Growth, biochemical, and ultrastructural analyses of Acacia modesta callus Wall. in response to different concentrations of growth regulators and heavy metals

Growth, biochemical, and ultrastructural analyses of Acacia modesta callus Wall. in response to different concentrations of growth regulators and heavy metals | 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 Growth, biochemical, and ultrastructural analyses of Acacia modesta callus Wall. in response to different concentrations of growth regulators and heavy metals Tarek Ahmed Radwan, Rita Victor Metry, Emad Eldin Ewais, Amal Ali Awadalla Mohamed This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6314982/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 Four concentrations (2, 4, 6, and 8 mg/L) of benzyladenine (BA) and kinetin (Kn) along with three concentrations (5, 10, and 15 mg/L) of 2,4-dichlorophenoxyacetic acid (2.4-D) were incorporated into Murashige and Skoog (MS) medium to investigate their effectiveness in inducing callus formation from axillary buds of A. modesta . It was found that the 2,4-D concentration of 10 and 15 mg/L had the most favorable effect on the induction rates and growth characteristics of A. modesta. Afterwards, the impacts of various concentrations (0, 0.125, 0.25, 0.5, and 1 ppm) of cadmium (Cd), nickel (Ni), and lead (Pb), on the morphological attributes and primary metabolites were measured in A. modesta calli grown at MS medium supplemented with 10 and 15 mg/L 2,4-D. In addition, ultrastructural attributes were investigated using transmission electron microscopy (TEM) in A. modesta calli grown at the highest metal concentrations. At low concentrations of the three heavy metals, the calli exhibited minimal morphological changes. However, at the highest concentrations, callus growth was significantly reduced, as evidenced by lower fresh weights and altered morphological characteristics. High metal concentrations caused compact, dehydrated, and necrotic tissues. Cd exposure induced the most severe effects, including cell wall thickening, cytoplasmic shrinkage, and plasmolysis, which were observed. Ni treatment led to reduced vascular bundle size, mesophyll thickness, and chloroplast integrity, while Pb exposure resulted in extensive vacuolation and mitochondrial disruption. The concentrations (0.125 and 0.25 ppm) of the three metals exhibited the highest contents of carbohydrates and proteins. The highest concentrations (1 ppm) of the metals had the lowest contents of carbohydrates and proteins. A. modesta callus cultures displayed varying degrees of tolerance to Cd, Ni, and Pb indicated by its ability to form callus at low metal concentrations, as well as the morphological adaptations to high metal levels, suggests potential for phytoremediation applications. Acacia modesta Cadmium Callus Heavy metals Nickel Lead Phytoremediation Transmission electron microscopy (TEM) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Industrial activities, incessant urbanization, and agricultural development are the major reasons for increasing pollution of soil and water (Alloway 2012 ; Xu et al. 2020 ; Akram et al. 2021 ). Cadmium (Cd), nickel (Ni), and lead (Pb) are the most widespread heavy metal pollutants causing serious consequences for plant health due to their high toxicity. They also significantly decrease soil productivity and human health (Long et al. 2021 ). In addition, these metals persist in the environment, accumulate in plants, and enter food chains leading to a significant threat to the ecosystem and human life (Ali et al. 2021 ; Angon et al. 2024 ). Cadmium causes severe phytotoxicity affecting growth processes such as nutrient uptake, enzyme activity, and photosynthesis in plants (Zulfiqar et al. 2022 ). Nickel and lead also have been reported to cause negative effects on plants such as stunted growth, sclerosis, and cell damage (Hassan et al. 2019 ; Shankar 2020). Metallophytes are those plant species that have mechanisms for accumulating and tolerating high concentrations of certain metals, while, most crops are highly sensitive to the toxic effects of these metal (Clemens 2006 ; Shaw 2022 ). Phytoremediation is the biotechnology where plants are used to remediate contaminated soils. It is a quite sustainable solution for the improvement of heavy metals in the earth's crust (Gulzar and Mazumder 2022 ) Acacia modesta Wall. is a promising plant species which could be adapted under unfavorable conditions and as a prospective biomass producers (Ali et al. 2013 ). Previous studies conducted on related Acacia species for their metal absorption and accumulation potential suggested that A. modesta may also be a suitable candidate for phytoremediation (Yadav et al. 2016 ; Akhtar et al. 2023 ; Zainab et al. 2023 ), This study was undertaken to investigate the effect of the three heavy metals, Cd, Ni, and Pb, on growth, morphology, ultrastructure, and content of primary metabolites of A. modesta callus cultures. Furthermore, A. modesta callus was utilized to investigate the phytoremediation potential and underlying mechanisms responsible for this species' tolerance to toxic metals, while minimizing any interference with the surrounding environment. 2. Materials and Methods 2.1. Plant Material Acacia modesta plant material was obtained from a mature tree located in the Botanical Garden of Aswan Governorate, Egypt. Explants consisting of axillary buds with a 5-mm section of the shoot were collected during the active growing season (October to February). Prior to culturing, the explants were surface sterilized using a two-step process: they were first washed in a 2% (w/v) detergent solution for 15 min, followed by immersion in 70% ethanol for 5 min. The explants were then sterilized in a 0.1% (w/v) aqueous mercuric chloride solution for 5 min, and rinsed three times in sterile distilled water. 2.2. Callus Induction and Culture Media The explants were inoculated onto Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs) to induce callus formation. The MS medium contained 30 g/L sucrose, and 0.8% Difco agar, and the pH was adjusted to 5.7 before autoclaving at 121°C for 20 min. The following PGRs were tested for callus induction: benzyladenine (BA) at 2, 4, 6, and 8 mg/L, Kinetin (Kn) at 2, 4, 6, and 8 mg/L, and 2,4-Dichlorophenoxyacetic acid (2,4-D) at 5, 10, and 15 mg/L. The cultures were incubated at 27 ± 2°C under continuous light conditions. Callus growth was assessed at weekly intervals for 12 weeks. Callus induction and multiplication rates were recorded. 2.3. Callus Multiplication and Somatic Embryogenesis For callus multiplication, the initial calli were subcultured onto fresh MS medium containing the optimum concentration of 2,4-D (10 mg/L or 15 mg/L). Subculturing was performed every 4 weeks to allow for the multiplication of somatic embryos. 2.4. Heavy Metal Treatment Callus cultures were retransferred to MS medium supplemented with both concentrations of 2,4-D each of them with varying concentrations (0.125, 0.25, 0.5, and 1.0 mg/L) of cadmium (Cd), nickel (Ni), and (0.125, 0.25, and 0.5 mg/L) of lead (Pb) to assess their impact on callus growth. Control callus cultures were maintained on MS medium without the addition of heavy metals (Fig. 1 ). The cultures were incubated for 30 days, and the color and overall quality of the callus were monitored. At the end of the experiment, Callus samples were harvested; the dry weight was taken after drying in an oven at 70 o C to constant mass to calculate the biomass production. Then, it was powdered, and stored at 4°C. Powdered material was incinerated at 450 ⁰C. The resulting ash was digested according to Allen et al. ( 1986 ). The metals were measured by an Atomic Absorption Spectrophotometer (solar 969). The metal content was represented as mg /g DW. The bioaccumulation factor (BAF) was calculated using the following equation: BAF= \(\:\frac{\left[\text{M}\text{e}\text{t}\text{a}\text{l}\right]\:\text{t}\text{i}\text{s}\text{s}\text{u}\text{e}}{\left[\text{M}\text{e}\text{t}\text{a}\text{l}\right]\text{m}\text{e}\text{d}\text{i}\text{u}\text{m}}\) (Du et al. 2020 ) Where [Metal] tissue and [Metal] medium are the concentrations of HMs in the callus tissue and medium, respectively. 2.5. Electron Microscopy Ultrastructure Examination For ultrastructural analysis, control, and treated callus samples were fixed in 3% glutaraldehyde in phosphate buffer, post-fixed in potassium permanganate for 5 min, and then dehydrated through an ethanol series. The samples were infiltrated with epoxy resin and sectioned into very thin slices, which were placed on copper grids. The sections were double-stained with uranyl acetate and lead citrate and examined using a transmission electron microscope (TEM) (JEOL JEM-1010) at 70 kV. 2.6. Estimation of total carbohydrates and total proteins The total carbohydrates were determined using an anthrone reagent (Morris 1948 ; Dhumal et al. 2007 ). The total proteins (water soluble- and insoluble) were determined with the Folin-Ciocalteau reagent (Lowry et al. 1951 ; Peterson 1977 ). 2.7. Data Analysis Statistical analysis of the results was performed using analysis of variance (ANOVA). Significant differences between treatments were determined using the least significant difference (LSD) test at a p-value of ≤ 0.05. 3. Results 3.1. Effect of Different Plant Growth Regulators on Induction, Characteristics and Morphology of Callus The callus induction rates and shooting of A. modesta were significantly influenced by the different concentrations of BA in the media (F-value= 1736.54; P-value= 0.000 and F-value= 2275.36; P-value= 0.000, respectively). The callus initiation started from 6 to 9 weeks (Table 1). The lowest induction and shooting rates were recorded under 4 mg/l of BA recording (18± 0.16 and 12± 0.21%, respectively) (Table 1). At the end of the experiment after 12 weeks, the callus covered the entire explant surface. The resulting callus appeared whitish and friable in all BA concentrations (Fig. 2). The callus induction and shooting rates were significantly influenced by the different concentrations of Kn in the media (F-value= 1324.35; P-value= 0.000 and F-value= 3681.26; P-value= 0.000, respectively) (Table 1). The callus initiation started from 6 to 10 weeks. The lowest concentration of Kn (2 mg/l) recorded the lowest callus induction rates and shooting, while the largest callus induction rates and shooting were reported in (4 mg/l of Kn) (Table 1). At the end of the experiment, the callus covered the entire explant surface. The resulting callus appeared in the concentration (2 and 4 mg/l) of Kn yellowish friable, while, in high concentrations (6 and 8 mg/l) of Kn, the resulting callus appeared compact and yellowish (Fig. 3). The callus induction rate in the calli of A. modesta was significantly influenced by the different concentrations of 2,4-D in the media (F-value= 103982.15; P-value= 0.000). The callus initiation started from 3 to 5 weeks. The lowest callus induction rate was reported in the lowest concentration of 2,4-D recording (43± 0.16%). As the concentration of 2,4-D increased in the media, the callus rate increased till reached the highest concentration of 2,4-D (15 mg/l) which recorded the highest callus induction rate (100 %±0) (Table 1). On the other hand, the shooting induction rate was 00.0±0.0 in the media supplemented with 2,4-D (Table 1). At the end of the experiment, the quality of the resulting callus was the best in 2,4-D treatment at all concentrations. The resulting callus appeared whitish friable in the lowest concentration of 2,4-D (5 mg/l), compact yellowish in (2,4-D 10 mg/l), and compact brownish in the highest concentration of 2,4-D (15 mg/l) (Fig. 4). 