A Comparative in Vitro Study of Pb Tolerance in Three Sugarcane Genotypes

A Comparative in Vitro Study of Pb Tolerance in Three Sugarcane Genotypes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Comparative in Vitro Study of Pb Tolerance in Three Sugarcane Genotypes yasmeen saleem, Aamir Ali, Shagufta Naz, Muhammad Jamil This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2170005/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Heavy metal accumulation in agriculture land is one of the major agriculture challenges affecting the crop production and threatens the food safety. Comparative study for Lead (Pb) tolerance in three sugarcane ( Saccharum officinarum L.) genotypes was carried out under in vitro conditions. Fresh weight, dry weight, callus morphology, shoot morphology, protein contents, catalase, superoxide dismutase and peroxidase, ascorbic acid contents, relative growth rate, stress tolerance index, Pb contents, bioconcentration factor were compared at 6 different concentrations of Pb (0mM, 0.1, 0.2, 0.3, 0.4, 0.5, 1mM). Lead affected the callus and its regeneration in all three genotypes while root regeneration did not affect considerably. All genotypes significantly vary at each treatment and parameter except BCF and protein contents. Genotype YT-53 proved most resistant toward Pb stress while CP-77400 was next to it and NSG-59 showed least resistance toward Pb stress. Most resistant genotypes can be used in heavy metal effected area for better yield and phytoremediation in Pb affected soil and in other breeding programs. sugarcane lead nitrate heavy metal toxicity heavy metal resistance phytoremediation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Heavy metal accumulation in agriculture land is one of the major challenges around the world. It affects crop production and threatens sustainable global food demands for increasing population (Frona et al., 2019 ). Various sites of the world land used for crop cultivation have been contaminated with heavy metals while very small net change in cultivated land in world over decades confound the problem (Gupta et al., 2021 ). It is estimated that the use of heavy metal contaminated water is over 20 million hectares in 50 countries (Khalid et al., 2017 ). Therefore, recent studies are targeting heavy metals like lead, cadmium, aluminum which cause soil contamination and is growing problems on global scale (Rai et al., 2019 ). Among these heavy metals lead (Pb) is the second most harmful pollutant and hence considered the metal of great concern. It affects the crop growth by disturbing the normal plant morphological and physiological functions (Ashraf et al. , 2017). Therefore, Pb induced phytotoxicity needs urgent attention (Rizvi et al., 2020 ). Sugarcane being the important agro-industrial crop in the world. It is cultivated in around 110 countries. Beside the world 80% of sugar production, it is also associated with other industries. But the potential yield of this crop is less than actual yield. Major reasons behind this gap are conventional breeding, wrong selection of genotypes, soil quality, biotic and abiotic stress. Moreover, many local genotypes are losing their potential to resist against biotic and abiotic stress over the years (Raza and Amir, 2021 ). The agriculture area of Paksitan is also being affected by heavy metals over the years. In modern world it is hard to control the spread of heavy metals and the only solution is to replace the traditional genotypes to resistant genotypes (Shakir et al., 2016 ). Pakistan sugarcane breeding programs are not sufficient to develop such genotypes and thus relay on exotic genotypes (Ahmad et al., 2017 ). Therefore new genotypes should screen for their resistance toward heavy metals. To evaluate the response heavy metal stress and to achieve stress resistant regenerants, Plant tissue culture is a reliable tool. It facilitates the rapid and precise evaluations of plant response under stress (Purohit et al., 2020 ). CP-77400 is cultivated since 1996 in Pakistan with yield of while YT-53 and NSG-59 have China and USA origin respectively have higher yield than CP-77-400 (khan et al., 2012 ). Yield of CP-77400 is 10 ton/hec, YT-53 is 13.42 while NSG-59 cane yield is 13.20 ton/hec (Afghan et al., 2015 ; Ahmad et al., 2017 ). The present work was designed to screen Pd toxicity in callus cultures of three sugarcane genotypes subjected to six different concentration of Pb(NO 3 ) 2 under in vitro conditions. Morphological and biochemical markers associated with Pd stress were evaluated. Regeneration potential and root induction of Pb treated calli were also observed to find out Pb resistant the survival rate of callus and regeneration. The objective of this study is to find the most resistant genotype of sugarcane. Information obtained can be helpful for the development of cultivar improvement programs by for choice of parents and breeding crosses in breeding program. Resistant genotype could be used in heavy metal effected areas. MATERIALS AND METHODS 2.1 Explant and media preparation The sugarcane genotypes (YT-53, CP-77-400 and NSG-59) were collected from the Shakerganj Sugarcane Research Institute Jhang Pakistan. The inner most layer of 3-4 months old sugarcane plant was source of explants. Plant material was washed with tap water and cleaned with 95 % ethyl alcohol and inoculated on sterilized culture vessels containing MS media with their respective growth regulator (3.5mg/l 2,4-D for YT-53 and NSG-59 and 5mg/l CP-77-400)and kept in suitable conditions (temperature, light) for callus growth and regeneration and recultured when required. The pH of culture media was kept 5.65-5.75 with 1N HCl and 1N NaOH. 2.2 Callus induction Well developed calli of each genotype were shifted to MS media containing their respective growth regulator (3.5mg/l 2,4-D for YT-53 and NSG-59 and 5mg/l CP-77-400)at 6 different concentration of (Pb(NO 3 ) 2 (0.1, 0.2, 0.3, 0.4, 0.5 and 1mM). Callus growth in term callus morphology, callus fresh and dry weight were noted after 30 days. Proliferated calli (after 60 days) were shifted to already standardized MS medium provided with 6 different levels (T0-T6) of Pb(NO 3 ) 2 while MS media without Pb(NO 3 ) 2 was used as control (T0). Callus cultures (0.5g) exposed to 6 different levels of Pb (NO 3 ) 2 were shifted on regeneration media to evaluate their regeneration potential. Numbers of shoots per culture vessels, regeneration frequency was noted after 30 days and maximum shoot length per plant was noted after 60 day of treatment 2.3 Regeneration For regeneration Pb treated calli were shifted on their respective regeneration media (2mg/l kinetin for YT-53 and NSG-59 and 1mg/l BAP for CP-77-400) under 16h photoperiod at 27 ± 2 °C after 30 days. Regeneration potential in terms of shoot morphology, shoot numbers, shoot length were noted after 30 days. 2.4 Root induction For root induction the well devloped plants were shifted into already standerized rooting media (3mg/l NAA for YT-53 while 1mg/l NAA +1mg/l IBA for CP-77-400 and NSG-59). Root induction poteniatl in term of root number, root length were noted after 30 days. 2.6 Estimation of Antioxidant Enzymes The catalase, peroxidase and superoxide dismutase activities and soluble protein contents were estimated with the help of spectrophotometer (UV 4000) after day 30, 60 and 90. After 150 days Pb treated callus were transferred to regeneration media.For extraction, two-gram callus was crushed with 0.1g PVP and 4ml of 0.1 M phosphate buffer with pH 7.2 in ice-chilled pestle and mortar. The mixture was centrifuged at 14000 rpm for 10 minutes at 4°C. The supernatant attained after centrifugation was used for enzyme analysis. 2.6.1 Specific activity of Catalase estimated by Beers and Sizer (1952) method some changes. Where reagents (50mM Phosphate Buffer and 3ml (0.036 % H 2 O 2 solution)) pipetted (in milliliters) into cuvettes. The temperature of the cuvettes was maintained 25 °C. Absorbance was recorded at 240 nm on spectrophotometer and Catalase extract was added. Reagents were mixed immediately and a decrease in Absorbance was recorded. Time required to reduce the absorbance from 0.45 to 0.40 was noted for the absorbance 240nm. 2.6.2 Specific activity of Peroxidase estimated by Racusen and Foote (1965) method. Where Guaiacol was used as substrate. Peroxidase activity was estimated by pipetting the reagents (Enzyme extract 10µl into cuvettes, 0.1 M phosphate buffer (2.5ml), 1 % Guaiacol (0.2ml), Distilled H2O(0.2ml)). After keeping the reactants for 30 minutes 0.1ml hydrogen peroxide was added in both tubes. Absorbance was monitored at 470 nm. 2.6.3 Superoxide dismutase (SOD) activity was assessed by Maral et al., (1977) method with modification that was based on principle that “Under specific condition one unit of SOD reduced the 50 % of the maximum reduction of NBT”. For SOD analysis two samples were prepared. Experimental sample contained 5µl enzyme extract and 2ml reaction mixture while control contain 2ml reaction mixture only. Both tubes were covered with black paper. These tubes were then immediately exposed to 40W fluorescent lamps for 10 minutes. The absorbance was measured at 560 nm. Superoxide dismutase activity was calculated as the percentage inhibition of NBT with the help of the following formula: 2.6.4 Total soluble Protein Contents estimatedby Racusen and Johnstone, (1961) method. Two samples were prepared for control and experiment. The experimental sample was provided with 0.2ml protein extract and 2ml Biuret reagent while control had 0.2ml distilled H 2 O and 2ml Biuret reagent.Constituents were mixed by shaking both tubes and left for 30 minutes at room temperature. When the reaction was completed, the optical density was recorded at 545nm on spectrophotometer (UV4000). The following formula was used for protein estimation. 2.7 Bio Concentration Factor (BCF) BCF= Lead accumulation in callus / Pb concentration in medium mg/kg x 100 mg/kg (Kulkarni et al., 2014). 2.8 Stress Tolrance Index STI =Average dry weight of callus(pretreated)/Average dry weight of callus (notpretreated) x100 (Kristin et al., 1997). 2.9 Statistical Analysis Descriptive statistical analysis (mean and standard error of the means) was performed for two factors (genotypes and treatments). Two-way full factorial design of experiment was applied. Then the data was subjected to univariate two-way analysis of variance (AONVA) using Statistix-8.1 software. As a post-hoc, the least significant difference (LSD) test was applied using 95% level of confidence (p-value ≤ 0.05). Results Callus Morphology Callus morphology aftre 30, 60, 90 days of inoculation is represented in Fig. 5 at different concentration of Pb(NO 3 ) 2 . It can be clearly observed that that callus morphology of all three genotypes was affected by elevated Pb concentration. All the calli started necroing. NSG-59 showed early necrosis than YT-53 and CP-77400. Callus of CP-77-400 and NSG-59 were completely dead at day 90 at 1mM Pb(NO 3 ) 2 . While YT-53 survived at this concentration. Callus Fresh Weight and Dry Weight Figure 1 (a ,b) illustrates that callus weight was significantly affected by Pb concentration as compared to control. Fresh and dry weight of callus of all three genotypes decreased by increasing the concentration of Pb(NO 3 ) 2 in MS media as compared to control. Callus exposed to (1mg/L )T6 did not show any increase in net fresh and dry weight thus proved most toxic for callus culture in sugarcane genotype CP-77-400 and NSG-59. Fresh weight and dry weight of callus in YT-53 was highest on all six lead concentrations while fresh weight and dry weight of callus in NSG-59 was lowest among all the tested genotypes of sugarcane at all levels of Pb(NO 3 ) 2 . All the genotypes show different in callus at weight at same treatment. YT-53 show highest callus weight at each concentration and CP-77400 was next to it. Callus dry weight and fresh weight of NSG-59 was lowest among all three genotypes. 