30 Exponential population growth has caused significant damage to the environment and ecosystems. One of the many factors affecting the environment is related to some heavy metals 'HMs' (Khan et al., 2021). There are several toxic HMs, including Cadmium 'Cd' (Saini & Dhania, 2020) that poses a threat to biota even at a very low concentration. Consumption of food contaminated with Cd is hazardous to human health (Khan et al., 2017). Accumulation of Cd can cause renal tubular dysfunction (Bernard, 2004) and affects the reproductive systems (Kumar & Sharma, 2019). Additionally, the presence of Cd affects mineral nutrient uptake in plants and their growth. This is associated with growth inhibition and low dry matter yield (Meena et al., 2018; Fattahi et al., 2019), photosynthesis and respiration inhibition (Navarro-León et al., 2019), and chlorosis (Chun et al., 2020). There are various methods, including physical, chemical, and biological approaches, being used and developed for remediation of HMs. One promising method that has the potential to save energy and cost is called 'phytoremediation' which involves using plants and plant processes to remove, contain, or reduce pollutants in the environment (Berti & Cunningham, 2000). In vitro selection and characterization of cadmium- tolerant calli of Tagetes erecta and Gomphrena globosa Ornamental plants (OPs) are beneficial to remove, control and reduce heavy metals (HMs) in a process called 'phytoremediation'. This study evaluated the in vitro system- based phytoremediation properties of Tagetes erecta and Gomphrena globosa calli. Leaves from in vitro seed-grown T. erecta and G. globosa were used as an explant source for callus culture. Callus culture was found optimal in MS medium supplemented with 8 μM BAP + NAA for T. erecta and 2 μ M 2, 4-D for G. globosa. These plants were grown in their respective optimized (controlled) medium enriched with different amounts (50, 100, 150, 200, and 250 μM) of Cadmium (Cd) added in the form of Cadmium chloride (CdCl2). The in vitro calli developed were evaluated for Cd stress tolerance based on callus diameter, growth tolerance index (GTI) and catalase (CAT) activity. Over four weeks, the callus diameters of T. erecta and G. globosa grown in different concentrations of Cd had lower growth than that of the controlled one. On the other hand, the GTI measured were greatest at 150 μM of Cd for both T. erecta (130.95%) and G. globosa (149.32%) suggesting a potential Cd tolerance. However, the CAT activity in T. erecta callus increased with the Cd concentration peaking at 150 µM then started declining while G. globosa callus showed the highest CAT activity at 50 μM of Cd. Thus, T. erecta callus showed greater Cd tolerance with the prospect of utilizing it for phytoremediation. The study also suggests growing T. erecta at 150 μM Cd for tolerant calli. Keywords: Cadmium tolerance, callus, catalase activity, growth tolerance index, ornamental plants. G. Lama 1, B. Shrestha 1, S. Limbu 1, P. R. Gurung 1, & K. K. Pant 1* Received: 4, March 2023 Revised: 20, May 2024 Accepted: 24, May 2024 Published: 31, May 2024 1 Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal. *E-mail: krishna.k.pant@gmail.com Banko Janakari, Vol 34 No. 1, 2024 Pp 30‒39https://doi.org/10.3126/banko.v34i1.66289 https://orcid.org/0009-0009-8772-7459 Banko Janakari, Vol 34 No. 1 31 Lama et al. The mechanism of HMs tolerance of plants has been the subject of numerous studies aimed at improving phytoremediation performance (Yan et al., 2020). Tissue culture-based in vitro breeding technique is a practical and economical way to improve plants. During the tissue culture process, callus cells can change due to mutagenic conditions (Phillips et al., 1994; Wang & Wang, 2012), leading to somaclonal variation (Skirvin et al., 1993). The culture stress process can also induce genetic and epigenetic changes (Gao et al., 2010), ultimately improving the genetics of the plant. The application of ornamental plants (OPs) for phytoremediation of HMs as well as beautification may be an attractive option (Khan et al., 