3.2. Effect of Cadmium, Nickel, and Lead on Callus Growth and their accumulation in the callus tissue The quality of the callus was reduced in the media treated with increasing concentration of Cd. The calli color was changed from a whitish friable in the lowest concentration of Cd (0.125 ppm) to a dehydrated compact brownish in the highest concentration of Cd (1 ppm) (Table 2). The highest callus biomass was observed in the control recording 4.65 and 4.6 mg/l DW for 10, and 15 2,4-D, respectively, followed by the lowest concentration of Cd (0.125 ppm), recording 3.25 and 3.15 mg/l DW for 10, and 15 2,4-D, respectively. As the concentration of Cd increased, the callus biomass decreased (Table 2). A severe reduction of callus biomass was observed in the highest concentration of Cd (1 ppm), recording 0.45 and 0.6 mg/l DW for 10, and 15 2,4-D, respectively (Table 2). The BAF of Cd in the calli of A. modesta was significantly influenced by the different concentrations of Cd in the media The highest BAF (0.072 for 10 2,4-D) was obtained in the highest concentration of Cd (1 ppm). The lowest concentration of Cd (0.125 ppm) corresponded to the lowest BAF (0.039 and 0.017 for 10 2,4-D and 15 2,4-D, respectively) (Table 2). The color of the callus varies from a friable whitish and yellowish nodular in low concentrations of Ni to a compact brownish in the highest concentration of Ni (1 ppm) (Table 2). The highest callus biomass was observed in control, recording 4.65 and 4.6 mg/l DW for 10, and 15 2,4-D, respectively, followed by the lowest concentration of Ni (0.125 ppm), recording 2.85 and 3.65 mg/l DW for 10, and 15 2,4-D, respectively (Table 2). The more the concentration of Ni increases in culture media, the calli biomass were reduced. The highest concentration of Ni (1 ppm) causes inhibition of callus growth and contains the lowest callus biomass, recording 0.65 and 0.8 mg/l DW for 10, and 15 2,4-D respectively. The BAF of Ni in the calli of A. modesta was significantly influenced by the different concentrations of Ni in the media. The highest BAFs (0.888 and 0.794 for 10 2,4-D and 15 2,4-D, respectively) were obtained under the influence of the highest Ni concentration (1 ppm) (Table 2). The addition of Pb in various concentrations to the culture media results in the inhibition of callus growth. As the concentrations of Pb increased the more hazardous effects were caused to the callus. The color of calli became compact yellowish in the low concentration of Pb and turned into a dehydrated compact dark brownish with necrotic in the highest concentration of Pb (1 ppm) (Table 2). High concentrations of Cd and Pb in the culture media resulted in a severe reduction in the mucilaginous material, resulting in the dehydration of calli with necrotic. The highest callus biomass was observed in control, recording 4.65 and 4.6 mg/l DW for 10, and 15 2,4-D, respectively, followed by the lowest concentration of Pb (0.125 ppm), recording 0.85 and 0.9 mg/l DW for 10, and 15 2,4-D respectively. The lowest callus biomass was observed in the highest Pb concentration (0.5 ppm), recording 0.6 and 0.65 mg/l DW for 10, and 15 2,4-D, respectively. The BAF was significantly influenced by the different concentrations of Pb in the calli of A. modesta (Table 2). The highest BAFs were found in the highest concentration of Pb (0.5 ppm). (1.464 and 2.06 for 10 2,4-D and 15 2,4-D, respectively (Table 2). 3.3. Callus Ultra-structural Changes under Heavy Metal Treatments The ultrastructure of Acacia modesta callus cells was observed under transmission electron microscopy (TEM) to examine cellular components and structural changes under treatment of 1 ppm of Cd, Pb, and Ni compared to control (Fig. 6). Control cells display well-organized organelles. Larger cells contained large vacuoles with electron-dense regions and thin cytoplasmic cell wall segments with few plasmodesmata. The walls appeared normal, cytoplasm contained all the cellular organelles including mitochondria, endoplasmic reticulum with ribosomes, chloroplasts, nucleus, nucleolus, and well-defined cell walls and membranes as indicative of healthy metabolic activity (Fig. 6, A). Significant ultra-structural changes in the callus tissue exposed to 1 ppm of Cd where cells were characterized by increased vacuolation, mitochondrial reduction, and formation of electron-dense granules. In addition, disruption of chloroplast, plasmolytic shrinkage, disintegration of the nucleus, shrinkage of cytoplasm, abnormal structures of organelle, thickening and constriction of cell wall and cell collapse, and disintegration were recorded (Fig. 6, B). The most significant negative impact on the tissue of A. modesta was observed with Pb compared to other metals. It was found to be accumulated in higher concentrations within the cells Compared to Ni and Cd, suggesting it is more readily absorbed or retained by the cells. This higher accumulation likely contributes to its stronger adverse effects, resulting in more severe tissue damage, with variations noted in chloroplast morphology, mitochondrial integrity, and membrane structure under metal exposure. These findings underscore the critical role of lead in causing cellular stress and damage, making it the most detrimental metal analyzed in this study (Fig. 6, C) Significant changes in the cellular anatomy of A. modesta in response to nickel exposure, including cell wall thickening and structural irregularities at higher concentrations, chloroplasts showed signs of damage, including disrupted thylakoid membranes and reduced granal stacking, and vacuoles appeared enlarged and contained electron-dense materials, indicating possible accumulation of nickel or secondary metabolites as a response to stress (Fig. 6, D). 3.4. Effect of Cadmium, Nickel, and Lead on Content of Total Carbohydrates and Total Proteins The content of total carbohydrates in the calli of A. modesta was significantly influenced by the different concentrations of HMs in the media (Table 2). The content of carbohydrates in the lowest concentration of Cd (0.125 ppm) was the highest compared to the other concentrations, it was two-fold than that of the control recording. The content of carbohydrates decreased with increasing Cd concentration in the media (Table 2). The lowest concentration of Ni (0.125 ppm) resulted in the highest content of carbohydrates (178.72±5.10 and 137.16±3.05 mg/g DW for 10 2,4-D and 15 2,4-D respectively). In addition, the calli grown in the largest concentration of Ni (1 ppm) contained the lowest carbohydrate contents (96.5±0.408 and 72.78±1.035 mg/g DW for 10 2,4-D and 15 2,4-D respectively) (Table 2). the lowest concentration of Pb in the calli of A. modesta treated with (control 0 ppm of 10 2,4-D) and (0.125 ppm of 15 2,4-D) contained the highest content of carbohydrates (121.942±1.4 and 297.47±10.85 mg/g DW respectively). In addition, the lowest carbohydrate contents were reported in (0.5 ppm of 10 2,4-D) and (the control 0 ppm of 15 2,4-D) recording 69.62±0.54 and 79.74±4.38 mg/g DW, respectively (Table 2). The content of total proteins in the calli of A. modesta was significantly influenced by the different concentrations of both Cd and Ni in the media (Table 2). The highest content of protein for the calli grown in 10 2,4-D was measured in the (0.25 ppm) Cd concentration (34.48±0.53 mg/g DW) while the calli grown in the control (0 ppm Cd) exhibited the highest protein content for 15 2,4-D of values equal to (43.38±0.85 mg/g DW). The highest concentration of Cd (1ppm) had the lowest content of protein (20.05±0.6 and 18.35±0.45 mg/g DW for 10 2,4-D and 15 2,4-D, respectively) (Table 2). The lowest concentration of Ni (0.125 ppm), protein content was the highest concentration (38.949±0.98 and 43.68±0.67 mg/g DW for 10 2,4-D and 15 2,4-D respectively), and exhibited approximately the same content of protein of the control (31.43±0. and 43.68±0.851 mg/g DW for 10 2,4-D and 15 2,4-D respectively) (Table 2). The total proteins in calli of A. modesta was not significantly different in the calli growing in different concentrations of Pb in 10 2,4-D ( F-value= 2.74; P-value= 0.113) while it was significantly affected by Pb in 15 2,4-D (F-value= 166.35; P-value= 0.00) (Table 2). Protein content was the highest concentration in the control (43.68±0.851 mg/g DW of 15 2,4-D). As the concentration of Pb increased, the protein content gradually decreased. the lowest protein contents were reported in (the control 0 ppm of 10 2,4-D) and (0.5 ppm of 15 2,4-D) recording 31.43±0.45 and 22.184±1.17 mg/g DW, respectively. Multivariate data analysis such as hierarchicalclustering analysis (HCA) and principal component analysis (PCA) were performed by using Minitab (version 18.1) to show the different patterns (Fig. 6). Fig (6, A) shows similarities or dissimilarities between different variables. Biomass and protein had similar pattern , while, content of metal in the tissue and BAF had similar pattern (Fig. 6, A). The first component of PCA plot (PC1) accounts for 58.0% of the variance; and it was correlated content of metal in callus tissue, BAF, and color and texture of callus (Fig. 6, B). The second component of PCA plot (PC2) accounted for 14.4% of the variance; it was correlated to texture . The third component of PCA plot (PC3) accounted for 12.6 % of the variance; it was correlated to carbohydrates, proteins, metal in tissue, and BAF. Exposure of callus to elevated concentrations of Pb (0.25-0.5) was positively linked to accumulation of metal in the tissue and affecting the color of the tissue. Low concentration of Ni as well as control treatments was positively linked to enhancement of the protein content and the biomass of the callus (Fig. 6, B). 4. Discussion Despite the heavy metal-induced stress, A. modesta callus cultures displayed varying degrees of tolerance to Cd, Ni, and Pb. The species’ ability to form callus at low metal concentrations, as well as the morphological adaptations to high metal levels, suggests the potential for phytoremediation applications. Previous studies on Acacia species, including A. catechu and A. mangium , have indicated their potential for phytoremediation, particularly in heavy metal-contaminated soils (Ghosh and Singh 2005). 4.1. Effect of Plant Growth Regulators on Callus Induction Our study showed that 2,4-D was the most effective plant growth regulator for inducing callus formation in A. modesta , consistent with previous reports on Acacia species and other leguminous and medicinal plants (Mousa 2023 ; Shahrour et al. 2024 ). While BA and Kn also promoted callus formation, their efficacy was lower than 2,4-D. This could be due to the specific nature of 2,4-D, which induces rapid cell division and differentiation, particularly in Acacia species (Reséndiz 2014 ; Gantait et al. 2018 ). At higher concentrations of 2,4-D, however, callus quality decreased, as evidenced by the shift to compact, brownish callus. This indicates that excessive hormone concentrations may induce stress, reducing callus vitality and regeneration potential (Al Gethami and El Sayed 2020 ; Li et al. 2024 ). 4.2. Impact of Cadmium, Nickel, and Lead on Callus Growth Exposure to Cd, Ni, and Pb significantly reduced callus growth and biomass production, with Pb showing the most pronounced toxicity. These results are consistent with the established understanding that Pb is highly toxic to plant cells, even at low concentrations (Riyazuddin et al. 2021 ). Lead disrupts critical plant processes such as photosynthesis and nutrient uptake, leading to stunted growth and necrosis (Tarragó and Brown 2017). Similarly, Cd and Ni exposure led to a concentration-dependent reduction in fresh and dry weight, with high concentrations causing necrosis and discoloration of the callus. These findings are in line with studies on other species, such as Brassica juncea and Solanum lycopersicum , where high metal concentrations inhibited callus growth and led to morphological changes (Kumari et al. 2022). 