INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025
Page 856
Physiological and Biochemical Attributes of Chromium Detoxification Are
Regulated by Root synthesized Organic Acids in Rice Varieties
Dr. A.K.M. Nazmul Huda*, Md. Habibur Rahman, Md. Imran Hossain, Md. Shadiqul Islam
Dept. Of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh
DOI: https://dx.doi.org/10.51244/IJRSI.2025.1210000076
Received: 02 October 2025; Accepted: 08 October 2025; Published: 04 November 2025
ABSTRACT
To know effective physiological response against chromium toxicity, 25 rice varieties were cultivated on the
hydroponic solution treated with 100µM chromium and physiological as well as biochemical features were
evaluated compared with non-treated control plants. In this investigation, it was found that the concentration of
citric acid synthesized and secreted by roots influences the mitigation of chromium toxicity. In varieties BR-58,
BR-63 and BR-68 chromium uptake were significantly higher than the control plant but their translocation to
shoot was restricted indicating elevated Cr retention in roots. This retention was facilitated by root secreted citric
acid which was assured by significant rhizospheric pH reduction (15%, 18.5% and 20.9% respectively) under
chromium stress. Furthermore, BR-73 showed an efficient exclusion mechanism keeping down metal uptake by
citric acid ensured by 15% rhizosphere pH reduction. In contrast, varieties of rhizospheres with a pH reduction
of less than 10% were unable to withstand chromium toxicity. The findings indicate that a reduction of 15% or
more in rhizospheric pH serves as the benchmark for the necessary level of organic acid secretion required for
chromium tolerance. Moreover, the strategies employed for tolerance differ based on genotypes rather than
species. Furthermore, it offers an efficient screening technique for metal tolerant rice plants.
Key word: rhizospheric pH, chromium tolerant, organic acids, adsorption, Oryza sativa
INTRODUCTION
Chromium (Cr), a hazardous heavy component found in the outer layers of the Earth, has adverse effects on the
environment. It is thoroughly used in leather tanning, electroplating process, steel production, metal finishing,
catalyst uses and pigmentation. The relevant sources of Chromium (Cr) exposure in the environment are
industrial discharges and domestic sewage (Nath et al. 2008;). In plants, accumulated Cr inhibits growth by
limiting the absorption of nutrients (Ullah et al., 2023; Shanker et al., 2005). It also causes chlorosis in young
plants, lowers pigment content, alters enzymatic functions, damages root cells, and induce ultrastructural
changes to the cell membrane and chloroplast (Panda and Choudhury 2008). Cr also significantly impairs the
development of stems and leaves during the early growth stage of the plant, as well as the formation of dry matter
in seedlings (Dey et al., 2023; Nematshahi et al., 2012). The toxicity of Cr is dependent on the metal species that
determine Cr's absorption, transport, and accumulation. Numerous investigations on the chemistry of chromium
in soil and its uptake by plants have indicated that Cr is harmful for plant growth (Arun et al. 2005).
In order to withstand the toxicity of heavy metals, plants have an intricate and interrelated system of defense
mechanisms. Plant’s physical barriers, which include cell walls, physiologically active tissues like trichomes,
and morphological features like thick cuticle, are their first line of protection against metals (Al-Khayri et.al.,
2023, Wong et.al., 2004, Harada et.al., 2010). To counteract and lessen the negative effects of HMs, plants
activate several cellular defense mechanisms when the metal ions penetrate biophysical barriers and enter tissues
and cells. Tolerating or neutralizing metal toxicity mostly involves the biogenesis of several cellular
macromolecules, including asnicotianamine, putrescine, spermine, mugineic acids, organic acids,
phytochelatins, metallothioneins, cellular exudates, heat shock proteins, certain amino acids, and hormones
(Viehweger 2014, Dalvi and Bhalerao 2013, Sharma and Dietz 2006). Ineffectiveness of the aforementioned
approaches in plants increases the production of ROS (Mourato et.al. 2012). In order to eliminate the free
radicals, plants then increase their enzymatic antioxidants, which includes superoxide dismutase (SOD), catalase
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025
Page 857
(CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR), as well as
their nonenzymatic antioxidants, such as ascorbate (AsA), glutathione (GSH), carotenoids, alkaloids,
tocopherols, proline, and phenolic compounds (Sharma et.al., 2012, Rastgoo et.al., 2011). However, genetic
potential of plant species as well as the metal type are the key determinant of metal tolerance level (Solanki and
Dhankhar 2011).
Root excreted low molecular weight organic acid (LMWOA) also known to regulate different stress especially
oxidative stress (Airaki et al., 2012). Synthesized LMWOA released to the rhizosphere through increased efflux
(De La Fuente et al., 1997; López-Bucio et al., 2000a) but not authenticated for every plant (RRRRR). Root
excretion organic acid and its impact on physiology was studied enough in previous. Most of this OA detoxify
heavy metal in two ways. First one in the internal chelating of heavy metals both in photosynthetic and non-
photosynthetic tissues (Fernando et al., 2010). Second one is the insoluble complex formation of organic acid
with heavy metal and release phosphorus ion (Pi) from the bound complex. Here positively charged cation of
heavy metal react with negatively charged carboxylic group of organic acids (OAs) to form insoluble complexes
that not absorbed by plant (Zhang et al., 2018).
However, these LMWOAs are synthesized in mitochondria and enzymes participate in glycolysis, TCA and
glyoxylate cycles are also responsible for the synthesis (Igamberdiev and Eprintsev, 2016). Stress condition
enhanced LMWOA synthesis by hampering the standard pathway of these cycles. Furthermore, anaplerotic
reactions (chemical reactions that form inter- mediates of the TCA cycle) rat enhance OAs synthesis to cope
with stress (Dong et al., 2004). Thus, higher level of transcription of genes encoding TCA cycle enzymes as well
as their enhanced enzymatic activities upregulate the LMWOA synthesis and help plants to cope with metal
toxicity (Zhou et al., 2018, Uhde-Stone et al., 2003a).
