts the uptake of water and nutrients in plants and suppresses cell division (Liu et al. 2003). Kabir et al. (2008) and Farooqi et al. (2009) suggested that inhibition in germination percentage, seedling length, tolerance index and dry mass of root and shoot is due to heavy metal. The reason behind reduc- tion in germination percentage under Cd stress might be due to escalated breakdown of reserved food material in seed embryo. Depletion in survival may be due to dif- ferent cytological and physiological disturbances (Girija et al. 2013) and inability to maintain balance between growth regulators and promoters (Meherchandani 1975). The descending fertility is an outcome of chromosomal breakages and anomalies which affect microsporogenesis leading to generation of non-viable gametes and decreas- ing plant fertility (Kumar and Singh, 2020). 4.1.2 Root and shoot length In the present investigation, root and shoot lengths minimized linearly as Cd doses increased. Similar result was also reported by Choudhary et al. (2012). Decrease in seedling length following metal treatment might be due to reduction in meristematic cells and also due to alteration in hydrolytic enzymes; sufficient food does not reach the developing radical and plumule, resulting in stunting of seedlings (Shafiq et al. 2008). According to Elloumi et al. (2007), effect of Cd exposure on root growth was more compared to shoot growth since roots are the first organ to contact the heavy metal and carry out the process of absorption (Guilherme et al. 2015) 4.1.3 Plant height and yield attributing traits In the present work, metal treated plants exhibited linearly declined plant height in comparison to the con- trol plants and this depletion was due to chromosomal damage. Reason behind the yield depletion was meiot- icturbulences whichaffected the production of normal microspores and megaspores resulting in low fruit set. Higher concentration of Cd causes growth inhibition which ascribes to cell division or various desecrations in the plant genome. Thilagavathi and Mullainathan (2011) reported that adecrease in quantitative traits have been ascribed to the physiological perturbation or due to chromosomal breakage. Yield is considered an impor- tant agronomical parameter in breeding program.Data regarding yield and related traits, exhibited significant decrease in yield at higher concentrationwhich might be due to metal induced genotoxicity resulting in altera- tions of physiological mechanisms, chromosomal aberra- tions and high pollen sterility. Similar results were recorded in soyabean (Pavadi and Dhanavel, 2004), cotton (Sundaravadivelu et al. 2006) Trigonella (Choudhary et al. 2012), Vicia faba (Shahwar et al 2016) and Capsicum annum (Aslam et al. 2017). 4.2. Physio and biochemical aspects 4.2.1 Photosynthetic pigment Photosynthetic pigment is an important param- eter directly correlated with plant growth and biomass (Acosta-Motos et al. 2017). In our study, photosynthetic pigment was inversely proportional to cadmium doses, their content decreased with enhancing concentration of cadmium relative to the control. Zengin and Munzuro- glu (2006) and Elloumi et al. (2007) demonstrated the same result in sunflower and almonds, respectively. The decline in the chlorophyll content in plants might be due to suppression of enzymes such as δ-aminolevulinic acid dehydratase and protochlorophyllide reductase (Van Ass- che and Clijsters 1990), which are necessary for chloro- phyll biosynthesis. Leeet al. (2004) and Siler et al. (2007) while working on Paspalum vaginatum (L.) and Centau- rium erythraea (L.) respectively reported that total chlo- rophyll diminished along with the enhanced metal con- centration. Carotenoids are an important constituent of photosynthetic pigments which absorb light energy to make food for plant. Carotenoids also save chlorophyll from photo damage. In the present study, photosynthetic pigment, stomatal length and width reduce by cadmium treatment. This reduction is probably due to nutritional imbalance (Wong and Wong 1990). 60 Durre Shahwar, Zeba Khan, Mohd. Khalil Yunus Ansari 4.2.2 Proline content Proline, a non-enzymatic antioxidant, scavenger of ROS, which accumulates in plants when exposed to abiotic stress (Saradhi et al. 1993). Itis considered as stress signaling molecule having capability to act as an antioxidative defense molecule. (Maggaio et al. 2002). It was reported by researchers that proline accumulation might act as compatible osmolyte in cells, maintains the configuration of macromolecule and organelles and its enhanced production confirms the osmo-tolerance in plants (Nanjo et al. 1999; Junaid et al. 2008). Dhir et al. (2004) demonstrated that proline accumulates in shoots of higher plants such as B. juncea, T. aestivum and Vigna radiata in response to Cd toxicity. 4.2.3 Protein content In present investigation, it was observed that cad- mium treatments affected greatly protein synthesis. A significantnegative difference was seen between treated plants and control. Similar results were also found by Bavi et al. (2011) in pea plants and Choudhary et al. (2012) in Trigonella. Balestrasse et al. (2003) reported that decline in protein content might be due to inhibi- tion in protein synthesis or an increase in the rate of protein degradation. Higher concentration of cadmium inhibits protease activity and total protein content. This shows toxic effect of cadmium concentration on mecha- nism of protein synthesis resulting in decreased protein content. Despite of these Chen et al. (2007) found that protein content decreased in Vigna unguiculata under the salt stress (sodium chloride). 