4.2.2.1 Shoot Morphology Regenerated shoots maintained on MS media provided with 0 mg/L (T0) didnot shown any necrosis even after 90 days in all three genotype (YT-53, CP-77-400, NSG-59) (Fig. 5 ). In YT-53 regenerated shoots become necrotic after 90 days and were completely dead at 0.5mM Pb(NO 3 ) 2 (T5). In CP-77-400 necrosis was observed after 60 and 90 days. In NSG-59 plant show early necroing even at 0.5mM Pb(NO 3 ) 2 (T1). Complete necrosis was observed in shoots regenerated from callus treated with T3 treatment after 90 days. Regeneration Potential in Lead Treated Callus Regeneration potential of plants developed from lead treated calli were significantly affected as compared to control in all three genotypes. All three genotypes show significant difference in regeneration potential at each level of Pb(NO 3 ) 2 . (Fig. 1 d, e, f). In YT-53 no regeneration was recorded in calli treated with 0.5mM Pb(NO 3 ) 2 (T5) and 1mM Pb(NO 3 ) 2 (T6). Calli of YT -53 in control cultures show 80% regeneration that reduced to 60% and 40% at 0.1mM Pb(NO 3 ) 2 (T1) and 0.2mM Pb(NO 3 ) 2 (T2) respectively. Length of shoots regenerated from lead treated callus were also less as compared to control. In CP-77-400 no shoot induction was recorded in calli treated with 0.5mM Pb(NO 3 ) 2 (T5) and 1mM Pb(NO 3 ) 2 (T6) while average number and average shoot length were also effected in Pb(NO 3 ) 2 treated calli. In NSG-59 regeneration in callus cultures were not observed at 0.4mM-1mM (T4, T5, T6). While reduction average number of shoots and shoot length was recorded after 30 days in lead treated calli. Effect of Pb(NO 3 ) 2 on Root induction Average length of root, average numbers of root, root frequency was recorded in plants regenerated from Pb(NO 3 ) 2 treated callus with or without different concentration of lead Pb(NO 3 ) 2 after 30 days. Maximum root numbers and root length was achieved in control i.e without Pb(NO 3 ) 2 treatment. Average numbers of roots, root length and root frequency decreased in all three genotypes regenerated from Pb(NO 3 ) 2 treated callus at different concentrations. NSG-59 exhibited highest root numbers among all genotypes under study (Fig. 1 -g, h, i). Antioxidant Enzyme Activities Effect different concentrations of Pb(NO 3 ) 2 on specific activity of POD, CAT, SOD in callus cultures of three sugarcane genotypes under six different concentrations of Pb(NO 3 ) 2 after 30, 60, 90 days (first, second, third month) is represented in Fig. 2 . Specific activity of Catalase (CAT) Specific CAT activity in all three genotypes increased significantly by increasing the concentration of Pb(NO 3 ) 2 in callus culture media that started to decline with time. In YT-53 specific activity of CAT was highest at all concentration of Pb(NO 3 ) 2 . Highest specific activity of catalase was recorded in YT-53 at T6 at day 90. While this activity was lowest in NSG-59 among all three tested genotypes. 4.4.2.2 Specific activity of Superoxide dismutase (SOD) Specific SOD activity in all three genotypes increased significantly by increasing the concentration of Pb(NO 3 ) 2 in callus culture media that started to decline with time. In YT-53 specific activity of SOD was highest at all concentration of Pb(NO 3 ) 2 . Highest specific activity of SOD was recorded in YT-53 at T6 at day 90. While this activity was lowest in NSG-59 among all three tested genotypes (Fig. 2 ). Specific activity of Peroxidase (POD) The effect of different concentrations of Pb(NO 3 ) 2 on specific POD activity in callus cultures of three sugarcane genotypes under six different concentrations of Pb(NO 3 ) 2 after 30, 60, 90 days (first, second, third month) is represented in Fig. 2 . In YT-53 specific activity of POD increased by increasing the concentration of Pb(NO 3 ) 2 in callus culture media as compared to control. This activity started to decline with time. YT-53 showed the highest POD activity among three genotypes. Total soluble Protein Contents In all three genotypes total soluble protein contents decreased gradually by increasing concentration of Pb(NO 3 ) 2 in callus culture media and as compared to control. Total soluble protein of three genotypes calli did not show any significant difference at all the levels of Pb(NO 3 ) 2 (Fig. 2 ). Ascorbic acid Effect of 6 different concentrations of ascorbic acid pretreatment on ascorbic acid in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO 3 ) 2 after 90 days is represented in Fig. 1 -j. In YT-53 ascorbic acid concentration in callus increased with increasing concentration od Pb(NO 3 ) 2 in media while the ascorbic acid concentration in ascorbic acid pretreated calli were higher than non-pretreated calli. Ascorbic acid concentration was highest at 1mM Pb(NO 3 ) 2 (T6) in ascorbic acid pretreated calli. STI (Stress Tolerance Index) The effect of six different concentration of Pb(NO 3 ) 2 and ascorbic acid pretreatment on stress tolerance index in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO 3 ) 2 after 90 day is represented in Fig. 1 -f. In YT-53 STI of callus decreased with increasing the Pb(NO 3 ) 2 in growth media while STI of YT-53 was highest among all three tested genotypes. RGR (Relative Growth Rate) Effect of different concentrations of Pb(NO 3 ) 2 on RGR(Relative Growth Rate) in callus of three sugarcane genotypes under 6 different concentrations of Pb 2 (NO 3 ) 2 after 90 day is represented in Fig. 4.107.as the concentration of in media increases the RGR decreased in all three genotypes that was more prominent in NSG-59 while CP-77400 was next to it.This reduction was lowest in YT-53 that means YT-53 show highest growth rate than other two genotypes at elevated Pb concentration (Fig. 1 -c). BCF (Bio Concentration Factor) Bio Concentration Factor (BCF) in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO 3 ) 2 after 90 day is represented in Fig. 1 -i. In YT-53 BCF of callus decreased with increasing the concentration of Pb(NO 3 ) 2 . Pb (Lead) Contents In Callus Pb (Lead) contents in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO 3 ) 2 after 90 days is represented in Fig. 1 -k. Lead(Pb) contents in control calli were found to be zero. Lead contents in three genotypes increases as concentration of Pb(NO 3 ) 2 increased in callus growing media. Maximum lead contents were found in callus maintained onT6 in YT-53. Principal component analysis (PCA) Principal component analysis (PCA) revealed two main components with eigenvalue above one. First principal component (PC1) along x-axis explained 68.7% overall variation with 10.31 eigenvalue and second principal component (PC2) along y-axis highlighted 16.6% variation with 2.49 eigenvalue (Fig. 4 ). Overall 71.19 percent variation was explained by the first two PCs (Fig. 4 , top-left). Due to the high contribution percentage, traits CAT, POD, AsA and BCF were grouped at PC2 while the other traits were at PC1 (Fig. 4 , top-right). The 25 observations were classified into three groups by three successive PCs (Fig. 4 , bottom-left). Controls of three genotypes can be seen in a single cluster in green. Genotype YT-53 showed higher values for POD, AsA, CAT than the other two genotypes. Higher fresh and dry weight, shoot length, root length, number of sheets and STI were observed in NSG-59. Stress indicators such as POD, AsA, CAT, SOD is positioned in same section of the plot (Fig. 4 bottom-right) while RGR, Roots, FW, DW, SL, STI, RL, Shoots are in other section of the plot showing positive correlation with each other. This PCA clearly classified the genotypes with respect to the treatments (Fig bottom-left) and well grouped the parameters (Fig. 4 bottom-right) according to the strength of correlation among variables. Discussion Callus browning rate indicates the callus sensitivity and its health under stress conditions Callus morphology of control calli of three genotypes of sugarcane were yellow, granular, nodular but calli those were treated with different concentrations of Pb(NO 3 ) 2 start browning in the presence of higher lead concentration gradually (Fig. 5 ). As necrosis increases with lead concentration and time, calli appear brown to dark brown. Necrosis due to tissue damage results in browning of callus (Sukma, 2020). Hyperactivity of oxidative enzymes involve in synthesis of phenolic compound further intensify callus browning (Koc et al., 2009 ). The observations are in line with the findings of previous studies where calli appeared brown when exposed to abiotic stress (Atabaki et al., 2018 ; Nawrot-Chorabik, 2017 ; Rattana and Bunning, 2015). To assess the response of metal stress on growth of callus was recorded in term of fresh and dry weight during present investigation. It is well known that plant growth attributes are inhibited by higher lead concentration. Suppressed growth of callus under Pb stress was also reported by the Waoo et al., 2017 in Datura inoxia and by Tahtamouni and Al-Qudah ( 2020 ) in Lantana Camara while reduced net fresh weight of callus accompanied with an increase of browning intensity in Phoenix dactylifera L. was reported by Abbas, (2016). Reduced growth of callus in terms of dry weight and fresh weight in all three cultivars were also observed during this study. Chabhal and Virk ( 2017 ) explained that reduced growth of callus due to metal toxicity might disrupt physiological processes. Pb ions align with nucleic acids that condense the chromatin and inhibit the replication, transcription processes that hampered the normal cell division thus leading in reduced plant growth. Pb ions bind with the cell wall that strengthens the cross-linking of pectin’s which hindered growth of cell in plants (Aslam et al., 2021 ). At cellular level Pb ions bind with oxygen, nitrogen, and sulfur atoms that damage thylakoid membranes. Moreover disturbance in nutrient uptake and electron transport chain make the situation more worst (Ali et al., 2014 ; Pirzadah et al., 2020 ). These impairing of physiological and biochemical processes might lead to reduced growth of callus. Responses of callus under stress conditions depended upon plant physiology, its genotype, concentration and type of stress agent and in-vitro manipulation (Purohit et al., 2020 ). It was observed that low concentrations of lead nitrate (0.1, 0.2mM) did not affect the callus fresh weight and dry weight significantly in all three sugarcane genotypes (YT-53, CP-77-400, NSG-59). but as the concentration of Pb increased in media the callus net fresh weight and callus dry weight start declining. Decrease in biomass depends upon concentration of Pb also explicated by earlier studies (Zhou et al., 2018 ; Souri et al., 2019 ; Khan et al., 2019 ; Amari et al., 2017 ). Usman et al., ( 2020 ) stated that plants can tolerate 100mg/l lead concentration in vivo conditions but in vitro conditions calli are more sensitive to wards stressor as each cell directly interact with the stress agents (Terletskaya et al., 2017 ). Krishania and Agarwal, 2012 stated that 100µM(0.1mM) Pb concentration become toxic for callus. But according to our observation sugarcane calli can survive at 0.5mM Pb(NO 3 ) 2 in case of CP-77-400 and NSG-59 but could not withstand 1mM concentration of Pb(NO 3 ) 2 while calli of YT-53 were survived at this level. Surviving ability and regeneration rate of sugarcane calli under abiotic stress depend upon genotypes (Mahmud et al., 2021 ). According to our work callus treated with Pb(NO 3 ) 2 have reduced regeneration potential even loss of regeneration ability in all three genotypes of sugarcane. No shoot induction was recorded in callus treated with high levels (0.4, 0.5, 1mM) of Pb(NO 3 ) 2 in these genotypes. Loss of regeneration potential in callus derived from high salt concentration oat calli was also reported by Mamun et al., ( 2020 ) and in potato Hassanein and Saleem, (2017). Reduction in regeneration potential perhaps due to damaged cells at proliferation stage. Atwell et al., ( 1999 ). stated that declining in shoot regeneration potential as a result Cd exposure was because callus cell lost its totipotency via physical damage of DNA and DNA methylation that made the regeneration difficult. Reduced shoot length from seeds under lead stress reported earlier (Zhou et al., ( 2018 ); Boi et al. , (2019) were similar to our observation where Pb toxicity also reduced the regenerated shoot length in all three genotypes of sugarcane. Shoot induction frequency under lead stress was also affected. Shoot induction frequency reduced to 40% from 80% in YT-53 and from 70–40% in CP-77-400 while in NSG-59 shoot induction frequency was 70% in control calli and was 30% at 0.3mM Pb(NO 3 ) 2 . Inhibitory effect of abiotic stress on regeneration frequency was also documented by Haque (2017) and Dogan ( 2020 ) . Heavy metal stress interferes with physiological and biochemical pathways during callus formation and also results in reduction of root length. Root growth was mainly affected through tissue damage. Root growth, length and numbers depend upon concentration of heavy metals and sensitivity of genotype (Kundu et al., 2018 ). Pb ions binds with spindles of cell wall during cell expansion that distract the normal cell division which ultimately inhibit root