Physiological and Biochemical Responses of Chrysanthemum × morifolium to Salinity Stress under In-Vitro Conditions

Physiological and Biochemical Responses of Chrysanthemum × morifolium to Salinity Stress under In-Vitro Conditions | 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 Physiological and Biochemical Responses of Chrysanthemum × morifolium to Salinity Stress under In-Vitro Conditions Rohit Mishra, Rakesh Kumar, Aditi Gupta, Vishal Chugh, Ajay kumar, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6636160/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 Salinity is a critical abiotic stress that significantly limits plant growth and productivity by disrupting physiological and biochemical processes. This study investigated the effects of NaCl-induced salinity on the in vitro culture of Chrysanthemum × morifolium Ramat. Explants were cultured under varying concentrations of NaCl (0–300 mmol/L) to assess its impact on callus formation, shoot regeneration, physiological attributes, and biochemical responses. Results showed that increasing NaCl concentrations reduced callus formation percentages, shoot regeneration frequency, and shoot length, with the highest reduction observed at 300 mmol/L NaCl. The number of shoots per explant decreased from 13.03 under non-saline conditions to 0.90 at 300 mmol/L. Chlorophyll content, carotenoids, and protein levels declined significantly with increasing salinity, whereas the proline content increased, indicating its role in osmotic adjustment and stress tolerance. Antioxidant enzyme activities, including catalase and superoxide dismutase, were enhanced under salt stress, with maximum activity recorded at 300 mmol/L NaCl, suggesting their involvement in mitigating oxidative damage. Lipid peroxidation and protein oxidative damage also increased, further indicating the detrimental effects of salinity. During the hardening phase, optimal survival was achieved using a potting mixture of coco peat, sand, vermicompost, and garden soil (2:1:1:1 ratio). Plants grown under in vivo saline conditions exhibited reduced biomass, root growth, and shoot development, with severe effects at 400 mmol/L NaCl. These findings provide insights into the physiological and biochemical responses of C. morifolium under salt stress, contributing to the development of salt-tolerant varieties and improving in vitro propagation techniques. Salinity stress Plant Tissue Culture Chrysanthemum × morifolium Physiological and Biochemical response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The global floriculture industry, one of the fastest growing agricultural sector, plays a significant economic role, with countries like the Netherlands, the United States, Japan and Germany being leaders in the production and export of cut flowers (Getu 2009 ). Chrysanthamum x morifolium species predominantly originate from East Asia, with China, Japan, Thailand, and India being major producers. With its diverse agro-climatic zones, India is well-suited for floriculture, with states like Tamil Nadu, Karnataka, Madhya Pradesh, and West Bengal leading in cultivation (Kumar et al. 2023 ). As India's floriculture industry expands, the area under Chrysanthemum cultivation has grown by 225.33%, with production increased by 391.77% from 2014 to 2024 (Kumar et al. 2024 ). Chrysanthemum ( Chrysanthemum × morifolium ), commonly called as the "Queen of the East", belongs to the family Asteraceae. It is a highly attractive, short-day plant, often treated as a seasonal bloomer in late autumn despite being perennial. Chrysanthemum cut flowers are valued for floral arrangements due to their extended vase life and are cultivated for various decorative uses, including garlands, bouquets and floral decorations for social and religious events (Bohra and Kumar 2014 , Patil et al. 2017 ). Apart from its economic value, Chrysanthemum also holds therapeutic importance in traditional medicine, used for treating inflammation, bruises, snake bites, and even as a remedy for fever (Ryu et al. 2019 ). Additionally, the petals are used in Chinese cuisine and for making herbal teas (Collins et al. 1997 ). Abiotic stress factors such as salinity, drought, and extreme temperatures significantly affect C. morifolium , reducing its growth, flower longevity, and aesthetic value (Vinocur and Altman 2005 ). By 2050, salinity is predicted to affect about 33% of the world's irrigated agricultural land (Shao et al ., 2008). High salinity impacts various physiological processes in plants, inhibiting growth and damaging key metabolic activities like photosynthesis and protein synthesis (Munns and Tester 2008 , Acosta-Motos et al. 2015 ). Salinity stress induces the accumulation of toxic products, such as reactive oxygen species (ROS), in plants, disrupting their metabolic balance and causing oxidative damage (Ahanger et al. 2017 ). In response to salt stress, plants undergo a series of physiological changes, including increased activity of antioxidant enzymes, and elevated proline content (AbdElgawad et al. 2016 , Sarabi et al. 2017 ). These changes reflect the plant's response to salt stress at various levels, with some mechanisms protecting against oxidative damage and enhancing salt stress tolerance. Analysis of stress responses in plants grown through traditional propagation methods is slow and labour-intensive. Plant tissue culture and micro-propagation techniques offer faster, more efficient alternatives for studying plant responses to stress conditions. This method enables the rapid production of large numbers of plants or herbal products from plant cells or tissues (explants) cultured on artificial nutrient media (Oseni, Pande and Nailwal 2018 , Dogan, Karatas and Aasim 2018 ). Salt stress can be simulated in plant tissue culture by adding sodium chloride (NaCl) to the media, offering a controlled environment to study plant responses at the cellular level (Shibli et al. 2007 ). This study was aimed to optimize NaCl concentrations for tissue-cultured Chrysanthemums under in-vitro conditions and subsequent application of these optimized concentrations to in vitro-developed plants under in vivo conditions within a polyhouse. The experiment involved the treating plants with varying NaCl concentrations to evaluate their impact on plant growth, stress tolerance, and to monitor associated physiological and biochemical changes during development. 2. Materials and Methods The experiment was conducted in Plant Tissue Culture Laboratory, College of Forestry at Banda University of Agriculture and Technology, Banda, Uttar Pradesh during the years 2022-2024. The climate in Banda is semi-arid/tropical, with dry, hot summers (reaching 49°C) and cold winters (down to 2°C). Annual rainfall averages between 800 and 910 mm and mostly occurring from mid-June to September. 2.1. Explant Preparation: Nodal segments from healthy mother plants were treated with biocides (Hydroxyquinoline), fungicides (Carbendazim, Mancozeb), and a bleaching solution (sodium hypochlorite) before being sterilized with HgCl 2 for in-vitro culturing (Table S1). 2.2. Culture Media: The Murashige and Skoog (MS) medium(Murashige and Skoog 1962) was used which has composition as sucrose (30 g/L), vitamins, growth regulators such as kinetin (Kin), 6-benzylaminopurine (BAP), and thidiazuron (TDZ) adjusting pH to 5.7–5.8, and adding agar powder (7 g/L). Sterilization of the media and other necessary glassware, equipment was done by autoclaving at 121°C for 20 minutes at 15 psi pressure. 2.3. Culture Media for Growth Stages: For shoot induction, MS media with BAP (0.5-2 mg/L) was used. For shoot proliferation of microshoots were transferred to MS media with varying concentrations of BAP, kinetin, or TDZ for shoot proliferation and rooting of elongated shoots were transferred to half-strength MS media with IBA and IAA for root induction. Sterilized explants were inoculated in culture media inside a laminar airflow chamber, and incubated at 25±1°C under a 16/8 hour light/dark photoperiod. Observations were recorded for shoot induction (%), number of shoots, shoot length (cm), root induction (%), number of roots per shoot and root length (cm). 2.4. Sub-culturing In Vitro plants on MS medium supplemented with NaCl To subculture in vitro plants in MS media supplemented with NaCl (25 mM to 300 mM), first sterile NaCl stock solutions were prepared for each concentration. MS media was prepared according to the standard protocol, and NaCl stock solutions were added to the media. The pH of the NaCl-supplemented MS media was adjusted to 5.7–5.8, then autoclaved. After autoclaving, media allowed to cool. The plant material (explants) is surface sterilized and subcultured into the NaCl-enriched media. Plant growth was regularly monitored for signs of salt stress, including reduced growth rate, physiological changes like chlorophyll and carotenoid content, biochemical changes such as protein, proline levels, antioxidant enzyme activities, or altered root development, and these parameters were recorded to analyze the effects of NaCl stress on the plants. 2.5. Establishment of In-vitro propagated plants for hardening Primary hardening of Chrysanthemum morifolium plants was carried out by transferring in vitro plants to a mixture of cocopeat, perlite and vermiculite (2:1:1) in trays under fiberglass house conditions for two weeks. For secondary hardening, plants were transferred to a mixture of cocopeat, sand and vermicompost (2:1:1) and maintained under shade net conditions for four weeks. During this period, physiological and biochemical changes were monitored under both in vitro stress and control conditions. These changes included alterations in growth rate, chlorophyll and carotenoid content, protein levels, proline accumulation, and antioxidant enzyme activity, providing insights into the plants' stress response mechanisms during hardening. 2.6. Physiological parameters 2.6.1.Chlorophyll and carotenoid contents of leaves To analyse chlorophyll and carotenoid contents, fully matured open leaves were taken from both control and stressed conditions. The chlorophyll content (chlorophyll a , b and total chlorophyll) of the leaves was estimated using the method described by Hiscox and Israelstam (Hiscox and Israelstam 1979) . A sample of 50 mg of leaves was placed in 10 ml of dimethyl sulfoxide (DMSO, Analytical grade) and incubated at 65°C for 4 hours. Post-incubation, the tubes were cooled to room temperature, and the absorbance was measured at 663, 645, and 470 nm using a UV-VIS spectrophotometer (5704 ECIL, India) against pure DMSO as the blank. The chlorophyll and carotenoid contents were calculated using the following formulas: Chlorophyll a (Chl-a) = 12.21 A663 − 2.81 A645 Chlorophyll b (Chl-b) = 20.13 A645 − 5.03 A663 Total Chlorophyll (mg g⁻¹ DW) = (20.7 × OD645) + (8.02 × OD663) × volume × dilution / (1000 × weight of the sample) Total Carotenoids = [1000 A470 - (3.27 Chl-a + 104 Chl-b)] / 229 All chlorophyll values were expressed in µg/g of fresh weight (fw). The ratio of chlorophyll a and b was calculated by dividing chlorophyll a by chlorophyll b to assess the balance between different chlorophyll types under stress and control conditions. The ratio of carotenoid to chlorophyll was calculated by dividing total carotenoids by total chlorophyll to analyze how stress conditions affected pigment composition. 2.6.2. Biochemical Parameters 2.6.2.1. Antioxidant Enzyme Activity: The antioxidant enzyme activities were analyzed to assess oxidative stress in C. morifolium under in-vitro conditions. Leaf samples from each treatment were collected in an ice-box to prevent proteolytic degradation. One gram of leaf tissue was homogenized in a pre-chilled mortar and pestle with 5 ml of chilled phosphate buffer (50 mM, pH 7.0). The homogenate was centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant was used for the following enzyme assays: 2.6.2.2. Superoxide Dismutase (SOD) Activity SOD activity was determined according to Fridovich (Fridovich 1975) . The reaction was based on the ability of SOD to inhibit the photochemical reduction of nitro blue tetrazolium (NBT). Absorbance was recorded at 560 nm, and enzyme activity was expressed as unit/min/mg of protein. 2.6.2.3. Catalase (CAT) Activity Catalase activity was measured using the method of Luck (Lück 1965) . Residual hydrogen peroxide (H₂O₂) was quantified by titration with potassium permanganate. Results were expressed as µmol of H₂O₂ decomposed per minute per mg of protein. 2.6.3. H₂O₂ Content Estimation H₂O₂ content was determined following the method of Alexieva et al. (Alexieva et al. 2001) . Using 0.1% TCA for extraction, the H₂O₂ was reacted with potassium iodide (KI), and the absorbance was measured at 390 nm. Results were expressed as µmol g⁻¹ of fresh weight. 2.6.4. Proline Content Proline was quantified following the method of Bates et al. (Bates, Waldren and Teare 1973). The leaves were homogenized in 3% sulpho-salicylic acid, and the proline content was determined by the formation of a pink chromophore with acid ninhydrin, followed by toluene extraction. Absorbance was recorded at 520 nm, and proline content was expressed as µg/g of dry weight. 2.6.5. Protein Content Protein content was estimated using Lowry's method (Lowry et al. 1951). The protein was precipitated using TCA, and absorbance of blue color developed after treatment with Folin-Ciocalteu reagent was measured at 660 nm. Protein content was calculated based on a standard curve prepared using bovine serum albumin (BSA). 2.6.6. Histochemical detection of superoxide and H₂O₂ by NBT and DAB staining Superoxide and H₂O₂ were detected in leaf tissues using NBT and DAB staining techniques following Kumar et al. (Kumar et al. 2014). Theleaves were stained overnight, cleared with ethanol and visualized against a contrast background. Superoxide appeared as dark blue spots, while H₂O₂ as reddish-brown spots. 3. Results The study investigated the sterilizing agent, the effects of basal media composition, phytohormones, and NaCl treatments on shoot induction, regeneration frequency, and shoot growth. Surface sterilization using an optimized combination of 2% NaOCl, 0.10% fungicide, 0.02% biocide, and 0.10% HgCl₂ resulted in the highest survival of Chrysanthemum × morifolium nodal explants, whereas higher concentrations significantly reduced viability (Table S1). Full-strength and half-strength MS media were supplemented with different concentrations of phytohormones, including BAP, TDZ, and kinetin, and the results demonstrated significant variations in shoot induction and growth. In full-strength MS medium, the addition of BAP at 2 mg/L yielded the highest shoot induction (69.67%) with an average of 3.57 shoots per explant and a mean shoot length of 5.43 cm. TDZ at 0.5 mg/L resulted in 59% shoot induction with 2.97 shoots and a shoot length of 2.77 cm. Among kinetin treatments, 1 mg/L produced the highest induction rate (50.33%), with an average of 3.23 shoots and a shoot length of 3.67 cm. However, higher concentrations of TDZ and kinetin reduced shoot induction and growth, indicating a concentration-dependent response. Half-strength MS medium supplemented with 2 mg/L BAP was the most effective for shoot induction and growth. It achieved the highest shoot regeneration frequency (89.67%), producing an average of 13.03 shoots per explant with a mean shoot length of 8.9 cm. BAP at 0.5 mg/L also showed high efficacy, with an 80.33% induction rate and 11.67 shoots per explant (Figure S1a&b and Table 1). The addition of NaCl to half-strength MS medium supplemented with 2 mg/L BAP significantly reduced shoot regeneration frequency and growth. Under control conditions (T0, 0 mM NaCl), the regeneration frequency was 89.33%, with a mean shoot length of 7.83 cm. At 25 mM NaCl (T1), the regeneration frequency decreased to 79.87%, and shoot length reduced to 5.97 cm. With increasing NaCl concentrations, the inhibitory effect became more pronounced. At 100 mM NaCl (T4), regeneration frequency dropped to 54.73%, and shoot length decreased to 3.27 cm. At 300 mM NaCl (T8), the lowest regeneration frequency (5.33%) and shoot length (0.9 cm) were observed, reflecting the severe impact of salt stress on shoot development. The result showed that the BAP was the most effective phytohormone for promoting shoot induction and elongation under both normal and stress conditions. However, salt stress caused a substantial decline in regeneration frequency and shoot length, with the inhibitory effects increasing with higher NaCl concentrations. These findings highlight the importance of optimizing phytohormone concentrations and managing salt stress for successful in vitro shoot regeneration ( Figure 1 a & b; Table 1). 