2021). Tagetes erecta (locally called 'Sayapatri') and Gomphrena globosa (locally called 'Makhamali') are culturally important OPs in Nepal. These two plant species possess industrial value with extensive markets during 'Tihar festival' (i.e. festival of lights observed in Oct/Nov) in Nepal. It has been demonstrated that these plants can effectively accumulate Chromium (Coelho et al., 2017) and Arsenic (Signes-Pastor et al., 2015). In vitro testing systems based on OPs can be a quintessential tool for the characterization of possible phytoremediation of different HMs (Khan et al., 2021). In light of this knowledge, this study aimed to evaluate the potential phytoremediation properties of in vitro calli developed from T. erecta and G. globosa (see Figure 1). Materials and methods In vitro seed germination and callus culture The seeds of T. erecta and G. globosa, obtained from the Puspa Bikash Kendra, Godawari, Lalitpur, Nepal, were separated from other floral parts and cleaned under running water with added 'Tween 20 solution' for 30 minutes. Sterilization was performed inside a laminar flow cabinet using 1% Sodium hypochlorite (NaOCl) solution and 70% ethanol (CH3CH2OH). The seeds were then germinated and grown on a hormone- free MS medium as described by Murashige & Skoog (1962). Later on, the leaves from the seed-grown plants (size: 1cm × 0.5cm) were removed and used as explants for growing calli in culture. The hormones 6-Benzylaminopurine (BAP), Naphthaleneacetic acid (NAA), and 2, 4-Dichlorophenoxyacetic acid (2, 4-D) were added to the MS medium for callus culture (Belarmino et al., 1992; Bodhipadma et al., 2017). Callus growth in Cd-enriched media The type of medium used for growing callus was selected based on the percentage of callus induction and the average weight of the callus. The selected medium was enriched with different quantities of Cd (50, 100, 150, 200, and 250 μM) by adding Cadmium chloride (CdCl2) solution as described by Nehnevajova et al. (2007). The Figure 1: The in vitro germinated plantlets from seeds: (A) T. erecta and (B) G. globosa. Banko Janakari, Vol 34 No. 1 32 Lama et al. effects of Cd on callus growth were monitored by measuring the diameter of the callus using the Diameter = √Length×√Width (Compton, 1994; Chenar et al., 2016). formula: The callus growth tolerance index (GTI) was used to select calli that are tolerant to HMs exposure (Samantaray et al., 2001). It was calculated using the formula: Catalase activity Catalase activity was evaluated from the callius developed on a Cd-enriched medium as described by Zhang et al. (2007). To accomplish this, 1.0 gram of callus was combined with 3 ml of a buffer solution (50 mmol/L sodium phosphate buffer at pH 7.8 with 1.0 mmol/L Ethylene diamine Tetra acetic Acid (EDTA) and 2% polyvinylpyrrolidone) and then centrifuged for 10 minutes at 5000g (relative centrifugal force). The resulting enzyme extract was added to a reaction mixture containing phosphate buffer (pH 7.0), 0.1 mmol/L EDTA and 20 mmol/L hydrogen peroxide (H2O2), and the reaction was monitored by measuring the depletion of H2O2 at 240 nanometers applying the Beer-Lambert law and using the Molar extinction coefficient (ε) of 36 mol/L cm. As per the Beer-Lambert law, the absorbance (A) of H2O2 was calculated as: A = εLc, Where, ε = Molar extinction coefficient; L = Length of light path; and c = Concentration of sample. The results were reported as the amount of enzyme activity per milliliter of sample per gram of callus as described by Swinehart (1962). Statistical analysis The study collected data on the growth parameters (percentage callus induction, diameter, and GTI) from five different samples, and data on the activity of the catalase enzyme from three different samples. These were measured in the form of 'mean' and 'Standard Error'. A statistical test called 'Spearman's Correlation Test' was used to investigate the relationship between the concentration of Cd in the growth media and the activity of the catalase enzyme. Results Selection of callus induction media The T. erecta leaf explants showed the earliest