In japonica rice, OsFRDL4 and OsFRDL2 are upregulated under Aluminum stress to secrete citric acid from
roots (Yokosho et al., 2011). But types and amount of root secreted organic acids are the lack of clear evidence
though it is assumed that surrounding environment may responsible for this specificity (RRRRRR).
Consumption of carbon sources for production and efflux of OAs under stress utilize significant proportion of
carbon imposing an energy cost to plants which is economically important for plants especially fast-growing
annual crops (Koyama et al., 2000; Herz et al., 2018). Plants optimize its carbon loss through tissue-specific or
location specific exudation of OAs from the roots firstly. Secondly, it limits the amount of OAs release by
negative regulators of OA exudation like GABA (Ramesh et al., 2015). But belowground rhizosphere deposition
was not even considered in any models for carbon allocation (Reyes et al., 2020). So, it demands future studies
on this significant issue. Moreover, the regulatory mechanisms regarding types and amount of root secreted
organic acids remain largely elusive.
However, effective physiological responses against toxic chromium in rice plants remain poorly understood.
These types of responses against metal toxicity are classified as either internal or external (Kochian et al., 2004).
Local rice variety, Pokkali develop tolerant against chromium by limiting its Fe reductase and Fe transporter
activities (Kabir, 2016). Furthermore, Zeng Fanrong et.al., (2008) reported that rice plants released oxalic, malic,
and citric acid at the rhizosphere and enhanced Cr accumulation. But there are no reports on how organic acids
work or if they have any effective role against chromium toxicity in rice. Moreover, external mechanisms, such
as root exudation-mediated heavy metal avoidance or tolerance, remain ill-defined. Therefore, different high-
yielding rice varieties were exposed to Cr stress and evaluated the physiological and biochemical features to
gain new insights into metal tolerance in rice.
MATERIALS AND METHODS
2.1 Plant cultivation
In this study, seeds from 25 authentic rice varieties (Bangladesh Rice Research Institute variety BRRI 50 to 73
and 22) were initially collected at the germplasm center of the Bangladesh Rice Research Institute. After being
sterilized with 95% (v/v) ethanol, water-washed seeds were allowed to germinate in wet filter paper Petri dishes
for two to three days at room temperature in the dark. The plants that germinated uniformly were transferred to
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025
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the Hoagland and Arnon (1950) hydroponic solution (100 ml). For rice seedlings under chromium stress, a
hydroponic solution containing 100 µM K2Cr2O7 (a source of the heavy metal chromium) was used, while the
solution for the control plant did not contain any chromium addition. The hydroponic solution's pH was adjusted
to 6.0. the chromium-treated and untreated control seedlings were cultured in a nutrient solution in a growth
chamber (Temp. 260-280 , Humidity 70%-80%) with 10 hours of light and 14 hours of darkness (550–560 mmol
s-1 per mA). The nutritional solution was guaranteed to be continuously aerated. After seven days of culture,
seedlings treated with and without chromium were collected, and distinct roots and shoots were used for each
experiment.
2.2 Evaluation of morphological characteristics
Seven-day-old plant’s root length and shoot height were measured in centimeters. The roots and shoots were
then dried in an oven set to 80o C for two days in order to calculate their dry weight.
2.3 Measurement of electrolyte leakage
Electrolyte leakage (EL) was measured using the Lutts et al. (1996) methodology.
2.4 Determination of chlorophyll concentration:
A pre-chilled mortar and pestle was used to homogenize 100 mg of fresh leaf tissue with 5–10 mL of 90% (v/v)
acetone and then centrifuged for five to ten minutes at 3000 rpm. To perform a spectrophotometric analysis, the
clear supernatant was collected. The concentration of chlorophyll in leaves was measured with 90% (v/v) acetone
based on the Lichtenthaler and Wellburn (1985) procedure.
2.5 Determination of Cr and Fe by AAS (atomic absorption spectroscopy)
Roots and shoots were first cleaned with CaSO4 and deionized water and then dried in an oven at 80o C for
three days. The samples were digested for 15 minutes at 100º C using mixes of HNO3 and H2O2 (3:1), and
atomic absorption spectroscopy was used for investigation.
2.6 Determination of total soluble proteins
Fresh plant tissue was homogenized in 1-2 milliliters of ice-cold 50 mM phosphate buffer (pH 7.0). The
homogenate was centrifuged for 15 minutes at 4°C at 12,000 rpm and Quantification of proteins was done using
the supernatant. 100 µL of the sample and 1.0 mL of Bradford reagent were mixed and allowed to sit at room
temperature for 5 to 10 minutes. The absorbance was measured with a UV-Visible spectrophotometer at 595 nm.
Absorbance measurements were plotted against known BSA concentrations to create a standard calibration
curve. Each sample protein concentration was determined by extrapolating the absorbance values from the
standard curve.
2.7 Estimation of lipid peroxidation
The roots and shoots were homogenized with 5% (w/v) trichloroacetic acid (TCA) and centrifuged at 11,500×g
for 15 minutes. Following centrifugation, thiobarbituric acid (TBA) was added to the separated supernatant, then
mixture was heated in a water bath for 30 minutes at 95 °C. As the mixture cooled, absorbance was measured at
532 nm. Malondialdehyde was measured and reported as nmol of MDA mg⁻¹ FW using an extinction value of
155 mM⁻¹ cm⁻¹ (Heath and Packer 1968).