3.1. Role of different NaCl concentrations on root regeneration Root regeneration in C. morifolium was most effective with 0.2 mg/L IBA in half-strength MS medium, yielding the highest root induction (90.4%) and the maximum roots per shoot (11.66). The longest roots (12.30 cm) were observed at 0.4 mg/L IBA. IAA showed lower efficiency, with 70.43% root induction, 10 roots per shoot, and a maximum root length of 11.40 cm at optimal concentrations. Higher auxin levels (1.0 mg/L) significantly reduced root induction, number, and length. These results underscore the efficacy of low IBA concentrations for root regeneration (Figure S2; Table S2). 3.2. Physiological and biochemical changes in C. morifolium under in-vitro salt stress : The study examined the physiological and biochemical responses of C. morifolium under varying salinity stress. Chlorophyll A and B content decreased with increasing salt levels, with the highest chlorophyll A (113.34 µg/g) and B (72.30 µg/g) observed at 25 mmol/L NaCl, and the lowest at 300 mmol/L NaCl. The chlorophyll A/B ratio increased with salinity, while total chlorophyll and carotenoid content also declined, with the highest total chlorophyll (207.49 µg/g) and carotenoid (38.13 µg/g) at 25 mmol/L NaCl ( Figure 2a). Proline content, a key stress marker, significantly increased with salinity, peaking at 300 mmol/L NaCl (92.33 µg/g) (Figure 2b ). Superoxide Dismutase (SOD) and Catalase (CAT) activities elevated under high salt stress, with the highest SOD activity (107.20 unit/min/mg protein) and CAT activity (137.86 µmoles of H 2 O 2 decomposed min-1 mg-1 protein) recorded at 300 mmol/L NaCl. Hydrogen Peroxide (H 2 O 2 ) levels also significantly increased under salinity, reaching a maximum of 133.52 µmoles g-1 fw in 300 mmol/L NaCl (Figure: 2c-2e). These findings highlight how C. morifolium responds to salinity stress by activating stress-responsive biochemical pathways, including protein degradation, proline accumulation, and enhanced antioxidant enzyme activity (Figure 2 a-e). In contrast, at 25 mmol/L NaCl (T1), ROS accumulation was much lower. These findings align with increased H₂O₂ and SOD levels, confirming that oxidative stress increases with salinity (Figure 2). 3.3. Histochemical detection of Superoxide and H 2 O 2 by NBT and DAB staining of Chrysanthemum leaves under NaCl treatment The histochemical analysis of C. morifolium leaves under NaCl-induced salinity showed significant accumulation of ROS, including hydrogen peroxide (H₂O₂) and superoxide anions. At 300 mmol/L NaCl (T8), DAB staining revealed intense brown pigmentation, indicating high H₂O₂ accumulation (Figure 3a), while NBT staining showed dark blue spots, indicating superoxide build up (Figure 3b). 3.4. Principle component analysis The Principal Component Analysis (PCA) biplot (Figure S3) provides a clear visualization of the impact of various treatments on plant growth and stress responses. The first principal component (F1), accounting for 98.78% of the variance, is primarily influenced by growth-promoting factors such as shoot regeneration frequency, total chlorophyll, chlorophyll A, chlorophyll B, carotenoids, protein content, and shoot length. Treatments T1, T2, T3, and T0, positioned on the right side of the biplot, show strong positive associations with these variables, indicating favourable conditions for growth, chlorophyll synthesis, protein accumulation, and shoot regeneration. These treatments likely provided optimal environmental or nutrient conditions, enabling the plants to allocate energy toward growth and development. In contrast, the second principal component (F2), which explains only 0.80% of the variance, is associated with stress-related variables like superoxide dismutase (SOD), hydrogen peroxide (H₂O₂), and proline. Treatments T4 and T5, positioned closer to these stress markers, indicate a moderate level of stress, activating antioxidant defense mechanisms to counteract oxidative and osmotic stress. Proline accumulation suggests osmotic stress responses. Conversely, treatments T8, T6, and T7, positioned on the far-left side of F1, are negatively correlated with growth-related variables, indicating detrimental effects on plant growth. The large separation between these treatments and growth parameters suggests that they inhibited shoot regeneration and reduced biomass accumulation, possibly due to poor environmental conditions, nutrient deficiencies, or other stress factors. ( Figure S3 ). The biplot clearly shows the trade-off between growth and stress responses. Treatments such as T1, T2, T3, and T0 promoted growth, enhancing shoot regeneration and chlorophyll synthesis. In contrast, treatments like T4 and T5 activated stress-related pathways, redirecting resources toward antioxidant and osmotic adjustments, which limited biomass accumulation. This is reflected by the increased SOD, H₂O₂, and proline levels, indicating oxidative and osmotic stress. The PCA biplot effectively differentiates between treatments that favor growth and those that trigger stress responses. Treatments T1, T2, T3, and T0 provided optimal conditions for plant development, while treatments like T4 and T5 activated defense mechanisms. Treatments T8, T6, and T7 were the least effective, resulting in suppressed growth. The biplot emphasizes the need to balance growth and stress responses for successful regeneration and development under varying conditions. 3.5. Establishment of In-vitro propagated plants for hardening The tissue culture-raised C. morifolium plants under salt stress were successfully acclimatized and hardened through a sequential process. In the primary hardening stage, the plants exhibited significant growth in a medium comprising cocopeat, perlite, and vermiculite in a 2:1:1 ratio. During secondary hardening, the plants continued to grow well in a mix of cocopeat, sand, and vermicompost (2:1:1). Finally, the acclimatized plants were transplanted into a healthy potting medium of vermicompost, garden soil, sand, and cocopeat in a 2:1:1:1 ratio, where they thrived under controlled conditions in a polyhouse (Figure S4; Figure 4a and b). The impact of varying NaCl concentrations on tissue-cultured C. morifolium plants under in-vivo conditions was evaluated at 15, 30, and 45 DAS. Significant effects were observed on parameters such as the number of leaves (Figure 4c), plant height (Figure 4d), secondary shoots (Figure 4e), and plant spread (Figure 4f), (Figure 4 c-f and Table 2). The highest number of leaves and plant height were recorded in treatments S2 and S3 (Figure 4c). Secondary shoots peaked at S3 (19.25 at 30 DAS) and S5 (14 at 15 DAS), while plant spread was greatest in S1 (18.45 cm at 30 DAS). Shoot diameters were maximized under S4 and S3, respectively (Table 2). The higher NaCl concentrations generally suppressed plant growth, with S1 and S3 performing best across most parameters. Negative controls consistently showed inferior results compared to positive controls. Root length measurements at the end of the experiment (45 DAS) revealed the longest roots under S1 salt treatment (15.22 cm), followed by S2. Negative controls produced shorter roots compared to positive controls (Figures 5a,b). Soil conductivity (E.C.) and pH were also influenced by NaCl levels. At 45 DAS, S5 had the highest E.C. (25.10), while S1 recorded the lowest (8.13). Soil pH was highest in S1 and S2 (7.69), and lowest in S5 (7.49). Negative controls showed consistently poorer performance compared to positive controls in both plant and soil parameters (Table S3). 3.6. Effect of NaCl on physiology and biochemical changes of tissue cultured raised C. morifolium under in vivo salt stress This study investigated the impact of different NaCl concentrations on tissue-cultured C . morifolium under in-vivo conditions, highlighting significant physiological and biochemical changes. Chlorophyll A and B contents decreased progressively with increasing NaCl concentrations, with the highest levels recorded at S1 (105.71 µg/g for chlorophyll A and 61.57 µg/g for chlorophyll B), while the lowest were observed at S5 (39.27 µg/g for chlorophyll A and 18.74 µg/g for chlorophyll B) (Fig. 6a). The chlorophyll A/B ratio increased under higher salt stress, peaking at S5 (2.09) (Fig. 6a). Total chlorophyll content also showed a decreasing trend, with S1 exhibiting the highest (167.28 µg/g) and S5 the lowest (58.01 µg/g) carotenoid content similarly declined with increasing NaCl, reaching the highest in S1 (33.16 µg/g) and the lowest in S5 (6.49 µg/g), resulting in a carotenoid/chlorophyll ratio of 0.19 in S1 and 0.11 in S5 (Figure 6a). Proline content, an osmoprotectant, increased markedly under high salt stress, peaking at S5 with 98.63 µg/g of fw, compared to the lowest content in S1 (40.88 µg/g of fw) (Figure 6b). Antioxidant enzyme activities, including SOD and catalase, were significantly elevated in response to salt stress, with catalase activity peaking at 143.87 µmoles of H2O2 decomposed/min/mg of protein in S5 (Figure 6c), and SOD activity reaching a maximum of 113.19 unit/min/mg of protein in S5 (Figure 6d). H2O2 accumulation also increased under higher salt conditions, with the highest content recorded in S5 (139.14 µmoles/g of fw), compared to the lowest in S1 (74.37 µmoles/g of fw) (Figure 6e). Negative controls consistently showed lower values compared to positive controls across all parameters, confirming the detrimental effects of higher NaCl concentrations on plant growth, chlorophyll synthesis, protein content, and antioxidant defense mechanisms. 3.7. Histochemical Detection of superoxide and H 2 O 2 by NBT and DAB Staining of In-vivo raised tissue cultured plants Histochemical analysis of C. morifolium leaves under different NaCl concentrations (0–400 mmol) revealed hydrogen peroxide (H 2 O 2 ) and superoxide anion accumulation. In leaves collected 45 DAS, DAB staining showed brown spots indicating H 2 O 2 accumulation, while NBT staining revealed blue pigments, signifying superoxide anion presence. Severe cell damage was observed at 400 mmol NaCl (S5), with intense brown and blue pigmentation, while fewer symptoms were seen at 25 mmol NaCl (S1). These findings align with biochemical analysis, where higher H 2 O 2 and SOD activity were recorded in S5 compared to the control (Figure S5). 3.8. Principle component analysis: The Principal Component Analysis (PCA) biplot (Figure S6) reveals the relationships between physiological and biochemical variables (active variables in red) and treatments (active observations in blue), explaining a total variance of 84.04% through two principal components, F1 and F2. The F1 accounts for 71.06% of the variance and is strongly influenced by stress-related variables like proline, hydrogen peroxide (H 2 O 2 ), and superoxide dismutase (SOD), which are prominently associated with higher salinity treatments such as S5 (400 mM) and S4 (200 mM). These treatments show a positive correlation with oxidative stress and osmotic regulation responses, indicating that they induce significant stress in the plant. Conversely, F2, which explains 12.99% of the variance, is dominated by growth-related variables such as plant height at 30 and 45 days, root length, and the number of leaves at 15 days. These growth parameters are positively associated with lower salinity treatments like S1 (25 mM) and S2 (50 mM), demonstrating that mild salinity levels promote plant growth. The negative control (So) is centered near the origin, showing a moderate response across all variables, while the positive control (So+) clusters near chlorophyll content (Chl A and Chl B), highlighting optimal conditions for photosynthesis and plant health under non-stress conditions. The biplot distinctly separates growth-related responses, aligned with the positive side of F2, from stress-related responses, positioned on the positive side of F1. This separation emphasizes that higher salinity levels hinder growth while activating stress defense mechanisms. In summary, the PCA biplot effectively visualizes the differential impacts of salinity treatments on plant growth and stress, demonstrating that lower salinity fosters growth, whereas elevated salinity triggers biochemical stress responses. 4. Discussion 4.1. Regeneration and multiplication of Chrysanthemum under different phytohormones condition This study focuses on identifying optimal NaCl concentrations that do not adversely affect Chrysanthemum growth in both in-vitro and in-vivo (pot culture) conditions. Field-grown explants face microbial contamination challenges in in-vitro cultures. Pre-treatments with 0.1% Carbendazim, 0.02% 8-HQC (30 min), and 0.1% HgCl₂ (3 min) resulted in 51.00% survival. Higher survival rates (75.56%) were reported with 0.2% Mancozeb, 0.2% Carbendazim, and 200 mg/L 8-HQC (Verma et al. 2012). Similarly, 0.1% HgCl₂ was most effective in Stevia (Sharuti Verma, Kuldeep Yadav and Narender Singh 2011) and yielded 65.08% survival in Chrysanthemum (Anjum et al. 2023). For shoot induction, half-strength of MS medium with 30 g/L sucrose, 2.0 mg/L BAP, and 7 g/L agar achieved 89.66% induction. Comparable rates were reported in Chrysanthemum (76.66%, 2.0 mg/L BAP; (Yesmin et al. 2014), 83.30% with 2.0 mg/L BAP + 0.5 mg/L IAA (Kashif Waseem et al. 2011) and 84.74% in Xanthosoma sagittifolium (Bansal et al. 2023). Jahan et al. (Jahan et al. 2021) recorded 100% regeneration with 2.0 mg/L BAP, while Boonkamjat et al. (Boonkamjat, Saetiew and Teerarak) reported 93.33% with 1 mg/L BA + 0.1 mg/L IAA in Chrysanthemum . Shoot proliferation was highest (13.03 shoots) in half-strength MS medium supplemented with 2.0 mg/L BAP. Similar findings were reported in Chrysanthemum (7.7 shoots) (Kashif Waseem et al. 2011) and Xanthosoma sagittifolium (13.44 shoots) (Bansal et al. 2023). Shoot length (8.90 cm) was optimal with 2.0 mg/L BAP, consistent with Ghosh et al.(Ghosh et al. 2021). Root induction was highest (90.40%) with half-strength MS medium + 0.2 mg/L IBA as supported by studies of researchers (Yesmin et al. 2014), (Rashid et al. 2009), (Kashif Waseem et al. 2011), and (Sushmarani, Venkatesha Murthy and Deeksha Raj 2021). Higher auxin concentrations inhibited rooting (Kaul et al. 1990) and longest roots (12.30 cm) were obtained with 0.4 mg/L IBA (Komalavalli and Rao 2000), while maximum roots (11.66 per shoot) were observed with 0.2 mg/L IBA, aligning with other researchers (Yesmin et al. 2014, Deltalab et al. 2024). The development of salt-tolerant plants through in-vitro techniques, such as callus and shoot cultures is a non-transgenic route. Since, salt stress typically reduces plant growth (Queiros et al. 2007), as slower growth linked to improved stress survival. In our study, the highest shoot regeneration frequency (79.86%) was recorded at 25 mmol/L NaCl, with the lowest observed at 300 mmol/L. Salt stress reduced shoot regeneration frequency due to impaired cell division and photosynthesis, as reported in similar studies on Chrysanthemum (Thongpukdee et al. 2012) and Bacopa monnieri (Sable et al. 2018). 4.2. Physiological, biochemical and histological analysis of in-vitro regenerated plant under NaCl stress Under salinity stress, C. morifolium displayed significant physiological and biochemical alterations compared to control conditions. Salinity treatment influenced photosynthetic pigments, including chlorophyll and carotenoids. The highest levels of chlorophyll A, chlorophyll B, total chlorophyll, and carotenoids were observed at 25 mmol/L NaCl (T1). However, the chlorophyll A/B ratio and carotenoid/chlorophyll ratio (0.231) at 200 mmol/L NaCl (T7). This decline in chlorophyll is attributed to pigment-protein complex instability under stress, as noted in previous studies (Dogan 2020, Rai et al. 2020). Moreover, the proline and protein levels varied significantly under salt stress. Maximum protein content was observed at 25 mmol/L NaCl, while proline accumulation peaked at 300 mmol/L, reflecting the role of osmolites in stress mitigation. Antioxidative enzymes showed increased activity, with SOD, CAT, and H₂O₂ scavengers rising by 2.3-, 2.8-, and 3.1-fold, respectively, at 300 mmol/L NaCl, supporting previous findings in Chrysanhemum (Guan et al. 2012). NBT and DAB staining confirmed increased O₂⁻ and H₂O₂ accumulation under salt stress, leading to oxidative damage, reduced membrane integrity, and necrotic lesions at 300 mmol/L NaCl, consistent with studies on Brassica napus and Chrysanthemum (Banerjee and Roychoudhury 2017, Ahmad et al. 2019). 