callus initiation on medium supplemented with 8 μM BAP + NAA, with 80% of the explants forming callus in the first week as shown in Table 1. Table 1: Effects of BAP, NAA, and 2, 4-D supplemented MS media on callus initiation of T. erecta observed during a four-week period BAP (μM) NAA (μM) 2, 4-D (μM) Week 1 Week 2 Week 3 Week 4 Weight measured in Week 4 (gm) 0 0 0 0% 0% 0% 0% 0 2 2 0 0% 0% 40% 100% 0.85±0.12 4 4 0 0% 0% 0% 20% 0.33±0.14 6 6 0 0% 0% 0% 80% 0.77±0.27 8 8 0 80% 80% 80% 100% 1.03±0.20 10 10 0 0% 80% 80% 100% 1.25±0.35 0 0 10 0% 40% 60% 60% 0.82±0.32 0 0 11 40% 60% 60% 80% 0.64±0.33 0 0 12 0% 40% 40% 60% 0.91±0.40 0 0 13 0% 60% 80% 80% 0.67±0.29 0 0 14 60% 60% 80% 80% 0.83±0.3 Banko Janakari, Vol 34 No. 1 33 Lama et al. After four weeks, 2 μM, 8 μM, and 10 μM BAP + NAA showed 100% callus formation, but none of the concentrations of 2, 4-D showed 100% callus formation. After four weeks, calli were weighed where 10 μM BAP + NAA gave the highest final weight. All the calli obtained from the combination of BAP and NAA were greenish-yellow (see Figure 2A), whereas the calli added to the 2, 4-D supplemented medium were brownish and granular. Since the search was directed towards rapid callus initiation, the MS medium supplemented with 8 μM BAP + NAA was selected as a controlled growth medium for T. erecta calli. On the other hand, G. globosa showed callus initiation in the first week, with all the concentrations of 2, 4-D; however, 100% initiation was shown only in the media supplemented with 2 and 8 μM 2, 4-D. All the calli were pale yellow (see Figure 2C). As shown in Table 2, the MS medium containing 2 μM 2, 4-D showed the fastest callus initiation and better growth, and was thus used as a controlled growth medium for G. globosa calli. Figure 2: Callus culture of T. erecta and G. globosa: (A) callus developed from in vitro leaf explants of T. erecta in MS medium supplemented with 8 μM BAP+NAA; (B) T. erecta callus developed in 150 µM Cd enriched MS medium supplemented with 8 μM BAP+NAA; (C) Callus developed from in vitro leaf explants of G. globosa in MS medium supplemented with 2 μM 2,4-D; and (D) G. globosa callus developed in 150 µM Cd enriched MS medium supplemented with 2 μM 2,4-D. Banko Janakari, Vol 34 No. 1 34 Lama et al. Table 2: Effects of 2, 4-D supplemented media on callus growth of G. globosa observed during a four-week period 2, 4-D (μM) Week 1 Week 2 Week 3 Week 4 Weight measured in Week 4 (gm) 2 100% 100% 100% 100% 0.93±0.4 4 80% 80% 80% 80% 1.08±0.26 6 80% 100% 100% 100% 0.31±0.1 8 100% 100% 100% 100% 0.65±0.21 16 80% 100% 100% 100% 0.65±0.2 Callus diameter and GTI in Cd-enriched media In the first week, all of the Tagetes erecta calli samples grown in the controlled medium and those containing 100 and 150 μM of CdCl2 were successfully initiated. After two weeks, the calli grown in a medium with 100 μM CdCl2 had the greater diameter, followed by those with 50 μM CdCl2 (see Figure 3). In the third week too, the calli grown in a medium with 100 μM CdCl2 had the largest diameter. The highest increase in overall diameter was seen in the controlled calli, followed by those with 200 and 150 μM CdCl2. The T. erecta calli turned brown when exposed to all concentrations of Cd treatment, with the most severe browning observed with 250 μM CdCl2. Figure 3: Effects of CdCl2 concentrations (50, 100, 150, 200, and 250 μM) on the callus diameter of T. erecta. In the case of G. globosa, the calli grown on the controlled medium were found to have the highest diameter followed by the medium supplemented with 150 µM CdCl2 throughout all the four weeks of callus growth (Figure 4). The browning of the calli was observed at the concentration of 150 μM CdCl2 and above. Figure 4: Effects of CdCl2 concentrations (50, 100, 150, 200, and 250 μM) on the callus diameter of G. globosa. The GTI of both G. globosa and T. erecta calli were found to be the highest when the medium contained 150 μM of CdCl2 (Figure 5). Similarly, the second highest growth rate was observed in the medium containing 200 μM of CdCl2. The GTI for G. globosa was 149.32% while