growth (Zulfiqar et al., 2019 ; Kanwal et al., 2020 ). Average root length and frequency in lead treated callus were also declined with respect to control during present study. Inhibited growth of roots under abiotic stress was also observed by Kiełkowsk (2017), Ahmed, e t al. , (2020) and Alkhateeb, ( 2015 ) under in vitro conditions. Conclusion It was concluded that Pb ions negatively affected callus growth, callus induction, regeneration and rooting of sugarcane genotypes YT-53, CP-77400, NSG-59. Regeneration potential of calli at elevated levels of heavy metals can be used to assess sensitivity of plants. Above results also suggested that Pb stress during callus proliferation stage persist during later developmental stages at higher concentrations while at lower concentration these toxic effects were not determinantal. All the genotypes behave differently towards different treatment and show significant difference from each other at different parameters and toward different concentration of Pb toxicity. Moreover, these toxic effects were more prominent in CP-77-400 and NSG-59 as compared to YT-53 that show the lead toxicity greatly influenced by genotype. YT-53 found more resistant toward Pb stress as show better performance at each parameter should be used in Pb contaminated area because of its high yield and resistance toward metal stress. This information could also be used by farmers to choose the genotype in heavy metal contaminated area. According to best of our knowledge no information about the effect of metal on later developmental stages under in vitro conditions in sugarcane callus culture have been found therefore above information can be used to develop metal tolerant genotypes and for phytoremediation studies. Declarations A author Contributions Y.S.: Conceptualization Methodology, Writing-original draft. A.A; Supervision: S.N; Project administration: M.J Softwar, Formal analysis, Data curation, All authors have read and agreed to the published version of the manuscript. Funding This research was not funded by any organization (Not Applicable) Conflicts of Interest The authors declare no conflict of interest. CONFLICT OF INTEREST DECLARATION AND AUTHOR AGREEMENT This statement is to certify that all authors have seen and approved the manuscript being submitted. We assure you that article is the Authors' original work. On behalf of all Co-Authors, the corresponding Author shall bear full responsibility for the submission. We attest to the fact that all authors listed on the title page have contributed significantly to the work, have read the manuscript, attest to the validity and legitimacy of the data and its interpretation, and agree to its submission to the “Environmental Science and Pollution Research” Ethics approval/declarations Not Applicable This work not involve any ethic approval study Consent to participate This study does not involve humans or animals, provide statement that it is not applicable or write (Not applicable) Consent for publication (Not applicable) Data Availability Statement The data presented in this study are available on request from the authors. Institutional Review Board Statement Not applicable. Informed Consent Statement Not Applicable Code availability Not applicable References Abass MH (2016) Responses of date palm ( Phoenix dactylifera L.) callus to biotic and abiotic stresses. Emirates J Food Agric 28(1):66–74 Afghan S, Asad M, Farooq U, Anwar MB, Shahzad A, Choudhary FA (2015) Investigating sugarcane genetic material inherited capability for agronomic attributes under agro-ecological conditions of Jhang. Pakistan Sugar Journal, 30 (1) Ahmad S, Unar GS, Gujar N, Panhwar RN, Soomro AF, Chattha AA (2017) Performance of new promising sugarcane variety yt-55 under different agro-climatic conditions of Pakistan. Pakistan Sugar Journal 32(1):8 Ahmed MS, Gardezi DA, Batley J, Hayashi S, Zander M, Javid A, Awan SI (2019) Somaclonal variations for red rot and sugarcane mosaic virus resistance and candidate genes integrity assessment in somaclones of selected sugarcane varieties ( Saccharum officinarum L). Pakistan J Agricultural Sci 56(1):15–27 Ali B, Xu X, Gill RA, Yang S, Ali S, Tahir M, Zhou W (2014) Promotive role of 5-aminolevulinic acid on mineral nutrients and antioxidative defense system under lead toxicity in Brassica napus . Ind Crops Prod 52:617–626 Alkhateeb (2015) Amari T, Ghnaya T, Abdelly C (2017) Nickel, cadmium and lead phytotoxicity and potential of halophytic plants in heavy metal extraction. South Afr J Bot 111:99–110 Ashraf U, Tang X (2017) Yield and quality responses, plant metabolism and metal distribution pattern in aromatic rice under lead (Pb) toxicity. Chemosphere 176:141–155 Aslam M, Aslam A, Sheraz M, Ali B, Ulhassan Z, Najeeb U, Gill RA (2021) Lead toxicity in cereals: mechanistic insight into toxicity, mode of action, and management. Front Plant Sci 11:2248–2268 Atabaki N, Rosimah N, Kalhori N, Lasumin N, Sahebi M, Abiri R (2018) In vitro selection and development of Malaysian salt-tolerant rice ( Oryza sativa L. cv. MR263) under salinity. Acta Scientific Agriculture , 2(8):8–17 Atwell BJ, Kriedemann PE, Turnbull CG (1999) Plants in action: adaptation in nature, performance in cultivation. Ann Botany 84(5):685–687 Beers R, Sizer I (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxidase by catalase. J Biol Chem 195:133–140 Boi ME, Porceddu M, Cappai G, De Giudici G, Bacchetta G (2020) Effects of zinc and lead on seed germination of Helichrysum microphyllum subsp. tyrrhenicum, a metal-tolerant plant. Int J Environ Sci Technol 17(4):1917–1928 Chabhal A, Virk GS (2017) The effect of nickel chloride (NiCl 2 ) on various callus growth dynamics of Coleus blumei (benth.) cultures. Int J Adv Res 5:863–868 Dogan M (2020) Effect of salt stress on in vitro organogenesis from nodal explant of Limnophila aromatica (Lamk.) Merr. and Bacopa monnieri (L.) Wettst. and their physio-morphological and biochemical responses. Physiology and Molecular Biology of Plants , 26(4): 803–816 Frona D, Szenderak J, Harangi-Rakos M (2019) The challenge of feeding the world. Sustainability 11(20):5816–5834 Gupta N, Yadav KK, Kumar V, Krishnan S, Kumar S, Nejad ZD, Alam J (2021) Evaluating heavy metals contamination in soil and vegetables in the region of North India: Levels, transfer and potential human health risk analysis. Environ Toxicol Pharmacol 82:1–10 Haque M, Islam SS, Subramaniam S (2017) Effects of salt and heat pre-treatment factors on efficient regeneration in barley ( Hordeum vulgare L). 3 Biotech 7(63):1–9 Hassanein AM, Salem J (2017) Rise potassium content of the medium improved survival, multiplication, growth and scavenging system of in vitro grown potato under salt stress. Egypt J Bot 57(1):259–275 Kanwal A, Farhan M, Sharif F, Hayyat MU, Shahzad L, Ghafoor GZ (2020) Effect of industrial wastewater on wheat germination, growth, yield, nutrients and bioaccumulation of lead. Sci Rep 10(1):1–9 Khalid S, Shahid M, khanniazi N, Murtaza B, Bibi I, Dumat C (2017) A comparison of technologies for remediation of heavy metal contaminated soils. J Geochem Explor 182:247–268 Khan MNM, Islam MM, Islam MS, Uddin MI (2019) Studies on in vitro response to callus induction and gene transfer technique of five high yielding indica rice varieties. J Sci Agric 3:41–45 Khan MR, Muhammad A, Hussain I, Shah SH, Kumar T, Inam S, Ali GM (2012) Rapid in vitro multiplication of sugarcane elite genotypes and detection of sugarcane mosaic virus through two steps RT-PCR. Int J Agric Biology, 14 (6) Kielkowska A (2017) Allium cepa root meristem cells under osmotic (sorbitol) and salt (NaCl) stress in vitro . Acta Bot Croatica 76(2):146–153 Koc NK, Baș B, Koc M, Küsek M (2009) Investigations of in vitro selection for salt tolerant lines in sour orange ( Citrus aurantium L). Biotechnology 8(1):155–159 Kristin AS, Senra RR, Perez FI, Enriquez BC, Gallegos JA, Vallego PR, Wassimi N, Kelley JD (1997) Improving common bean performance under drought stress. Crop Sci 37:43–50 Kulkarni DK, Delbari AS, Mahajan DM (2014) Bio concentration factor (BCF) for heavy metals detection and selection of hyper-accumulator plants: Case study of Pune-India and Tehran–Iran. Indian J Fundamental Appl Life Sci 4(1):163–170 Kundu D, Dey S, Raychaudhuri SS (2018) Chromium (VI)–induced stress response in the plant Plantago ovata Forsk in vitro . Genes and Environment 40(1):21–34 Mahmud K, Islam N, Kumar A, Azim GMA, Rajib MRR, Islam MJ (2021) Regeneration of sugarcane genotypes under different level of Sodium Chloride Salt. Acta Sci Agric 5(3):47–54 Mamun AA, Islam MM, Alam NEK, Noushin S (2020) In vitro callus induction under NaCl salt stress and subsequent plant regeneration in oats ( Avena sativa L). Plant Tissue Culture and Biotechnology 30(2):253–266 Maral J, Puget K, Micheson AM (1977) Comparative study of superoxide dismutase, catalase, and glutathione peroxidase levels in erythrocytes of different animals. Biochem Biophys Res Commun 77:1525–1535 Nawrot-Chorabik K (2017) Response of the callus cells of fir ( Abies nordmanniana ) to in vitro heavy metal stress. Folia Forestalia Polonica 59(1):25–33 Pirzadah TB, Malik B, Tahir I, Hakeem KR, Alharby HF, Rehman RU (2020) Lead toxicity alters the antioxidant defense machinery and modulate the biomarkers in Tartary buckwheat plants. Int Biodeterior Biodegrad 151:1–11 Purohit S, Gupta S, Jain R (2020) Effect of chromium on in-vitro growth and antioxidant potential of Dianthus caryophyllus L. Int J Pharm Sci Res 11(9):4420–4425 Racusen D, Foote M (1965) Protein synthesis in dark grown bean leaves. Can J Bot 43:817–824 Racusen D, Johnstone DB (1961) Estimation of protein in cellular material. Nature 191:492–493 Rai PK, Lee SS, Zhang M, Tsang YF, Kim KH (2019) Heavy metals in food crops: Health risks, fate, mechanisms, and management. Environ Int 125:365–385 Rattana K, Bunnag S (2015) Differential salinity tolerance in calli and shoots of four rice cultivars. Asian J Crop Sci 7(1):48–60 Raza HA, Amir RM (2021) Analysis of sugarcane production in Punjab, Pakistan; constraints and yield nexus. Humanities, (3), 350–362 Rizvi A, Zaidi A, Ameen F, Ahmed B, AlKahtani MD, Khan MS (2020) Heavy metal induced stress on wheat: phytotoxicity and microbiological management. Royal Society of Shakir SK, Azizullah A, Murad W, Daud MK, Nabeela F, Rahman H, Häder DP (2016) Toxic metal pollution in Pakistan and its possible risks to public health. Reviews of Environmental Contamination and Toxicology Volume 242:1–6 Souri MK, Hatamian M, Tesfamariam T (2019) Plant growth stage influences heavy metal accumulation in leafy vegetables of garden cress and sweet basil. Chem Biol Technol Agric 6(1):1–7 Sukma KPW, Daryono BS, Suprapti I (2020) Callus response of hybrid and Madura local corn to salt stress. In IOP Conference Series: Earth and Environmental Science , 3(457): 1–6 Tahtamouni P, R. W., Al-Qudah PTS (2020) Responses of Lantana Camara Linn. Callus cultures to heavy metals added to the culture media. Jordan J Biol Sci 13(4):551–557 Terletskaya et al (2017) Usman K, Abu-Dieyeh MH, Zouari N, Al-Ghouti MA (2020) Lead (Pb) bioaccumulation and antioxidative responses in Tetraena qataranse . Sci Rep 10(1):1–10 Waoo AA, Khare S, Ganguly S (2017) Comparative analysis of effect of Nickel and Cadmium on in vitro cultures of Datura inoxia . J Innov Appl Res 1(1):35–38 Zhou J, Zhang Z, Zhang Y, Wei Y, Jiang Z (2018) Effects of lead stress on the growth, physiology, and cellular structure of privet seedlings. PLoS ONE 13(3):1–17 Zhou J, Zhang Z, Zhang Y, Wei Y, Jiang Z (2018) Effects of lead stress on the growth, physiology, and cellular structure of privet seedlings. PLoS ONE 13(3):1–17 Zulfiqar U, Farooq M, Hussain S, Maqsood M, Hussain M, Ishfaq M, Anjum MZ (2019) Lead toxicity in plants: Impacts and remediation. J Environ Manage 250(15):1–21 Table Table 1. Two-way full factorial (3x7) analysis of variance (ANOVA) of studied parameters in which contribution of variation (%) of each source of variation (SOV) for each parameter is calculated as proportion of total sum of square (SS) Contribution in variation (%) b SOV a FW DW RGR Roots Shoots Genotype (V) c 11.89 05.83 09.17 11.80 03.01 Treatment (T) 84.08 84.93 88.33 75.52 91.63 V x T 03.68 08.02 02.19 11.53 03.20 Error 00.35 01.24 00.31 01.15 02.16 d CV% 04.21 06.36 02.82 10.42 14.70 STI RL SL BCF AsA Genotype (V) 05.82 02.24 08.20 06.16 39.40 Treatment (T) 84.93 89.41 84.44 52.51 46.86 V x T 08.01 06.15 05.81 41.14 12.65 Error 01.24 02.21 01.55 00.16 01.09 CV% 06.36 13.69 12.80 06.53 03.58 First month Pb SOD CAT POD TPC Genotype (V) 14.13 28.24 00.70 6.83 10.16 Treatment (T) 82.55 63.65 92.33 85.34 85.81 V x T 02.80 06.27 04.91 03.84 02.07 Error 00.52 01.84 02.06 03.99 01.96 CV% 05.11 09.08 03.30 10.50 01.46 Second month Genotype (V) 13.52 04.30 30.75 12.84 Treatment (T) 83.50 90.47 62.04 74.56 V x T 01.84 03.68 05.70 11.79 Error 01.13 01.55 01.51 00.82 CV% 05.97 04.81 07.18 01.32 Third month Genotype (V) 25.27 42.53 57.04 12.08 Treatment (T) 63.39 23.96 20.56 75.61 V x T 10.66 32.60 22.37 11.99 Error 00.69 00.91 00.05 00.32 CV% 06.32 06.65 01.67 02.78 a. Fresh weight (FW), Dry weight (DW), Relative growth rate (RGR), Number of roots (Roots), Number of shoots (Shoots), Stress tolerance index (STI), Root length (RL), Shoot length (SL), Bio-concentration factor, Ascorbic acid (AsA), Lead (Pb), Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Total protein contents (TPC) b. Source of variation (SOV), degree of freedom (df) for genotype, treatment, V x T, and error was 2, 6, 12 and 189 respectively for FW, DW and STI; error df for RGR was 80 and for roots, shoots, RL, SL was 84; error df for BCF, AsA, Pb, SOD, CAT, POD and TPC was 42 because c. Contribution in variation (%) was significant (p-value <.01) for all the studied parameters d. Coefficient of variation (%) calculated as ratio of overall standard deviation (square root of MSE) and grand mean Supplementary Files supementry.file.