2.8 Determination of rhizospheric pH change
A digital pH meter was employed to measure the media's pH both before and after the seedlings were cultured.
Using these data, the pH reduction was calculated and expressed as a percentage.
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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2.9 Silver nitrate precipitation test:
First, 0.1 M silver nitrate (AgNO₃) was prepared. Then a few drops of silver nitrate were added to each tube
holding the culture fluid. The organic acid indication, white precipitate, was seen.
2.10 Thin-layer chromatography (TLC)
Rice plant root exudates (BR-58, BR-63, BR-68, and BR-73), treated and untreated with Cr, were analyzed using
thin-layer chromatography (TLC). The mobile phase was made up of ethanol, NH4OH, and H2O in the ratio
75.5:12.5:12.5. The TLC plate was then placed in the TLC chamber after the placement of the samples and
standards of known organic acids on it. The TLC plate is removed from the chamber and allowed to dry when
the solvent has travelled a sufficient distance. The TLC plate was then examined under a UV light, and the spots
were noted. The sample's organic acids were determined by comparing them with known benchmarks.
2.11 Enzymatic analysis
The enzymes CAT (EC. 1.11.1.6), POD (EC. 1.11.1.7), SOD (EC. 1.15.1.1), and GR (EC. 1.6.4.2) were extracted
from one-week-old plants using a modified version of Goud and Kachole's (2012) approach. After being crushed
in 100 mM phosphate buffer, the roots and shoots tissues were centrifuged for 10 minutes at 13000×g. For the
Catalase (EC. 1.11.1.6) analysis, the reaction mixture (2 ml) consisted of 400 µL of 6% (v/v) H2O2, 100 µL of
root extract and 100 mM potassium phosphate buffer (pH 7.0). A UV spectrophotometer was used to measure
the absorbance at 240 nm (extinction coefficient of 0.036 mM-1 cm-1) at 30-second to one-minute intervals after
the addition of root or shoot extract. The unit of measurement for CAT activity is mmol of H2O2 oxidized min-
1 (mg protein 1).
Similarly, two ml reaction mixture was prepared to measure peroxidase (EC 1.11.1.7) activity. Here 100 mM
potassium phosphate buffer (pH 6.5), 1ml of 50 mM pyrogallol, 400 µL of 200 mM H₂O₂, and 100 µL of root
extract as the enzyme source were all included in the combination. In the spectrophotometer, the absorbance
variations were measured from 30 seconds to 1.5 minutes at 430 nm (extinction coefficient 12 mM-1 cm-1). The
concentration of peroxidase is measured in mmol pyrogallol oxidized min-1 (mg protein-1). Additionally, 50
mM sodium carbonate/bicarbonate buffer (pH 9.8), 0.1 mM EDTA, 0.6 mM epinephrine, and enzyme made up
the SOD (EC. 1.15.1.1) assay combination. Then adrenochrome formation was observed at 475 nm using a UV-
Vis spectrophotometer. The quantity of enzyme required for 50% inhibition of epinephrine oxidation is
established as per unit SOD activity. In order to perform glutathione reductase (EC. 1.6.4.2) analysis, 100 µL of
root extract was added to the reaction mixture, which also contained 1 mL of 0.2 M phosphate buffer (pH 7.0),
1 mM EDTA, 0.75 ml of distilled water, 0.1 mL of 20 mM oxidized glutathione (GSSG), and 0.1 mL of 2 mM
NADPH. At 340 nm, the oxidation of NADPH by GR was then measured. The extinction coefficient of 6.12
mM-1 cm-1 was then used to calculate the rate of GR activity (nmol min-1).
2.12 Determination of hydrogen peroxide and superoxide (O2-)
Tissues were centrifuged at 10,000×g for 15 minutes, after being pulverized in 0.1% (v/w) trichloroacetic acid
(TCA). Before reading the absorbance at 390 nm, the reaction mixture was made by mixing potassium iodide
(M) and phosphate buffer (10mM, pH7.0) with the supernatant. It was left in the dark for an hour (Alexieva et
al., 2001). A standard calibration curve with known H2O2 levels was used for quantification.
For superoxide (O2-) measurement, plant samples were centrifuged at 5000 rpm for 7 minutes at 4 0C after they
had been crushed in 1 milliliter of 65 mM potassium phosphate buffer (pH 7.8). 50µl of 10 mM hydroxylamine
hydrochloride, 0.5 ml of supernatant, and 450µl of 65 mM potassium phosphate buffer (pH 7.8) were used to
create the reaction mixture, which was then incubated for 30 minutes at 25 °C. After that, 125µl of 7mM alpha-
napthyl amine and 10mM sulfanilamide were added to the mixture, and then incubated for 20 minutes at 25 °C.
The absorbance at 530 nm was measured using spectrophotometry. Superoxide (O2-) levels were determined
using a standard curve built with known NO values.
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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2.1.3 Estimation of metabolites (Glutathione, phytochelatin and proline) content:
The method developed by Anderson et al. (1992) was used to extract glutathione. In order to determine total
glutathione, GSSG was first converted to GSH by mixing the leaf extract with 130 mM sodium phosphate buffer
(pH 7.4) and one unit of glutathione reductase. The mixture was then kept at 30°C for 10 minutes. The final
reaction mixture was then made by adding 50 mM of NADPH and 7 mM of sodium phosphate buffer (pH 6.8)
that contained 6 mM of DTNB. Absorbance was measured at 412 nm after this reaction mixture was held at
30°C for 10 minutes. To estimate glutathione, a standard curve of known quantities of GSH was employed
(Griffith 1980). Phytochelatin content was determined using the previously described procedure by Mahmud et
al. (2018).