4.3. The Bioaccumulation Factor (BAF) of Cadmium, Nickel, and Lead Negative effects on ecosystems can arise from the bioaccumulation of pollutants within living organisms. This process, in which organisms absorb and hold harmful pollutants from their surrounding contaminated environment, is referred to as BAF (Isensee 2024 ). Plants are categorized as hyperaccumulators if their BAF greater than 1 (Bashir et al. 2014 ; Cui et al. 2023 ). It was reported in this study that the BAF of these HMs in A. modesta calli rose in proportion to the concentration of HMs in the culture media. In the current study, it was found that the BAF of Cd in A. modesta calli was less than 1. Similarly, potatoes also exhibited a BAF of Cd below 1 (Liu et al. 2021 ). In contrast, higher BAF values exceeding 1 for Cd were observed in Artemisia selengensis , Brassica juncea , and tea (Xu et al. 2020 ; Nepal et al. 2024 ; Ju et al. 2024 ). All Ni treatments in the present study resulted in BAFs exceeding 1 in A. modesta calli. Similarly, the BAF of Ni was found to be greater than 1 in both Acacia saligna and Acacia polyacantha , based on a comparable line of analysis by Masvodza et al. ( 2013 ). But in Eichhornia crassipes , the BAF on Ni was below1 (Mahamood et al. 2023 ). The BAF of Pb in A. modesta calli was greater than 1. In a similar study, Mahdavi et al. ( 2014 ) observed that the BAF of Pb in Acacia victoria was less than 1. Likewise, research by Mohamed et al. ( 2020 ) demonstrated that Jatropha curcas also exhibited a BAF of Pb below 1. According to the current investigation, the BAF of the three HMs was discovered in the declining order Pb > Ni > Cd. 4.4. Ultrastructural Changes Induced by Cadmium, Nickel, and Lead Transmission electron microscopy (TEM) provided detailed insights into the cellular changes induced by heavy metals. Control callus cells exhibited healthy organelles, including intact chloroplasts and mitochondria, while heavy metal exposure caused significant ultrastructural damage. Cd exposure resulted in cytoplasmic shrinkage, plasmolysis, and thickening of the cell walls, suggesting a response to osmotic stress and cell wall fortification as a protective mechanism against toxicity (Shanying et al. 2017 ). Nickel exposure led to reductions in vascular bundle size and mesophyll thickness, which likely impaired the transport of nutrients and water, contributing to growth inhibition (Hassan et al. 2019 ; Rehman et al. 2021 ). Pb caused severe vacuolation, mitochondrial disruption, and necrosis, indicating its high toxicity and potential to disrupt cellular energy metabolism (Malar et al. 2016; Kohli et al. 2020 ). These structural changes highlight the severe impact of heavy metals on cellular integrity and function, which could contribute to the reduced growth and viability of callus tissues (Jorjani and Karakaş 2024 ). 4.4. Effect of Cadmium, Nickel, and Lead on Total Carbohydrates and Total Proteins Total carbohydrates. There is a strong influence of HMs on carbohydrate contents in plants (Yang et al. 2023 ). Cadmium affects carbohydrate metabolism in various ways and leads to changes in their content, which can result from the inactivation and alteration of certain enzymes involved in carbohydrate synthesis (Latef 2018 ). In the present study, the content of total carbohydrates was increased at a low concentration of Cd, while it decreased with increasing metal concentration. In the same context, an increase in soluble sugars at low concentrations of metal stress and a decrease at higher concentrations as in rice seedlings, Pisum sativum , and Lemna polyrrhiza (John et al. 2008 ). The digestion and mobilization of reserved food have been reported to be affected by Ni pollution such as carbohydrates and proteins in sprouted seeds (Mustafa et al. 2023 ). In the present study, a low concentration of Ni increased the content of total carbohydrates, while it decreased with increasing metal concentration. Similarly, in wheat seedlings, carbohydrate content was increased at low concentrations of Ni (Loreti et al. 2003 ). Reducing and non-reducing sugars were reduced under high concentrations of Ni due to decreasing the activity of α-amylase in two sunflower cultivars (Ashraf et al. 2011 ). In contrast, the content of soluble sugars is increased under Ni toxicity in Eruca sativa (Kamran et al. 2016 ), tomato (Okunlola et al. 2017 ), Catharanthus roseus (Yasin et al. 2018 ), and Cucurbita pepo (Valivand et al. 2019 ). Tolerance to Pb toxicity involves slowing the generation of radicals through increased protein and carbohydrate content and affecting the peroxidase activity and polyphenol oxidases (Singh et al. 2011 ). In the present study, a low concentration of Pb increased the content of total carbohydrates, while it decreased with increasing metal concentration. Vegetables grown under different Pb concentrations had lower glucose and sucrose content with higher Pb doses than vegetables and crops grown on control substrates (Mishra and Dubey 2013 ). On contrary, total carbohydrate and starch levels increased under Cd and Pb stress in Vicia faba (Abu-Muriefah 2015 ) and in Coronopus didymus (Sidhu et al. 2017 ). Total proteins. Heavy metals cause low protein content in plants grown on metal-contaminated soil due to changes in plant physiology as reduction of nitrate reductase activity (Widowati 2012 ). In the present study, the protein content was reduced with increasing Cd dose. These results are line with the results of Galal ( 2016 ) and Galal et al. ( 2021 ) who reported that proteins decreased in Cucurbita pepo and Pisum sativum tissues grown on contaminated media with Cd. Likewise, the total protein content showed a significant reduction in dose-dependent in Catharanthus Roseus calli exposed to Cd different concentrations (Abnosi et al. 2015 ). In contrast, the total content of protein was increased due to the irrigation of two poplar species ( Populus nigra and Populus alba ) with treated wastewater containing Cd, Ni, and Pb (Houda et al. 2016 ). High Ni levels result in inhibition of key enzymes involved in the digestion of food reserves (proteases and α- and β-amylase), protein synthesis, carbohydrate metabolism, and reserve mobilization (Chaki et al. 2020 ). In the present study, the protein content was reduced with increasing Ni dose. Similarly, Ashraf et al. ( 2011 ) found that exposure to Ni stress significantly inhibited protease activity and thus the conversion of stored proteins into amino acids. In addition, Ni can cause the reduction of proteins, especially low molecular weight proteins, which increases oxidative stress in many plant species (Silva et al. 2020 ). In contrast, a slight increase in total protein content was found in plants subjected to Ni stress (Maheshwari and Dubey 2008 ). The effect of Pb on total protein concentration is unclear, although high concentrations can decrease the protein content (Piotrowska et al. 2009 ), some amino acids, such as proline, increase under lead stress (Qureshi et al. 2007 ). These proteins play an important role in plant tolerance to Pb. The present study revealed that compared to the control, protein decreased at all Pb concentrations. The same results were revealed by Gupta et al. ( 2009 ) and Zhang et al. ( 2020 ) when studying the effect of three HMs on the total content of protein in Zea mays and Hydrilla verticillata ; it was concluded that an increase in the concentration of three HMs leads to a decrease in the total protein content and the highest content was detected in the control. In contrast, due to Pb exposure, the soluble protein content showed a significant increasing with increasing Pb concentration in c oleoptiles of wheat (Lamhamdi et al. 2011 ) and Coronopus didymus (Sidhu et al. 2017 ). Conclusion The heavy metals caused significant reductions in callus biomass, changes in morphology, and ultrastructural damage, highlighting the phytotoxic nature of these metals. While all three metals inhibited callus growth, the ability of A. modesta to form callus and exhibit morphological adaptations at lower metal concentrations suggests its potential for phytoremediation. Further research is needed to explore the genetic and biochemical mechanisms underlying A. modesta tolerance to heavy metals, with a particular focus on metal accumulation and detoxification pathways. Understanding these mechanisms could pave the way for developing more efficient and sustainable phytoremediation strategies for the environmental cleanup of heavy metal-contaminated soils. The present study focused on callus cultures; however, further research is needed to evaluate the performance of intact plants in phytoremediation efforts, particularly in the context of metal hyperaccumulation and detoxification mechanisms. Declarations Conflicts of Interest : The authors declare no conflict of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contributions: Conceptualization, T.A.A.R.; methodology, R.V., T.A.A.R., A.A.A.M; software, R.V., T.A.A.R, A.A.A.M, E,E; formal analysis, R.V., A.A.A.M; investigation, T.A.A.R, R.V., A.A.A.M; resources, T.A.A.R; data curation, T.A.A.R, R.V., A.A.A.M; writing—original draft preparation, TAARR.V., A.A.A.M; writing—review and editing, TAAR, A.A.A.M. E.E; supervision, E.E. All authors have read and agreed to the published version of the manuscript. Acknowledgments: The authors appreciate of the facilities that were provided by the Department of Botany at Aswan University. References Abnosi MH, Amirjani M, Mahdiyeh M, Moradipoor H (2015) Biochemical and cellular response of Catharanthus roseus callus cells to cadmium toxicity. J Genet Resour 1:101–114. Abu-Muriefah SS (2015) Effects of silicon on Faba bean ( Vicia faba L.) plants grown under heavy metal stress conditions. 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J soil Sci plant Nutr 22:212–269 Tables Table 1 Effect of MS basal media supplemented with different growth regulators (BA, Kinetin, and 2,4-D) on induction of callus and characteristic of callus proliferated from nodal cutting of Acacia modesta Growth regulator (mg/l) Weeks to Callus Initiation Callus Callus induction rate (%) Degree of callus Color Shooting induction rate (%) BA Kn 2,4-D 2 8-9 19 c±0.092 + WF 25 b±0.354 4 7-9 18 d±0.156 + WF 12 d±0.212 6 6-7 24 b±0.156 ++ WF 18 c±0.170 8 7-8 30 a±0.283 ++ WF 30 a±0.141 2 8-10 18 d±0.148 + YF 11 d±0.092 4 8-10 24 a±0.099 + YF 24 a±0.014 6 6-8 23 b±0.078 + CY 15 c±0.226 8 7-9 20 c±0.092 ++ CY 20 b±0.106 5 4-6 43 c±0.177 +++ YF 0.0 ±0.0 10 3-5 60 b±0.099 ++++ CY 0.0 ±0.0 15 4-5 100 a±0.0 ++++ CB 0.0 ±0.0 *Note: Data were recorded after 12 weeks of culture when the callus can be seen clearly by the naked eye. WF=whitish friable; YF=yellowish friable; YG=yellowish granular; CY=compact yellowish; CN=compact nodular; NB=nodular brownish; CGN=compact greenish nodular; CY=compact yellowish nodular; CB=compact brownish. - No Callus, + Very poor callus, ++ Poor callus, +++ Good callus, ++++ Very good callus. Table 2 Influence of 2,4-D and Heavy Metal Concentrations (Cd, Ni, and Pb) on Biomass Production, Callus Characteristics, and Metal Uptake in Plant Tissue Cultures Growth hormone (mg/L) Growth characteristics Metal (ppm) Biomass (mg/l DW) Color Texture Metal in tissue mg/kg DW) BAF 10 (2,4-D) Cd 0 4.65 a whitish friable 0.000 e 0.000 e 0.125 3.25 b whitish friable 0.005 d 0.039 d 0.25 0.75 c Yellowish granular 0.010 c 0.049 c 0.5 0.65 d Yellowish Compact 0.031 b 0.063 b 1 0.45 e Brownish Compact 0.072 a 0.072 a 15 (2,4-D) 0 4.6 a whitish friable 0.000 e 0.000 d 0.125 3.15 b whitish friable 0.002 d 0.017 c 0.25 0.9 c Yellowish granular 0.006 c 0.022 b 0.5 0.8 c Yellowish Compact 0.021 b 0.042 a 1 0.12 d Brownish Compact 0.04 a 0.04 a Ni 10 (2,4-D) 0 4.65 a whitish friable 0.000 e 0.000 e 0.125 2.85 b whitish friable 0.026 d 0.204 d 0.25 1.75 c Yellowish nodular 0.076 c 0.306 c 0.5 1.4 d Yellowish Compact 0.238 b 0.475 b 1 0.65 e Brownish Compact 0.888 a 0.888 a 15 (2,4-D) 0 4.6 a whitish friable 0.000 e 0.000 e 0.125 3.65 b whitish friable 0.018 d 0.147 d 0.25 2.3 c Yellowish nodular 0.071 c 0.283 c 0.5 1.6 d Yellowish Compact 0.214 b 0.427 b 1 0.8 e Brownish Compact 0.794 a 0.794 a Pb 10 (2,4-D) 0 4.65 a whitish friable 0.000 d 0.000 d 0.125 0.65 b Yellowish Compact 0.14 c 1.085 c 0.25 0.65 b Brownish Compact 0.346 b 1.385 b 0.5 0.6 b Dark Brownish Compact 0.732 a 1.464 a 15 (2,4-D) 0 4.6 a whitish friable 0.000 d 0.000 c 0.125 0.9 b Yellowish Compact 0.142 c 1.136 b 0.25 0.8 b Brownish Compact 0.513 b 2.051 a 0.5 0.065 c Dark Brownish Compact 1.032 a 2.061 a Table 3. Impact of growth hormones and heavy metal exposure on primary metabolite production in calli of Acacia modesta Growth hormone (mg/L) Primary metabolite (mg/g DW) Metal (ppm) Carbohydrates Protein Cd 10 (2,4-D) 0 121.94 c ±1.4 31.43 b* ±0.45 0.125 239.26 a ±2.06 26.98 c ±0.52 0.25 188.52 b ±11.2 34.48 a ±0.53 0.5 179.87 b ±5.05 32.48 b ±0.52 1 126.79 c ±0.3 20.05 d ±0.6 15 (2,4-D) 0 79.74 CD ±4.38 43.38 A ±0.85 0.125 177.07 A ±7.74 36.67 B ±0.91 0.25 114.12 B ±1.55 29.60 C ±0.45 0.5 89.18 C ±1.06 31.48 C ±0.98 1 76.04 D ±2.12 18.35 D ±0.45 Ni 10 (2,4-D) 0 121.94 b ±1.4 31.43 c ±0.45 0.125 178.72 a ±5.1 38.95 a ±0.98 0.25 175.38 a ±16.91 33.18 b ±0.2 0.5 119.90 b ±2.15 29.38 d ±0.3 1 96.50 c ±0.41 16.99 e ±0.77 15 (2,4-D) 0 79.74 D ±4.38 43.38 A ±0.85 0.125 137.16 A ±3.05 43.68 A ±0.67 0.25 123.16 B ±0.12 36.40 B ±1.83 0.5 98.61 C ±0.12 29.51 C ±0.99 1 72.77 E ±1.04 25.94 D ±0.69 Pb 10 (2,4-D) 0 121.94 a ±1.4 31.43 a ±0.45 0.125 104.97 b ±1.61 32.43 a ±1.89 0.25 77.40 c ±6.09 36.75 a ±4.68 0.5 69.62 c ±0.54 31.74 a ±1.07 15 (2,4-D) 0 79.74 C ±4.38 43.38 A ±0.85 0.125 297.47 A ±10.85 32.96 B ±1.67 0.25 157.90 B ±2.53 29.64 C ±0.84 0.5 81.32 C ±2.68 22.18 D ±1.17 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. 