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2170005","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":210015051,"identity":"d11cda19-119a-46c8-b4e0-88069619772e","order_by":0,"name":"yasmeen saleem","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYFADdhBRUSPHwMBDrBZmEHHmmDGJWhjbmBMbCGkxOH786YYPFXfs+ZuZDz66cYYtfcPxswcffGCwk9NtwKHlTI7ZzRlnniXOOMyWbJxTIZO74UxesuEMhmRjswM4tBzIYbvN23Y4geEwj5l0zhm23A0HcsykeRgOJG7DpeX882e3//47bC9/mP/779w25nSD828IaLmRYHabseEw44bDPGzMQC0JBjcI2CJ5443ZzZ5jhxM3HmYzBjrsmOHMG2+MDWcY4PYL3/n0Zzd+1By2lzve/PBzTkWNPN/5HMMHHyrs5HBpUcAQh4gYYFcOAvINhEVGwSgYBaNgpAMA3tBo0V9qcUQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4695-8447","institution":"University of Sargodha","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"yasmeen","middleName":"","lastName":"saleem","suffix":""},{"id":210015052,"identity":"743c5ebe-d947-46c3-9251-fe0d34cd98a7","order_by":1,"name":"Aamir Ali","email":"","orcid":"","institution":"University of Sargodha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aamir","middleName":"","lastName":"Ali","suffix":""},{"id":210015053,"identity":"7f7c50bf-6090-4efc-acb2-d242f89d827b","order_by":2,"name":"Shagufta Naz","email":"","orcid":"","institution":"Lahore College for Women University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shagufta","middleName":"","lastName":"Naz","suffix":""},{"id":210015054,"identity":"8de3f2b1-3e7a-4159-b861-aefdeb9f078d","order_by":3,"name":"Muhammad Jamil","email":"","orcid":"","institution":"Islamia University of Bahawalpur: The Islamia University of Bahawalpur Pakistan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Jamil","suffix":""}],"badges":[],"createdAt":"2022-10-15 19:11:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2170005/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2170005/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":39085026,"identity":"454ae51d-9bdb-4a55-804d-a0f373a803a6","added_by":"auto","created_at":"2023-06-26 14:32:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":387170,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of treatments on morphological traits (a, b, d, e, g, h), relative growth rate (c), stress tolerance index (f), bio-concentration factor (i), ascorbic acid (j) and lead (k) along with multiple comparison of genotype means using Honest Significant Difference (HSD) test at alpha 0.05. Dissimilar letters indicate the significant difference among genotype means. Red dotted horizontal line is the reference line indicating the grand mean in each plot (a to k). Bars are means and whiskers are standard error calculated from three replicates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/d933b0e5839bf01e07d8b52f.png"},{"id":39086279,"identity":"e26bad8c-ce84-404e-bd18-3c57b08adfcc","added_by":"auto","created_at":"2023-06-26 14:40:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":354814,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of treatments on three enzymes and total protein contents (top to bottom) for three months (left to right) with multiple comparison of genotype means using Honest Significant Difference (HSD) test at alpha 0.05. Dissimilar letters indicate the significant difference among genotype means. Red dotted horizontal line is the reference line indicating the grand mean in each plot. Total 63 (seven treatments x three genotypes x three replicates) observations were recorded for each parameter.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/21ff9d0adf3b930d1d13d33a.png"},{"id":39085031,"identity":"17fc9cf8-8884-42d6-9f9c-7fe61f33ee22","added_by":"auto","created_at":"2023-06-26 14:32:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":176592,"visible":true,"origin":"","legend":"\u003cp\u003ePearson’s Correlation coefficient (r) among the studied parameters shows variables which have been ordered by agglomerative hierarchical clustering (AHC) method and the absolute r-value ± 0.62 has been calculated as significant at alpha 0.001. Four rectangle boxes at the diagonal indicate the related clusters of the studied variables. Total observations in the panel used correlation analysis (N = 25) include seven treatment means of each genotype, three genotype means and one grand mean (7 x 3 =21 | 21 + 3 + 1 = 25). Long form of variables is given in the legends of table 1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/435d8c0865f2790924d4f4b0.png"},{"id":39085028,"identity":"1edcef8f-7c2b-4391-9eb8-3ecaac74752c","added_by":"auto","created_at":"2023-06-26 14:32:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":241852,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of 15 parameters and 25 observations, summary of PCs with eigenvalue and variation explained per each principal component (top-left), variation contribution (%) of each parameter in first three selective PCs (top-right), score plot of observations (bottom-left) grouped into three PCs (color-coded) and loading plot of parameters (bottom-right). Values in the bracts (bottom left and right) are eigenvalues of and percentage of variation explained by principal components (PC1 on x-axis and PC2 on y-axis).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/0b9365dbfce758ae4719a90a.png"},{"id":39086968,"identity":"16794115-d9a0-422c-82a6-aec426b0a52b","added_by":"auto","created_at":"2023-06-26 14:48:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1213135,"visible":true,"origin":"","legend":"\u003cp\u003eComparative morphology of callus three genotype of sugarcane under different Pb concentrations\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/924a859e0a92564c5c9c5c62.png"},{"id":39086281,"identity":"507e1cd2-5f3e-43c8-8c4f-621062597494","added_by":"auto","created_at":"2023-06-26 14:40:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":152754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Shoots of YT-53 regenerated from 0.5mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2 \u003c/sub\u003etreated callus on MS+2mg/l Kin at day \u003cstrong\u003e(a)\u003c/strong\u003e30 \u003cstrong\u003e(b)\u003c/strong\u003e60 \u003cstrong\u003e(c)\u003c/strong\u003e90 days \u0026nbsp;(1x)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/9620d40aa56d7b51a2bf1216.png"},{"id":39086967,"identity":"a15ba196-d14d-4956-b62a-02b0654b4afb","added_by":"auto","created_at":"2023-06-26 14:48:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":169107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Shoots of CP-77-400 regenerated from 0.3mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2 \u003c/sub\u003etreated callus on MS+1mg/l BAP at day\u0026nbsp; \u003cstrong\u003e(a)\u003c/strong\u003e30\u0026nbsp; \u003cstrong\u003e(b) \u003c/strong\u003e60 \u003cstrong\u003e(c) \u003c/strong\u003e90 days (1x)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/8f663861f70a043cd2df0a9d.png"},{"id":39086283,"identity":"d6a07305-d8d2-4547-bd11-4c7aa3d1a26a","added_by":"auto","created_at":"2023-06-26 14:40:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":184680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Shoots of NSG-59 regenerated from 0.3mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2 \u003c/sub\u003etreated callu+2mg/l Kin. at \u003cstrong\u003e(a)\u003c/strong\u003e30\u0026nbsp; \u003cstrong\u003e(b)\u003c/strong\u003e60\u003cstrong\u003e(c)\u003c/strong\u003e90 days (1x)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/02fb5a864029920ea828820f.png"},{"id":41513452,"identity":"a7b2b7ee-7ecc-4be6-8edc-dc8adf6b62f6","added_by":"auto","created_at":"2023-08-14 07:39:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3774662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/3dcb275f-c97b-48d1-929b-212cf3c2521b.pdf"},{"id":39085034,"identity":"8cc4a3ff-7030-4b0b-9251-749e438e6ab6","added_by":"auto","created_at":"2023-06-26 14:32:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37366547,"visible":true,"origin":"","legend":"","description":"","filename":"supementry.file.docx","url":"https://assets-eu.researchsquare.com/files/rs-2170005/v1/43599116843e92f116d2b40f.docx"}],"financialInterests":"","formattedTitle":"A Comparative in Vitro Study of Pb Tolerance in Three Sugarcane Genotypes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeavy metal accumulation in agriculture land is one of the major challenges around the world. It affects crop production and threatens sustainable global food demands for increasing population (Frona et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Various sites of the world land used for crop cultivation have been contaminated with heavy metals while very small net change in cultivated land in world over decades confound the problem (Gupta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is estimated that the use of heavy metal contaminated water is over 20\u0026nbsp;million hectares in 50 countries (Khalid et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, recent studies are targeting heavy metals like lead, cadmium, aluminum which cause soil contamination and is growing problems on global scale (Rai et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among these heavy metals lead (Pb) is the second most harmful pollutant and hence considered the metal of great concern. It affects the crop growth by disturbing the normal plant morphological and physiological functions (Ashraf \u003cem\u003eet al.\u003c/em\u003e, 2017). Therefore, Pb induced phytotoxicity needs urgent attention (Rizvi et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSugarcane being the important agro-industrial crop in the world. It is cultivated in around 110 countries. Beside the world 80% of sugar production, it is also associated with other industries. But the potential yield of this crop is less than actual yield. Major reasons behind this gap are conventional breeding, wrong selection of genotypes, soil quality, biotic and abiotic stress. Moreover, many local genotypes are losing their potential to resist against biotic and abiotic stress over the years (Raza and Amir, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe agriculture area of Paksitan is also being affected by heavy metals over the years. In modern world it is hard to control the spread of heavy metals and the only solution is to replace the traditional genotypes to resistant genotypes (Shakir et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Pakistan sugarcane breeding programs are not sufficient to develop such genotypes and thus relay on exotic genotypes (Ahmad et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore new genotypes should screen for their resistance toward heavy metals.