To measure the amount of proline samples of leaves and roots were centrifuged at 11,500×g for 12 minutes after
being homogenized in 3% (v/w) sulfosalicylic acid. Next, the 100 μL plant extract supernatant was mixed with
200 μL glacial acetic acid, 100 μL of 3% (v/w) sulfosalicylic acid, and 200 μL acidic ninhydrin. The mixture
was then heated for 60 minutes at 96◦C and immediately chilled on ice. Spectrophotometer was used to take a
reading at 520 nm (Bates et al., 1973). Calculations were performed using a standard curve with known proline
concentrations.
2.1.4 Statistical analysis
Completely randomized block design with four independent replications was adopted in each experiment.t-test
at 0.05% significance level with the help of Microsoft Excel 2007 was performed as statistical analysis.
Moreover, Graph Pad Prism was applied to prepare graphical presentations.
RESULT
3.1 Morpho-physiological parameters:
Seedlings grown on the hydroponic solution containing Cr showed retardation of growth in most of the rice
varieties except BR-58, BR-63, BRRI-68, and BR-73. No significant shoot and root length as well as root and
shoot dry weight reduction were observed in these four varieties for chromium toxicity compared with the control
(table. 1). Moreover, chlorophyll concentrations in the shoot of these four varieties remained unchanged under
chromium stress, whereas in other varieties, it decreased meaningfully under chromium stress.
Furthermore, no meaningful differences between control and chromium treated plant was observed in these four
varieties (BR-58, BR-63, BRRI-68, and BR-73) in case of electrolyte leakage. But in remaining varieties
electrolyte leakage was found to be increased significantly under chromium stress equated to control.
Considering the above-mentioned parameters BRRI-58, BRRI-63, BRRI-68 and BRRI-73 found to cope with
chromium toxicity.
Table 1. Morpho-physiological features of 25 high yielding rice varieties grown in absence or presence of
Chromium on hydroponic solution. Different letters indicate significance difference between means of
treatments (number of replications is 4) followed by t-test. Data were from one week plats.
Variet
y
Treatmen
t
Root
length
(cm)
Root dry
wt
(mg)
Shoot
length
(cm)
Shoot dry
wt
(mg)
Total
Chlorophyll
(µgm/mg)
Electrolyte
leakage in
root (µs cm-
1 g-1)
BRRI-
22
Control 4.83±0.29a 2.67±0.58
a
7.5±.5a 7.33±0.58a 130.58±5.56a 0.955±0.08a
Treatment 3.5±0.5b 0.77±0.25
b
4.83±0.29b 5.33±0.58b 79.27±5.77b 1.750±0.23b
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BRRI-
50
Control 6±0.87a 2±0a 10.5±0.87a 5.33±0.58a 255.18±47.22a 0.915±0.60a
Treatment 2.17±0.58b 0.36±0.13
b
3.17±0.76b 2.33±0.58b 162.30±20.14b 1.962±0.86b
BRRI-
51
Control 7.83±1.44a 2±0a 8.5±0.5a 6.83±0.29a 290.57±8.92a 1.263±0.15a
Treatment 3.67±1.15b 0.83±0.29
b
7.17±0.29b 5.5±0.5b 268.09±20.79b 2.568±0.81b
BRRI-
52
Control 9.67±0.58a 4.33±0.58
a
11.33±0.58
a
12.67±0.58
a
200.24±11.85a 0.531±0.41a
Treatment 5.83±0.29b 1.77±0.87
b
8.17±0.76b 10.67±0.58
b
180.93±3.28b 1.502±0.58b
BRRI-
53
Control 8±1a 3.33±0.58
a
11±0.5a 9.33±0.58a 309.21±3.32a 0.789±0.18a
Treatment 6.17±0.29b 2±0b 9±0.5b 7.67±0.58b 288.94±3.17b 1.669±0.59b
BRRI-
54
Control 5±0.87a 3.83±0.29
a
9±0.5a 8.67±1.15a 424.33±42.49a 0.869±0.29a
Treatment 3.5±0b 3±0b 9±0a 8.83±1.04a 220.44±78.13b 1.718±0.307
b
BRRI-
55
Control 7.33±0.29a 3.83±0.29
a
13.83±0.76
a
10.17±0.29
a
227.89±31.12a 0.955±0.21a
Treatment 6.33±0.58b 2.67±0.29
b
11.33±0.58
b
8.66±0.58b 216.90±20.56a 1.483±0.48b
BRRI-
56
Control 6.67±0.58a 4±0.5a 10.67±0.58
a
8.67±0.29a 416.20±170.96
a
1.203±0.08a
Treatment 5.33±0.29b 3±0b 9.33±0.58b 7.33±0.29b 387.21±48.55a 1.758±0.19b
BRRI-
57
Control 8.33±0.58a 1.83±0.29
a
9.83±0.58a 7.83±0.29a 264.99±44.99a 1.083±0.14a
Treatment 6.5±0.5b 0.93±0.12
b
8±0.87b 6.67±0.58b 232.14±58.09a 2.145±0.74b
BRRI-
58
Control 9.17±2.02a 3.33±0.58
a
10.83±0.76
a
8.5±0.5a 262.39±78.84a 1.735±0.23a
Treatment 7.33±2.31a 2.67±1.15
a
10.5±0.5a 8.33±0.58a 300.94±42.66a 1.902±0.81a
BRRI-
59
Control 8.17±0.76a 2.83±0.29
a
8±1a 8.67±0.29a 454.60±54.07a 1.259±0.35a