4.3. The impact of varying NaCl concentrations on tissue-cultured C. morifolium plants under in-vivo conditions Salinity stress significantly impacted plant biomass by altering water potential, increasing ion toxicity, and inhibiting cell wall expansion, leading to reduced root growth and plant height. A decline in growth parameters was observed with increasing NaCl concentrations, with the most significant biomass reduction at 45 DAS in S5 (400 mmol/L NaCl). Leaf number, plant height, and secondary shoot numbers varied across treatments, with S2, S3, and S1 showing the highest values at different stages. Plant spread was highest in S1, while primary stem diameter peaked in S4 and secondary shoot diameter in S3, S4, and S2 at different DAS intervals. These results align with previous studies on ornamental plants like Tagetes erecta (Sayyed et al. 2014), Chrysanthemum (Bañón et al. 2010), and marigold (Bahmanzadegan and Aboutalebi 2013), where salt stress-induced growth reduction was attributed to osmotic and ionic imbalances, inhibited cell division and elongation, and decreased cell wall extensibility. Prolonged salinity exposure led to severe symptoms such as leaf scorching, necrosis, and leaf drop, potentially resulting in plant death. Salinity significantly affects root growth by limiting nutrient and water uptake, ultimately impacting plant health and yield. In our study, maximum root length was recorded under S1 (25 mmol/L NaCl), while the negative control exhibited the least growth. Similar reductions in root length under increasing salinity have been reported in Chrysanthemum indicum (Ranganayakulu, Veeranagamallaiah and Chinta Sudhakar 2013, Mazhar et al. 2012). Additionally, soil EC, a key indicator of soil health, increased with higher NaCl concentrations, peaking in S5 (400 mmol/L NaCl) at 45 DAS. In contrast, soil pH remained highest in S1 and S2 at different time points. 4.4. Effect of NaCl on physiology and biochemical changes of tissue cultured raised C. morifolium under In vivo salt stress Salinity stress significantly impacts photosynthesis by inducing chlorosis, reducing pigment synthesis, and increasing oxidative stress(Shaw 1995, Parida and Das 2005). Carotenoids, which protect against ROS, decline under salt stress, leading to chlorophyll degradation (Rao and Rao 1981). In our study, the highest chlorophyll A (105.71 µg/g), chlorophyll B (61.57 µg/g), total chlorophyll (167.28 µg/g), and carotenoids (33.16 µg/g) were observed in S1 (25 mmol/L NaCl), while the highest chlorophyll A/B ratio (2.09 µg/g) was found in S5 (400 mmol/L NaCl), consistent with previous reports in corn (Molazem, Qurbanov and Dunyamaliyev 2010), cucumber (Malik et al. 2010), and purslane (Parvaneh, Shahrokh and Meysam 2012). Protein content, crucial for osmotic adjustment, was highest in S1 (47.87 µg/g), indicating a balance between synthesis and degradation under moderate stress (Dogan 2020, Shatnawi et al. 2010). Proline, a key osmo-protectant, peaked in S5 (98.63 µg/g), supporting its role in salinity tolerance as reported by other researchers (Ashraf and Tufail 1995, Mansour et al. 2005). ROS scavenging enzymes, such as SOD and CAT, play a vital role in mitigating oxidative stress (Yasemin et al. 2021, Parvin et al. 2019). The highest SOD (113.19 unit/min/mg), CAT (143.87 µmoles H 2 O 2 decomposed min-1 mg-1), and H 2 O 2 content (139.14 µmoles g-1 FW) were observed in S5, indicating increased ROS detoxification at higher salinity (Zandalinas et al. 2017, Laxa et al. 2019). High ROS levels, including superoxide anions (O 2 – ) and H 2 O 2 , lead to oxidative damage and plasma membrane disruption, as observed in C. morifolium under severe salt stress (Banerjee and Roychoudhury 2017, Su et al. 2019). Similar trends have been reported in Brassica napus (Huang et al. 2022), where enhanced CAT and GR activity contributed to stress tolerance (Bor, Özdemir and Türkan 2003, Yaşar and Ellialtıoğlu 2013). 5. Conclusion The present study established a standardization protocol that can be used to produce true-to-type plants, which are otherwise challenging to obtain for this species. This well-standardized protocol may facilitate large-scale multiplication of desired types, thereby meeting the demand for high-quality planting material of C. morifolium . The second main focus of the present investigation was to analyze the morphological, physiological, and biochemical responses of Chrysanthemum to NaCl-induced salinity stress under In-vitro and In-vivo (pot culture) raised tissue-cultured plants. Based on the findings, it is concluded that salinity imposes both osmotic and ionic stress, impairing plant cell functions, damaging cell membranes, and affecting the photosynthetic apparatus. Salt stress reduces biomass, plant growth, chlorophyll content, carotenoid content, and restricts the accumulation of beneficial nutrients in plant tissues. Plants adapted to salt stress through mechanisms such as upregulation of antioxidant enzyme activities and accumulation of osmolytes like proline, which protect plant cells from the harmful effects of Na + and Cl - ions. Plants that performed better under varying NaCl concentrations likely employed these mechanisms to maintain ionic homeostasis and protect against reactive oxygen species generated under salinity stress. These findings are commercially valuable for developing salt-tolerant varieties, ensuring better survival and productivity in saline environments. Declarations Acknowledgement The plant tissue culture work was performed in Plant Tissue Culture Laboratory developed under Rashtriya Krishi Vikas Yojana (UP/RKVY-AGRE/2019/861). Biochemical work conducted at Biochemistry laboratory developed under ICAR fund at BUAT, Banda. Author contributions: SP, RK: conceived the idea and drafted the manuscript; RM: performed Plant tissue culture work; VC: analysis of physiological and biochemical work and editing work; SK edited the manuscript. Declaration of competing interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability statement: The data that support the findings of this study are available upon request. Conflict of Interest : The authors declared that no conflict of interest exists. Ethics approval and Consent to participate: Not Applicable. References AbdElgawad, H., G. Zinta, M. M. Hegab, R. Pandey, H. Asard & W. Abuelsoud (2016) High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Frontiers in plant science, 7 , 276. Acosta-Motos, J.-R., P. Diaz-Vivancos, S. Alvarez, N. Fernández-García, M. J. Sanchez-Blanco & J. A. Hernández (2015) Physiological and biochemical mechanisms of the ornamental Eugenia myrtifolia L. plants for coping with NaCl stress and recovery. Planta, 242 , 829-846. Ahanger, M. A., N. S. Tomar, M. Tittal, S. Argal & R. Agarwal (2017) Plant growth under water/salt stress: ROS production; antioxidants and significance of added potassium under such conditions. Physiology and Molecular Biology of Plants, 23 , 731-744. Ahmad, R., S. Hussain, M. A. Anjum, M. F. Khalid, M. Saqib, I. Zakir, A. Hassan, S. Fahad & S. Ahmad (2019) Oxidative stress and antioxidant defense mechanisms in plants under salt stress. Plant abiotic stress tolerance: Agronomic, molecular and biotechnological approaches , 191-205. Alexieva, V., I. Sergiev, S. Mapelli & E. Karanov (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell & Environment, 24 , 1337-1344. Anjum, N., S. Dogra, R. Pandey, P. Pandotra, N. Laishram, A. Singh, S. Kashyap & A. H. Shah (2023) Optimization the sterilization and acclimatization protocol for micropropagation of commercial cultivar chrysanthemum ‘Maghi White’. Ashraf, M. & M. Tufail (1995) Variation in salinity tolerance in sunflower (Helianthus annum L.). Journal of Agronomy and Crop Science, 174 , 351-362. Bahmanzadegan, M. & A. Aboutalebi (2013) Interaction between ammonium nitrate and salinity on germination rate and vegetative growth of French marigold (Tageta patula). Banerjee, A. & A. Roychoudhury (2017) Abiotic stress, generation of reactive oxygen species, and their consequences: an overview. Reactive Oxygen Species in Plants: Boon or Bane‐Revisiting the Role of ROS , 23-50. Bañón, S., E. Conesa, R. Valdés, J. Miralles, J. Martínez & M. Sánchez Blanco. 2010. Effects of saline irrigation on phytoregulator-treated chrysanthemum plants. In XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): International Symposium on 937 , 307-312. Bansal, S., M. K. Sharma, S. Singh, P. Joshi, P. Pathania, E. V. Malhotra, S. Rajkumar & P. Misra (2023) Histological and molecular insights in to in vitro regeneration pattern of Xanthosoma sagittifolium. Scientific Reports, 13 , 5806. Bates, L. S., R. Waldren & I. Teare (1973) Rapid determination of free proline for water-stress studies. Plant and soil, 39 , 205-207. Bohra, M. & A. Kumar (2014) Studies on effect of organic manure and bioinoculants on vegetative and floral attributes of chrysanthemum cv. Little darling. The Bioscan, 9 , 1007-1010. Boonkamjat, T., K. Saetiew & M. Teerarak Influence of plant growth regulators on shoot development of Chrysanthemums. Bor, M., F. Özdemir & I. Türkan (2003) The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant science, 164 , 77-84. Collins, R., T. Ng, W. Fong, C. Wan & H. Yeung (1997) A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of Chinese medicinal herbs. Life Sciences, 60 , PL345-PL351. Deltalab, B., B. Kaviani, D. Kulus & S. A. Sajjadi (2024) Optimization of shoot multiplication and root induction in Saintpaulia ionantha H. Wendl. using thiamine (vitamin B1) and IBA: A promising approach for economically important African violet propagation. Plant Cell, Tissue and Organ Culture (PCTOC), 156 , 74. Dogan, M. (2020) Effect of salt stress on in vitro organogenesis from nodal explant of Limnophila aromatica (Lamk.) Merr. and Bacopa monnieri (L.) Wettst. and their physio-morphological and biochemical responses. Physiology and molecular biology of plants, 26 , 803-816. Dogan, M., M. Karatas & M. Aasim (2018) Cadmium and lead bioaccumulation potentials of an aquatic macrophyte Ceratophyllum demersum L.: a laboratory study. Ecotoxicology and Environmental Safety, 148 , 431-440. Fridovich, I. (1975) Superoxide dismutases. Annual review of biochemistry, 44 , 147-159. Getu, M. (2009) Ethiopian floriculture and its impact on the environment. Mizan law review, 3 , 240-270. Ghosh, U. K., M. N. Islam, M. N. Siddiqui & M. A. R. Khan (2021) Understanding the roles of osmolytes for acclimatizing plants to changing environment: a review of potential mechanism. Plant Signaling & Behavior, 16 , 1913306. Guan, Z., S. Chen, F. Chen, Z. Liu, W. Fang & J. Tang (2012) Comparison of stress effect of NaCl, Na+ and Cl-on two Chrysanthemum species. Acta Horticulturae , 369. Hiscox, J. & G. Israelstam (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian journal of botany, 57 , 1332-1334. Huang, Q., M. A. Farooq, F. Hannan, W. Chen, A. Ayyaz, K. Zhang, W. Zhou & F. Islam (2022) Endogenous nitric oxide contributes to chloride and sulphate salinity tolerance by modulation of ion transporter expression and reestablishment of redox balance in Brassica napus cultivars. Environmental and Experimental Botany, 194 , 104734. Jahan, M., M. Islam, S. Islam, P. Das, M. M. Islam, M. Kabir & A. Mamun (2021) Clonal propagation of Chrysanthemum morifolium ramat using various explants obtained from field grown plants. GSC Biol. Pharm. Sci, 16 , 87-93. Kashif Waseem, K. W., M. Jilani, M. Khan, M. K. Mehwish Kiran & G. K. Ghazanfarullah Khan (2011) Efficient in vitro regeneration of chrysanthemum (Chrysanthemum morifolium L.) plantlets from nodal segments. Kaul, V., R. M. Miller, J. F. Hutchinson & D. Richards (1990) Shoot regeneration from stem and leaf explants of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.). Plant Cell, Tissue and Organ Culture, 21 , 21-30. Komalavalli, N. & M. V. Rao (2000) In vitro micropropagation of Gymnema sylvestre–A multipurpose medicinal plant. Plant cell, tissue and organ culture, 61 , 97-105. Kumar, A., S. Pathania, B. Kashyap, R. Dhiman & Y. Gupta (2024) A decade analysis of flower area, production and instability index-A review. Journal of Ornamental Horticulture, 27 , 1-10. Kumar, A., S. Pathania, B. Kashyap, S. Dhiman & Y. Gupta (2023) Indian floriculture: Current issues and initiatives-A review paper. Journal of Ornamental Horticulture, 26 , 1-9. Kumar, D., M. A. Yusuf, P. Singh, M. Sardar & N. B. Sarin (2014) Histochemical detection of superoxide and H2O2 accumulation in Brassica juncea seedlings. Bio-protocol, 4 , e1108-e1108. Laxa, M., M. Liebthal, W. Telman, K. Chibani & K.-J. Dietz (2019) The role of the plant antioxidant system in drought tolerance. Antioxidants, 8 , 94. Lowry, O. H., N. J. Rosebrough, A. L. Farr & R. J. Randall (1951) Protein measurement with the Folin phenol reagent. J biol Chem, 193 , 265-275. Lück, H. 1965. Catalase. In Methods of enzymatic analysis , 885-894. Elsevier. Malik, A. A., W.-G. Li, L.-N. Lou, J.-H. Weng & J.-F. Chen (2010) Biochemical/physiological characterization and evaluation of in vitro salt tolerance in cucumber. African Journal of Biotechnology, 9 , 3298-3302. Mansour, M., K. Salama, F. Ali & A. Abou Hadid (2005) Cell and plant responses to NaCl in Zea mays L. cultivars differing in salt tolerance. Gen. Appl. Plant Physiol, 31 , 29-41. Mazhar, A. A., S. I. Shedeed, N. G. Abdel-Aziz & M. Mahgoub (2012) Growth, flowering and chemical constituents of Chrysanthemum indicum L. plant in response to different levels of humic acid and salinity. Molazem, D., E. Qurbanov & S. Dunyamaliyev (2010) Role of proline, Na and chlorophyll content in salt tolerance of corn (Zea mays L.). American-Eurasian J. Agric. & Environ. Sci, 9 , 319-324. Munns, R. & M. Tester (2008) Mechanisms of salinity tolerance. Annu. Rev. Plant Biol., 59 , 651-681. Murashige, T. & F. Skoog (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15 , 473-497. Oseni, O. M., V. Pande & T. K. Nailwal (2018) A review on plant tissue culture, a technique for propagation and conservation of endangered plant species. International journal of current microbiology and applied sciences, 7 , 3778-3786. Parida, A. K. & A. B. Das (2005) Salt tolerance and salinity effects on plants: a review. Ecotoxicology and environmental safety, 60 , 324-349. Parvaneh, R., T. Shahrokh & H. S. Meysam (2012) Studying of salinity stress effect on germination, proline, sugar, protein, lipid and chlorophyll content in purslane (Portulaca oleracea L.) leaves. Journal of Stress Physiology & Biochemistry, 8 , 182-193. Parvin, K., M. Hasanuzzaman, M. B. Bhuyan, K. Nahar, S. M. Mohsin & M. Fujita (2019) Comparative physiological and biochemical changes in tomato (Solanum lycopersicum L.) under salt stress and recovery: role of antioxidant defense and glyoxalase systems. Antioxidants, 8 , 350. Patil, U., A. Karale, S. Katwate & M. Patil (2017) Mutation breeding in chrysanthemum (Dendranthema grandiflora T.). Journal of Pharmacognosy and Phytochemistry, 6 , 230-232. Queiros, F., F. Fidalgo, I. Santos & R. Salema (2007) In vitro selection of salt tolerant cell lines in Solanum tuberosum L. Biologia plantarum, 51 , 728-734. Rai, H., N. NAMITA, D. Raju, M. Singh, K. P. Singh, G. Kumar, S. K. Sinha, S. Lekshmy, R. Pandey & B. Poulose (2020) In vitro screening of chrysanthemum (Chrysanthemum morifolium) varieties for salt tolerance. The Indian Journal of Agricultural Sciences, 90 , 2138-2144. Ranganayakulu, G., G. Veeranagamallaiah & C. S. Chinta Sudhakar (2013) Effect of salt stress on osmolyte accumulation in two groundnut cultivars (Arachis hypogaea L.) with contrasting salt tolerance. Rao, G. & G. Rao. 1981. PIGMENT COMPOSITION AND CHLOROPHYLLASE ACTIVITY IN PIGEON PEA (CAJANUS-INDICUS SPRENG) AND GINGELLEY (SESAMUM-INDICUM L) UNDER NACL SALINITY. 