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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-6314982","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454196812,"identity":"41641d14-de91-4142-ba19-a7daa2f5c9ab","order_by":0,"name":"Tarek Ahmed Radwan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYHACAyjNxvgATQSvFhBmYzZAEiFOC5sEUVrM2w9vk/i544+8eXtbWsWPP4cTG9ibt0kwVNTi1CJzJq1MsveMgeGcM8eO3extA2rhOVYmwXDmOE4tEgw5Zjd42wwYZ0ikt93gbbid2CCRYybB2HYMtxb+N2Y3/7YZ2M+Qf95W+OcPUIv8G6CWf3i0AM28DbQlcYYE2zFmHjaQLTxALQ01eLQ8K/8t22acPIMnLVlatu2/cRtPWrFFwrEDeByWvNnwbZuc7Qz2Y4Yf3/xJk+1nP7zxxoeaOpxaMAEbiEhgOEyCFiggxZZRMApGwSgY5gAAqKhUePmTfZEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4278-4961","institution":"Aswan University","correspondingAuthor":true,"prefix":"","firstName":"Tarek","middleName":"Ahmed","lastName":"Radwan","suffix":""},{"id":454196813,"identity":"9eb8b15d-c06f-49bb-9625-bfed3be7b613","order_by":1,"name":"Rita Victor Metry","email":"","orcid":"","institution":"Aswan University","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"Victor","lastName":"Metry","suffix":""},{"id":454196814,"identity":"70130cfc-7207-4cfb-8da8-af668c5e9b3d","order_by":2,"name":"Emad Eldin Ewais","email":"","orcid":"","institution":"Al-Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Emad","middleName":"Eldin","lastName":"Ewais","suffix":""},{"id":454196815,"identity":"578af73e-b4e8-4eaa-888d-370c16ed4c05","order_by":3,"name":"Amal Ali Awadalla Mohamed","email":"","orcid":"","institution":"Aswan University","correspondingAuthor":false,"prefix":"","firstName":"Amal","middleName":"Ali Awadalla","lastName":"Mohamed","suffix":""}],"badges":[],"createdAt":"2025-03-26 19:27:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6314982/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6314982/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82638929,"identity":"e7bac3b5-4681-4613-8aab-cd94ca03fa5c","added_by":"auto","created_at":"2025-05-13 14:54:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107780,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental layout for callus induction from \u003cem\u003eA. modesta\u003c/em\u003eaxillary buds using different growth regulators and heavy metal treatments (Cd, Ni, and Pb) in combination with 2,4-D\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/4a8df1e8ddc250b1867120dc.jpg"},{"id":82640758,"identity":"c05f8978-1075-4a9d-adc1-7d182dc02321","added_by":"auto","created_at":"2025-05-13 15:10:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227792,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of whitish friable calli proliferated from explants of \u003cem\u003eA. modesta\u003c/em\u003e obtained after 12 weeks of culture on: (A) MS medium + 2 mg/l BA, (B) MS medium + 4 mg/l BA, (C) MS medium + 6 mg/l BA (showing shoot buds initiation obtained from calli), (D) MS medium + BA 8 mg/l (showing shoots multiplication). (bar =1cm)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/b9037d35ced6a2ca2b5ab5b2.png"},{"id":82639859,"identity":"17b34638-ca28-4d68-a2dc-07c2531cd78b","added_by":"auto","created_at":"2025-05-13 15:02:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":380147,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of compact and yellowish calli proliferation from explants of \u003cem\u003eA. modesta\u003c/em\u003e obtained after 12 weeks of culture on: (A) MS medium + 2 mg/l kinetin, (B) MS medium + 4 mg/l kinetin, (C) MS medium + 6 mg/l kinetin (showing shoots initiation from axillary buds), (D) MS medium + 8 mg/l kinetin. (bar =1cm)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/29e4a1dfc148a1d29a9ca311.png"},{"id":82639857,"identity":"def82f5b-1c58-4f5e-ba6b-f99d601f72af","added_by":"auto","created_at":"2025-05-13 15:02:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349912,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of calli proliferated from nodal cutting explants of \u003cem\u003eA. modesta\u003c/em\u003e obtained after 12 weeks of culture at varying concentrations of MS medium + 2,4-D (A) yellowish friable callus on MS medium +2,4-D 5 mg/l, (B) compact yellowish callus on MS medium +2,4-D 10 mg/l (C) and (D) compact brownish callus on MS medium +2,4-D 15mg/l. (bar =1cm)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/c55c7cf9930dfef31de792a6.png"},{"id":82638932,"identity":"65aa517a-4f32-4d03-bb8e-ce7f471774e0","added_by":"auto","created_at":"2025-05-13 14:54:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":415505,"visible":true,"origin":"","legend":"\u003cp\u003eThe ultrastructure of typical parenchymatous callus cells of \u003cem\u003eA. modesta\u003c/em\u003e; control (A) and all treatments 1 PPM of Cd (B), Pb (C) and Ni (D) under the transmission electron microscope (×2500), Showing CW cell wall, CP chloroplast, CM cell membrane, M mitochondrion. All scale bars = 500 nm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/e6f5dee09ae1d3bcc24bdecc.png"},{"id":82638936,"identity":"f0eab9ca-7f9c-43f7-8675-0fee763fc9c8","added_by":"auto","created_at":"2025-05-13 14:54:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":127679,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical clustering analysis (HCA) between variables (A) and principal component analysis (PCA) \u0026nbsp;(B) of the morphological, primary metabolites , bioaccumulation of metals in the \u0026nbsp;callus \u0026nbsp;of \u003cem\u003eA. modesta\u003c/em\u003e;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/810f1ea389908f17ef0d375d.png"},{"id":109799983,"identity":"6b040e75-6872-4111-83f5-f7e8fd7179f1","added_by":"auto","created_at":"2026-05-22 15:35:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2121305,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6314982/v1/0ad98e2d-73d4-4506-a5f3-3bd803f7abc0.pdf"}],"financialInterests":"","formattedTitle":"Growth, biochemical, and ultrastructural analyses of Acacia modesta callus Wall. in response to different concentrations of growth regulators and heavy metals","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIndustrial activities, incessant urbanization, and agricultural development are the major reasons for increasing pollution of soil and water (Alloway \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Akram et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cadmium (Cd), nickel (Ni), and lead (Pb) are the most widespread heavy metal pollutants causing serious consequences for plant health due to their high toxicity. They also significantly decrease soil productivity and human health (Long et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, these metals persist in the environment, accumulate in plants, and enter food chains leading to a significant threat to the ecosystem and human life (Ali et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Angon et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cadmium causes severe phytotoxicity affecting growth processes such as nutrient uptake, enzyme activity, and photosynthesis in plants (Zulfiqar et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nickel and lead also have been reported to cause negative effects on plants such as stunted growth, sclerosis, and cell damage (Hassan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shankar 2020).\u003c/p\u003e \u003cp\u003eMetallophytes are those plant species that have mechanisms for accumulating and tolerating high concentrations of certain metals, while, most crops are highly sensitive to the toxic effects of these metal (Clemens \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Shaw \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhytoremediation is the biotechnology where plants are used to remediate contaminated soils. It is a quite sustainable solution for the improvement of heavy metals in the earth's crust (Gulzar and Mazumder \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cem\u003eAcacia modesta\u003c/em\u003e Wall. is a promising plant species which could be adapted under unfavorable conditions and as a prospective biomass producers (Ali et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Previous studies conducted on related Acacia species for their metal absorption and accumulation potential suggested that \u003cem\u003eA. modesta\u003c/em\u003e may also be a suitable candidate for phytoremediation (Yadav et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Akhtar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zainab et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e),\u003c/p\u003e \u003cp\u003eThis study was undertaken to investigate the effect of the three heavy metals, Cd, Ni, and Pb, on growth, morphology, ultrastructure, and content of primary metabolites of \u003cem\u003eA. modesta\u003c/em\u003e callus cultures. Furthermore, \u003cem\u003eA. modesta\u003c/em\u003e callus was utilized to investigate the phytoremediation potential and underlying mechanisms responsible for this species' tolerance to toxic metals, while minimizing any interference with the surrounding environment.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant Material\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAcacia modesta\u003c/em\u003e plant material was obtained from a mature tree located in the Botanical Garden of Aswan Governorate, Egypt. Explants consisting of axillary buds with a 5-mm section of the shoot were collected during the active growing season (October to February). Prior to culturing, the explants were surface sterilized using a two-step process: they were first washed in a 2% (w/v) detergent solution for 15 min, followed by immersion in 70% ethanol for 5 min. The explants were then sterilized in a 0.1% (w/v) aqueous mercuric chloride solution for 5 min, and rinsed three times in sterile distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Callus Induction and Culture Media\u003c/h2\u003e \u003cp\u003eThe explants were inoculated onto Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs) to induce callus formation. The MS medium contained 30 g/L sucrose, and 0.8% Difco agar, and the pH was adjusted to 5.7 before autoclaving at 121\u0026deg;C for 20 min.\u003c/p\u003e \u003cp\u003eThe following PGRs were tested for callus induction: benzyladenine (BA) at 2, 4, 6, and 8 mg/L, Kinetin (Kn) at 2, 4, 6, and 8 mg/L, and 2,4-Dichlorophenoxyacetic acid (2,4-D) at 5, 10, and 15 mg/L. The cultures were incubated at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under continuous light conditions. Callus growth was assessed at weekly intervals for 12 weeks. Callus induction and multiplication rates were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Callus Multiplication and Somatic Embryogenesis\u003c/h2\u003e \u003cp\u003eFor callus multiplication, the initial calli were subcultured onto fresh MS medium containing the optimum concentration of 2,4-D (10 mg/L or 15 mg/L). Subculturing was performed every 4 weeks to allow for the multiplication of somatic embryos.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Heavy Metal Treatment\u003c/h2\u003e \u003cp\u003eCallus cultures were retransferred to MS medium supplemented with both concentrations of 2,4-D each of them with varying concentrations (0.125, 0.25, 0.5, and 1.0 mg/L) of cadmium (Cd), nickel (Ni), and (0.125, 0.25, and 0.5 mg/L) of lead (Pb) to assess their impact on callus growth. Control callus cultures were maintained on MS medium without the addition of heavy metals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The cultures were incubated for 30 days, and the color and overall quality of the callus were monitored. At the end of the experiment, Callus samples were harvested; the dry weight was taken after drying in an oven at 70 \u003csup\u003eo\u003c/sup\u003eC to constant mass to calculate the biomass production. Then, it was powdered, and stored at 4\u0026deg;C. Powdered material was incinerated at 450 ⁰C. The resulting ash was digested according to Allen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). The metals were measured by an Atomic Absorption Spectrophotometer (solar 969). The metal content was represented as mg /g DW.