Treatment 5.67±0.58b 2±0b 6±1b 7.5±0.5b 211.31±52.26b 2.185±0.16b
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BRRI-
60
Control 9±0a 2.33±0.58
a
13.83±0.29
a
10.33±0.58
a
347.63±8.61a 1.865±0.56a
Treatment 7.67±0.58b 1.67±0.58
b
11.5±0.87b 9.17±1.15b 316.30±40.03a 2.026±0.46a
BRRI-
61
Control 6.67±0.58a 3.67±0.58
a
9±0a 8±0a 274.89±6.00a 0.997±0.58a
Treatment 4±1.73b 2.33±0.58
b
7.67±0.58b 6.67±0.58b 165.69±57.69b 1.941±0.49b
BR-62 Control 6.33±2.25a 2.67±0.58
a
9±2.783a 6.67±2.52a 363.50±40.25a 0.819±0.18a
Treatment 6.33±3.21a 3±1a 9.5±2.179a 6.66±2.08a 282.39±38.00b 0.925±0.44a
BRRI-
63
Control 3.33±0.28a 3.33±0.58
a
10.83±0.58
a
8±1a 310.99±88.42a 1.932±0.913
a
Treatment 4.5±0.87a 2.67±0.58
a
10.17±1.89
a
7.33±0.58a 306.85±26.59a 1.944±0.48a
BRRI-
64
Control 9.33±0.58a 4±0a 10.67±0.76
a
10.33±0.58
a
465.75±10.17a 1±0a
Treatment 6.67±0.58b 3.17±0.29
b
9.33±0.29b 9.17±0.29b 382.09±15.69b 1.825±0.72b
BRRI-
65
Control 3.33±1.15a 4.17±1.26
a
11.5±1.80a 10±0a 336.40±30.52a 1.189±0.27a
Treatment 5.5±1.80a 3.67±0.58
a
13.67±1.61
a
9.33±0.58a 173.67±44.59b 1.653±0.74a
BRRI-
66
Control 8.33±1.53a 3.67±0.58
a
9±1a 7±0a 414.54±38.93a 0.766±0.12a
Treatment 4.67±0.58b 2.5±0.5b 7.17±0.29b 5.67±0.58b 263.36±20.61b 1.516±0.41b
BRRI-
67
Control 5.67±0.58a 4±1.73a 11±0.5a 9.33±0.58a 512.35±43.78a 0.976±0.24a
Treatment 4.13±0.55b 0.93±0.12
b
9.17±0.29b 8.33±0.29b 209.97±89.81b 2.222±0.48b
BRRI-
68
Control 4.33±1.53a 2.33±0.58
a
7.17±2.84a 9.67±1.53a 251.98±22.95a 0.869±0.10a
Treatment 4.83±1.89a 2.83±0.29
a
7.5±3.04a 9.33±0.58a 268.57±76.08a 0.942±0.64a
BRRI-
69
Control 5.17±1.04a 3.33±0.58
a
9.5±0a 7.33±0.58a 430.47±53.04a 1.136±0.460
a
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Treatment 6.17±2.57a 4.33±3.21
a
8.83±2.08a 7.33±2.08a 262.21±42.71b 1.031±0.540
a
BRRI-
70
Control 4.33±2.57a 1.67±0.29
a
10.67±1.15
a
6.83±0.58a 829.50±93.83a 1.018±0.57a
Treatment 3±0b 0.87±0.12
b
6.5±0.5b 4.33±0.58b 424.15±79.69b 1.361±0.591
a
BRRI-
71
Control 7.5±0.5a 3.17±0.76
a
10±0.87a 9.33±0.58a 459.82±48.06a 0.892±0.11a
Treatment 6.17±0.29b 2.17±0.29
b
7±0.87b 7.5±0.5b 326.48±75.24b 1.761±0.540
b
BRRI-
72
Control 11.33±0.58
a
3.5±0.5a 9.17±0.76a 9±1a 393.12±112.24
a
1.253 ±0.60a
Treatment 7.33±1.15b 2.17±0.29
b
7.33±0.29b 5.33±0.58b 375.90±68.46a 2.142±0.618
b
BRRI-
73
Control 8.33±0.29a 3.5±0.5a 9.33±1.53a 8±1a 352.32±87.64a 0.989±0.23a
Treatment 7±0b 2.17±0.29
b
11±1a 9±1a 232.54±28.91b 1.538±0.3a
3.2 Chromium and Iron content:
As the initial investigation indicates that BR-58, BR-63, BRRI-68, and BR-73 varieties owned the detoxification
mechanism, analysis of chromium concentration in root and shoot was conducted in these four varieties. Root's
chromium content of rice varieties BR-58, BR-63, and BR-68 was significantly higher under chromium stress
compared with the control plant. But in the shoot, no meaningful differences in chromium content between
control and chromium-stressed plants of the varieties were observed (Fig. 1). This analysis concludes that
chromium translocation from root to shoot was inhibited in varieties BR-58, BR-63, and BR-68 under the
stressed condition.
Furthermore, significant iron content compared with the control plant was found only in the root of varieties BR-
58 and BR-63 and in the shoot of BR-58 among the varieties grown under chromium stress.
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Figure 1. Chromium (A) and Iron (B) concentrations in rice seedlings grown under chromium stress and non-
stress condition. Significant deviations among the treatments were denoted by different letters followed by t-test
at 5% (P<0.05) significance level (number of replications is 4)
3.3 Lipid peroxidation and total soluble protein:
Malondialdehyde (MDA) content both in root and shoot of varieties BR-58, BR-63, BR-68 and BR-73 showed
no significant differences between non treated control and treatments indicating the tolerance of these varieties
against chromium induced oxidative damage (Fig. 2).
Moreover, no significant difference of total soluble protein content between control and chromium stressed
plants except in the root of variety BR-63.