768-770. COUNCIL SCIENTIFIC INDUSTRIAL RESEARCH PUBL & INFO DIRECTORATE, NEW DELHI …. Rashid, M., M. Khalekuzzaman, M. Hasan, R. Das, M. Hossain & S. Mahabbat-E-Khoda (2009) Establishment of an efficient method for micropropagation of an important medicinal herb (Scoparia dulcis L.) from shoot tips and nodal segments. Int. J. Sustain. Crop Prod, 4 , 5-9. Ryu, J., B. Nam, B.-R. Kim, S. H. Kim, Y. D. Jo, J.-W. Ahn, J.-B. Kim, C. H. Jin & A.-R. Han (2019) Comparative analysis of phytochemical composition of gamma-irradiated mutant cultivars of Chrysanthemum morifolium. Molecules, 24 , 3003. Sable, A. D., P. B. Kardile, A. D. Sable & A. V. Kharde (2018) Studies on effect of different concentration of NaCI on bacoside production from brahmi (Bacopa monnieri) under in vitro condition. J Pharmacogn Phytochem, 7 , 1386-1389. Sarabi, B., S. Bolandnazar, N. Ghaderi & J. Ghashghaie (2017) Genotypic differences in physiological and biochemical responses to salinity stress in melon (Cucumis melo L.) plants: Prospects for selection of salt tolerant landraces. Plant physiology and biochemistry, 119 , 294-311. Sayyed, A., H. Gul, Z. Ullah & M. Hamayun (2014) Effect of salt stress on growth of Tagetes erecta L. Pakhtunkhwa Journal of Life Science, 2 , 96-106. Sharuti Verma, S. V., K. Y. Kuldeep Yadav & N. S. Narender Singh (2011) Optimization of the protocols for surface sterilization, regeneration and acclimatization of Stevia rebaudiana Bertoni. Shatnawi, M., A. Al-Fauri, H. Megdadi, M. K. Al-Shatnawi, R. Shibli, S. Abu-Romman & A. Al-Ghzawi (2010) In vitro multiplication of Chrysanthemum morifolium Ramat and it is responses to NaCl induced salinity. Jordan Journal of Biological Sciences, 3 , 101-110. Shaw, B. (1995) Changes in the levels of photosynthetic pigments in Phaseolus aureus Roxb. exposed to Hg and Cd at two stages of development: a comparative study. Bulletin of environmental contamination and toxicology, 55 , 574-580. Shibli, R. A., M. Kushad, G. G. Yousef & M. A. Lila (2007) Physiological and biochemical responses of tomato microshoots to induced salinity stress with associated ethylene accumulation. Plant growth regulation, 51 , 159-169. Su, L.-J., J.-H. Zhang, H. Gomez, R. Murugan, X. Hong, D. Xu, F. Jiang & Z.-Y. Peng (2019) Reactive oxygen species‐induced lipid peroxidation in apoptosis, autophagy, and ferroptosis. Oxidative medicine and cellular longevity, 2019 , 5080843. Sushmarani, Y., P. Venkatesha Murthy & N. Deeksha Raj (2021) Influence of BAP with TDZ growth regulators on in vitro regeneration in chrysanthemum ( T.) Dendranthema grandiflora cv. Marigold. Journal of Pharmacognosy and Phytochemistry, 10 , 1171-1176. Thongpukdee, A., K. Chanjirakul, C. Thepsithar, K. Obsuwan & R. Chantadech. 2012. In vitro salt tolerance of chrysanthemum'Money Maker Improve'. In International Symposium on Orchids and Ornamental Plants 1025 , 173-178. Verma, A. K., K. Prasad, T. Anakiram & S. Kumar (2012) Standardization of protocol for pre-treatment, surface sterilization, regeneration, elongation and acclimatization of Chrysanthemum morifolium Ramat. International Journal of Horticulture, 2. Vinocur, B. & A. Altman (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Current opinion in biotechnology, 16 , 123-132. Yaşar, F. & Ş. Ellialtıoğlu (2013) Antioxidative responses of some eggplant genotypes to salinity stress. Yuzuncu Yıl University Journal of Agricultural Sciences, 23 , 215-221. Yasemin, S., A. G. Değer, S. Çevik & N. Köksal (2021) Benchmarking of the effects of salinity on antioxidant enzymes activities, lipid peroxidation and H2O2 levels in the leaves of two zinnia species. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, 24 , 31-39. Yesmin, S., A. Hashem, K. Das, M. Hasan & M. Islam (2014) Efficient in vitro regeneration of chrysanthemum (Chrysanthemum morifolium Ramat.) through nodal explant culture. Nuclear science and applications, 23 , 47-50. Zandalinas, S. I., D. Balfagón, V. Arbona & A. Gómez-Cadenas (2017) Modulation of antioxidant defense system is associated with combined drought and heat stress tolerance in citrus. Frontiers in Plant Science, 8 , 953. Tables Tables 1 and 2 are available in the Supplementary Files section. Supplementary Material The Supplementary Tables are not available with this version. Supplementary Files FigureS1.tif FigureS2.tif FigureS3.tif FigureS4.tif FigureS5.tif FigureS6.tif Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6636160","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466572248,"identity":"ce6f783d-7b67-4926-8028-0cd236a9f0b8","order_by":0,"name":"Rohit Mishra","email":"","orcid":"","institution":"Banda University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Rohit","middleName":"","lastName":"Mishra","suffix":""},{"id":466572249,"identity":"f555c0a5-49b7-4652-b95f-87a919c5c172","order_by":1,"name":"Rakesh Kumar","email":"","orcid":"","institution":"Banda University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"","lastName":"Kumar","suffix":""},{"id":466572250,"identity":"db106926-3a12-441c-807c-c8ee32084e71","order_by":2,"name":"Aditi Gupta","email":"","orcid":"","institution":"Banda University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Aditi","middleName":"","lastName":"Gupta","suffix":""},{"id":466572251,"identity":"8b9afeb1-e6ec-475e-8616-ed4640a2f15c","order_by":3,"name":"Vishal Chugh","email":"","orcid":"","institution":"Banda University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Vishal","middleName":"","lastName":"Chugh","suffix":""},{"id":466572252,"identity":"4fd3b79c-ea78-47a2-92c0-b053be53e2aa","order_by":4,"name":"Ajay kumar","email":"","orcid":"","institution":"Banda University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"kumar","suffix":""},{"id":466572253,"identity":"ea9a8e6b-0c65-483b-8cab-58bca29dd3a9","order_by":5,"name":"salim khan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYFAC5oYDDAcOAGn+jw8YGA5ARQ3waWGEamFnMDYgWgsDWAs/g5kEQgseIN9+sPHQjTN35M2ZGdKqeWruyPEzMD98wFBwB6cWgzOJDYdzbjwz3NnMcOw2z7FnxpINbEAXGjzDrYUBpOXDYcYNhxnbbvOwHU7ccICHTYLB4DBuh/U/BGux33CYma2Y5x8RWhhugB0GVHmYjY2Zt40ILQY3QLacOZy84TAPs+TcvsPGks1AvyTgdVjy4c85xw7bbjh/hvHDm2+H5fjZmx8++PAHj8OQARMPiGQG4gTiNABj9QexKkfBKBgFo2BEAQD9yGHJRNRwHQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1582-0266","institution":"King Saud University","correspondingAuthor":true,"prefix":"","firstName":"salim","middleName":"","lastName":"khan","suffix":""},{"id":466572254,"identity":"a02dc063-31a8-4054-b8a6-aee24387eff4","order_by":6,"name":"Shalini Purwar","email":"","orcid":"https://orcid.org/0000-0002-0471-7207","institution":"Banda University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shalini","middleName":"","lastName":"Purwar","suffix":""}],"badges":[],"createdAt":"2025-05-10 17:36:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6636160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6636160/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84294585,"identity":"c733bd8c-ef34-4592-9485-8ad12d64cef9","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":788887,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea)\u003c/strong\u003e Shoot multiplication in Murashige and Skoog (MS) media under different concentrations of NaCl salt stress: 25 mM (A - 10 days, B - 20 days, C - 30 days), 50 mM (D - 10 days, E - 20 days, F - 30 days), 75 mM (G - 10 days, H - 20 days, I - 30 days), 100 mM (J - 10 days, K - 20 days, L - 30 days), 125 mM (M - 10 days, N - 20 days, O - 30 days), 150 mM (P - 10 days, Q - 20 days, R - 30 days), 200 mM (S - 10 days, T - 20 days, U - 30 days), and 300 mM (V - 10 days, W - 20 days, X - 30 days). Figure demonstrates the effect of varying NaCl concentrations on shoot growth and multiplication over time. \u003cstrong\u003eb) \u003c/strong\u003eShoot regeneration frequency at different concentration of NaCl (25mM, 50mM, 75mM,100mM,125mM,150mM, 200mM,200mM) in half MS media. \u003cstrong\u003ec)\u003c/strong\u003e Shoot length at different concentration of NaCl (25mM, 50mM, 75mM,100mM,125mM,150mM, 200mM,200mM) in half MS media.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/723b148fcb3e96458694fe36.png"},{"id":84295634,"identity":"1f1ffc37-ef63-4a9e-8e97-5ef5c25f37d5","added_by":"auto","created_at":"2025-06-10 09:24:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":296717,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological and Biochemical analysis of leaves collected from tissue culture raised plants at different concentration of NaCl (Control, 25mM, 50mM, 75mM,100mM,125mM,150mM, 200mMand300mM) in half MS media. (\u003cstrong\u003eA)\u0026nbsp; \u003c/strong\u003eChlorophyll and Carotenoid; (\u003cstrong\u003eB)\u003c/strong\u003e Protein; (\u003cstrong\u003eC)\u003c/strong\u003e Proline : (\u003cstrong\u003eD)\u003c/strong\u003e CAT; \u0026nbsp;(\u003cstrong\u003eE)\u003c/strong\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2; \u003c/sub\u003e\u0026nbsp;(\u003cstrong\u003eF) \u003c/strong\u003eSOD\u0026nbsp; estimation by spectrophotometer methods.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/a77cbc79ab0899a2f0102098.png"},{"id":84294579,"identity":"8e41cdd7-618d-4b52-9dcd-b5decf4ad39a","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":981989,"visible":true,"origin":"","legend":"\u003cp\u003eThe histochemical analysis of \u003cem\u003eC. morifolium\u003c/em\u003e leaves grown under tissue culture half MS media supplemented with different concentration of NaCl\u003cstrong\u003e :\u003c/strong\u003e Control (T\u003csub\u003e0\u003c/sub\u003e)\u0026nbsp; 25mM (T\u003csub\u003e1\u003c/sub\u003e), 50mM (T\u003csub\u003e2\u003c/sub\u003e)75mM(T\u003csub\u003e3\u003c/sub\u003e),100mM(T\u003csub\u003e4\u003c/sub\u003e),125mM(T\u003csub\u003e5\u003c/sub\u003e),150mM(T\u003csub\u003e6\u003c/sub\u003e), 200mM(T\u003csub\u003e7\u003c/sub\u003e),300mM(T\u003csub\u003e8\u003c/sub\u003e); \u003cstrong\u003e\u0026nbsp;(a) \u003c/strong\u003eDAB Staining showed the accumulation of H2O2; \u0026nbsp;(\u003cstrong\u003eb)\u003c/strong\u003e NBT Staining showed the accumulation of SOD\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/091edef96e76531f55aa37f6.png"},{"id":84294577,"identity":"65cbbc34-6132-4fa6-a794-a5f89ae0e7ae","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":712660,"visible":true,"origin":"","legend":"\u003cp\u003eTCR-\u003cem\u003eC. morifolium plants\u003c/em\u003e transferred to media having different concentration of NaCl (0 – 400 mmol) under \u003cem\u003eIn-vivo\u003c/em\u003econditions a) after 15 days b) after 45 days; \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003ec)\u003c/strong\u003eNumber of leaves; (\u003cstrong\u003ed)\u003c/strong\u003e Plant height (cm); ( e\u003cstrong\u003e)\u003c/strong\u003e Number of secondary shoots; (\u003cstrong\u003ef)\u003c/strong\u003e Plant spread (cm) at 15, 30 and 45 DAS.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/1cdf8c4f7a2a06f0f0f8e572.png"},{"id":84294583,"identity":"7e36713e-92e7-4ff8-9724-863339c6f0ba","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":561450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRoot observation after different salt stress under \u003cem\u003eIn-vivo\u003c/em\u003econditions at 45 DAS and b) Graphical representation of the root length (cm) at 45 DAS\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/0d6c310eafb41faad1508f3c.png"},{"id":84295641,"identity":"283e283f-0a97-439a-bd49-cd88d4ac83d1","added_by":"auto","created_at":"2025-06-10 09:24:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in (A) Phytopigments (B) Proline, (C) CAT, (D) SOD and (E) H2O2 content of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. morifolium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIn-vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e salt stress and control conditions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/76a5cf836bd03add2c400e73.png"},{"id":88762005,"identity":"3a645f39-8425-47c7-8fdd-ea238470d18a","added_by":"auto","created_at":"2025-08-11 08:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5218266,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/0e60c7b5-1600-4978-be46-055b6c852df1.pdf"},{"id":84294580,"identity":"e7f3e616-a313-4dc9-a1e2-165c0dfa5b1a","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":409202,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/4a4e3e668fdeac5fda5b5212.tif"},{"id":84294575,"identity":"421c1cc7-ec96-4e3d-b1c3-1991a7dbe50e","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":480150,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/6d7144e70007025f6225ea9d.tif"},{"id":84294578,"identity":"3ce8809d-481f-453c-9916-d32075a7e578","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":62716,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/0b5b27b510adc75f336da9bb.tif"},{"id":84294598,"identity":"a39658d1-cb46-4468-98cc-68aeb430b7da","added_by":"auto","created_at":"2025-06-10 09:16:28","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":702708,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/b4f91ad6fd95dbf4eeccbae2.tif"},{"id":84295645,"identity":"0e128e30-cc92-4893-bc38-51d4435dc734","added_by":"auto","created_at":"2025-06-10 09:24:28","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":524906,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/2f5570c47bed9a4956d78585.tif"},{"id":84297208,"identity":"c5d516eb-d7eb-4cc2-bec4-4e74bd6f624a","added_by":"auto","created_at":"2025-06-10 09:40:28","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":70674,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/5be8bbe1466cd40dbb0ef514.tif"},{"id":84294587,"identity":"3567251b-da91-4f1c-b7e1-e3079e919417","added_by":"auto","created_at":"2025-06-10 09:16:27","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":45595,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6636160/v1/56becfc30434c309833cb1a6.docx"}],"financialInterests":"","formattedTitle":"Physiological and Biochemical Responses of Chrysanthemum × morifolium to Salinity Stress under In-Vitro Conditions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe global floriculture industry, one of the fastest growing agricultural sector, plays a significant economic role, with countries like the Netherlands, the United States, Japan and Germany being leaders in the production and export of cut flowers (Getu \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eChrysanthamum x morifolium\u003c/em\u003e species predominantly originate from East Asia, with China, Japan, Thailand, and India being major producers. With its diverse agro-climatic zones, India is well-suited for floriculture, with states like Tamil Nadu, Karnataka, Madhya Pradesh, and West Bengal leading in cultivation (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As India's floriculture industry expands, the area under \u003cem\u003eChrysanthemum\u003c/em\u003e cultivation has grown by 225.33%, with production increased by 391.77% from 2014 to 2024 (Kumar et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChrysanthemum (\u003cem\u003eChrysanthemum \u0026times; morifolium\u003c/em\u003e), commonly called as the \"Queen of the East\", belongs to the family Asteraceae. It is a highly attractive, short-day plant, often treated as a seasonal bloomer in late autumn despite being perennial. Chrysanthemum cut flowers are valued for floral arrangements due to their extended vase life and are cultivated for various decorative uses, including garlands, bouquets and floral decorations for social and religious events (Bohra and Kumar \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Patil et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Apart from its economic value, Chrysanthemum also holds therapeutic importance in traditional medicine, used for treating inflammation, bruises, snake bites, and even as a remedy for fever (Ryu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, the petals are used in Chinese cuisine and for making herbal teas (Collins et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAbiotic stress factors such as salinity, drought, and extreme temperatures significantly affect \u003cem\u003eC. morifolium\u003c/em\u003e, reducing its growth, flower longevity, and aesthetic value (Vinocur and Altman \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). By 2050, salinity is predicted to affect about 33% of the world's irrigated agricultural land (Shao \u003cem\u003eet al\u003c/em\u003e., 2008). High salinity impacts various physiological processes in plants, inhibiting growth and damaging key metabolic activities like photosynthesis and protein synthesis (Munns and Tester \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Acosta-Motos et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Salinity stress induces the accumulation of toxic products, such as reactive oxygen species (ROS), in plants, disrupting their metabolic balance and causing oxidative damage (Ahanger et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In response to salt stress, plants undergo a series of physiological changes, including increased activity of antioxidant enzymes, and elevated proline content (AbdElgawad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Sarabi et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These changes reflect the plant's response to salt stress at various levels, with some mechanisms protecting against oxidative damage and enhancing salt stress tolerance.