4.2.4 Antioxidant and lipid peroxidation Heavy metal stress may have detrimental effects on plant stress machinery. Andre et al. (2010) suggested that antioxidant enzymes are considered an essential defense element against stress and improve the activity of antioxidant system to overcome stress generated by ROS. ROS are known as the natural by-products of aero- bic organisms and are generated during mitochondrial electron transport (Debnath et al. 2021). In the present investigation, dose-dependent enhancements in anti- oxidant enzyme activity were recorded, suggesting ROS production due to severity of Cd stress. Salama et al. (2009) and Shehab et al. (2010) observed that antioxidant activity elevates as concentration increases but decreases at higher concentrations, probably due to chronic stress exposure. SOD plays a crucial role to safeguard plants against stressby converting O2 - to H2O2 with the help of POX and subsequently reducing it into H2O (Alscher et al. 2002). The results are supported by Arleta et al. (2001); Dixit et al. (2001); Choudhary et al. (2012). Ele- vated malondialdehyde (MDA) levels indicated enhanced lipid peroxidationincreasing concentration of Cd con- firming metal induced oxidative stress in lentil plant. Similar results are recorded by Malecka et al. (2001); Unavyar et al. (2006). 4.3. DNA damage Chromosomal anomalies are induced due to factors that affect DNA synthesis and replication or on nucleo- proteins, resulting in chromosomal breakages or mal- functioning of spindle apparatus and abnormal chro- mosomal segregation (Sutan et al. 2018). In our inves- tigation, adverse effect of cadmium on the frequency of chromosomal anomalies were observed, presumably due to mutagenic effect of subject heavy metal in inducing alterations in DNA. While we observed normal mei- otic cells in control group, a spectrum of anomalies was observed in treated individuals. The frequency of chro- mosomal aberrations was directly proportional to the concentration of cadmium. The anomalies induced by cadmium nitrate were of broad spectrum and compara- tively included a higher proportion of sticky chromo- somes. Khan et al. (2012) suggested the occurrence of sticky chromosome as a result of improper folding of chromosome fibers and their intermingling. Jayabalan and Rao (1987) reported that stickiness was caused by the segregation of histone proteins and alterations in the pattern of cyto-chemically balanced reactions. Bhat et al. (2007) suggested that stray chromosomes may be due to spindle dysfunction and clustering of chromosomes. Anaphasic bridges originate due to unequal separation of dicentric chromosomes (Singh and Khanna, 1988) or presence of sticky chromosomes which remain con- nected by chromosome bridges during anaphase because of incomplete separation of the daughter chromosomes (Kabarity et al. 1974). Laggards were observed at ana- phase and telophase in Cd treated plants, and it origi- nates due to disruption of spindle. Das and Roy (1989) hold the view that spindle fibers fail to carry chromo- somes to their respective poles due to mutagen reac- tion leaving the chromosome behind as a lagging chro- mosome or laggard. Stickiness of chromosomal end, delayed terminalization and failure of chromosomes to move at opposite poles were also possible reasons behind laggard production (Verma et al. 2012). Disturbed polarity at anaphase and telophase might be attributed to disturbances in the spindle fibers (Bhat et al. 2007). 61Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Utsunomiya et al. (2002) had opinion that formation of micronuclei is because of non-oriented chromosomes which are unable to reach the pole. Ruan et al. (1992) suggested that micronuclei are kind of abnormality which culminates into loss of chromosomal material and is regarded as an indicator of mutagenicity. Our result suggested a close colinearity between the treatments and percentage of chromosomal anomalies, higher the concentration, more the damage chromo- some undergoes. Similar observations were also reported by treatment of different metals and chemicals by other workers such as Srivastava and Kapoor (2008); Khan et al. (2009b); Kumar and Yadav (2010);Tripathi and Kumar (2010); Jafri et al.(2011); Gulfishan et al. (2012); Shahwar et al. (2016, 2017, 2018, 2019, 2020); Aslam et al. (2017), Khan et al. (2019). 5. CONCLUSION During the present investigation, it was concluded that cadmium induced morphological, physiological, biochemical variation and DNA damage over control in Lens culinaris. Genotypes of lentils were greatly affect- ed due to the treatment of cadmium, recommending genetic variation in the subsequent generation. It was observed in this study that at their lower concentrations, cadmium was tolerable by the plant without losing via- bility, while higher concentrations were genotoxic and induce variation/mutation in the genotypes as well as phenotypes and causing more variation and developing variants/mutant of better quality and selected it. There- fore, plants with better characteristics should be isolated and selected for crop improvement programmes. Fur- ther molecular techniques or various genetic engineering techniques should be carried out to check the mutation at genic level as it will be acoherent tool to isolate the desired characters and produce a new variety of lentil through breeding program. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 3 - 2022 Firenze University Press Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques Kamika Sribenja1, Alongklod Tanomtong1, Nuntaporn Getlekha2,* Chromosome number of some Satureja species from Turkey Esra Kavcı1, Esra Martin1, Halil Erhan Eroğlu2,*, Fatih Serdar Yıldırım3 L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations Selma Tabur1,*, Nai̇me Büyükkaya Bayraktar2, Serkan Özmen1 Characterization of the chromosomes of sotol (Dasylirion cedrosanum Trel.) using cytogenetic banding techniques Kristel Ramírez-Matadamas1, Elva Irene Cortés-Gutiérrez2, Sergio Moreno-Limón2, Catalina García-Vielma1,* Contributions of species Rineloricaria pentamaculata (Loricariidae:Loricariinae) in a karyoevolutionary context A Cius¹, CA Lorscheider2, LM Barbosa¹, AC Prizon¹, CH Zawadzki3, LA Borin-Carvalho¹, FE Porto4, ALB Portela-Castro1,4 Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Durre Shahwar1,2,*, Zeba Khan3, Mohammad Yunus Khalil Ansari1 Biogenic synthesis of noble metal nanoparticles using Melissa officinalis L. and Salvia officinalis L. extracts and evaluation of their biosafety potential Denisa Manolescu1,2, Georgiana Uță1,2,*, Anca Șuțan3, Cătălin Ducu1, Alin Din1, Sorin Moga1, Denis Negrea1, Andrei Biță4, Ludovic Bejenaru4, Cornelia Bejenaru5, Speranța Avram2 Polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (Foeniculum vulgare Mill.) 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