\u003c/p\u003e \u003cp\u003eTo evaluate the response heavy metal stress and to achieve stress resistant regenerants, Plant tissue culture is a reliable tool. It facilitates the rapid and precise evaluations of plant response under stress (Purohit et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCP-77400 is cultivated since 1996 in Pakistan with yield of while YT-53 and NSG-59 have China and USA origin respectively have higher yield than CP-77-400 (khan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Yield of CP-77400 is 10 ton/hec, YT-53 is 13.42 while NSG-59 cane yield is 13.20 ton/hec (Afghan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ahmad et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present work was designed to screen Pd toxicity in callus cultures of three sugarcane genotypes subjected to six different concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions. Morphological and biochemical markers associated with Pd stress were evaluated. Regeneration potential and root induction of Pb treated calli were also observed to find out Pb resistant the survival rate of callus and regeneration. The objective of this study is to find the most resistant genotype of sugarcane. Information obtained can be helpful for the development of cultivar improvement programs by for choice of parents and breeding crosses in breeding program. Resistant genotype could be used in heavy metal effected areas.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1 Explant and media preparation\u0026nbsp;\u003c/strong\u003eThe sugarcane genotypes (YT-53, CP-77-400 and NSG-59) were collected from the Shakerganj Sugarcane Research Institute Jhang Pakistan. The inner most layer of 3-4 months old sugarcane plant was source of explants. Plant material was washed with tap water and cleaned with 95 % ethyl alcohol and inoculated on sterilized culture vessels containing MS media with their respective growth regulator (3.5mg/l 2,4-D for YT-53 and NSG-59 and 5mg/l CP-77-400)and kept in suitable conditions (temperature, light) for callus growth and regeneration and recultured when required. The pH of culture media was kept 5.65-5.75 with 1N HCl and 1N NaOH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Callus induction\u0026nbsp;\u003c/strong\u003eWell developed calli of each genotype were shifted to MS media containing their respective growth regulator (3.5mg/l 2,4-D for YT-53 and NSG-59 and 5mg/l CP-77-400)at 6 different concentration of (Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (0.1, 0.2, 0.3, 0.4, 0.5 and 1mM). Callus growth in term callus morphology, callus fresh and dry weight were noted after 30 days.\u003c/p\u003e\n\u003cp\u003eProliferated calli (after 60 days) were shifted to already standardized MS medium provided with 6 different levels (T0-T6) of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e while MS media without Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e was used as control (T0).\u003c/p\u003e\n\u003cp\u003eCallus cultures (0.5g) exposed to 6 different levels of Pb (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewere shifted on regeneration media to evaluate their regeneration potential. Numbers of shoots per culture vessels, regeneration frequency was noted after 30 days and maximum shoot length per plant was noted after 60 day of treatment\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Regeneration\u003c/strong\u003e For regeneration Pb treated calli were shifted on their respective regeneration media (2mg/l kinetin for YT-53 and NSG-59 and 1mg/l BAP for CP-77-400) under 16h photoperiod at 27 ± 2 °C after 30 days.\u0026nbsp;Regeneration potential in terms of shoot morphology, shoot numbers, shoot length were noted after 30 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Root induction\u003c/strong\u003e For root induction the well devloped plants were shifted into already standerized rooting media\u0026nbsp;(3mg/l NAA for YT-53 while 1mg/l NAA +1mg/l IBA for CP-77-400 and NSG-59). Root induction poteniatl in term of root number, root length were noted after 30 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Estimation of Antioxidant Enzymes\u0026nbsp;\u003c/strong\u003eThe catalase, peroxidase and superoxide dismutase activities and soluble protein contents were estimated with the help of spectrophotometer (UV 4000) after day 30, 60 and 90. After 150 days Pb treated callus were transferred to regeneration media.For extraction, two-gram callus was crushed with 0.1g PVP and 4ml of 0.1 M phosphate buffer with pH 7.2 in ice-chilled pestle and mortar. The mixture was centrifuged at 14000 rpm for 10 minutes at 4°C. The supernatant attained after centrifugation was used for enzyme analysis. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.1 Specific activity of Catalase\u003c/strong\u003e estimated by Beers and Sizer (1952) method some changes. \u0026nbsp;Where reagents (50mM Phosphate Buffer and 3ml (0.036 % H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution)) pipetted (in milliliters) into cuvettes. \u0026nbsp;The temperature of the cuvettes was maintained 25 °C. Absorbance was recorded at 240 nm on spectrophotometer and Catalase extract was added. Reagents were mixed immediately and a decrease in Absorbance was recorded. Time required to reduce the absorbance from 0.45 to 0.40 was noted for the absorbance 240nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2 Specific activity of Peroxidase\u003c/strong\u003e estimated by Racusen and Foote (1965) method. Where Guaiacol was used as substrate. Peroxidase activity was estimated by pipetting the reagents (Enzyme extract 10µl into cuvettes, 0.1 M phosphate buffer (2.5ml), 1 % Guaiacol (0.2ml), Distilled H2O(0.2ml)). After keeping the reactants for 30 minutes 0.1ml hydrogen peroxide was added in both tubes. Absorbance was monitored at 470 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.3 Superoxide dismutase (SOD)\u003c/strong\u003e activity was assessed by Maral \u003cem\u003eet al.,\u003c/em\u003e (1977) method with modification that was based on principle that “Under specific condition one unit of SOD reduced the 50 % of the maximum reduction of NBT”. For SOD analysis two samples were prepared. Experimental sample contained 5µl enzyme extract and 2ml reaction mixture while control contain 2ml reaction mixture only. Both tubes were covered with black paper. These tubes were then immediately exposed to 40W fluorescent lamps for 10 minutes. The absorbance was measured at 560 nm. Superoxide dismutase activity was calculated as the percentage inhibition of NBT with the help of the following formula:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.4 Total soluble\u003c/strong\u003e \u003cstrong\u003eProtein Contents\u0026nbsp;\u003c/strong\u003eestimatedby Racusen and Johnstone, (1961) method. Two samples were prepared for control and experiment. The experimental sample was provided with 0.2ml protein extract and 2ml Biuret reagent while control had 0.2ml distilled H\u003csub\u003e2\u003c/sub\u003eO and 2ml Biuret reagent.Constituents were mixed by shaking both tubes and left for 30 minutes at room temperature. When the reaction was completed, the optical density was recorded at 545nm on spectrophotometer (UV4000). The following formula was used for protein estimation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Bio Concentration Factor (BCF) BCF=\u003c/strong\u003eLead accumulation in callus / Pb concentration in medium mg/kg x 100 mg/kg (Kulkarni \u003cem\u003eet al.,\u003c/em\u003e 2014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Stress Tolrance Index STI\u003c/strong\u003e=Average dry weight of callus(pretreated)/Average dry weight of callus (notpretreated) x100 (Kristin \u003cem\u003eet al.,\u003c/em\u003e 1997).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Statistical Analysis\u0026nbsp;\u003c/strong\u003eDescriptive statistical analysis (mean and standard error of the means) was performed for two factors (genotypes and treatments). \u0026nbsp;Two-way full factorial design of experiment was applied. Then the data was subjected to univariate two-way analysis of variance (AONVA) using Statistix-8.1 software. As a post-hoc, the least significant difference (LSD) test was applied using 95% level of confidence (p-value ≤ 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCallus Morphology\u003c/h2\u003e \u003cp\u003eCallus morphology aftre 30, 60, 90 days of inoculation is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003e at different concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. It can be clearly observed that that callus morphology of all three genotypes was affected by elevated Pb concentration. All the calli started necroing.\u003c/p\u003e \u003cp\u003eNSG-59 showed early necrosis than YT-53 and CP-77400. Callus of CP-77-400 and NSG-59 were completely dead at day 90 at 1mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. While YT-53 survived at this concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCallus Fresh Weight and Dry Weight\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a ,b) illustrates that callus weight was significantly affected by Pb concentration as compared to control. Fresh and dry weight of callus of all three genotypes decreased by increasing the concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in MS media as compared to control. Callus exposed to (1mg/L )T6 did not show any increase in net fresh and dry weight thus proved most toxic for callus culture in sugarcane genotype CP-77-400 and NSG-59. Fresh weight and dry weight of callus in YT-53 was highest on all six lead concentrations while fresh weight and dry weight of callus in NSG-59 was lowest among all the tested genotypes of sugarcane at all levels of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. All the genotypes show different in callus at weight at same treatment. YT-53 show highest callus weight at each concentration and CP-77400 was next to it. Callus dry weight and fresh weight of NSG-59 was lowest among all three genotypes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.2.2.1 Shoot Morphology\u003c/h2\u003e \u003cp\u003eRegenerated shoots maintained on MS media provided with 0 mg/L (T0) didnot shown any necrosis even after 90 days in all three genotype (YT-53, CP-77-400, NSG-59) (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In YT-53 regenerated shoots become necrotic after 90 days and were completely dead at 0.5mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T5). In CP-77-400 necrosis was observed after 60 and 90 days. In NSG-59 plant show early necroing even at 0.5mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T1). Complete necrosis was observed in shoots regenerated from callus treated with T3 treatment after 90 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRegeneration Potential in Lead Treated Callus\u003c/h2\u003e \u003cp\u003eRegeneration potential of plants developed from lead treated calli were significantly affected as compared to control in all three genotypes. All three genotypes show significant difference in regeneration potential at each level of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e, f).\u003c/p\u003e \u003cp\u003eIn YT-53 no regeneration was recorded in calli treated with 0.5mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T5) and 1mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T6). Calli of YT -53 in control cultures show 80% regeneration that reduced to 60% and 40% at 0.1mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T1) and 0.2mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T2) respectively. Length of shoots regenerated from lead treated callus were also less as compared to control.\u003c/p\u003e \u003cp\u003eIn CP-77-400 no shoot induction was recorded in calli treated with 0.5mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T5) and 1mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T6) while average number and average shoot length were also effected in Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treated calli.\u003c/p\u003e \u003cp\u003eIn NSG-59 regeneration in callus cultures were not observed at 0.4mM-1mM (T4, T5, T6). While reduction average number of shoots and shoot length was recorded after 30 days in lead treated calli.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e on Root induction\u003c/h2\u003e \u003cp\u003eAverage length of root, average numbers of root, root frequency was recorded in plants regenerated from Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treated callus with or without different concentration of lead Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 30 days. Maximum root numbers and root length was achieved in control i.e without Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treatment.\u003c/p\u003e \u003cp\u003eAverage numbers of roots, root length and root frequency decreased in all three genotypes regenerated from Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e treated callus at different concentrations. NSG-59 exhibited highest root numbers among all genotypes under study (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e-g, h, i).