\u003c/p\u003e \u003cp\u003eAnalysis of stress responses in plants grown through traditional propagation methods is slow and labour-intensive. Plant tissue culture and micro-propagation techniques offer faster, more efficient alternatives for studying plant responses to stress conditions. This method enables the rapid production of large numbers of plants or herbal products from plant cells or tissues (explants) cultured on artificial nutrient media (Oseni, Pande and Nailwal \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Dogan, Karatas and Aasim \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalt stress can be simulated in plant tissue culture by adding sodium chloride (NaCl) to the media, offering a controlled environment to study plant responses at the cellular level (Shibli et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This study was aimed to optimize NaCl concentrations for tissue-cultured Chrysanthemums under \u003cem\u003ein-vitro\u003c/em\u003e conditions and subsequent application of these optimized concentrations to in vitro-developed plants under in vivo conditions within a polyhouse. The experiment involved the treating plants with varying NaCl concentrations to evaluate their impact on plant growth, stress tolerance, and to monitor associated physiological and biochemical changes during development.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThe experiment was conducted in Plant Tissue Culture Laboratory, College of Forestry at Banda University of Agriculture and Technology, Banda, Uttar Pradesh during the years 2022-2024. The climate in Banda is semi-arid/tropical, with dry, hot summers (reaching 49°C) and cold winters (down to 2°C). Annual rainfall averages between 800 and 910 mm and mostly occurring from mid-June to September.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1. Explant Preparation: \u003c/strong\u003eNodal segments from healthy mother plants were treated with biocides (Hydroxyquinoline), fungicides (Carbendazim, Mancozeb), and a bleaching solution (sodium hypochlorite) before being sterilized with HgCl\u003csub\u003e2\u003c/sub\u003e for in-vitro culturing (Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Culture Media: \u003c/strong\u003eThe Murashige and Skoog (MS) medium(Murashige and Skoog 1962) was used which has composition as sucrose (30 g/L), vitamins, growth regulators such as kinetin (Kin), 6-benzylaminopurine (BAP), and thidiazuron (TDZ) adjusting pH to 5.7–5.8, and adding agar powder (7 g/L). Sterilization of the media and other necessary glassware, equipment was done by autoclaving at 121°C for 20 minutes at 15 psi pressure. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Culture Media for Growth Stages:\u003c/strong\u003e For shoot induction, MS media with BAP (0.5-2 mg/L) was used. For shoot proliferation of microshoots were transferred to MS media with varying concentrations of BAP, kinetin, or TDZ for shoot proliferation and rooting of elongated shoots were transferred to half-strength MS media with IBA and IAA for root induction. Sterilized explants were inoculated in culture media inside a laminar airflow chamber, and incubated at 25±1°C under a 16/8 hour light/dark photoperiod. Observations were recorded for shoot induction (%), number of shoots, shoot length (cm), root induction (%), number of roots per shoot and root length (cm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Sub-culturing \u003cem\u003eIn Vitro\u003c/em\u003e plants on MS medium supplemented with NaCl\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo subculture \u003cem\u003ein vitro\u003c/em\u003e plants in MS media supplemented with NaCl (25 mM to 300 mM), first sterile NaCl stock solutions were prepared for each concentration. MS media was prepared according to the standard protocol, and NaCl stock solutions were added to the media. The pH of the NaCl-supplemented MS media was adjusted to 5.7–5.8, then autoclaved. After autoclaving, media allowed to cool. The plant material (explants) is surface sterilized and subcultured into the NaCl-enriched media. Plant growth was regularly monitored for signs of salt stress, including reduced growth rate, physiological changes like chlorophyll and carotenoid content, biochemical changes such as protein, proline levels, antioxidant enzyme activities, or altered root development, and these parameters were recorded to analyze the effects of NaCl stress on the plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Establishment of In-vitro propagated plants for hardening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary hardening of \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e plants was carried out by transferring \u003cem\u003ein vitro\u003c/em\u003e plants to a mixture of cocopeat, perlite and vermiculite (2:1:1) in trays under fiberglass house conditions for two weeks. For secondary hardening, plants were transferred to a mixture of cocopeat, sand and vermicompost (2:1:1) and maintained under shade net conditions for four weeks. During this period, physiological and biochemical changes were monitored under both \u003cem\u003ein vitro\u003c/em\u003e stress and control conditions. These changes included alterations in growth rate, chlorophyll and carotenoid content, protein levels, proline accumulation, and antioxidant enzyme activity, providing insights into the plants' stress response mechanisms during hardening.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Physiological parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.1.Chlorophyll and carotenoid contents of leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyse chlorophyll and carotenoid contents, fully matured open leaves were taken from both control and stressed conditions. The chlorophyll content (chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e and total chlorophyll) of the leaves was estimated using the method described by \u003cstrong\u003eHiscox and Israelstam \u003c/strong\u003e\u003cstrong\u003e(Hiscox and Israelstam 1979)\u003c/strong\u003e\u003cstrong\u003e. A\u003c/strong\u003e sample of 50 mg of leaves was placed in 10 ml of dimethyl sulfoxide (DMSO, Analytical grade) and incubated at 65°C for 4 hours. Post-incubation, the tubes were cooled to room temperature, and the absorbance was measured at 663, 645, and 470 nm using a UV-VIS spectrophotometer (5704 ECIL, India) against pure DMSO as the blank. The chlorophyll and carotenoid contents were calculated using the following formulas:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e (Chl-a)\u003c/strong\u003e = 12.21 A663 − 2.81 A645\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChlorophyll \u003cem\u003eb\u003c/em\u003e (Chl-b)\u003c/strong\u003e = 20.13 A645 − 5.03 A663\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Chlorophyll (mg g⁻¹ DW)\u003c/strong\u003e = (20.7 × OD645) + (8.02 × OD663) × volume × dilution / (1000 × weight of the sample)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Carotenoids\u003c/strong\u003e = [1000 A470 - (3.27 Chl-a + 104 Chl-b)] / 229\u003c/p\u003e\n\u003cp\u003eAll chlorophyll values were expressed in µg/g of fresh weight (fw). The ratio of chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e was calculated by dividing chlorophyll \u003cem\u003ea\u003c/em\u003e by chlorophyll \u003cem\u003eb\u003c/em\u003e to assess the balance between different chlorophyll types under stress and control conditions. The ratio of carotenoid to chlorophyll was calculated by dividing total carotenoids by total chlorophyll to analyze how stress conditions affected pigment composition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2. Biochemical Parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2.1. Antioxidant Enzyme Activity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant enzyme activities were analyzed to assess oxidative stress in \u003cem\u003eC. morifolium\u003c/em\u003e under in-vitro conditions. Leaf samples from each treatment were collected in an ice-box to prevent proteolytic degradation. One gram of leaf tissue was homogenized in a pre-chilled mortar and pestle with 5 ml of chilled phosphate buffer (50 mM, pH 7.0). The homogenate was centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant was used for the following enzyme assays:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2.2. Superoxide Dismutase (SOD) Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOD activity was determined according to \u003cstrong\u003eFridovich \u003c/strong\u003e\u003cstrong\u003e(Fridovich 1975)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The reaction was based on the ability of SOD to inhibit the photochemical reduction of nitro blue tetrazolium (NBT). Absorbance was recorded at 560 nm, and enzyme activity was expressed as unit/min/mg of protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2.3. Catalase (CAT) Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCatalase activity was measured using the method of \u003cstrong\u003eLuck \u003c/strong\u003e\u003cstrong\u003e(Lück 1965)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Residual hydrogen peroxide (H₂O₂) was quantified by titration with potassium permanganate. Results were expressed as µmol of H₂O₂ decomposed per minute per mg of protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.3. H₂O₂ Content Estimation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH₂O₂ content was determined following the method of \u003cstrong\u003eAlexieva \u003cem\u003eet al.\u003c/em\u003e \u003c/strong\u003e\u003cstrong\u003e(Alexieva et al. 2001)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Using 0.1% TCA for extraction, the H₂O₂ was reacted with potassium iodide (KI), and the absorbance was measured at 390 nm. Results were expressed as µmol g⁻¹ of fresh weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.4. Proline Content \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProline was quantified following the method of Bates et al. (Bates, Waldren and Teare 1973). The leaves were homogenized in 3% sulpho-salicylic acid, and the proline content was determined by the formation of a pink chromophore with acid ninhydrin, followed by toluene extraction. Absorbance was recorded at 520 nm, and proline content was expressed as µg/g of dry weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.5. Protein Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein content was estimated using Lowry's method (Lowry et al. 1951). The protein was precipitated using TCA, and absorbance of blue color developed after treatment with Folin-Ciocalteu reagent was measured at 660 nm. Protein content was calculated based on a standard curve prepared using bovine serum albumin (BSA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.6. Histochemical detection of superoxide and H₂O₂ by NBT and DAB staining \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide and H₂O₂ were detected in leaf tissues using NBT and DAB staining techniques following Kumar et al. (Kumar et al. 2014). Theleaves were stained overnight, cleared with ethanol and visualized against a contrast background. Superoxide appeared as dark blue spots, while H₂O₂ as reddish-brown spots.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe study investigated the sterilizing agent, the effects of basal media composition, phytohormones, and NaCl treatments on shoot induction, regeneration frequency, and shoot growth.\u0026nbsp;Surface sterilization using an optimized combination of 2% NaOCl, 0.10% fungicide, 0.02% biocide, and 0.10% HgCl₂ resulted in the highest survival of \u003cem\u003eChrysanthemum \u0026times; morifolium\u003c/em\u003e nodal explants, whereas higher concentrations significantly reduced viability (Table S1).\u0026nbsp;Full-strength and half-strength MS media were supplemented with different concentrations of phytohormones, including BAP, TDZ, and kinetin, and the results demonstrated significant variations in shoot induction and growth. In full-strength MS medium, the addition of BAP at 2 mg/L yielded the highest shoot induction (69.67%) with an average of 3.57 shoots per explant and a mean shoot length of 5.43 cm. TDZ at 0.5 mg/L resulted in 59% shoot induction with 2.97 shoots and a shoot length of 2.77 cm. Among kinetin treatments, 1 mg/L produced the highest induction rate (50.33%), with an average of 3.23 shoots and a shoot length of 3.67 cm. However, higher concentrations of TDZ and kinetin reduced shoot induction and growth, indicating a concentration-dependent response. Half-strength MS medium supplemented with 2 mg/L BAP was the most effective for shoot induction and growth. It achieved the highest shoot regeneration frequency (89.67%), producing an average of 13.03 shoots per explant with a mean shoot length of 8.9 cm. BAP at 0.5 mg/L also showed high efficacy, with an 80.33% induction rate and 11.67 shoots per explant (Figure S1a\u0026amp;b and Table 1).\u003c/p\u003e\n\u003cp\u003eThe addition of NaCl to half-strength MS medium supplemented with 2 mg/L BAP significantly reduced shoot regeneration frequency and growth. Under control conditions (T0, 0 mM NaCl), the regeneration frequency was 89.33%, with a mean shoot length of 7.83 cm. At 25 mM NaCl (T1), the regeneration frequency decreased to 79.87%, and shoot length reduced to 5.97 cm. With increasing NaCl concentrations, the inhibitory effect became more pronounced. At 100 mM NaCl (T4), regeneration frequency dropped to 54.73%, and shoot length decreased to 3.27 cm. At 300 mM NaCl (T8), the lowest regeneration frequency (5.33%) and shoot length (0.9 cm) were observed, reflecting the severe impact of salt stress on shoot development. The result showed that the BAP was the most effective phytohormone for promoting shoot induction and elongation under both normal and stress conditions. However, salt stress caused a substantial decline in regeneration frequency and shoot length, with the inhibitory effects increasing with higher NaCl concentrations. These findings highlight the importance of optimizing phytohormone concentrations and managing salt stress for successful in vitro shoot regeneration (\u003cstrong\u003eFigure 1 a \u0026amp; b;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTable 1).