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe bioaccumulation factor (BAF) was calculated using the following equation:\u003c/p\u003e \u003cp\u003eBAF=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left[\\text{M}\\text{e}\\text{t}\\text{a}\\text{l}\\right]\\:\\text{t}\\text{i}\\text{s}\\text{s}\\text{u}\\text{e}}{\\left[\\text{M}\\text{e}\\text{t}\\text{a}\\text{l}\\right]\\text{m}\\text{e}\\text{d}\\text{i}\\text{u}\\text{m}}\\)\u003c/span\u003e\u003c/span\u003e (Du et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWhere [Metal] tissue and [Metal] medium are the concentrations of HMs in the callus tissue and medium, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Electron Microscopy Ultrastructure Examination\u003c/h2\u003e \u003cp\u003eFor ultrastructural analysis, control, and treated callus samples were fixed in 3% glutaraldehyde in phosphate buffer, post-fixed in potassium permanganate for 5 min, and then dehydrated through an ethanol series. The samples were infiltrated with epoxy resin and sectioned into very thin slices, which were placed on copper grids. The sections were double-stained with uranyl acetate and lead citrate and examined using a transmission electron microscope (TEM) (JEOL JEM-1010) at 70 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Estimation of total carbohydrates and total proteins\u003c/h2\u003e \u003cp\u003eThe total carbohydrates were determined using an anthrone reagent (Morris \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1948\u003c/span\u003e; Dhumal et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The total proteins (water soluble- and insoluble) were determined with the Folin-Ciocalteau reagent (Lowry et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1951\u003c/span\u003e; Peterson \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1977\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Data Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the results was performed using analysis of variance (ANOVA). Significant differences between treatments were determined using the least significant difference (LSD) test at a p-value of \u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Effect of Different Plant Growth Regulators on Induction, Characteristics and Morphology of Callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe callus induction rates and shooting of \u003cem\u003eA. modesta\u003c/em\u003e were significantly influenced by the different concentrations of BA in the media (F-value= 1736.54; P-value= 0.000 and F-value= 2275.36; P-value= 0.000, respectively). The callus initiation started from 6 to 9 weeks (Table 1). The lowest induction and shooting rates were recorded under 4 mg/l of BA recording (18\u0026plusmn;\u0026nbsp;0.16 and 12\u0026plusmn;\u0026nbsp;0.21%, respectively) (Table 1).\u0026nbsp;At the end of the experiment after 12 weeks, the callus covered the entire explant surface. The resulting callus appeared whitish and friable in all BA concentrations (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe callus induction and shooting rates were significantly influenced by the different concentrations of Kn in the media (F-value= 1324.35; P-value= 0.000 and F-value= 3681.26; P-value= 0.000, respectively) (Table 1). The callus initiation started from 6 to 10 weeks. The lowest concentration of Kn (2 mg/l) recorded the lowest callus induction rates and shooting, while the largest callus induction rates and shooting were reported in (4 mg/l of Kn) (Table 1). At the end of the experiment, the callus covered the entire explant surface. The resulting callus appeared in the concentration (2 and 4 mg/l) of Kn yellowish friable, while, in high concentrations (6 and 8 mg/l) of Kn, the resulting callus appeared compact and yellowish (Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe callus induction rate in the calli of \u003cem\u003eA. modesta\u003c/em\u003e was significantly influenced by the different concentrations of 2,4-D in the media (F-value= 103982.15; P-value= 0.000). The callus initiation started from 3 to 5 weeks. The lowest callus induction rate was reported in the lowest concentration of 2,4-D recording (43\u0026plusmn;\u0026nbsp;0.16%). As the concentration of 2,4-D increased in the media, the callus rate increased till reached the highest concentration of 2,4-D (15 mg/l) which recorded the highest\u0026nbsp;callus induction rate (100 %\u0026plusmn;0) (Table 1).\u0026nbsp;On the other hand,\u0026nbsp;the shooting induction rate was 00.0\u0026plusmn;0.0 in the media supplemented with 2,4-D (Table 1).\u0026nbsp;At the end of the experiment, the quality of the resulting callus was the best in 2,4-D treatment at all concentrations. The resulting callus appeared whitish friable in the lowest concentration of 2,4-D (5 mg/l), compact yellowish in (2,4-D 10 mg/l), and compact brownish in the highest concentration of 2,4-D (15 mg/l) (Fig. 4). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.2. Effect of Cadmium, Nickel, and Lead\u0026nbsp;on Callus Growth and their accumulation in the callus tissue\u003c/h2\u003e\n\u003cp\u003eThe quality of the callus was reduced in the media treated with increasing concentration of Cd. The calli color was changed from a whitish friable in the lowest concentration of Cd (0.125 ppm) to a dehydrated compact brownish in the highest concentration of Cd (1 ppm) (Table 2). The highest callus biomass was observed in the control recording 4.65 and 4.6\u0026nbsp;mg/l DW for 10, and 15\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e2,4-D, respectively, followed by the lowest concentration of Cd (0.125 ppm), recording 3.25 and 3.15 mg/l\u0026nbsp;DW for 10, and 15 2,4-D, respectively. As the concentration of Cd increased, the callus biomass decreased (Table 2). A severe reduction of callus biomass was observed in the highest concentration of Cd (1 ppm), recording 0.45 and 0.6\u0026nbsp;mg/l\u0026nbsp;DW for 10, and 15 2,4-D, respectively (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe BAF of Cd in the calli of \u003cem\u003eA. modesta\u003c/em\u003e was significantly influenced by the different concentrations of Cd in the media\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe highest BAF (0.072 for 10 2,4-D) was obtained in the highest concentration of Cd (1 ppm). The lowest concentration of Cd (0.125 ppm) corresponded to the lowest BAF (0.039 and 0.017 for 10 2,4-D\u0026nbsp;and 15 2,4-D, respectively) (Table 2).\u003c/p\u003e\n\u003cp\u003eThe color of the callus varies from a friable whitish and yellowish nodular in low concentrations of Ni to a compact brownish in the highest concentration of Ni (1 ppm) (Table 2). The highest callus biomass was observed in control, recording 4.65 and 4.6\u0026nbsp;mg/l DW for 10, and 15\u0026nbsp;2,4-D, respectively, followed by\u0026nbsp;the lowest concentration of Ni (0.125 ppm), recording 2.85 and 3.65\u0026nbsp;mg/l DW for 10, and 15\u0026nbsp;2,4-D, respectively (Table 2). The more the concentration of Ni increases in culture media, the calli biomass were reduced. The highest concentration of Ni (1 ppm) causes inhibition of callus growth and contains the lowest callus biomass, recording 0.65 and 0.8\u0026nbsp;mg/l DW for 10, and 15\u0026nbsp;2,4-D respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe BAF of Ni in the calli of \u003cem\u003eA. modesta\u003c/em\u003e was significantly influenced by the different concentrations of Ni in the media. The highest BAFs (0.888 and 0.794 for 10 2,4-D and 15 2,4-D, respectively) were obtained under the influence of the highest Ni concentration (1 ppm) (Table 2).\u003c/p\u003e\n\u003cp\u003eThe addition of Pb in various concentrations to the culture media results in the inhibition of callus growth. As the concentrations of Pb increased the more hazardous effects were caused to the callus. The color of calli became compact yellowish in the low concentration of Pb and turned into a dehydrated compact dark brownish with necrotic in the highest concentration of Pb (1 ppm) (Table 2). High concentrations of Cd and Pb in the culture media resulted in a severe reduction in the mucilaginous material, resulting in the dehydration of calli with necrotic. The highest callus biomass was observed in control, recording 4.65 and 4.6 mg/l\u0026nbsp;DW for 10, and 15 2,4-D, respectively, followed by the lowest concentration of Pb (0.125 ppm), recording 0.85 and 0.9 mg/l\u0026nbsp;DW for 10, and 15 2,4-D respectively. The lowest callus biomass was observed in the highest Pb concentration (0.5 ppm), recording 0.6 and 0.65 mg/l\u0026nbsp;DW for 10, and 15 2,4-D, respectively.\u003c/p\u003e\n\u003cp\u003eThe BAF was significantly influenced by the different concentrations of Pb in the calli of \u003cem\u003eA. modesta\u003c/em\u003e (Table 2). The highest BAFs were found in the highest concentration of Pb (0.5 ppm). (1.464 and 2.06 for 10 2,4-D and 15 2,4-D, respectively (Table 2).\u003c/p\u003e\n\u003ch2\u003e3.3. Callus Ultra-structural Changes under Heavy Metal Treatments\u003c/h2\u003e\n\u003cp\u003eThe ultrastructure of \u003cem\u003eAcacia modesta\u003c/em\u003e callus cells was observed under transmission electron microscopy (TEM) to examine cellular components and structural changes under treatment of 1 ppm of Cd, Pb, and Ni compared to control (Fig. 6). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eControl cells display well-organized organelles. Larger cells contained large vacuoles with electron-dense regions and thin cytoplasmic cell wall segments with few plasmodesmata. The walls appeared normal, cytoplasm contained all the cellular organelles including mitochondria, endoplasmic reticulum with\u0026nbsp;ribosomes, chloroplasts, nucleus, nucleolus, and well-defined cell walls and membranes as indicative of healthy metabolic activity (Fig. 6, A).\u003c/p\u003e\n\u003cp\u003eSignificant ultra-structural changes in the callus tissue exposed to 1 ppm of Cd where cells were characterized by increased vacuolation, mitochondrial reduction, and formation of electron-dense granules. In addition, disruption of chloroplast, plasmolytic shrinkage, disintegration of the nucleus, shrinkage of cytoplasm, abnormal structures of organelle, thickening and constriction of cell wall and cell collapse, and disintegration were recorded (Fig. 6, B).\u003c/p\u003e\n\u003cp\u003eThe most significant negative impact on the tissue of \u003cem\u003eA. modesta\u003c/em\u003e was observed with Pb compared to other metals. It was found to be accumulated in higher concentrations within the cells Compared to Ni and Cd, suggesting it is more readily absorbed or retained by the cells. This higher accumulation likely contributes to its stronger adverse effects, resulting in more severe tissue damage, with variations noted in chloroplast morphology, mitochondrial integrity, and membrane structure under metal exposure. These findings underscore the critical role of lead in causing cellular stress and damage, making it the most detrimental metal analyzed in this study (Fig. 6, C)\u003c/p\u003e\n\u003cp\u003eSignificant changes in the cellular anatomy of \u003cem\u003eA. modesta\u003c/em\u003e in response to nickel exposure, including cell wall thickening and structural irregularities at higher concentrations, chloroplasts showed signs of damage, including disrupted thylakoid membranes and reduced granal stacking, and vacuoles appeared enlarged and contained electron-dense materials, indicating possible accumulation of nickel or secondary metabolites as a response to stress (Fig. 6, D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Effect of Cadmium, Nickel, and Lead on\u003c/strong\u003e \u003cstrong\u003eContent of Total Carbohydrates and Total Proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe content of total carbohydrates in the calli of\u0026nbsp;\u003cem\u003eA. modesta\u003c/em\u003e was significantly influenced by the different concentrations of HMs in the media (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe content of carbohydrates in the lowest concentration of Cd (0.125 ppm) was the highest compared to the other concentrations, it was two-fold than that of the control recording. The content of carbohydrates decreased with increasing Cd concentration in the media (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe lowest concentration of Ni (0.125 ppm) resulted in the highest content of carbohydrates (178.72\u0026plusmn;5.10 and 137.16\u0026plusmn;3.05 mg/g DW for 10 2,4-D and 15 2,4-D respectively). In addition, the calli grown in the largest concentration of Ni (1 ppm) contained the lowest carbohydrate contents (96.5\u0026plusmn;0.408 and 72.78\u0026plusmn;1.035 mg/g DW for 10 2,4-D and 15 2,4-D respectively) (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ethe lowest concentration of Pb in the calli of \u003cem\u003eA. modesta\u003c/em\u003e treated with (control 0 ppm of 10 2,4-D) and (0.125 ppm of 15 2,4-D) contained the highest content of carbohydrates (121.942\u0026plusmn;1.4 and 297.47\u0026plusmn;10.85 mg/g DW respectively). In addition, the lowest carbohydrate contents were reported in (0.5 ppm of 10 2,4-D) and (the control 0 ppm of 15 2,4-D) recording 69.62\u0026plusmn;0.54\u0026nbsp;and 79.74\u0026plusmn;4.38\u0026nbsp;mg/g DW, respectively (Table 2).