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Figure 2: Malondialdehyde (A) and total soluble protein content (B) in the roots and shoots of 7 days old rice
plants (BR-58,63,68,73) grown under Chromium stress and without chromium stress. Different letters in each
column indicate significant differences between means of treatments followed by t-test at 5% significance level
(number of replications is 4).
3.4 Determination of root secreted organic acids and rhizospherepHreduction rate:
Rhizosphere pH decrease after culture (due to low molecular weight organic acids released by rice roots) on the
Hongland solution was mentioned in all rice varieties, including the chromium-sensitive variety BR-51, in which
it was less than 10% in both stress and non-stress conditions (fig. 3). But in BR-58, BR-63, BR-68 and BR-73
(treated as chromium tolerant) this reduction rate was over 15% under chromium stress conditions. The required
amount of low molecular weight organic acids (LMWOA) release is assured by more than 15% rhizospheric pH
reduction to avoid chromium toxicity. Furthermore, rhizospheres with less than 10% pH reduction could not
release a sufficient amount of LMWOA to cope with chromium toxicity.
However, a white precipitate in the silver nitrate test supports that the root of rice seedlings releases organic acid.
Furthermore, thin-layer chromatographic analysis confirmed that both the Cr-treated and non-treated rice
seedlings secreted citric acid.
Figure-3: A) Rhizosphoric pH reduction rate (%) under Chromium stress and without chromium stress. B) TLC
paper with spots of organic acid C) precipitation of organic acids in AgNO₃ test
3.5 Enzymatic activity:
CAT and POD activities in the roots of the varieties BR-58, BR-63 and BR-68 grown under chromium stress
were significantly increased when compared with the control plant (Table. 2). GR activities in the roots of
varieties BR-58 and BR-68 were also increased due to chromium. Moreover, enhancement of SOD activities
was observed only in the root of variety BR-68 for toxic chromium. Moreover, no mentionable changes of
A B
C
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enzymatic activities (CAT, POD, SOD and GR) were observed in roots between control and treated plants of
variety BR-73.
Furthermore, no mentionable enhancement of CAT, POD, and SOD activities was followed in the shoots of
varieties BR-58, BR-63, BR-68, and BR-73 under chromium stress. But GR activity in the shoot of varieties
BR-58, BR-68 and BR-73 grown under chromium stress was found to be increased significantly compared with
the control plant.
Furthermore, reactive oxygen species (ROS) especially superoxide (O2
-) and hydrogen peroxide (H2O2) were
also estimated. H2O2 content in the root of varieties BR-58, 63 and 68 was enhanced meaningfully than that of
control plant but in the shoot of these varieties, no meaningful changes were observed. One the other hand no
significant differences of superoxide (O2
-) concentration was discovered except in the root of BR-68.
Table 2 : Changes in enzyme activity in roots and shoots of rice varieties grown in the presence or absence of
chromium. Different letters indicate significant differences between mean (including standard deviation) of
treatments (number of replication is 4) followed by t-test. Data were from one week old plants.
BR-58 BR-63 BR-68 BR-73
R
O
O
T
CAT (min-1protein-
1)
Cr- 4.27±1.01a 4.29±1.37a 1.02±0.87a 0.64±0.53a
Cr+ 7.08±0.51b 7.80±0.35b 2.34±0.38b 0.62±0.48a
POD (min-1protein-
1)
Cr- 4.01±0.02a 1.36±0.79a 1.45±0.22a 0.94±0.57a
Cr+ 7.90±0.75b 3.80±0.54b 2.51±0.44b 0.64±0.35a
SOD (min-1protein-
1)
Cr- 8.0x10 -
2±1.0x10 -2a
6.0x10-2±7.0x10 -
2a
1.0x10-
2±1.0x10 -2a
4.0x10-
2±1.0x10 -2a
Cr+ 8.0x10 -2±0a 3.0x10-2±1.0x10 -
2a
5.0x10-
2±1.0x10 -2b
2.0x10-2±0a
GR (nmol.NADH.
min-1gm protein-1)
Cr- 2.0x10 -
2±1.0x10 -2a
8.0x10-2±2.0x10 -
2a
4.0x10-2±0a 5.0x10-
2±2.0x10 -2a
Cr+ 3.0x10 -
2±1.0x10 -2b
6.0x10-2±1.0x10 -
2a
5.0x10-
2±1.0x10 -2b
5.0x10-
2±1.0x10 -2a
Super Oxide(O2-)
(µg/mg)
Cr- 0.526±o.24a 1.011±0.32a 0.706±0.12a 0.528±0.09a
Cr+ 0.606±0.16a 1.469±0.32a 0.476±0.03b 0.569±0.08a
H2O2 (µg/mg) Cr- 1.2x10 -
2±0.3x10 -2a
2.7x10-2±1.0x10 -
2a
2.9x10-
2±1.0x10 -2a
8.1x10-
2±1.0x10 -2a
Cr+ 4.3x10 -
2±0.8x10 -2b
79.8x10-2±3.0x10
-2b
7.4x10-
2±1.0x10 -2b
10.2x10-
2±2.0x10 -2a
S
H
CAT (min-1protein-
1)
Cr- 0.55±0.27a 2.66±0.39a 0.88±0.68a 0.56±0.34a
Cr+ 0.79±0.31a 2.81±1.98a 0.95±0.05a 0.82±0.19a
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O
O
T
POD (min-1protein-
1)
Cr- 1.31±0.18a 1.04±0.16a 1.86±0.21a 0.46±0.06a
Cr+ 1.79±0.38a 1.59±0.35a 2.02±0.12a 0.61±0.09a
SOD (min-1protein-
1)
Cr- 2.0x10 -
2±1.0x10 -2a
2.0x10-2±0a 2.0x10-
2±1.0x10 -2a
2.0x10-
2±1.0x10 -2a
Cr+ 3.0x10 -2±0a 2.0x10-2±1.0x10 -
2a
2.0x10-2±0a 3.0x10-2±0a
GR (nmol.NADH.