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. Role\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof different NaCl concentrations on root regeneration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRoot regeneration in \u003cem\u003eC. morifolium\u003c/em\u003e was most effective with 0.2 mg/L IBA in half-strength MS medium, yielding the highest root induction (90.4%) and the maximum roots per shoot (11.66). The longest roots (12.30 cm) were observed at 0.4 mg/L IBA. IAA showed lower efficiency, with 70.43% root induction, 10 roots per shoot, and a maximum root length of 11.40 cm at optimal concentrations. Higher auxin levels (1.0 mg/L) significantly reduced root induction, number, and length. These results underscore the efficacy of low IBA concentrations for root regeneration\u0026nbsp;\u003cstrong\u003e(Figure S2; Table S2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Physiological and biochemical changes in \u003cem\u003eC. morifolium\u003c/em\u003e under in-vitro salt stress\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe study examined the physiological and biochemical responses of \u003cem\u003eC. morifolium\u003c/em\u003e under varying salinity stress. Chlorophyll A and B content decreased with increasing salt levels, with the highest chlorophyll A (113.34 \u0026micro;g/g) and B (72.30 \u0026micro;g/g) observed at 25 mmol/L NaCl, and the lowest at 300 mmol/L NaCl. The chlorophyll A/B ratio increased with salinity, while total chlorophyll and carotenoid content also declined, with the highest total chlorophyll (207.49 \u0026micro;g/g) and carotenoid (38.13 \u0026micro;g/g) at 25 mmol/L NaCl ( Figure 2a). Proline content, a key stress marker, significantly increased with salinity, peaking at 300 mmol/L NaCl (92.33 \u0026micro;g/g) \u003cstrong\u003e(Figure 2b\u003c/strong\u003e). Superoxide Dismutase (SOD) and Catalase (CAT) activities elevated under high salt stress, with the highest SOD activity (107.20 unit/min/mg protein) and CAT activity (137.86 \u0026micro;moles of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposed min-1 mg-1 protein) recorded at 300 mmol/L NaCl. Hydrogen Peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels also significantly increased under salinity, reaching a maximum of 133.52 \u0026micro;moles g-1 fw in 300 mmol/L NaCl \u003cstrong\u003e(Figure: 2c-2e).\u003c/strong\u003e These findings highlight how \u003cem\u003eC. morifolium\u003c/em\u003e responds to salinity stress by activating stress-responsive biochemical pathways, including protein degradation, proline accumulation, and enhanced antioxidant enzyme activity \u003cstrong\u003e(Figure 2 a-e).\u0026nbsp;\u003c/strong\u003eIn contrast, at 25 mmol/L NaCl (T1), ROS accumulation was much lower. These findings align with increased H₂O₂ and SOD levels, confirming that oxidative stress increases with salinity \u003cstrong\u003e(Figure 2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Histochemical detection of Superoxide and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eby NBT and DAB staining of \u003cem\u003eChrysanthemum\u003c/em\u003e leaves under NaCl treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe histochemical analysis of \u003cem\u003eC. morifolium\u003c/em\u003e leaves under NaCl-induced salinity showed significant accumulation of ROS, including hydrogen peroxide (H₂O₂) and superoxide anions. At 300 mmol/L NaCl (T8), DAB staining revealed intense brown pigmentation, indicating high H₂O₂ accumulation \u003cstrong\u003e(Figure 3a),\u003c/strong\u003e while NBT staining showed dark blue spots, indicating superoxide build up \u003cstrong\u003e(Figure 3b).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.4. Principle component analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Principal Component Analysis (PCA) biplot \u003cstrong\u003e(Figure S3)\u003c/strong\u003e provides a clear visualization of the impact of various treatments on plant growth and stress responses. The first principal component (F1), accounting for 98.78% of the variance, is primarily influenced by growth-promoting factors such as shoot regeneration frequency, total chlorophyll, chlorophyll A, chlorophyll B, carotenoids, protein content, and shoot length. Treatments T1, T2, T3, and T0, positioned on the right side of the biplot, show strong positive associations with these variables, indicating favourable conditions for growth, chlorophyll synthesis, protein accumulation, and shoot regeneration. These treatments likely provided optimal environmental or nutrient conditions, enabling the plants to allocate energy toward growth and development. In contrast, the second principal component (F2), which explains only 0.80% of the variance, is associated with stress-related variables like superoxide dismutase (SOD), hydrogen peroxide (H₂O₂), and proline. Treatments T4 and T5, positioned closer to these stress markers, indicate a moderate level of stress, activating antioxidant defense mechanisms to counteract oxidative and osmotic stress. Proline accumulation suggests osmotic stress responses. Conversely, treatments T8, T6, and T7, positioned on the far-left side of F1, are negatively correlated with growth-related variables, indicating detrimental effects on plant growth. The large separation between these treatments and growth parameters suggests that they inhibited shoot regeneration and reduced biomass accumulation, possibly due to poor environmental conditions, nutrient deficiencies, or other stress factors. (\u003cstrong\u003eFigure S3\u003c/strong\u003e). The biplot clearly shows the trade-off between growth and stress responses. Treatments such as T1, T2, T3, and T0 promoted growth, enhancing shoot regeneration and chlorophyll synthesis. In contrast, treatments like T4 and T5 activated stress-related pathways, redirecting resources toward antioxidant and osmotic adjustments, which limited biomass accumulation. This is reflected by the increased SOD, H₂O₂, and proline levels, indicating oxidative and osmotic stress. The PCA biplot effectively differentiates between treatments that favor growth and those that trigger stress responses. Treatments T1, T2, T3, and T0 provided optimal conditions for plant development, while treatments like T4 and T5 activated defense mechanisms. Treatments T8, T6, and T7 were the least effective, resulting in suppressed growth. The biplot emphasizes the need to balance growth and stress responses for successful regeneration and development under varying conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Establishment of \u003cem\u003eIn-vitro\u003c/em\u003e propagated plants for hardening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue culture-raised \u003cem\u003eC. morifolium\u003c/em\u003e plants under salt stress were successfully acclimatized and hardened through a sequential process. In the primary hardening stage, the plants exhibited significant growth in a medium comprising cocopeat, perlite, and vermiculite in a 2:1:1 ratio. During secondary hardening, the plants continued to grow well in a mix of cocopeat, sand, and vermicompost (2:1:1). Finally, the acclimatized plants were transplanted into a healthy potting medium of vermicompost, garden soil, sand, and cocopeat in a 2:1:1:1 ratio, where they thrived under controlled conditions in a polyhouse (Figure S4; Figure 4a and b).\u003c/p\u003e\n\u003cp\u003eThe impact of varying NaCl concentrations on tissue-cultured \u003cem\u003eC. morifolium\u0026nbsp;\u003c/em\u003eplants under in-vivo conditions was evaluated at 15, 30, and 45 DAS. Significant effects were observed on parameters such as the number of leaves (Figure 4c), plant height (Figure 4d), secondary shoots (Figure 4e), and plant spread (Figure 4f), (Figure 4 c-f and Table 2). The highest number of leaves and plant height were recorded in treatments S2 and S3 (Figure 4c). Secondary shoots peaked at S3 (19.25 at 30 DAS) and S5 (14 at 15 DAS), while plant spread was greatest in S1 (18.45 cm at 30 DAS). Shoot diameters were maximized under S4 and S3, respectively (Table 2). The higher NaCl concentrations generally suppressed plant growth, with S1 and S3 performing best across most parameters. Negative controls consistently showed inferior results compared to positive controls. Root length measurements at the end of the experiment (45 DAS) revealed the longest roots under S1 salt treatment (15.22 cm), followed by S2. Negative controls produced shorter roots compared to positive controls (Figures 5a,b). Soil conductivity (E.C.) and pH were also influenced by NaCl levels. At 45 DAS, S5 had the highest E.C. (25.10), while S1 recorded the lowest (8.13). Soil pH was highest in S1 and S2 (7.69), and lowest in S5 (7.49). Negative controls showed consistently poorer performance compared to positive controls in both plant and soil parameters (Table S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Effect of NaCl on physiology and biochemical changes of tissue cultured raised \u003cem\u003eC. morifolium\u003c/em\u003e under in vivo salt stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study investigated the impact of different NaCl concentrations on tissue-cultured \u003cem\u003eC\u003c/em\u003e. \u003cem\u003emorifolium\u003c/em\u003e under in-vivo conditions, highlighting significant physiological and biochemical changes. Chlorophyll A and B contents decreased progressively with increasing NaCl concentrations, with the highest levels recorded at S1 (105.71 \u0026micro;g/g for chlorophyll A and 61.57 \u0026micro;g/g for chlorophyll B), while the lowest were observed at S5 (39.27 \u0026micro;g/g for chlorophyll A and 18.74 \u0026micro;g/g for chlorophyll B) (Fig. 6a). The chlorophyll A/B ratio increased under higher salt stress, peaking at S5 (2.09) (Fig. 6a). Total chlorophyll content also showed a decreasing trend, with S1 exhibiting the highest (167.28 \u0026micro;g/g) and S5 the lowest (58.01 \u0026micro;g/g) carotenoid content similarly declined with increasing NaCl, reaching the highest in S1 (33.16 \u0026micro;g/g) and the lowest in S5 (6.49 \u0026micro;g/g), resulting in a carotenoid/chlorophyll ratio of 0.19 in S1 and 0.11 in S5 (Figure 6a). Proline content, an osmoprotectant, increased markedly under high salt stress, peaking at S5 with 98.63 \u0026micro;g/g of fw, compared to the lowest content in S1 (40.88 \u0026micro;g/g of fw) (Figure 6b). Antioxidant enzyme activities, including SOD and catalase, were significantly elevated in response to salt stress, with catalase activity peaking at 143.87 \u0026micro;moles of H2O2 decomposed/min/mg of protein in S5 (Figure 6c), and SOD activity reaching a maximum of 113.19 unit/min/mg of protein in S5 (Figure 6d). H2O2 accumulation also increased under higher salt conditions, with the highest content recorded in S5 (139.14 \u0026micro;moles/g of fw), compared to the lowest in S1 (74.37 \u0026micro;moles/g of fw) (Figure 6e). Negative controls consistently showed lower values compared to positive controls across all parameters, confirming the detrimental effects of higher NaCl concentrations on plant growth, chlorophyll synthesis, protein content, and antioxidant defense mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. Histochemical Detection of superoxide and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eby NBT and DAB Staining of \u003cem\u003eIn-vivo\u003c/em\u003e raised tissue cultured plants\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistochemical analysis of \u003cem\u003eC. morifolium\u003c/em\u003e leaves under different NaCl concentrations (0\u0026ndash;400 mmol) revealed hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and superoxide anion accumulation. In leaves collected 45 DAS, DAB staining showed brown spots indicating H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation, while NBT staining revealed blue pigments, signifying superoxide anion presence. Severe cell damage was observed at 400 mmol NaCl (S5), with intense brown and blue pigmentation, while fewer symptoms were seen at 25 mmol NaCl (S1). These findings align with biochemical analysis, where higher H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and SOD activity were recorded in S5 compared to the control \u003cstrong\u003e(Figure S5).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8. Principle component analysis:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Principal Component Analysis (PCA) biplot \u003cstrong\u003e(Figure S6)\u003c/strong\u003e reveals the relationships between physiological and biochemical variables (active variables in red) and treatments (active observations in blue), explaining a total variance of 84.04% through two principal components, F1 and F2. \u0026nbsp;The F1 accounts for 71.06% of the variance and is strongly influenced by stress-related variables like proline, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and superoxide dismutase (SOD), which are prominently associated with higher salinity treatments such as S5 (400 mM) and S4 (200 mM). These treatments show a positive correlation with oxidative stress and osmotic regulation responses, indicating that they induce significant stress in the plant. Conversely, F2, which explains 12.99% of the variance, is dominated by growth-related variables such as plant height at 30 and 45 days, root length, and the number of leaves at 15 days. These growth parameters are positively associated with lower salinity treatments like S1 (25 mM) and S2 (50 mM), demonstrating that mild salinity levels promote plant growth. The negative control (So) is centered near the origin, showing a moderate response across all variables, while the positive control (So+) clusters near chlorophyll content (Chl A and Chl B), highlighting optimal conditions for photosynthesis and plant health under non-stress conditions. The biplot distinctly separates growth-related responses, aligned with the positive side of F2, from stress-related responses, positioned on the positive side of F1. This separation emphasizes that higher salinity levels hinder growth while activating stress defense mechanisms. In summary, the PCA biplot effectively visualizes the differential impacts of salinity treatments on plant growth and stress, demonstrating that lower salinity fosters growth, whereas elevated salinity triggers biochemical stress responses.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e4.1. Regeneration and multiplication of \u003cem\u003eChrysanthemum\u003c/em\u003e under different phytohormones condition\u003c/h2\u003e\n \u003cp\u003eThis study focuses on identifying optimal NaCl concentrations that do not adversely affect \u003cem\u003eChrysanthemum\u003c/em\u003e growth in both \u003cem\u003ein-vitro\u003c/em\u003e and \u003cem\u003ein-vivo\u003c/em\u003e (pot culture) conditions. Field-grown explants face microbial contamination challenges in in-vitro cultures. Pre-treatments with 0.1% Carbendazim, 0.02% 8-HQC (30 min), and 0.1% HgCl₂ (3 min) resulted in 51.00% survival. Higher survival rates (75.56%) were reported with 0.2% Mancozeb, 0.2% Carbendazim, and 200 mg/L 8-HQC (Verma et al. 2012). Similarly, 0.1% HgCl₂ was most effective in \u003cem\u003eStevia\u003c/em\u003e (Sharuti Verma, Kuldeep Yadav and Narender Singh 2011) and yielded 65.08% survival in \u003cem\u003eChrysanthemum\u003c/em\u003e (Anjum et al. 2023). For shoot induction, half-strength of MS medium with 30 g/L sucrose, 2.0 mg/L BAP, and 7 g/L agar achieved 89.66% induction. Comparable rates were reported in \u003cem\u003eChrysanthemum\u003c/em\u003e (76.66%, 2.0 mg/L BAP; (Yesmin et al. 2014), 83.30% with 2.0 mg/L BAP\u0026thinsp;+\u0026thinsp;0.5 mg/L IAA (Kashif Waseem et al. 2011) and 84.74% in \u003cem\u003eXanthosoma sagittifolium\u003c/em\u003e (Bansal et al. 2023). Jahan et al. (Jahan et al. 2021) recorded 100% regeneration with 2.0 mg/L BAP, while Boonkamjat et al. (Boonkamjat, Saetiew and Teerarak) reported 93.33% with 1 mg/L BA\u0026thinsp;+\u0026thinsp;0.1 mg/L IAA in \u003cem\u003eChrysanthemum\u003c/em\u003e. Shoot proliferation was highest (13.03 shoots) in half-strength MS medium supplemented with 2.0 mg/L BAP. Similar findings were reported in \u003cem\u003eChrysanthemum\u003c/em\u003e (7.7 shoots) (Kashif Waseem et al. 2011) and \u003cem\u003eXanthosoma sagittifolium\u003c/em\u003e (13.44 shoots) (Bansal et al. 2023). Shoot length (8.90 cm) was optimal with 2.0 mg/L BAP, consistent with Ghosh et al.(Ghosh et al. 2021). Root induction was highest (90.40%) with half-strength MS medium\u0026thinsp;+\u0026thinsp;0.2 mg/L IBA as supported by studies of researchers (Yesmin et al. 2014), (Rashid et al. 2009), (Kashif Waseem et al. 2011), and (Sushmarani, Venkatesha Murthy and Deeksha Raj 2021). Higher auxin concentrations inhibited rooting (Kaul et al. 1990) and longest roots (12.30 cm) were obtained with 0.4 mg/L IBA (Komalavalli and Rao 2000), while maximum roots (11.66 per shoot) were observed with 0.2 mg/L IBA, aligning with other researchers (Yesmin et al. 2014, Deltalab et al. 2024).\u003c/p\u003e\n \u003cp\u003eThe development of salt-tolerant plants through in-vitro techniques, such as callus and shoot cultures is a non-transgenic route. Since, salt stress typically reduces plant growth (Queiros et al. 2007), as slower growth linked to improved stress survival. In our study, the highest shoot regeneration frequency (79.86%) was recorded at 25 mmol/L NaCl, with the lowest observed at 300 mmol/L. Salt stress reduced shoot regeneration frequency due to impaired cell division and photosynthesis, as reported in similar studies on Chrysanthemum (Thongpukdee et al. 2012) and \u003cem\u003eBacopa monnieri\u003c/em\u003e (Sable et al. 2018).