\u003c/p\u003e\n\u003cp\u003eThe content of total proteins in the calli of \u003cem\u003eA. modesta\u003c/em\u003e was significantly influenced by the different concentrations of both Cd and Ni in the media (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe highest content of protein for the calli grown in 10 2,4-D was measured in the (0.25 ppm) Cd concentration (34.48\u0026plusmn;0.53 mg/g DW) while the calli grown in the control (0 ppm Cd) exhibited the highest protein content for 15 2,4-D of values equal to (43.38\u0026plusmn;0.85 mg/g DW). The highest concentration of Cd (1ppm) had the lowest content of protein (20.05\u0026plusmn;0.6 and 18.35\u0026plusmn;0.45 mg/g DW for 10 2,4-D and 15 2,4-D, respectively) (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe lowest concentration of Ni (0.125 ppm), protein content was the highest concentration (38.949\u0026plusmn;0.98 and 43.68\u0026plusmn;0.67 mg/g DW for 10 2,4-D and 15 2,4-D respectively), and exhibited approximately the same content of protein of the control (31.43\u0026plusmn;0. and 43.68\u0026plusmn;0.851 mg/g DW for 10 2,4-D and 15 2,4-D respectively) (Table 2).\u003c/p\u003e\n\u003cp\u003eThe total proteins in calli of \u003cem\u003eA. modesta\u003c/em\u003e was not significantly different in the calli growing in different concentrations of Pb in 10 2,4-D \u003cem\u003e(\u003c/em\u003eF-value= 2.74; P-value= 0.113) while it was significantly affected by Pb in 15 2,4-D (F-value= 166.35; P-value= 0.00) (Table 2). Protein content was the highest concentration in the control (43.68\u0026plusmn;0.851 mg/g DW of 15 2,4-D). As the concentration of Pb increased, the protein content gradually decreased. the lowest protein contents were reported in (the control 0 ppm of 10 2,4-D) and (0.5 ppm of 15 2,4-D) recording 31.43\u0026plusmn;0.45\u0026nbsp;and\u0026nbsp;22.184\u0026plusmn;1.17\u0026nbsp;mg/g DW, respectively.\u003c/p\u003e\n\u003cp\u003eMultivariate data analysis such as hierarchicalclustering analysis (HCA) and principal\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003ecomponent analysis (PCA) were performed by using Minitab (version\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e18.1) to show the different patterns (Fig. 6). Fig (6, A) shows similarities or dissimilarities between different variables. \u0026nbsp; Biomass and protein had similar pattern , while, content of metal in the tissue and BAF had similar pattern \u0026nbsp;(Fig. 6, A). The first component of PCA plot (PC1) accounts for 58.0% of the variance; and it was correlated content of metal in callus tissue, BAF, and color and texture of callus (Fig. 6, B). \u0026nbsp;The second component of PCA plot (PC2) accounted for 14.4% of the variance; it was correlated to texture\u003cem\u003e.\u003c/em\u003e The third component of PCA plot (PC3) accounted for 12.6 % of the variance; it was correlated to carbohydrates, proteins, metal in tissue, and BAF. Exposure of callus to elevated concentrations of Pb (0.25-0.5) was positively linked to accumulation of metal in the tissue and affecting the color of the tissue. Low concentration of Ni as well as control treatments was positively linked to enhancement of the protein content and\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe biomass of the callus (Fig. 6, B).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDespite the heavy metal-induced stress, \u003cem\u003eA. modesta\u003c/em\u003e callus cultures displayed varying degrees of tolerance to Cd, Ni, and Pb. The species’ ability to form callus at low metal concentrations, as well as the morphological adaptations to high metal levels, suggests the potential for phytoremediation applications.\u003c/p\u003e \u003cp\u003ePrevious studies on \u003cem\u003eAcacia\u003c/em\u003e species, including \u003cem\u003eA. catechu\u003c/em\u003e and \u003cem\u003eA. mangium\u003c/em\u003e, have indicated their potential for phytoremediation, particularly in heavy metal-contaminated soils (Ghosh and Singh 2005).\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Effect of Plant Growth Regulators on Callus Induction\u003c/h2\u003e \u003cp\u003eOur study showed that 2,4-D was the most effective plant growth regulator for inducing callus formation in \u003cem\u003eA. modesta\u003c/em\u003e, consistent with previous reports on \u003cem\u003eAcacia\u003c/em\u003e species and other leguminous and medicinal plants (Mousa \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shahrour et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While BA and Kn also promoted callus formation, their efficacy was lower than 2,4-D. This could be due to the specific nature of 2,4-D, which induces rapid cell division and differentiation, particularly in \u003cem\u003eAcacia\u003c/em\u003e species (Reséndiz \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gantait et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). At higher concentrations of 2,4-D, however, callus quality decreased, as evidenced by the shift to compact, brownish callus. This indicates that excessive hormone concentrations may induce stress, reducing callus vitality and regeneration potential (Al Gethami and El Sayed \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Impact of Cadmium, Nickel, and Lead on Callus Growth\u003c/h2\u003e \u003cp\u003eExposure to Cd, Ni, and Pb significantly reduced callus growth and biomass production, with Pb showing the most pronounced toxicity. These results are consistent with the established understanding that Pb is highly toxic to plant cells, even at low concentrations (Riyazuddin et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lead disrupts critical plant processes such as photosynthesis and nutrient uptake, leading to stunted growth and necrosis (Tarragó and Brown 2017). Similarly, Cd and Ni exposure led to a concentration-dependent reduction in fresh and dry weight, with high concentrations causing necrosis and discoloration of the callus. These findings are in line with studies on other species, such as \u003cem\u003eBrassica juncea\u003c/em\u003e and \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, where high metal concentrations inhibited callus growth and led to morphological changes (Kumari et al. 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.3. The Bioaccumulation Factor (BAF) of Cadmium, Nickel, and Lead\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eNegative effects on ecosystems can arise from the bioaccumulation of pollutants within living organisms. This process, in which organisms absorb and hold harmful pollutants from their surrounding contaminated environment, is referred to as BAF (Isensee \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Plants are categorized as hyperaccumulators if their BAF greater than 1 (Bashir et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It was reported in this study that the BAF of these HMs in \u003cem\u003eA. modesta\u003c/em\u003e calli rose in proportion to the concentration of HMs in the culture media.\u003c/p\u003e \u003cp\u003eIn the current study, it was found that the BAF of Cd in \u003cem\u003eA. modesta\u003c/em\u003e calli was less than 1. Similarly, potatoes also exhibited a BAF of Cd below 1 (Liu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, higher BAF values exceeding 1 for Cd were observed in \u003cem\u003eArtemisia selengensis\u003c/em\u003e, \u003cem\u003eBrassica juncea\u003c/em\u003e, and tea (Xu et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nepal et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ju et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll Ni treatments in the present study resulted in BAFs exceeding 1 in \u003cem\u003eA. modesta\u003c/em\u003e calli. Similarly, the BAF of Ni was found to be greater than 1 in both \u003cem\u003eAcacia saligna\u003c/em\u003e and \u003cem\u003eAcacia polyacantha\u003c/em\u003e, based on a comparable line of analysis by Masvodza et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). But in \u003cem\u003eEichhornia crassipes\u003c/em\u003e, the BAF on Ni was below1 (Mahamood et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe BAF of Pb in \u003cem\u003eA. modesta\u003c/em\u003e calli was greater than 1. In a similar study, Mahdavi et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) observed that the BAF of Pb in \u003cem\u003eAcacia victoria\u003c/em\u003e was less than 1. Likewise, research by Mohamed et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) demonstrated that \u003cem\u003eJatropha curcas\u003c/em\u003e also exhibited a BAF of Pb below 1.\u003c/p\u003e \u003cp\u003eAccording to the current investigation, the BAF of the three HMs was discovered in the declining order Pb \u0026gt; Ni \u0026gt; Cd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Ultrastructural Changes Induced by Cadmium, Nickel, and Lead\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) provided detailed insights into the cellular changes induced by heavy metals. Control callus cells exhibited healthy organelles, including intact chloroplasts and mitochondria, while heavy metal exposure caused significant ultrastructural damage. Cd exposure resulted in cytoplasmic shrinkage, plasmolysis, and thickening of the cell walls, suggesting a response to osmotic stress and cell wall fortification as a protective mechanism against toxicity (Shanying et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Nickel exposure led to reductions in vascular bundle size and mesophyll thickness, which likely impaired the transport of nutrients and water, contributing to growth inhibition (Hassan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rehman et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Pb caused severe vacuolation, mitochondrial disruption, and necrosis, indicating its high toxicity and potential to disrupt cellular energy metabolism (Malar et al. 2016; Kohli et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These structural changes highlight the severe impact of heavy metals on cellular integrity and function, which could contribute to the reduced growth and viability of callus tissues (Jorjani and Karakaş \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Effect of Cadmium, Nickel, and Lead on Total Carbohydrates and Total Proteins\u003c/h2\u003e \u003cp\u003e \u003cb\u003eTotal carbohydrates.\u003c/b\u003e There is a strong influence of HMs on carbohydrate contents in plants (Yang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cadmium affects carbohydrate metabolism in various ways and leads to changes in their content, which can result from the inactivation and alteration of certain enzymes involved in carbohydrate synthesis (Latef \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, the content of total carbohydrates was increased at a low concentration of Cd, while it decreased with increasing metal concentration. In the same context, an increase in soluble sugars at low concentrations of metal stress and a decrease at higher concentrations as in rice seedlings, \u003cem\u003ePisum sativum\u003c/em\u003e, and \u003cem\u003eLemna polyrrhiza\u003c/em\u003e (John et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e ).\u003c/p\u003e \u003cp\u003eThe digestion and mobilization of reserved food have been reported to be affected by Ni pollution such as carbohydrates and proteins in sprouted seeds (Mustafa et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the present study, a low concentration of Ni increased the content of total carbohydrates, while it decreased with increasing metal concentration. Similarly, in wheat seedlings, carbohydrate content was increased at low concentrations of Ni (Loreti et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Reducing and non-reducing sugars were reduced under high concentrations of Ni due to decreasing the activity of α-amylase in two sunflower cultivars (Ashraf et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In contrast, the content of soluble sugars is increased under Ni toxicity in \u003cem\u003eEruca sativa\u003c/em\u003e (Kamran et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), tomato (Okunlola et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eCatharanthus roseus\u003c/em\u003e (Yasin et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and \u003cem\u003eCucurbita pepo\u003c/em\u003e (Valivand et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTolerance to Pb toxicity involves slowing the generation of radicals through increased protein and carbohydrate content and affecting the peroxidase activity and polyphenol oxidases (Singh et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the present study, a low concentration of Pb increased the content of total carbohydrates, while it decreased with increasing metal concentration. Vegetables grown under different Pb concentrations had lower glucose and sucrose content with higher Pb doses than vegetables and crops grown on control substrates (Mishra and Dubey \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). On contrary, total carbohydrate and starch levels increased under Cd and Pb stress in \u003cem\u003eVicia faba\u003c/em\u003e (Abu-Muriefah \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and in \u003cem\u003eCoronopus didymus\u003c/em\u003e (Sidhu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal proteins.