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Enzyme Activities\u003c/h2\u003e \u003cp\u003eEffect different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e on specific activity of POD, CAT, SOD in callus cultures of three sugarcane genotypes under six different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 30, 60, 90 days (first, second, third month) is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSpecific activity of Catalase (CAT)\u003c/h2\u003e \u003cp\u003eSpecific CAT activity in all three genotypes increased significantly by increasing the concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in callus culture media that started to decline with time. In YT-53 specific activity of CAT was highest at all concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eHighest specific activity of catalase was recorded in YT-53 at T6 at day 90. While this activity was lowest in NSG-59 among all three tested genotypes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4.2.2 Specific activity of Superoxide dismutase (SOD)\u003c/h2\u003e \u003cp\u003eSpecific SOD activity in all three genotypes increased significantly by increasing the concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in callus culture media that started to decline with time. In YT-53 specific activity of SOD was highest at all concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eHighest specific activity of SOD was recorded in YT-53 at T6 at day 90. While this activity was lowest in NSG-59 among all three tested genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSpecific activity of Peroxidase (POD)\u003c/h2\u003e \u003cp\u003eThe effect of different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e on specific POD activity in callus cultures of three sugarcane genotypes under six different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 30, 60, 90 days (first, second, third month) is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn YT-53 specific activity of POD increased by increasing the concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in callus culture media as compared to control. This activity started to decline with time. YT-53 showed the highest POD activity among three genotypes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTotal soluble Protein Contents\u003c/h2\u003e \u003cp\u003eIn all three genotypes total soluble protein contents decreased gradually by increasing concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in callus culture media and as compared to control. Total soluble protein of three genotypes calli did not show any significant difference at all the levels of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAscorbic acid\u003c/h2\u003e \u003cp\u003eEffect of 6 different concentrations of ascorbic acid pretreatment on ascorbic acid in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 90 days is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e-j.\u003c/p\u003e \u003cp\u003eIn YT-53 ascorbic acid concentration in callus increased with increasing concentration od Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in media while the ascorbic acid concentration in ascorbic acid pretreated calli were higher than non-pretreated calli. Ascorbic acid concentration was highest at 1mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (T6) in ascorbic acid pretreated calli.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSTI (Stress Tolerance Index)\u003c/b\u003e The effect of six different concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e and ascorbic acid pretreatment on stress tolerance index in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 90 day is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e-f.\u003c/p\u003e \u003cp\u003eIn YT-53 STI of callus decreased with increasing the Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in growth media while STI of YT-53 was highest among all three tested genotypes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRGR (Relative Growth Rate)\u003c/b\u003e Effect of different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e on RGR(Relative Growth Rate) in callus of three sugarcane genotypes under 6 different concentrations of Pb\u003csub\u003e2\u003c/sub\u003e(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 90 day is represented in Fig.\u0026nbsp;4.107.as the concentration of in media increases the RGR decreased in all three genotypes that was more prominent in NSG-59 while CP-77400 was next to it.This reduction was lowest in YT-53 that means YT-53 show highest growth rate than other two genotypes at elevated Pb concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e-c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBCF (Bio Concentration Factor)\u003c/b\u003e Bio Concentration Factor (BCF) in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 90 day is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e-i. In YT-53 BCF of callus decreased with increasing the concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePb (Lead) Contents In Callus\u003c/b\u003e Pb (Lead) contents in callus of three sugarcane genotypes under 6 different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e after 90 days is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003e-k.\u003c/p\u003e \u003cp\u003eLead(Pb) contents in control calli were found to be zero. Lead contents in three genotypes increases as concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e increased in callus growing media. Maximum lead contents were found in callus maintained onT6 in YT-53.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis (PCA)\u003c/h2\u003e \u003cp\u003ePrincipal component analysis (PCA) revealed two main components with eigenvalue above one. First principal component (PC1) along x-axis explained 68.7% overall variation with 10.31 eigenvalue and second principal component (PC2) along y-axis highlighted 16.6% variation with 2.49 eigenvalue (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall 71.19 percent variation was explained by the first two PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e, top-left). Due to the high contribution percentage, traits CAT, POD, AsA and BCF were grouped at PC2 while the other traits were at PC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e, top-right). The 25 observations were classified into three groups by three successive PCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e, bottom-left). Controls of three genotypes can be seen in a single cluster in green. Genotype YT-53 showed higher values for POD, AsA, CAT than the other two genotypes. Higher fresh and dry weight, shoot length, root length, number of sheets and STI were observed in NSG-59. Stress indicators such as POD, AsA, CAT, SOD is positioned in same section of the plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e bottom-right) while RGR, Roots, FW, DW, SL, STI, RL, Shoots are in other section of the plot showing positive correlation with each other. This PCA clearly classified the genotypes with respect to the treatments (Fig bottom-left) and well grouped the parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e4\u003c/span\u003e bottom-right) according to the strength of correlation among variables.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCallus browning rate indicates the callus sensitivity and its health under stress conditions Callus morphology of control calli of three genotypes of sugarcane were yellow, granular, nodular but calli those were treated with different concentrations of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e start browning in the presence of higher lead concentration gradually (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As necrosis increases with lead concentration and time, calli appear brown to dark brown. Necrosis due to tissue damage results in browning of callus (Sukma, 2020). Hyperactivity of oxidative enzymes involve in synthesis of phenolic compound further intensify callus browning (Koc et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The observations are in line with the findings of previous studies where calli appeared brown when exposed to abiotic stress (Atabaki et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nawrot-Chorabik, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rattana and Bunning, 2015).\u003c/p\u003e \u003cp\u003eTo assess the response of metal stress on growth of callus was recorded in term of fresh and dry weight during present investigation. It is well known that plant growth attributes are inhibited by higher lead concentration. Suppressed growth of callus under Pb stress was also reported by the Waoo et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e in \u003cem\u003eDatura inoxia\u003c/em\u003e and by Tahtamouni and Al-Qudah (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in \u003cem\u003eLantana Camara\u003c/em\u003e while reduced net fresh weight of callus accompanied with an increase of browning intensity in \u003cem\u003ePhoenix dactylifera\u003c/em\u003e L. was reported by Abbas, (2016). Reduced growth of callus in terms of dry weight and fresh weight in all three cultivars were also observed during this study. Chabhal and Virk (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) explained that reduced growth of callus due to metal toxicity might disrupt physiological processes. Pb ions align with nucleic acids that condense the chromatin and inhibit the replication, transcription processes that hampered the normal cell division thus leading in reduced plant growth. Pb ions bind with the cell wall that strengthens the cross-linking of pectin\u0026rsquo;s which hindered growth of cell in plants (Aslam et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). At cellular level Pb ions bind with oxygen, nitrogen, and sulfur atoms that damage thylakoid membranes. Moreover disturbance in nutrient uptake and electron transport chain make the situation more worst (Ali et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pirzadah et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These impairing of physiological and biochemical processes might lead to reduced growth of callus.\u003c/p\u003e \u003cp\u003eResponses of callus under stress conditions depended upon plant physiology, \u003cem\u003eits\u003c/em\u003e genotype, concentration and type of stress agent and \u003cem\u003ein-vitro\u003c/em\u003e manipulation (Purohit et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It was observed that low concentrations of lead nitrate (0.1, 0.2mM) did not affect the callus fresh weight and dry weight significantly in all three sugarcane genotypes (YT-53, CP-77-400, NSG-59). but as the concentration of Pb increased in media the callus net fresh weight and callus dry weight start declining. Decrease in biomass depends upon concentration of Pb also explicated by earlier studies (Zhou et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Souri et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Amari et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUsman et al., (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) stated that plants can tolerate 100mg/l lead concentration in vivo conditions but \u003cem\u003ein vitro\u003c/em\u003e conditions calli are more sensitive to wards stressor as each cell directly interact with the stress agents (Terletskaya et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Krishania and Agarwal, 2012 stated that 100\u0026micro;M(0.1mM) Pb concentration become toxic for callus. But according to our observation sugarcane calli can survive at 0.5mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in case of CP-77-400 and NSG-59 but could not withstand 1mM concentration of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e while calli of YT-53 were survived at this level. Surviving ability and regeneration rate of sugarcane calli under abiotic stress depend upon genotypes (Mahmud et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to our work callus treated with Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e have reduced regeneration potential even loss of regeneration ability in all three genotypes of sugarcane. No shoot induction was recorded in callus treated with high levels (0.4, 0.5, 1mM) of Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in these genotypes. Loss of regeneration potential in callus derived from high salt concentration oat calli was also reported by Mamun et al., (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and in potato Hassanein and Saleem, (2017). Reduction in regeneration potential perhaps due to damaged cells at proliferation stage. Atwell et al., (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). stated that declining in shoot regeneration potential as a result Cd exposure was because callus cell lost its totipotency via physical damage of DNA and DNA methylation that made the regeneration difficult.