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e4.2. Physiological, biochemical and histological analysis of \u003cem\u003ein-vitro\u003c/em\u003e regenerated plant under NaCl stress\u003c/h2\u003e\n \u003cp\u003eUnder salinity stress, \u003cem\u003eC. morifolium\u003c/em\u003e displayed significant physiological and biochemical alterations compared to control conditions. Salinity treatment influenced photosynthetic pigments, including chlorophyll and carotenoids. The highest levels of chlorophyll A, chlorophyll B, total chlorophyll, and carotenoids were observed at 25 mmol/L NaCl (T1). However, the chlorophyll A/B ratio and carotenoid/chlorophyll ratio (0.231) at 200 mmol/L NaCl (T7). This decline in chlorophyll is attributed to pigment-protein complex instability under stress, as noted in previous studies (Dogan 2020, Rai et al. 2020). Moreover, the proline and protein levels varied significantly under salt stress. Maximum protein content was observed at 25 mmol/L NaCl, while proline accumulation peaked at 300 mmol/L, reflecting the role of osmolites in stress mitigation. Antioxidative enzymes showed increased activity, with SOD, CAT, and H₂O₂ scavengers rising by 2.3-, 2.8-, and 3.1-fold, respectively, at 300 mmol/L NaCl, supporting previous findings in \u003cem\u003eChrysanhemum\u003c/em\u003e (Guan et al. 2012). NBT and DAB staining confirmed increased O₂⁻ and H₂O₂ accumulation under salt stress, leading to oxidative damage, reduced membrane integrity, and necrotic lesions at 300 mmol/L NaCl, consistent with studies on \u003cem\u003eBrassica napus\u003c/em\u003e and \u003cem\u003eChrysanthemum\u003c/em\u003e (Banerjee and Roychoudhury 2017, Ahmad et al. 2019).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e4.3. The impact of varying NaCl concentrations on tissue-cultured \u003cem\u003eC. morifolium\u003c/em\u003e plants under in-vivo conditions\u003c/h2\u003e\n \u003cp\u003eSalinity stress significantly impacted plant biomass by altering water potential, increasing ion toxicity, and inhibiting cell wall expansion, leading to reduced root growth and plant height. A decline in growth parameters was observed with increasing NaCl concentrations, with the most significant biomass reduction at 45 DAS in S5 (400 mmol/L NaCl). Leaf number, plant height, and secondary shoot numbers varied across treatments, with S2, S3, and S1 showing the highest values at different stages. Plant spread was highest in S1, while primary stem diameter peaked in S4 and secondary shoot diameter in S3, S4, and S2 at different DAS intervals. These results align with previous studies on ornamental plants like \u003cem\u003eTagetes erecta\u003c/em\u003e (Sayyed et al. 2014), \u003cem\u003eChrysanthemum\u003c/em\u003e (Ba\u0026ntilde;\u0026oacute;n et al. 2010), and marigold (Bahmanzadegan and Aboutalebi 2013), where salt stress-induced growth reduction was attributed to osmotic and ionic imbalances, inhibited cell division and elongation, and decreased cell wall extensibility. Prolonged salinity exposure led to severe symptoms such as leaf scorching, necrosis, and leaf drop, potentially resulting in plant death.\u003c/p\u003e\n \u003cp\u003eSalinity significantly affects root growth by limiting nutrient and water uptake, ultimately impacting plant health and yield. In our study, maximum root length was recorded under S1 (25 mmol/L NaCl), while the negative control exhibited the least growth. Similar reductions in root length under increasing salinity have been reported in \u003cem\u003eChrysanthemum indicum\u003c/em\u003e (Ranganayakulu, Veeranagamallaiah and Chinta Sudhakar 2013, Mazhar et al. 2012). Additionally, soil EC, a key indicator of soil health, increased with higher NaCl concentrations, peaking in S5 (400 mmol/L NaCl) at 45 DAS. In contrast, soil pH remained highest in S1 and S2 at different time points.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4.4. Effect of NaCl on physiology and biochemical changes of tissue cultured raised\u003c/strong\u003e \u003cstrong\u003eC. morifolium\u003c/strong\u003e \u003cstrong\u003eunder\u003c/strong\u003e \u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003esalt stress\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSalinity stress significantly impacts photosynthesis by inducing chlorosis, reducing pigment synthesis, and increasing oxidative stress(Shaw 1995, Parida and Das 2005). Carotenoids, which protect against ROS, decline under salt stress, leading to chlorophyll degradation (Rao and Rao 1981). In our study, the highest chlorophyll A (105.71 \u0026micro;g/g), chlorophyll B (61.57 \u0026micro;g/g), total chlorophyll (167.28 \u0026micro;g/g), and carotenoids (33.16 \u0026micro;g/g) were observed in S1 (25 mmol/L NaCl), while the highest chlorophyll A/B ratio (2.09 \u0026micro;g/g) was found in S5 (400 mmol/L NaCl), consistent with previous reports in corn (Molazem, Qurbanov and Dunyamaliyev 2010), cucumber (Malik et al. 2010), and purslane (Parvaneh, Shahrokh and Meysam 2012). Protein content, crucial for osmotic adjustment, was highest in S1 (47.87 \u0026micro;g/g), indicating a balance between synthesis and degradation under moderate stress (Dogan 2020, Shatnawi et al. 2010). Proline, a key osmo-protectant, peaked in S5 (98.63 \u0026micro;g/g), supporting its role in salinity tolerance as reported by other researchers (Ashraf and Tufail 1995, Mansour et al. 2005).\u003c/p\u003e\n \u003cp\u003eROS scavenging enzymes, such as SOD and CAT, play a vital role in mitigating oxidative stress (Yasemin et al. 2021, Parvin et al. 2019). The highest SOD (113.19 unit/min/mg), CAT (143.87 \u0026micro;moles H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposed min-1 mg-1), and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content (139.14 \u0026micro;moles g-1 FW) were observed in S5, indicating increased ROS detoxification at higher salinity (Zandalinas et al. 2017, Laxa et al. 2019). High ROS levels, including superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, lead to oxidative damage and plasma membrane disruption, as observed in \u003cem\u003eC. morifolium\u003c/em\u003e under severe salt stress (Banerjee and Roychoudhury 2017, Su et al. 2019). Similar trends have been reported in \u003cem\u003eBrassica napus\u003c/em\u003e (Huang et al. 2022), where enhanced CAT and GR activity contributed to stress tolerance (Bor, \u0026Ouml;zdemir and T\u0026uuml;rkan 2003, Yaşar and Ellialtıoğlu 2013).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study established a standardization protocol that can be used to produce true-to-type plants, which are otherwise challenging to obtain for this species. This well-standardized protocol may facilitate large-scale multiplication of desired types, thereby meeting the demand for high-quality planting material of \u003cem\u003eC. morifolium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe second main focus of the present investigation was to analyze the morphological, physiological, and biochemical responses of \u003cem\u003eChrysanthemum\u003c/em\u003e to NaCl-induced salinity stress under \u003cem\u003eIn-vitro\u003c/em\u003e and \u003cem\u003eIn-vivo\u003c/em\u003e (pot culture) raised tissue-cultured plants. Based on the findings, it is concluded that salinity imposes both osmotic and ionic stress, impairing plant cell functions, damaging cell membranes, and affecting the photosynthetic apparatus. Salt stress reduces biomass, plant growth, chlorophyll content, carotenoid content, and restricts the accumulation of beneficial nutrients in plant tissues. Plants adapted to salt stress through mechanisms such as upregulation of antioxidant enzyme activities and accumulation of osmolytes like proline, which protect plant cells from the harmful effects of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e ions. Plants that performed better under varying NaCl concentrations likely employed these mechanisms to maintain ionic homeostasis and protect against reactive oxygen species generated under salinity stress. These findings are commercially valuable for developing salt-tolerant varieties, ensuring better survival and productivity in saline environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant tissue culture work was performed in Plant Tissue Culture Laboratory developed under Rashtriya Krishi Vikas Yojana (UP/RKVY-AGRE/2019/861). Biochemical work conducted at Biochemistry laboratory developed under ICAR fund at BUAT, Banda.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e SP, RK: conceived the idea and drafted the manuscript; RM: performed Plant tissue culture work; VC: analysis of physiological and biochemical work and editing work; SK edited the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u003c/strong\u003e\u0026nbsp; \u0026nbsp;The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available upon \u0026nbsp; request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e: The authors declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and Consent to participate:\u0026nbsp;\u003c/strong\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdElgawad, H., G. Zinta, M. M. Hegab, R. Pandey, H. Asard \u0026amp; W. Abuelsoud (2016) High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. \u003cem\u003eFrontiers in plant science,\u003c/em\u003e 7\u003cstrong\u003e,\u003c/strong\u003e 276.\u003c/li\u003e\n\u003cli\u003eAcosta-Motos, J.-R., P. Diaz-Vivancos, S. Alvarez, N. Fern\u0026aacute;ndez-Garc\u0026iacute;a, M. J. Sanchez-Blanco \u0026amp; J. A. Hern\u0026aacute;ndez (2015) Physiological and biochemical mechanisms of the ornamental Eugenia myrtifolia L. plants for coping with NaCl stress and recovery. \u003cem\u003ePlanta,\u003c/em\u003e 242\u003cstrong\u003e,\u003c/strong\u003e 829-846.\u003c/li\u003e\n\u003cli\u003eAhanger, M. A., N. S. Tomar, M. Tittal, S. Argal \u0026amp; R. Agarwal (2017) Plant growth under water/salt stress: ROS production; antioxidants and significance of added potassium under such conditions. \u003cem\u003ePhysiology and Molecular Biology of Plants,\u003c/em\u003e 23\u003cstrong\u003e,\u003c/strong\u003e 731-744.\u003c/li\u003e\n\u003cli\u003eAhmad, R., S. Hussain, M. A. Anjum, M. F. Khalid, M. Saqib, I. Zakir, A. Hassan, S. Fahad \u0026amp; S. Ahmad (2019) Oxidative stress and antioxidant defense mechanisms in plants under salt stress. \u003cem\u003ePlant abiotic stress tolerance: Agronomic, molecular and biotechnological approaches\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e 191-205.\u003c/li\u003e\n\u003cli\u003eAlexieva, V., I. Sergiev, S. Mapelli \u0026amp; E. Karanov (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. \u003cem\u003ePlant, Cell \u0026amp; Environment,\u003c/em\u003e 24\u003cstrong\u003e,\u003c/strong\u003e 1337-1344.\u003c/li\u003e\n\u003cli\u003eAnjum, N., S. Dogra, R. Pandey, P. Pandotra, N. Laishram, A. Singh, S. Kashyap \u0026amp; A. H. Shah (2023) Optimization the sterilization and acclimatization protocol for micropropagation of commercial cultivar chrysanthemum \u0026lsquo;Maghi White\u0026rsquo;.\u003c/li\u003e\n\u003cli\u003eAshraf, M. \u0026amp; M. Tufail (1995) Variation in salinity tolerance in sunflower (Helianthus annum L.). \u003cem\u003eJournal of Agronomy and Crop Science,\u003c/em\u003e 174\u003cstrong\u003e,\u003c/strong\u003e 351-362.\u003c/li\u003e\n\u003cli\u003eBahmanzadegan, M. \u0026amp; A. Aboutalebi (2013) Interaction between ammonium nitrate and salinity on germination rate and vegetative growth of French marigold (Tageta patula).\u003c/li\u003e\n\u003cli\u003eBanerjee, A. \u0026amp; A. Roychoudhury (2017) Abiotic stress, generation of reactive oxygen species, and their consequences: an overview. \u003cem\u003eReactive Oxygen Species in Plants: Boon or Bane‐Revisiting the Role of ROS\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e 23-50.\u003c/li\u003e\n\u003cli\u003eBa\u0026ntilde;\u0026oacute;n, S., E. Conesa, R. Vald\u0026eacute;s, J. Miralles, J. Mart\u0026iacute;nez \u0026amp; M. S\u0026aacute;nchez Blanco. 2010. Effects of saline irrigation on phytoregulator-treated chrysanthemum plants. In \u003cem\u003eXXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): International Symposium on 937\u003c/em\u003e, 307-312.\u003c/li\u003e\n\u003cli\u003eBansal, S., M. K. Sharma, S. Singh, P. Joshi, P. Pathania, E. V. Malhotra, S. Rajkumar \u0026amp; P. Misra (2023) Histological and molecular insights in to in vitro regeneration pattern of Xanthosoma sagittifolium. \u003cem\u003eScientific Reports,\u003c/em\u003e 13\u003cstrong\u003e,\u003c/strong\u003e 5806.\u003c/li\u003e\n\u003cli\u003eBates, L. S., R. Waldren \u0026amp; I. Teare (1973) Rapid determination of free proline for water-stress studies. \u003cem\u003ePlant and soil,\u003c/em\u003e 39\u003cstrong\u003e,\u003c/strong\u003e 205-207.\u003c/li\u003e\n\u003cli\u003eBohra, M. \u0026amp; A. Kumar (2014) Studies on effect of organic manure and bioinoculants on vegetative and floral attributes of chrysanthemum cv. Little darling. \u003cem\u003eThe Bioscan,\u003c/em\u003e 9\u003cstrong\u003e,\u003c/strong\u003e 1007-1010.\u003c/li\u003e\n\u003cli\u003eBoonkamjat, T., K. Saetiew \u0026amp; M. Teerarak Influence of plant growth regulators on shoot development of Chrysanthemums.\u003c/li\u003e\n\u003cli\u003eBor, M., F. \u0026Ouml;zdemir \u0026amp; I. T\u0026uuml;rkan (2003) The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. \u003cem\u003ePlant science,\u003c/em\u003e 164\u003cstrong\u003e,\u003c/strong\u003e 77-84.\u003c/li\u003e\n\u003cli\u003eCollins, R., T. Ng, W. Fong, C. Wan \u0026amp; H. Yeung (1997) A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of Chinese medicinal herbs. \u003cem\u003eLife Sciences,\u003c/em\u003e 60\u003cstrong\u003e,\u003c/strong\u003e PL345-PL351.\u003c/li\u003e\n\u003cli\u003eDeltalab, B., B. Kaviani, D. Kulus \u0026amp; S. A. Sajjadi (2024) Optimization of shoot multiplication and root induction in Saintpaulia ionantha H. Wendl. using thiamine (vitamin B1) and IBA: A promising approach for economically important African violet propagation. \u003cem\u003ePlant Cell, Tissue and Organ Culture (PCTOC),\u003c/em\u003e 156\u003cstrong\u003e,\u003c/strong\u003e 74.\u003c/li\u003e\n\u003cli\u003eDogan, M. (2020) Effect of salt stress on in vitro organogenesis from nodal explant of Limnophila aromatica (Lamk.) Merr. and Bacopa monnieri (L.) Wettst. and their physio-morphological and biochemical responses. \u003cem\u003ePhysiology and molecular biology of plants,\u003c/em\u003e 26\u003cstrong\u003e,\u003c/strong\u003e 803-816.\u003c/li\u003e\n\u003cli\u003eDogan, M., M. Karatas \u0026amp; M. Aasim (2018) Cadmium and lead bioaccumulation potentials of an aquatic macrophyte Ceratophyllum demersum L.: a laboratory study. \u003cem\u003eEcotoxicology and Environmental Safety,\u003c/em\u003e 148\u003cstrong\u003e,\u003c/strong\u003e 431-440.\u003c/li\u003e\n\u003cli\u003eFridovich, I. (1975) Superoxide dismutases. \u003cem\u003eAnnual review of biochemistry,\u003c/em\u003e 44\u003cstrong\u003e,\u003c/strong\u003e 147-159.\u003c/li\u003e\n\u003cli\u003eGetu, M. (2009) Ethiopian floriculture and its impact on the environment. \u003cem\u003eMizan law review,\u003c/em\u003e 3\u003cstrong\u003e,\u003c/strong\u003e 240-270.\u003c/li\u003e\n\u003cli\u003eGhosh, U. K., M. N. Islam, M. N. Siddiqui \u0026amp; M. A. R. Khan (2021) Understanding the roles of osmolytes for acclimatizing plants to changing environment: a review of potential mechanism. \u003cem\u003ePlant Signaling \u0026amp; Behavior,\u003c/em\u003e 16\u003cstrong\u003e,\u003c/strong\u003e 1913306.\u003c/li\u003e\n\u003cli\u003eGuan, Z., S. Chen, F. Chen, Z. Liu, W. Fang \u0026amp; J. Tang (2012) Comparison of stress effect of NaCl, Na+ and Cl-on two Chrysanthemum species. \u003cem\u003eActa Horticulturae\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e 369.\u003c/li\u003e\n\u003cli\u003eHiscox, J. \u0026amp; G. Israelstam (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. \u003cem\u003eCanadian journal of botany,\u003c/em\u003e 57\u003cstrong\u003e,\u003c/strong\u003e 1332-1334.