\u003c/b\u003e Heavy metals cause low protein content in plants grown on metal-contaminated soil due to changes in plant physiology as reduction of nitrate reductase activity (Widowati \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, the protein content was reduced with increasing Cd dose. These results are line with the results of Galal (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Galal et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) who reported that proteins decreased in \u003cem\u003eCucurbita pepo\u003c/em\u003e and \u003cem\u003ePisum sativum\u003c/em\u003e tissues grown on contaminated media with Cd. Likewise, the total protein content showed a significant reduction in dose-dependent in \u003cem\u003eCatharanthus Roseus\u003c/em\u003e calli exposed to Cd different concentrations (Abnosi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, the total content of protein was increased due to the irrigation of two poplar species (\u003cem\u003ePopulus nigra\u003c/em\u003e and \u003cem\u003ePopulus alba\u003c/em\u003e) with treated wastewater containing Cd, Ni, and Pb (Houda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHigh Ni levels result in inhibition of key enzymes involved in the digestion of food reserves (proteases and α- and β-amylase), protein synthesis, carbohydrate metabolism, and reserve mobilization (Chaki et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, the protein content was reduced with increasing Ni dose. Similarly, Ashraf et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) found that exposure to Ni stress significantly inhibited protease activity and thus the conversion of stored proteins into amino acids. In addition, Ni can cause the reduction of proteins, especially low molecular weight proteins, which increases oxidative stress in many plant species (Silva et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, a slight increase in total protein content was found in plants subjected to Ni stress (Maheshwari and Dubey \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effect of Pb on total protein concentration is unclear, although high concentrations can decrease the protein content (Piotrowska et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), some amino acids, such as proline, increase under lead stress (Qureshi et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These proteins play an important role in plant tolerance to Pb. The present study revealed that compared to the control, protein decreased at all Pb concentrations. The same results were revealed by Gupta et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Zhang et al. (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) when studying the effect of three HMs on the total content of protein in \u003cem\u003eZea mays\u003c/em\u003e and \u003cem\u003eHydrilla verticillata\u003c/em\u003e; it was concluded that an increase in the concentration of three HMs leads to a decrease in the total protein content and the highest content was detected in the control. In contrast, due to Pb exposure, the soluble protein content showed a significant increasing with increasing Pb concentration in c\u003cem\u003eoleoptiles\u003c/em\u003e of wheat (Lamhamdi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and \u003cem\u003eCoronopus didymus\u003c/em\u003e (Sidhu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe heavy metals caused significant reductions in callus biomass, changes in morphology, and ultrastructural damage, highlighting the phytotoxic nature of these metals. While all three metals inhibited callus growth, the ability of \u003cem\u003eA. modesta\u003c/em\u003e to form callus and exhibit morphological adaptations at lower metal concentrations suggests its potential for phytoremediation.\u003c/p\u003e\u003cp\u003eFurther research is needed to explore the genetic and biochemical mechanisms underlying \u003cem\u003eA. modesta\u003c/em\u003e tolerance to heavy metals, with a particular focus on metal accumulation and detoxification pathways. Understanding these mechanisms could pave the way for developing more efficient and sustainable phytoremediation strategies for the environmental cleanup of heavy metal-contaminated soils.\u003c/p\u003e\u003cp\u003eThe present study focused on callus cultures; however, further research is needed to evaluate the performance of intact plants in phytoremediation efforts, particularly in the context of metal hyperaccumulation and detoxification mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003e \u003cb\u003eConflicts of Interest\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eConceptualization, T.A.A.R.; methodology, R.V., T.A.A.R., A.A.A.M; software, R.V., T.A.A.R, A.A.A.M, E,E; formal analysis, R.V., A.A.A.M; investigation, T.A.A.R, R.V., A.A.A.M; resources, T.A.A.R; data curation, T.A.A.R, R.V., A.A.A.M; writing\u0026mdash;original draft preparation, TAARR.V., A.A.A.M; writing\u0026mdash;review and editing, TAAR, A.A.A.M. E.E; supervision, E.E. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThe authors appreciate of the facilities that were provided by the Department of Botany at Aswan University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbnosi MH, Amirjani M, Mahdiyeh M, Moradipoor H (2015) Biochemical and cellular response of \u003cem\u003eCatharanthus roseus \u003c/em\u003ecallus cells to cadmium toxicity. J Genet Resour 1:101\u0026ndash;114. \u003c/li\u003e\n\u003cli\u003eAbu-Muriefah SS (2015) Effects of silicon on Faba bean (\u003cem\u003eVicia faba\u003c/em\u003e L.) plants grown under heavy metal stress conditions. 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Environ Geochem Health 45:5441\u0026ndash;5466\u003c/li\u003e\n\u003cli\u003eZhang H, Zhang L-L, Li J, et al (2020) Comparative study on the bioaccumulation of lead, cadmium and nickel and their toxic effects on the growth and enzyme defence strategies of a heavy metal accumulator, \u003cem\u003eHydrilla verticillata\u003c/em\u003e (Lf) Royle. Environ Sci Pollut Res 27:9853\u0026ndash;9865\u003c/li\u003e\n\u003cli\u003eZulfiqar U, Ayub A, Hussain S, et al (2022) Cadmium toxicity in plants: Recent progress on morpho-physiological effects and remediation strategies. J soil Sci plant Nutr 22:212\u0026ndash;269\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Effect of MS basal media supplemented with different growth regulators (BA, Kinetin, and 2,4-D) on induction of callus and characteristic of callus proliferated from nodal cutting of \u003cem\u003eAcacia modesta\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"627\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth regulator\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg/l)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeeks to Callus Initiation Callus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCallus induction rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDegree of callus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eShooting induction rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e8-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e19\u003csup\u003ec\u0026plusmn;0.092\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eWF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e25\u003csup\u003eb\u0026plusmn;0.354\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e7-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e18\u003csup\u003ed\u0026plusmn;0.156\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eWF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e12\u003csup\u003ed\u0026plusmn;0.212\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e6-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e24\u003csup\u003eb\u0026plusmn;0.156\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eWF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e18\u003csup\u003ec\u0026plusmn;0.170\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e7-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e30\u003csup\u003ea\u0026plusmn;0.283\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eWF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e30\u003csup\u003ea\u0026plusmn;0.141\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u0026nbsp;8-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e18\u003csup\u003ed\u0026plusmn;0.148\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eYF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e11\u003csup\u003ed\u0026plusmn;0.092\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u0026nbsp;8-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e24\u003csup\u003ea\u0026plusmn;0.099\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eYF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e24\u003csup\u003ea\u0026plusmn;0.014\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e6-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e23\u003csup\u003eb\u0026plusmn;0.078\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e15\u003csup\u003ec\u0026plusmn;0.226\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e7-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e20\u003csup\u003ec\u0026plusmn;0.092\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e20\u003csup\u003eb\u0026plusmn;0.106\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e4-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e43\u003csup\u003ec\u0026plusmn;0.177\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eYF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u003csup\u003e\u0026plusmn;0.0\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e3-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e60\u003csup\u003eb\u0026plusmn;0.099\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u003csup\u003e\u0026plusmn;0.0\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 4-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003csup\u003ea\u0026plusmn;0.0\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e0.0\u003csup\u003e\u0026plusmn;0.0\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Note: Data were recorded after 12 weeks of culture when the callus can be seen clearly by the naked eye. WF=whitish friable; YF=yellowish friable; YG=yellowish granular; CY=compact yellowish; CN=compact nodular; NB=nodular brownish; CGN=compact greenish nodular; CY=compact yellowish nodular; CB=compact brownish. - No Callus, + Very poor callus, ++ Poor callus, +++ Good callus, ++++ Very good callus.