it was 130.95% for T. erecta, suggesting a potential tolerance level to Cd stress of calli in both the plants. Figure 5: GTI of T. erecta and G. globosa in media enriched with 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM CdCl2. Banko Janakari, Vol 34 No. 1 35 Lama et al. Catalase activity The CAT activity in the T. erecta calli were found to have increased with the increase in the concentration of CdCl2 in the medium, reaching a peak (nearly 60) at a concentration of 150 μM, before decreasing as shown in Figure 6. In contrast, the highest level of CAT activity (nearly 45) in G. globosa callus was observed at a concentration of 50 μM CdCl2. A positive correlation was seen between the concentration of CdCl2 and CAT activity in T. erecta callus up to 150 μM CdCl2 [Spearman’s rank correlation coefficient (1) = 0.54 & p = 0.18]. In contrast, G. globosa callus showed a significant negative correlation between the concentration of CdCl2 and CAT activity at 50 μM CdCl2 [Spearman’s rank correlation coefficient (1) = -0.51 & p = 0.5]. Figure6: CAT activity of calli of both T. erecta and G. globosa in controlled medium 'C' and the media supplemented with 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM CdCl2. Discussion The T. erecta callus were successfully induced with a combination of BAP and NAA at a concentration of 8 μM, similar to the results of previous studies by Benítez-García et al. (2014) and Munshi et al. (2021), although Belarmino et al. (1992) reported difficulty in regenerating callus from leaf explant. The browning of T. erecta callus in media supplemented with 2, 4-D could be caused by the oxidation of phenolic compounds, leading to cell death (Khosroushahi et al., 2011; Vijayalakhsmi & Shourie, 2017). In contrast, the G. globosa callus formation was the most effective at a low concentration of 2, 4-D, similar to the findings of Vieira et al. (1994) and Bodhipadma et al. (2017). The T. erecta callus grown in media containing 200 μM CdCl2 had the greatest diameter among all treatment groups, but it did not have the highest fresh weight. This phenomenon might be caused by the reduction in cell division and an increase in cell growth caused by the toxic effect of Cd (Zou et al., 2012). Labancová et al. (2020) found that poplar callus entered a linear phase characterized by decreased cell division rate and increased cell growth when exposed to 10 μM Cd. Additionally, the presence of chloride in the Cd source (i.e. CdCl2) has been reported to promote cell elongation, as it has a better ability to regulate osmosis and generate turgor pressure (Colmenero-Flores et al., 2019). In the case of G. globosa, both the growth in length and biomass were at the highest when the media contained 150 μM of CdCl2. This indicated that the growth in length and biomass of the callus had a similar pattern. Namjooyan et al. (2012) and Israr et al. (2006) both found that Cd reduced callus growth in Carthamus tinctorius and Gomphrena globosa, respectively, with the most significant reduction at higher concentrations (75 μM, 100 μM, and 250 μM, respectively). Our study also found that the callus growth was highest in the explants exposed to 150 μM Cd, which is suggested to be due to Cd accumulation. The GTI was higher in the calli exposed to 150 μM of CdCl2 for both T. erecta and G. globosa plants, indicating that these calli had a mechanism for tolerance to Cd (Bernabe´-Antonio et al., 2015). However, after a positive response to 150 μM CdCl2, the biomass declined again, which may be due to the phenomenon of hormesis which is an adaptive response where low levels of stress activate cellular and molecular pathways that enhance the ability of the cell and organism to withstand more severe stress (Calabrese et al., 2007; Bernebe-Antonio et al., 2015). A high level of antioxidant enzymes can improve tolerance to stress caused by heavy metals (Gechev et al., 2006). Tolerant calli have been found to have significantly higher CAT activity than non-tolerant calli (Rout & Sahoo, 2007). This study observed that as the concentration of CdCl2 increased, the