min-1gm protein-1)
Cr- 2.0x10 -2±0a 2.0x10-2±1.0x10 -
2a
2.0x10-2±0a 2.0x10-2±0a
Cr+ 3.0x10 -2±0b 2.0x10-2±1.0x10 -
2a
3.0x10-2±0b 3.0x10-
2±1.0x10 -2b
Super Oxide(O2-)
(µg/mg)
Cr- 0.542±0.08a 0.810±0.19a 0.152±0.04a 0.271±0.15a
Cr+ 0.579±0.17a 0.585±0.09a 0.237±0.04a 0.342±0.14a
H2O2 (µg/mg) Cr- 2.7±2.0x10 -2a 1.5x10-2±1.0x10 -
2a
1.5x10-
2±1.0x10 -2a
2.4x10-
2±1.0x10 -2a
Cr+ 2.3x10-
2±2.0x10 -2a
2.0x10-2±1.0x10 -
2a
1.4x10-
2±1.0x10 -2a
2.9±1.0x10 -2a
3.6 Metabolites (glutathione, proline and phytochelatin)
Amount of glutathione in the root of variety BR-58 and BR-68 as well as in shoot of varieties BR-58, BR-68
and BR-73 were significantly raised under chromium stress compared with non -treated control plant (Table 3).
However, no significant change of glutathione content was followed both in root and shoot of variety BR-63.
Moreover, secondary metabolite proline was significantly increased in roots of varieties BR-58, BR-63 and BR-
68 under chromium stress compared with control plants. But a meaningful difference of proline content in the
shoot of these varieties was not observed.
Furthermore, phytochelatin content in the root of BR-58 was significantly increased when grown on hydroponic
solution supplemented with chromium compared with non-treated control plants. But in the shoot of all varieties,
it was not increased significantly.
Table 3: Metabolites in roots and shoots of rice varieties grown in the presence or absence of chromium.
Different letters indicate significant differences between mean (including standard deviation) of treatments
followed by t-test (number of replications is 4). Data were from one week old plants.
Glutathione
(µgm/mg FW)
Proline
(µgm/mg FW)
Phytochelatine
(µgm/mg FW)
R
O
O
BR-58 Cr- 1.6x10-2±0.1x10 -2a 12.4x10-2±1.2x10 3.4x10-2±0.7x10 -2a
Cr+ 2.0x10-2±0.7x10 -3b 18.2x10-2±2.8x10 -2b 4.9x10-2±0.7x10 -2b
BR-63 Cr- 1.8x10-2±0.4x10 -2a 14.7x10-2±1.7x10 -2a 5.2x10-2±1.5x10 -2a
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T Cr+ 1.9x10-2±0.3x10 -2a 24.9x10-2±1.8x10 -2b 6.7x10-2±2.1x10 -2a
Br-68 Cr- 1.2x10 2±0.1x10 -2a 8.2x10-2±2.1x10 -2a 3.7x10-2±0.4x10 -2a
Cr+ 1.8x10-2±0.3x10 -3b 1.1x10-2±2.2x10 -2b 4.2x10-2±1.2x10 -2a
BR-73 Cr- 1.6x10-2±0.2x10 -2a 11.3x10-2±1.3x10 -2a 6.8x10-2±0.3x10 -2a
Cr+ 1.6x10-2±0.2x10 -2a 15.0x10-2±5.2x10 -2a 6.9x10-2±0.7x10 -2a
S
H
O
O
T
BR-58 Cr- 1.3x10-2±0.46x10 -2a 15.1x10-2±6.1x10 -2a 4.9x10-2±0.2x10 -2a
Cr+ 2.8x10-2±0.3x10 -2b 17.9x10-2±3.9x10 -2a 4.9x10-2±0.8x10 -2a
BR-63 Cr- 1.7x10-2±0.4x10 -2a 32.2x10-2±8.6x10 -2a 4.6x10-2±0.5x10 -2a
Cr+ 1.7x10-2±0.4x10 -2a 31.1x10-2±6.9x10 -2a 5.2x10-2±0.9x10 -2a
Br-68 Cr- 1.5x10-2±0.2x10 -3a 16.5x10-2±6.5x10 -2a 4.1x10-2±0.5x10 -3a
Cr+ 1.7x10-2±0.6x10 -3b 14.6x10-2±2.9x10 -22a 4.9x10-2±0.7x10 -2a
BR-73 Cr- 1.9x10-2±0.2x10 -2a 12.4x10-2±2.8x10 -2a 7.1x10-2±0.6x10 -2a
Cr+ 2.4x10-2±0.2x10 2b 9.7x10-2±1.4x10 -2a 7.0x10-2±1.4x10 -2a
DISCUSSION
Heavy metal stress significantly affects the physiology of plants (Rehman, et, al., 2021, Rolf et.al, 2004) and
crop production is reduced as a consequence (Jewell et al., 2010). On the other hand, plants also cope with heavy
metal toxicity through the mechanism of avoidance and tolerance (Yu et, al., 2019). Metal-tolerant rice varieties
are proving to be a boon for the farmers. Plant breeders employ selection method on a large number of plants
cultivated under metal stress to assess heavy metal-tolerant varieties. However, assessment of tolerance level is
very crucial for selecting a tolerant variety as it varies among the different species or varieties. In this
investigation, twenty-five authentic high-yielding rice varieties were developed in the chromium treated
hydroponic solution as treatment and without chromium as control. The result of chromium toxicity on different
morphological and physiological parameters such as root and shoot length, total chlorophyll content, and
electrolyte leakage in rice plants was identified by comparing with control plants, as these parameters are
significantly hampered in rice plant for chromium (Riaz et, al., 2024, Khatun, et. al, 2019). Fargasova (2001)
also mentioned that photosynthetic pigments as well as photosynthetic processes are damaged for toxic metals.