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Influence of 2,4-D and Heavy Metal Concentrations (Cd, Ni, and Pb) on Biomass Production, Callus Characteristics, and Metal Uptake in Plant Tissue Cultures\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth hormone (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 416px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth characteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMetal (ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiomass (mg/l DW)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColor\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTexture\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetal in tissue\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003emg/kg DW)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.005\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.039\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.75\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003egranular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.010\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.049\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.65\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.031\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.063\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.45\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.072\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.072\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4.6\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3.15\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.002\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.017\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.9\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003egranular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.006\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.022\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.8\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.021\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.042\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.12\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.04\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNi\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.65\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2.85\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.026\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.204\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.75\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003enodular\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.076\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.306\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.4\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.238\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.475\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.65\u003csup\u003e\u0026nbsp;e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.888\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.888\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.6\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3.65\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.018\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.147\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2.3\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003enodular\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.071\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.283\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.6\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.214\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.427\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.8\u003csup\u003e\u0026nbsp;e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.794\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.794\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4.65\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.65\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.085\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.65\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.346\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.385\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.6\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDark Brownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.732\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.464\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e4.6\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003ewhitish\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003efriable\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.9\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYellowish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.142\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.136\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.8\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.513\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.051\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.065\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eDark Brownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eCompact\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.032\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2.061\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eImpact of growth hormones and heavy metal exposure on primary metabolite production in calli of \u003cem\u003eAcacia modesta\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth hormone (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 304px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cstrong\u003ePrimary metabolite (mg/g DW)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetal (ppm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbohydrates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCd\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e121.94\u003csup\u003ec\u003c/sup\u003e\u003csup\u003e\u0026plusmn;1.4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e31.43\u003csup\u003eb*\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.45\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.125 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e239.26\u003csup\u003ea\u003c/sup\u003e\u003csup\u003e\u0026plusmn;2.06\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e26.98\u003csup\u003ec\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.52\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e188.52\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026plusmn;11.2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e34.48\u003csup\u003ea\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.53\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e179.87\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026plusmn;5.05\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e32.48\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.52\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e126.79\u003csup\u003ec\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e20.05\u003csup\u003ed\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e79.74\u003csup\u003eCD\u003c/sup\u003e\u003csup\u003e\u0026plusmn;4.38\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e43.38\u003csup\u003eA\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.85\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e177.07\u003csup\u003eA\u003c/sup\u003e\u003csup\u003e\u0026plusmn;7.74\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e36.67\u003csup\u003eB\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.91\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e114.12\u003csup\u003eB\u003c/sup\u003e\u003csup\u003e\u0026plusmn;1.55\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e29.60\u003csup\u003eC\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.45\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e89.18\u003csup\u003eC\u003c/sup\u003e\u003csup\u003e\u0026plusmn;1.06\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e31.48\u003csup\u003eC\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.98\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e76.04\u003csup\u003eD\u003c/sup\u003e\u003csup\u003e\u0026plusmn;2.12\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e18.35\u003csup\u003eD\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.45\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNi\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e121.94\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026plusmn;1.4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e31.43\u003csup\u003ec\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.45\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e178.72\u003csup\u003ea\u003c/sup\u003e\u003csup\u003e\u0026plusmn;5.1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e38.95\u003csup\u003ea\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.98\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e175.38\u003csup\u003ea\u003c/sup\u003e\u003csup\u003e\u0026plusmn;16.91\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e33.18\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e119.90\u003csup\u003eb\u003c/sup\u003e\u003csup\u003e\u0026plusmn;2.15\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e29.38\u003csup\u003ed\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e96.50\u003csup\u003ec\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.41\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e16.99\u003csup\u003ee\u003c/sup\u003e\u003csup\u003e\u0026plusmn;0.77\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15 (2,4-D)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u0026nbsp;0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003e79.74\u003csup\u003eD\u003c/sup\u003e\u003csup\u003e\u0026plusmn;4.38\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n 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\u003c/tbody\u003e\n\u003c/table\u003e"}],"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":false,"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":"Acacia modesta, Cadmium, Callus, Heavy metals, Nickel, Lead, Phytoremediation, Transmission electron microscopy (TEM)","lastPublishedDoi":"10.21203/rs.3.rs-6314982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6314982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFour concentrations (2, 4, 6, and 8 mg/L) of benzyladenine (BA) and kinetin (Kn) along with three concentrations (5, 10, and 15 mg/L) of 2,4-dichlorophenoxyacetic acid (2.4-D) were incorporated into Murashige and Skoog (MS) medium to investigate their effectiveness in inducing callus formation from axillary buds of \u003cem\u003eA. modesta\u003c/em\u003e. It was found that the 2,4-D concentration of 10 and 15 mg/L had the most favorable effect on the induction rates and growth characteristics of \u003cem\u003eA. modesta.\u003c/em\u003e Afterwards, the impacts of various concentrations (0, 0.125, 0.25, 0.5, and 1 ppm) of cadmium (Cd), nickel (Ni), and lead (Pb), on the morphological attributes and primary metabolites were measured in \u003cem\u003eA. modesta\u003c/em\u003e calli grown at MS medium supplemented with 10 and 15 mg/L 2,4-D. In addition, ultrastructural attributes were investigated using transmission electron microscopy (TEM) in \u003cem\u003eA. modesta\u003c/em\u003e calli grown at the highest metal concentrations.\u003c/p\u003e \u003cp\u003eAt low concentrations of the three heavy metals, the calli exhibited minimal morphological changes. However, at the highest concentrations, callus growth was significantly reduced, as evidenced by lower fresh weights and altered morphological characteristics. High metal concentrations caused compact, dehydrated, and necrotic tissues. Cd exposure induced the most severe effects, including cell wall thickening, cytoplasmic shrinkage, and plasmolysis, which were observed. Ni treatment led to reduced vascular bundle size, mesophyll thickness, and chloroplast integrity, while Pb exposure resulted in extensive vacuolation and mitochondrial disruption.\u003c/p\u003e \u003cp\u003eThe concentrations (0.125 and 0.25 ppm) of the three metals exhibited the highest contents of carbohydrates and proteins. The highest concentrations (1 ppm) of the metals had the lowest contents of carbohydrates and proteins.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. modesta\u003c/em\u003e callus cultures displayed varying degrees of tolerance to Cd, Ni, and Pb indicated by its ability to form callus at low metal concentrations, as well as the morphological adaptations to high metal levels, suggests potential for phytoremediation applications.\u003c/p\u003e","manuscriptTitle":"Growth, biochemical, and ultrastructural analyses of Acacia modesta callus Wall. in response to different concentrations of growth regulators and heavy metals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 14:54:00","doi":"10.21203/rs.3.rs-6314982/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":"aa597b19-ffb8-49e7-8ab6-f63a247644d0","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject, encouragement to resubmit","date":"2026-05-19T03:16:02+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T08:01:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 14:54:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6314982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6314982","identity":"rs-6314982","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}