\u003c/li\u003e\n\u003cli\u003eHuang, Q., M. A. Farooq, F. Hannan, W. Chen, A. Ayyaz, K. Zhang, W. Zhou \u0026amp; F. Islam (2022) Endogenous nitric oxide contributes to chloride and sulphate salinity tolerance by modulation of ion transporter expression and reestablishment of redox balance in Brassica napus cultivars. \u003cem\u003eEnvironmental and Experimental Botany,\u003c/em\u003e 194\u003cstrong\u003e,\u003c/strong\u003e 104734.\u003c/li\u003e\n\u003cli\u003eJahan, M., M. Islam, S. Islam, P. Das, M. M. Islam, M. Kabir \u0026amp; A. Mamun (2021) Clonal propagation of Chrysanthemum morifolium ramat using various explants obtained from field grown plants. \u003cem\u003eGSC Biol. Pharm. Sci,\u003c/em\u003e 16\u003cstrong\u003e,\u003c/strong\u003e 87-93.\u003c/li\u003e\n\u003cli\u003eKashif Waseem, K. W., M. Jilani, M. Khan, M. K. Mehwish Kiran \u0026amp; G. K. Ghazanfarullah Khan (2011) Efficient in vitro regeneration of chrysanthemum (Chrysanthemum morifolium L.) plantlets from nodal segments.\u003c/li\u003e\n\u003cli\u003eKaul, V., R. M. Miller, J. F. Hutchinson \u0026amp; D. Richards (1990) Shoot regeneration from stem and leaf explants of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.). \u003cem\u003ePlant Cell, Tissue and Organ Culture,\u003c/em\u003e 21\u003cstrong\u003e,\u003c/strong\u003e 21-30.\u003c/li\u003e\n\u003cli\u003eKomalavalli, N. \u0026amp; M. V. Rao (2000) In vitro micropropagation of Gymnema sylvestre\u0026ndash;A multipurpose medicinal plant. \u003cem\u003ePlant cell, tissue and organ culture,\u003c/em\u003e 61\u003cstrong\u003e,\u003c/strong\u003e 97-105.\u003c/li\u003e\n\u003cli\u003eKumar, A., S. Pathania, B. Kashyap, R. Dhiman \u0026amp; Y. Gupta (2024) A decade analysis of flower area, production and instability index-A review. \u003cem\u003eJournal of Ornamental Horticulture,\u003c/em\u003e 27\u003cstrong\u003e,\u003c/strong\u003e 1-10.\u003c/li\u003e\n\u003cli\u003eKumar, A., S. Pathania, B. Kashyap, S. Dhiman \u0026amp; Y. Gupta (2023) Indian floriculture: Current issues and initiatives-A review paper. \u003cem\u003eJournal of Ornamental Horticulture,\u003c/em\u003e 26\u003cstrong\u003e,\u003c/strong\u003e 1-9.\u003c/li\u003e\n\u003cli\u003eKumar, D., M. A. Yusuf, P. Singh, M. Sardar \u0026amp; N. B. Sarin (2014) Histochemical detection of superoxide and H2O2 accumulation in Brassica juncea seedlings. \u003cem\u003eBio-protocol,\u003c/em\u003e 4\u003cstrong\u003e,\u003c/strong\u003e e1108-e1108.\u003c/li\u003e\n\u003cli\u003eLaxa, M., M. Liebthal, W. Telman, K. Chibani \u0026amp; K.-J. Dietz (2019) The role of the plant antioxidant system in drought tolerance. \u003cem\u003eAntioxidants,\u003c/em\u003e 8\u003cstrong\u003e,\u003c/strong\u003e 94.\u003c/li\u003e\n\u003cli\u003eLowry, O. H., N. J. Rosebrough, A. L. Farr \u0026amp; R. J. Randall (1951) Protein measurement with the Folin phenol reagent. \u003cem\u003eJ biol Chem,\u003c/em\u003e 193\u003cstrong\u003e,\u003c/strong\u003e 265-275.\u003c/li\u003e\n\u003cli\u003eL\u0026uuml;ck, H. 1965. Catalase. In \u003cem\u003eMethods of enzymatic analysis\u003c/em\u003e, 885-894. Elsevier.\u003c/li\u003e\n\u003cli\u003eMalik, A. A., W.-G. Li, L.-N. Lou, J.-H. Weng \u0026amp; J.-F. Chen (2010) Biochemical/physiological characterization and evaluation of in vitro salt tolerance in cucumber. \u003cem\u003eAfrican Journal of Biotechnology,\u003c/em\u003e 9\u003cstrong\u003e,\u003c/strong\u003e 3298-3302.\u003c/li\u003e\n\u003cli\u003eMansour, M., K. Salama, F. Ali \u0026amp; A. Abou Hadid (2005) Cell and plant responses to NaCl in Zea mays L. cultivars differing in salt tolerance. \u003cem\u003eGen. Appl. Plant Physiol,\u003c/em\u003e 31\u003cstrong\u003e,\u003c/strong\u003e 29-41.\u003c/li\u003e\n\u003cli\u003eMazhar, A. A., S. I. Shedeed, N. G. Abdel-Aziz \u0026amp; M. Mahgoub (2012) Growth, flowering and chemical constituents of Chrysanthemum indicum L. plant in response to different levels of humic acid and salinity.\u003c/li\u003e\n\u003cli\u003eMolazem, D., E. Qurbanov \u0026amp; S. Dunyamaliyev (2010) Role of proline, Na and chlorophyll content in salt tolerance of corn (Zea mays L.). \u003cem\u003eAmerican-Eurasian J. Agric. \u0026amp; Environ. Sci,\u003c/em\u003e 9\u003cstrong\u003e,\u003c/strong\u003e 319-324.\u003c/li\u003e\n\u003cli\u003eMunns, R. \u0026amp; M. Tester (2008) Mechanisms of salinity tolerance. \u003cem\u003eAnnu. Rev. Plant Biol.,\u003c/em\u003e 59\u003cstrong\u003e,\u003c/strong\u003e 651-681.\u003c/li\u003e\n\u003cli\u003eMurashige, T. \u0026amp; F. Skoog (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. \u003cem\u003ePhysiologia plantarum,\u003c/em\u003e 15\u003cstrong\u003e,\u003c/strong\u003e 473-497.\u003c/li\u003e\n\u003cli\u003eOseni, O. M., V. Pande \u0026amp; T. K. Nailwal (2018) A review on plant tissue culture, a technique for propagation and conservation of endangered plant species. \u003cem\u003eInternational journal of current microbiology and applied sciences,\u003c/em\u003e 7\u003cstrong\u003e,\u003c/strong\u003e 3778-3786.\u003c/li\u003e\n\u003cli\u003eParida, A. K. \u0026amp; A. B. Das (2005) Salt tolerance and salinity effects on plants: a review. \u003cem\u003eEcotoxicology and environmental safety,\u003c/em\u003e 60\u003cstrong\u003e,\u003c/strong\u003e 324-349.\u003c/li\u003e\n\u003cli\u003eParvaneh, R., T. Shahrokh \u0026amp; H. S. Meysam (2012) Studying of salinity stress effect on germination, proline, sugar, protein, lipid and chlorophyll content in purslane (Portulaca oleracea L.) leaves. \u003cem\u003eJournal of Stress Physiology \u0026amp; Biochemistry,\u003c/em\u003e 8\u003cstrong\u003e,\u003c/strong\u003e 182-193.\u003c/li\u003e\n\u003cli\u003eParvin, K., M. Hasanuzzaman, M. B. Bhuyan, K. Nahar, S. M. Mohsin \u0026amp; M. Fujita (2019) Comparative physiological and biochemical changes in tomato (Solanum lycopersicum L.) under salt stress and recovery: role of antioxidant defense and glyoxalase systems. \u003cem\u003eAntioxidants,\u003c/em\u003e 8\u003cstrong\u003e,\u003c/strong\u003e 350.\u003c/li\u003e\n\u003cli\u003ePatil, U., A. Karale, S. Katwate \u0026amp; M. Patil (2017) Mutation breeding in chrysanthemum (Dendranthema grandiflora T.). \u003cem\u003eJournal of Pharmacognosy and Phytochemistry,\u003c/em\u003e 6\u003cstrong\u003e,\u003c/strong\u003e 230-232.\u003c/li\u003e\n\u003cli\u003eQueiros, F., F. Fidalgo, I. Santos \u0026amp; R. Salema (2007) In vitro selection of salt tolerant cell lines in Solanum tuberosum L. \u003cem\u003eBiologia plantarum,\u003c/em\u003e 51\u003cstrong\u003e,\u003c/strong\u003e 728-734.\u003c/li\u003e\n\u003cli\u003eRai, H., N. NAMITA, D. Raju, M. Singh, K. P. Singh, G. Kumar, S. K. Sinha, S. Lekshmy, R. Pandey \u0026amp; B. Poulose (2020) In vitro screening of chrysanthemum (Chrysanthemum morifolium) varieties for salt tolerance. \u003cem\u003eThe Indian Journal of Agricultural Sciences,\u003c/em\u003e 90\u003cstrong\u003e,\u003c/strong\u003e 2138-2144.\u003c/li\u003e\n\u003cli\u003eRanganayakulu, G., G. Veeranagamallaiah \u0026amp; C. S. Chinta Sudhakar (2013) Effect of salt stress on osmolyte accumulation in two groundnut cultivars (Arachis hypogaea L.) with contrasting salt tolerance.\u003c/li\u003e\n\u003cli\u003eRao, G. \u0026amp; G. Rao. 1981. PIGMENT COMPOSITION AND CHLOROPHYLLASE ACTIVITY IN PIGEON PEA (CAJANUS-INDICUS SPRENG) AND GINGELLEY (SESAMUM-INDICUM L) UNDER NACL SALINITY. 768-770. COUNCIL SCIENTIFIC INDUSTRIAL RESEARCH PUBL \u0026amp; INFO DIRECTORATE, NEW DELHI \u0026hellip;.\u003c/li\u003e\n\u003cli\u003eRashid, M., M. Khalekuzzaman, M. Hasan, R. Das, M. Hossain \u0026amp; S. Mahabbat-E-Khoda (2009) Establishment of an efficient method for micropropagation of an important medicinal herb (Scoparia dulcis L.) from shoot tips and nodal segments. \u003cem\u003eInt. J. Sustain. Crop Prod,\u003c/em\u003e 4\u003cstrong\u003e,\u003c/strong\u003e 5-9.\u003c/li\u003e\n\u003cli\u003eRyu, J., B. Nam, B.-R. Kim, S. H. Kim, Y. D. Jo, J.-W. Ahn, J.-B. Kim, C. H. Jin \u0026amp; A.-R. Han (2019) Comparative analysis of phytochemical composition of gamma-irradiated mutant cultivars of Chrysanthemum morifolium. \u003cem\u003eMolecules,\u003c/em\u003e 24\u003cstrong\u003e,\u003c/strong\u003e 3003.\u003c/li\u003e\n\u003cli\u003eSable, A. D., P. B. Kardile, A. D. Sable \u0026amp; A. V. Kharde (2018) Studies on effect of different concentration of NaCI on bacoside production from brahmi (Bacopa monnieri) under in vitro condition. \u003cem\u003eJ Pharmacogn Phytochem,\u003c/em\u003e 7\u003cstrong\u003e,\u003c/strong\u003e 1386-1389.\u003c/li\u003e\n\u003cli\u003eSarabi, B., S. Bolandnazar, N. Ghaderi \u0026amp; J. Ghashghaie (2017) Genotypic differences in physiological and biochemical responses to salinity stress in melon (Cucumis melo L.) plants: Prospects for selection of salt tolerant landraces. \u003cem\u003ePlant physiology and biochemistry,\u003c/em\u003e 119\u003cstrong\u003e,\u003c/strong\u003e 294-311.\u003c/li\u003e\n\u003cli\u003eSayyed, A., H. Gul, Z. Ullah \u0026amp; M. Hamayun (2014) Effect of salt stress on growth of Tagetes erecta L. \u003cem\u003ePakhtunkhwa Journal of Life Science,\u003c/em\u003e 2\u003cstrong\u003e,\u003c/strong\u003e 96-106.\u003c/li\u003e\n\u003cli\u003eSharuti Verma, S. V., K. Y. Kuldeep Yadav \u0026amp; N. S. Narender Singh (2011) Optimization of the protocols for surface sterilization, regeneration and acclimatization of Stevia rebaudiana Bertoni.\u003c/li\u003e\n\u003cli\u003eShatnawi, M., A. Al-Fauri, H. Megdadi, M. K. Al-Shatnawi, R. Shibli, S. Abu-Romman \u0026amp; A. Al-Ghzawi (2010) In vitro multiplication of Chrysanthemum morifolium Ramat and it is responses to NaCl induced salinity. \u003cem\u003eJordan Journal of Biological Sciences,\u003c/em\u003e 3\u003cstrong\u003e,\u003c/strong\u003e 101-110.\u003c/li\u003e\n\u003cli\u003eShaw, B. (1995) Changes in the levels of photosynthetic pigments in Phaseolus aureus Roxb. exposed to Hg and Cd at two stages of development: a comparative study. \u003cem\u003eBulletin of environmental contamination and toxicology,\u003c/em\u003e 55\u003cstrong\u003e,\u003c/strong\u003e 574-580.\u003c/li\u003e\n\u003cli\u003eShibli, R. A., M. Kushad, G. G. Yousef \u0026amp; M. A. Lila (2007) Physiological and biochemical responses of tomato microshoots to induced salinity stress with associated ethylene accumulation. \u003cem\u003ePlant growth regulation,\u003c/em\u003e 51\u003cstrong\u003e,\u003c/strong\u003e 159-169.\u003c/li\u003e\n\u003cli\u003eSu, L.-J., J.-H. Zhang, H. Gomez, R. Murugan, X. Hong, D. Xu, F. Jiang \u0026amp; Z.-Y. Peng (2019) Reactive oxygen species‐induced lipid peroxidation in apoptosis, autophagy, and ferroptosis. \u003cem\u003eOxidative medicine and cellular longevity,\u003c/em\u003e 2019\u003cstrong\u003e,\u003c/strong\u003e 5080843.\u003c/li\u003e\n\u003cli\u003eSushmarani, Y., P. Venkatesha Murthy \u0026amp; N. Deeksha Raj (2021) Influence of BAP with TDZ growth regulators on in vitro regeneration in chrysanthemum ( T.) Dendranthema grandiflora cv. Marigold. \u003cem\u003eJournal of Pharmacognosy and Phytochemistry,\u003c/em\u003e 10\u003cstrong\u003e,\u003c/strong\u003e 1171-1176.\u003c/li\u003e\n\u003cli\u003eThongpukdee, A., K. Chanjirakul, C. Thepsithar, K. Obsuwan \u0026amp; R. Chantadech. 2012. In vitro salt tolerance of chrysanthemum\u0026apos;Money Maker Improve\u0026apos;. In \u003cem\u003eInternational Symposium on Orchids and Ornamental Plants 1025\u003c/em\u003e, 173-178.\u003c/li\u003e\n\u003cli\u003eVerma, A. K., K. Prasad, T. Anakiram \u0026amp; S. Kumar (2012) Standardization of protocol for pre-treatment, surface sterilization, regeneration, elongation and acclimatization of Chrysanthemum morifolium Ramat. \u003cem\u003eInternational Journal of Horticulture,\u003c/em\u003e 2.\u003c/li\u003e\n\u003cli\u003eVinocur, B. \u0026amp; A. Altman (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. \u003cem\u003eCurrent opinion in biotechnology,\u003c/em\u003e 16\u003cstrong\u003e,\u003c/strong\u003e 123-132.\u003c/li\u003e\n\u003cli\u003eYaşar, F. \u0026amp; Ş. Ellialtıoğlu (2013) Antioxidative responses of some eggplant genotypes to salinity stress. \u003cem\u003eYuzuncu Yıl University Journal of Agricultural Sciences,\u003c/em\u003e 23\u003cstrong\u003e,\u003c/strong\u003e 215-221.\u003c/li\u003e\n\u003cli\u003eYasemin, S., A. G. Değer, S. \u0026Ccedil;evik \u0026amp; N. K\u0026ouml;ksal (2021) Benchmarking of the effects of salinity on antioxidant enzymes activities, lipid peroxidation and H2O2 levels in the leaves of two zinnia species. \u003cem\u003eKahramanmaraş S\u0026uuml;t\u0026ccedil;\u0026uuml; İmam \u0026Uuml;niversitesi Tarım ve Doğa Dergisi,\u003c/em\u003e 24\u003cstrong\u003e,\u003c/strong\u003e 31-39.\u003c/li\u003e\n\u003cli\u003eYesmin, S., A. Hashem, K. Das, M. Hasan \u0026amp; M. Islam (2014) Efficient in vitro regeneration of chrysanthemum (Chrysanthemum morifolium Ramat.) through nodal explant culture. \u003cem\u003eNuclear science and applications,\u003c/em\u003e 23\u003cstrong\u003e,\u003c/strong\u003e 47-50.\u003c/li\u003e\n\u003cli\u003eZandalinas, S. I., D. Balfag\u0026oacute;n, V. Arbona \u0026amp; A. G\u0026oacute;mez-Cadenas (2017) Modulation of antioxidant defense system is associated with combined drought and heat stress tolerance in citrus. \u003cem\u003eFrontiers in Plant Science,\u003c/em\u003e 8\u003cstrong\u003e,\u003c/strong\u003e 953.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Supplementary Material","content":"\u003cp\u003eThe Supplementary Tables are not available with this version.\u003c/p\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":"Salinity stress, Plant Tissue Culture, Chrysanthemum × morifolium, Physiological and Biochemical response","lastPublishedDoi":"10.21203/rs.3.rs-6636160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6636160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSalinity is a critical abiotic stress that significantly limits plant growth and productivity by disrupting physiological and biochemical processes. This study investigated the effects of NaCl-induced salinity on the in vitro culture of \u003cem\u003eChrysanthemum \u0026times; morifolium\u003c/em\u003e Ramat. Explants were cultured under varying concentrations of NaCl (0\u0026ndash;300 mmol/L) to assess its impact on callus formation, shoot regeneration, physiological attributes, and biochemical responses. Results showed that increasing NaCl concentrations reduced callus formation percentages, shoot regeneration frequency, and shoot length, with the highest reduction observed at 300 mmol/L NaCl. The number of shoots per explant decreased from 13.03 under non-saline conditions to 0.90 at 300 mmol/L. Chlorophyll content, carotenoids, and protein levels declined significantly with increasing salinity, whereas the proline content increased, indicating its role in osmotic adjustment and stress tolerance. Antioxidant enzyme activities, including catalase and superoxide dismutase, were enhanced under salt stress, with maximum activity recorded at 300 mmol/L NaCl, suggesting their involvement in mitigating oxidative damage. Lipid peroxidation and protein oxidative damage also increased, further indicating the detrimental effects of salinity. During the hardening phase, optimal survival was achieved using a potting mixture of coco peat, sand, vermicompost, and garden soil (2:1:1:1 ratio). Plants grown under in vivo saline conditions exhibited reduced biomass, root growth, and shoot development, with severe effects at 400 mmol/L NaCl. These findings provide insights into the physiological and biochemical responses of \u003cem\u003eC. morifolium\u003c/em\u003e under salt stress, contributing to the development of salt-tolerant varieties and improving in vitro propagation techniques.\u003c/p\u003e","manuscriptTitle":"Physiological and Biochemical Responses of Chrysanthemum × morifolium to Salinity Stress under In-Vitro Conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 09:16:22","doi":"10.21203/rs.3.rs-6636160/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":"e3d2ab5d-0423-4f2c-a478-7a95488885c8","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T08:02:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 09:16:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6636160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6636160","identity":"rs-6636160","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}