activity of CAT initially increased, but then decreased at toxicity levels Banko Janakari, Vol 34 No. 1 36 Lama et al. of CdCl2. This decline in CAT activity was accompanied by the browning of the calli, which is a sign of cell necrosis caused by the production of phenolic compounds (Sandalio et al., 2001; Shekhawat et al., 2010). Browning was more severe when CAT activity decreased, suggesting that phenolic compounds were replacing the scavenging role of Catalase on Hydrogen peroxide (Michalak, 2006). Phenolic compounds can act as antioxidants in their reduced form, but are cytotoxic in their oxidized form (Michalak et al., 2006; Khosroushahi et al., 2011; Vijayalakshmi & Shourie, 2017). It was suggested that the variation in the activity of the calli in different media with and without Cd was caused by mutation. The presence of Cd in culture media can lead to increased somaclonal variation and spontaneous mutations in some callus cells, resulting in regeneration with altered metal accumulation (Nehnevajova & Herzig, 2007). The use of phytohormones such as 2, 4-D and NAA can also lead to mutation through an increase in cytosine methylation in plant tissue cultures (Phillips et al., 1994). However, the use of constant hormone concentration across all Cd treatments suggested that the mutation is a result of changes in Cd concentration rather than hormone. Conclusion This study presents an initial step in the form of a callus culture to develop tolerant plants for phytoremediation of Cadmium. The results suggest that callus culture in MS media supplemented with 150 μM Cd is appropriate for developing Cd-tolerant calli for T. erecta. The toxicity symptoms could be profound above this level as detected in our study. Although the results for the tolerant calli of G. globosa did not materialize, the outcome obtained has given a toxicity level for its callus. The toxicity levels for the calli of these plants indicated that the calli of T. erecta were more tolerant than that of G. globosa. Our study suggests that the calli of T. erecta treated with 150 μM Cd can be used to develop its plantlets. Thus, the selection of tolerant calli and the toxicity level of HMs in ornamental plants can be determined by understanding the stress responses of their calli. This method of selection can be used in other plants as well, but its use, especially, in selecting tolerant ornamental plants can be beneficial as they are less likely to end up in their food-chain process and since their flower parts have an insignificant accumulation of heavy metals. Besides, the cut-flowers of these plants could still be used, and hence could be idle for phytoremediation. Acknowledgments We are grateful to the University Grants Commission, Sanothimi, Bhaktapur for providing us financial support to conduct this study. Similarly, we are thankful to the Central Department of Botany, Tribhuvan University, Kirtipur for providing us access to all the laboratory facilities. Last but not the least, we would like to acknowledge Associate Professor, Deepak Raj Pant for his valuable suggestions. Author's contribution The concept and design were developed by GL. KKP and PRG polished the designs whereas BS, SL and GL carried out experimentation. Data availability The data that support the findings of this study are available on request from the corresponding author. Conflict of interest The authors declare no conflict of interest Funding This project was funded by University Grants Commission, Sanothimi, Bhaktapur, Nepal. Ethical approval and consent No harm to any plants or animals was done. Banko Janakari, Vol 34 No. 1 37 Lama et al. References Belarmino, M.M., Toshinorians, A.B.E., & Sasahara, S. (1992). Callus induction and plant regeneration in African marigold (T. erecta L.). Japaneese Journal of Breeding, 42: 835-841. Benítez-García, I., Vanegas-Espinoza, P.E., Meléndez-Martínez, A.J., Heredia, F.J., Paredes- López, O., & Villar-Martínez, A.A.D. (2014). Callus culture development of two varieties of Tagetes erecta and carotenoid production. 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