\u003c/p\u003e \u003cp\u003eReduced shoot length from seeds under lead stress reported earlier (Zhou et al., (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); Boi \u003cem\u003eet al.\u003c/em\u003e, (2019) were similar to our observation where Pb toxicity also reduced the regenerated shoot length in all three genotypes of sugarcane. Shoot induction frequency under lead stress was also affected. Shoot induction frequency reduced to 40% from 80% in YT-53 and from 70\u0026ndash;40% in CP-77-400 while in NSG-59 shoot induction frequency was 70% in control calli and was 30% at 0.3mM Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. Inhibitory effect of abiotic stress on regeneration frequency was also documented by Haque (2017) and Dogan (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eHeavy metal stress interferes with physiological and biochemical pathways during callus formation and also results in reduction of root length. Root growth was mainly affected through tissue damage. Root growth, length and numbers depend upon concentration of heavy metals and sensitivity of genotype (Kundu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Pb ions binds with spindles of cell wall during cell expansion that distract the normal cell division which ultimately inhibit root growth (Zulfiqar et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kanwal et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Average root length and frequency in lead treated callus were also declined with respect to control during present study. Inhibited growth of roots under abiotic stress was also observed by Kiełkowsk (2017), Ahmed, \u003cem\u003ee t al.\u003c/em\u003e, (2020) and Alkhateeb, (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt was concluded that Pb ions negatively affected \u003cem\u003ecallus\u0026nbsp;\u003c/em\u003egrowth, callus induction, regeneration and rooting of sugarcane genotypes YT-53, CP-77400, NSG-59.\u0026nbsp;Regeneration potential of calli at elevated levels of heavy metals can be used to assess sensitivity of plants. Above results also suggested that Pb stress during callus proliferation stage persist during later developmental stages at higher concentrations while at lower concentration these toxic effects were not determinantal. All the genotypes behave differently towards different treatment and show significant difference from each other at different parameters and toward different concentration of Pb toxicity. Moreover, these toxic effects were more prominent in CP-77-400 and NSG-59 as compared to YT-53 that show the lead toxicity greatly influenced by genotype. YT-53 found more resistant toward Pb stress as show better performance at each parameter should be used in Pb contaminated area because of its high yield and resistance toward metal stress. This information could also be used by farmers to choose the genotype in heavy metal contaminated area. According to best of our knowledge no information about the effect of metal on later developmental stages under \u003cem\u003ein vitro\u003c/em\u003e conditions in sugarcane callus culture have been found therefore above information can be used to develop metal tolerant genotypes and for phytoremediation studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003eauthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.S.:\u0026nbsp;Conceptualization Methodology, Writing-original draft. A.A;\u0026nbsp;Supervision: S.N; Project administration:\u0026nbsp;M.J\u0026nbsp;Softwar,\u0026nbsp;Formal analysis,\u0026nbsp; Data curation,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was not funded by any organization\u003c/p\u003e\n\u003cp\u003e(Not Applicable)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCONFLICT OF INTEREST DECLARATION AND AUTHOR AGREEMENT\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThis statement is to certify that all authors have seen and approved the manuscript being submitted. We assure you that article is the Authors\u0026apos; original work. On behalf of all Co-Authors, the corresponding Author shall bear full responsibility for the submission. We attest to the fact that all authors listed on the title page have contributed significantly to the work, have read the manuscript, attest to the validity and legitimacy of the data and its interpretation, and agree to its submission to the \u0026ldquo;Environmental Science and Pollution Research\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval/declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work not involve any ethic approval study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent\u003c/strong\u003e \u003cstrong\u003eto participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This study does not involve humans or animals, provide statement that it is not applicable or write\u003c/p\u003e\n\u003cp\u003e(Not applicable)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e (Not applicable)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available on request from the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAbass MH (2016) Responses of date palm (\u003cem\u003ePhoenix dactylifera\u003c/em\u003e L.) callus to biotic and abiotic stresses. Emirates J Food Agric 28(1):66\u0026ndash;74\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAfghan S, Asad M, Farooq U, Anwar MB, Shahzad A, Choudhary FA (2015) Investigating sugarcane genetic material inherited capability for agronomic attributes under agro-ecological conditions of Jhang. Pakistan Sugar Journal, \u003cem\u003e30\u003c/em\u003e(1)\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAhmad S, Unar GS, Gujar N, Panhwar RN, Soomro AF, Chattha AA (2017) Performance of new promising sugarcane variety yt-55 under different agro-climatic conditions of Pakistan. Pakistan Sugar Journal 32(1):8\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAhmed MS, Gardezi DA, Batley J, Hayashi S, Zander M, Javid A, Awan SI (2019) Somaclonal variations for red rot and sugarcane mosaic virus resistance and candidate genes integrity assessment in somaclones of selected sugarcane varieties (\u003cem\u003eSaccharum officinarum\u003c/em\u003e L). Pakistan J Agricultural Sci 56(1):15\u0026ndash;27\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAli B, Xu X, Gill RA, Yang S, Ali S, Tahir M, Zhou W (2014) Promotive role of 5-aminolevulinic acid on mineral nutrients and antioxidative defense system under lead toxicity in \u003cem\u003eBrassica napus\u003c/em\u003e. Ind Crops Prod 52:617\u0026ndash;626\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAlkhateeb (2015)\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAmari T, Ghnaya T, Abdelly C (2017) Nickel, cadmium and lead phytotoxicity and potential of halophytic plants in heavy metal extraction. South Afr J Bot 111:99\u0026ndash;110\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAshraf U, Tang X (2017) Yield and quality responses, plant metabolism and metal distribution pattern in aromatic rice under lead (Pb) toxicity. Chemosphere 176:141\u0026ndash;155\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAslam M, Aslam A, Sheraz M, Ali B, Ulhassan Z, Najeeb U, Gill RA (2021) Lead toxicity in cereals: mechanistic insight into toxicity, mode of action, and management. Front Plant Sci 11:2248\u0026ndash;2268\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAtabaki N, Rosimah N, Kalhori N, Lasumin N, Sahebi M, Abiri R (2018) \u003cem\u003eIn vitro\u003c/em\u003e selection and development of Malaysian salt-tolerant rice (\u003cem\u003eOryza sativa\u003c/em\u003e L. cv. MR263) under salinity. \u003cem\u003eActa Scientific Agriculture\u003c/em\u003e, 2(8):8\u0026ndash;17\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAtwell BJ, Kriedemann PE, Turnbull CG (1999) Plants in action: adaptation in nature, performance in cultivation. Ann Botany 84(5):685\u0026ndash;687\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBeers R, Sizer I (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxidase by catalase. J Biol Chem 195:133\u0026ndash;140\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBoi ME, Porceddu M, Cappai G, De Giudici G, Bacchetta G (2020) Effects of zinc and lead on seed germination of Helichrysum microphyllum subsp. tyrrhenicum, a metal-tolerant plant. Int J Environ Sci Technol 17(4):1917\u0026ndash;1928\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChabhal A, Virk GS (2017) The effect of nickel chloride (NiCl\u003csub\u003e2\u003c/sub\u003e) on various callus growth dynamics of \u003cem\u003eColeus blumei\u003c/em\u003e (benth.) cultures. Int J Adv Res 5:863\u0026ndash;868\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDogan M (2020) Effect of salt stress on \u003cem\u003ein vitro\u003c/em\u003e organogenesis from nodal explant of \u003cem\u003eLimnophila aromatica\u003c/em\u003e (Lamk.) Merr. and \u003cem\u003eBacopa monnieri\u003c/em\u003e (L.) Wettst. and their physio-morphological and biochemical responses. \u003cem\u003ePhysiology and Molecular Biology of Plants\u003c/em\u003e, 26(4): 803\u0026ndash;816\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFrona D, Szenderak J, Harangi-Rakos M (2019) The challenge of feeding the world. Sustainability 11(20):5816\u0026ndash;5834\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGupta N, Yadav KK, Kumar V, Krishnan S, Kumar S, Nejad ZD, Alam J (2021) Evaluating heavy metals contamination in soil and vegetables in the region of North India: Levels, transfer and potential human health risk analysis. Environ Toxicol Pharmacol 82:1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHaque M, Islam SS, Subramaniam S (2017) Effects of salt and heat pre-treatment factors on efficient regeneration in barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L). 3 Biotech 7(63):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHassanein AM, Salem J (2017) Rise potassium content of the medium improved survival, multiplication, growth and scavenging system of \u003cem\u003ein vitro\u003c/em\u003e grown potato under salt stress. Egypt J Bot 57(1):259\u0026ndash;275\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKanwal A, Farhan M, Sharif F, Hayyat MU, Shahzad L, Ghafoor GZ (2020) Effect of industrial wastewater on wheat germination, growth, yield, nutrients and bioaccumulation of lead. Sci Rep 10(1):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKhalid S, Shahid M, khanniazi N, Murtaza B, Bibi I, Dumat C (2017) A comparison of technologies for remediation of heavy metal contaminated soils. J Geochem Explor 182:247\u0026ndash;268\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKhan MNM, Islam MM, Islam MS, Uddin MI (2019) Studies on \u003cem\u003ein vitro\u003c/em\u003e response to callus induction and gene transfer technique of five high yielding indica rice varieties. J Sci Agric 3:41\u0026ndash;45\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKhan MR, Muhammad A, Hussain I, Shah SH, Kumar T, Inam S, Ali GM (2012) Rapid in vitro multiplication of sugarcane elite genotypes and detection of sugarcane mosaic virus through two steps RT-PCR. Int J Agric Biology, \u003cem\u003e14\u003c/em\u003e(6)\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKielkowska A (2017) \u003cem\u003eAllium cepa\u003c/em\u003e root meristem cells under osmotic (sorbitol) and salt (NaCl) stress \u003cem\u003ein vitro\u003c/em\u003e. Acta Bot Croatica 76(2):146\u0026ndash;153\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKoc NK, Baș B, Koc M, K\u0026uuml;sek M (2009) Investigations of \u003cem\u003ein vitro\u003c/em\u003e selection for salt tolerant lines in sour orange (\u003cem\u003eCitrus aurantium\u003c/em\u003e L). Biotechnology 8(1):155\u0026ndash;159\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKristin AS, Senra RR, Perez FI, Enriquez BC, Gallegos JA, Vallego PR, Wassimi N, Kelley JD (1997) Improving common bean performance under drought stress. Crop Sci 37:43\u0026ndash;50\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKulkarni DK, Delbari AS, Mahajan DM (2014) Bio concentration factor (BCF) for heavy metals detection and selection of hyper-accumulator plants: Case study of Pune-India and Tehran\u0026ndash;Iran. Indian J Fundamental Appl Life Sci 4(1):163\u0026ndash;170\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKundu D, Dey S, Raychaudhuri SS (2018) Chromium (VI)\u0026ndash;induced stress response in the plant \u003cem\u003ePlantago ovata\u003c/em\u003e Forsk \u003cem\u003ein vitro\u003c/em\u003e. Genes and Environment 40(1):21\u0026ndash;34\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMahmud K, Islam N, Kumar A, Azim GMA, Rajib MRR, Islam MJ (2021) Regeneration of sugarcane genotypes under different level of Sodium Chloride Salt. Acta Sci Agric 5(3):47\u0026ndash;54\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMamun AA, Islam MM, Alam NEK, Noushin S (2020) \u003cem\u003eIn vitro\u003c/em\u003e callus induction under NaCl salt stress and subsequent plant regeneration in oats (\u003cem\u003eAvena sativa\u003c/em\u003e L). Plant Tissue Culture and Biotechnology 30(2):253\u0026ndash;266\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMaral J, Puget K, Micheson AM (1977) Comparative study of superoxide dismutase, catalase, and glutathione peroxidase levels in erythrocytes of different animals. Biochem Biophys Res Commun 77:1525\u0026ndash;1535\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNawrot-Chorabik K (2017) Response of the callus cells of fir (\u003cem\u003eAbies nordmanniana\u003c/em\u003e) to in vitro heavy metal stress. Folia Forestalia Polonica 59(1):25\u0026ndash;33\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePirzadah TB, Malik B, Tahir I, Hakeem KR, Alharby HF, Rehman RU (2020) Lead toxicity alters the antioxidant defense machinery and modulate the biomarkers in Tartary buckwheat plants. Int Biodeterior Biodegrad 151:1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePurohit S, Gupta S, Jain R (2020) Effect of chromium on \u003cem\u003ein-vitro\u003c/em\u003e growth and antioxidant potential of \u003cem\u003eDianthus caryophyllus\u003c/em\u003e L. Int J Pharm Sci Res 11(9):4420\u0026ndash;4425\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRacusen D, Foote M (1965) Protein synthesis in dark grown bean leaves. Can J Bot 43:817\u0026ndash;824\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRacusen D, Johnstone DB (1961) Estimation of protein in cellular material. Nature 191:492\u0026ndash;493\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRai PK, Lee SS, Zhang M, Tsang YF, Kim KH (2019) Heavy metals in food crops: Health risks, fate, mechanisms, and management. Environ Int 125:365\u0026ndash;385\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRattana K, Bunnag S (2015) Differential salinity tolerance in calli and shoots of four rice cultivars. Asian J Crop Sci 7(1):48\u0026ndash;60\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRaza HA, Amir RM (2021) Analysis of sugarcane production in Punjab, Pakistan; constraints and yield nexus. Humanities, (3), 350\u0026ndash;362\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRizvi A, Zaidi A, Ameen F, Ahmed B, AlKahtani MD, Khan MS (2020) Heavy metal induced stress on wheat: phytotoxicity and microbiological management. \u003cem\u003eRoyal Society of\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShakir SK, Azizullah A, Murad W, Daud MK, Nabeela F, Rahman H, H\u0026auml;der DP (2016) Toxic metal pollution in Pakistan and its possible risks to public health. Reviews of Environmental Contamination and Toxicology Volume 242:1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSouri MK, Hatamian M, Tesfamariam T (2019) Plant growth stage influences heavy metal accumulation in leafy vegetables of garden cress and sweet basil. Chem Biol Technol Agric 6(1):1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSukma KPW, Daryono BS, Suprapti I (2020) Callus response of hybrid and Madura local corn to salt stress. \u003cem\u003eIn IOP Conference Series: Earth and Environmental Science\u003c/em\u003e, 3(457): 1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTahtamouni P, R. W., Al-Qudah PTS (2020) Responses of \u003cem\u003eLantana Camara\u003c/em\u003e Linn. Callus cultures to heavy metals added to the culture media. Jordan J Biol Sci 13(4):551\u0026ndash;557\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTerletskaya et al (2017)\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eUsman K, Abu-Dieyeh MH, Zouari N, Al-Ghouti MA (2020) Lead (Pb) bioaccumulation and antioxidative responses in \u003cem\u003eTetraena qataranse\u003c/em\u003e. Sci Rep 10(1):1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWaoo AA, Khare S, Ganguly S (2017) Comparative analysis of effect of Nickel and Cadmium on \u003cem\u003ein vitro\u003c/em\u003e cultures of \u003cem\u003eDatura inoxia\u003c/em\u003e. J Innov Appl Res 1(1):35\u0026ndash;38\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhou J, Zhang Z, Zhang Y, Wei Y, Jiang Z (2018) Effects of lead stress on the growth, physiology, and cellular structure of privet seedlings. PLoS ONE 13(3):1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhou J, Zhang Z, Zhang Y, Wei Y, Jiang Z (2018) Effects of lead stress on the growth, physiology, and cellular structure of privet seedlings. PLoS ONE 13(3):1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZulfiqar U, Farooq M, Hussain S, Maqsood M, Hussain M, Ishfaq M, Anjum MZ (2019) Lead toxicity in plants: Impacts and remediation. J Environ Manage 250(15):1\u0026ndash;21\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Two-way full factorial (3x7) analysis of variance (ANOVA) of studied parameters in which contribution of variation (%) of each source of variation (SOV) for each parameter is calculated as proportion of total sum of square (SS)\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003eContribution in variation (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eSOV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eFW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRGR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRoots\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eShoots\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenotype (V)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e11.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e05.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e09.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatment (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e84.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e84.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e88.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eV x T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e08.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003csup\u003ed\u003c/sup\u003eCV%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e04.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSTI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBCF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenotype (V)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e05.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e08.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatment (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e84.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e84.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eV x T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e08.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e05.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e41.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCV%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003eFirst month\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePOD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTPC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenotype (V)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatment (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e63.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eV x T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e04.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCV%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e05.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e09.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003eSecond month\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenotype (V)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e04.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatment (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e83.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e62.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eV x T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e03.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e05.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCV%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e05.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e04.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e07.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003eThird month\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGenotype (V)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e57.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatment (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e63.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e23.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eV x T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e00.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCV%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e06.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e01.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e02.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ea. Fresh weight (FW), Dry weight (DW), Relative growth rate (RGR), Number of roots (Roots), Number of shoots (Shoots), Stress tolerance index (STI), Root length (RL), Shoot length (SL), Bio-concentration factor, Ascorbic acid (AsA), Lead (Pb), Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Total protein contents (TPC)\u003c/p\u003e\n\u003cp\u003eb. Source of variation (SOV), degree of freedom (df) for genotype, treatment, V x T, and error was 2, 6, 12 and 189 respectively for FW, DW and STI; error df for RGR was 80 and for roots, shoots, RL, SL was 84; error df for BCF, AsA, Pb, SOD, CAT, POD and TPC was 42 because\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ec. Contribution in variation (%) was significant (p-value \u0026lt;.01) for all the studied parameters\u003c/p\u003e\n\u003cp\u003ed. Coefficient of variation (%) calculated as ratio of overall standard deviation (square root of MSE) and grand mean\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sugarcane, lead nitrate, heavy metal toxicity, heavy metal resistance, phytoremediation","lastPublishedDoi":"10.21203/rs.3.rs-2170005/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2170005/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeavy metal accumulation in agriculture land is one of the major agriculture challenges affecting the crop production and threatens the food safety. Comparative study for Lead (Pb) tolerance in three sugarcane (\u003cem\u003eSaccharum officinarum\u003c/em\u003e L.) genotypes was carried out under \u003cem\u003ein vitro\u003c/em\u003e conditions. Fresh weight, dry weight, callus morphology, shoot morphology, protein contents, catalase, superoxide dismutase and peroxidase, ascorbic acid contents, relative growth rate, stress tolerance index, Pb contents, bioconcentration factor were compared at 6 different concentrations of Pb (0mM, 0.1, 0.2, 0.3, 0.4, 0.5, 1mM). Lead affected the callus and its regeneration in all three genotypes while root regeneration did not affect considerably. All genotypes significantly vary at each treatment and parameter except BCF and protein contents. Genotype YT-53 proved most resistant toward Pb stress while CP-77400 was next to it and NSG-59 showed least resistance toward Pb stress. Most resistant genotypes can be used in heavy metal effected area for better yield and phytoremediation in Pb affected soil and in other breeding programs.\u003c/p\u003e","manuscriptTitle":"A Comparative in Vitro Study of Pb Tolerance in Three Sugarcane Genotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-26 14:32:03","doi":"10.21203/rs.3.rs-2170005/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9e73399a-6ac4-489f-aec1-8174c882ce2d","owner":[],"postedDate":"June 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-08-14T07:31:05+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-26 14:32:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2170005","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2170005","identity":"rs-2170005","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}