However, in this study, varieties BR-58, BR-63, BR-68, and BR-73 were proved chromium tolerant based on
morpho-physiological features. Moreover, constant level of total protein and MDA content under chromium
stress compared with control plants, indicates resistance against chromium as higher lipid peroxidation, cut down
protein level, is well documented in rice against chromium toxicity (Khatun, et. al., 2019, Mukta, et.al, 2019)
Analysis of chromium content in root and shoot exhibits that chromium translocation to shoot is inhibited
compared with the control plant in variety BR-58, BR-63 and BR-68. Vacuolar sequestration can restrict heavy
metal chromium in root cells vacuoles (Huda et al., 2017) with the help of thiol (SH)-containing molecule
Phytochelatin and helps plants to survive under stress (Huda et al., 2017). In the present investigation, in variety
BR-58 and BR-68, a significant increase of phytochelatin content and its precursor glutathione equated with
control plants ensure the chromium sequestration in roots. However, in variety BR-63 no enhanced
phyochelatine ensure that low molecular weight citric acid play vital role on vacuolar sequestration. Because
citrate function as counterions being stored in the plant cell vacuole (Martinoia, et.al,1994 and Meyer et.al.,
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2011). A similar result was also found in rice under Cd stress due to exogenous silicon application (Bari, et.al,
2020).
Plant roots absorb metal ions stored in the xylem, form a complex with the chelator, and then transport them to
the shoot. The molecules that function as chelators inside the cell to sequester the heavy metals in the cell vacuole
are organic acids, amino acids, and phosphate derivatives (Rauser 1999). In rice, exogenous application of
organic acids enhances heavy metal uptake and transport to the aerial portion through the xylem (Khatun et. al.,
2019) as well as sequesters in cell vacuoles (Huda et. al., 2016). On the other hand, secreted organic acids by
plant roots form non-toxic compounds with heavy metals by their carboxyl groups and prevent their entrance to
the plant (Guan, et. al., 2024, Yu G et al 2019). Previous investigations indicate that plants release significant
amounts of low molecular organic acids (LMWOAs) in response to heavy metal toxicity, which is an exclusion
mechanism rendering metal uptake and also unique to each species (Guan, et, al., 2024, Montiel-Rozas et. al.,
2016). Lowering pH significantly influenced plant metal uptake that enhance phytoextraction strategy (Wang,
et. al., 2006). However, the release of organic acid was well documented by a white precipitate in the silver
nitrate test in this study. Moreover, thin-layer chromatographic analysis confirmed that both the Cr-treated and
non-treated rice seedlings secrete citric acid. In this investigation, reduction of rhizospheric pH (more than 15%),
as well as decreased translocation of Cr to shoots in varieties BR-58, BR-63 and BR-68 indicates secreted
LMWOAs confer metal tolerance by sequester mechanism. Moreover, in variety BR-73, reduced rhizospheric
pH (more than 15%) and reduced chromium uptake propose an effective exclusion mechanism. Zeng Fanrong
et.al., (2008) reported that rice plants released oxalic, malic, and citric acid at the rhizosphere and enhanced Cr
accumulation. But this report could not provide any evidence regarding the amount of low molecular organic
acids (LMWOAs) secretion to confer chromium tolerance as well as any other mechanism of chromium
detoxification rather than accumulation, as the investigation was limited to two rice genotypes.
However, stability constant of OA-metal complexes specifies the detoxification capacity of organic acids.
Chelating complex between Al and citrate in carrot form at the ratio 1:1 where oxalate form complex with Al at
the ratio of 1:3 (Kyoma et.al., 1990) indicating that stability constant is variable among different ratio. These
two organic acids ensure detoxification mechanism by preventing Al to bind ATP or other ligands. Moreover,
in aquatic solution, higher concentration of malate is required to alleviate Al rhizotoxicity through chelation
(Thomas et.al., 2005). Aluminum-OA complex is precipitated in apoplast and excrete more malate in Al-tolerant
wheat than the Al-sensitive wheat variety (Ryan et.al., 1995).
Furthermore, Consumption of carbon sources for production and efflux of Organic acids (OA) under stress
consumed significant proportion of carbon imposing an energy cost to plants which is economically important
for fast-growing annual crops like rice (Koyama et al., 2000; Herz et al., 2018). Furthermore, plants optimize its
carbon loss by limiting the amount of OAs release by negative regulators of OA exudation like GABA (Ramesh
et al., 2015).
But in our investigation, it was evident that the required amount of citric acid secretion to cope with chromium
toxicity was assured by more than 15% rhizospheric pH reduction, whereas sensitive rice plants reduce less than
10% rhizospheric pH.
It is the first report that provides rice varieties (BR-58, 63, 68 and 73) ignore its carbon optimization process to
produce citric acids for coping with heavy metal chromium. Moreover, rice plant release citric acid to such a
height that can neutralize chromium and it measured by its 15% rhizospheric pH reduction. Our investigation
also concludes that 15% and above rhizospheric pH reduction in rice plant is the benchmark for required amount
of citric acid secretion to be chromium tolerant. This investigation also discovered that under chromium stress,
root secreted LMWOAs adopted two mechanisms such as vacuolar sequestration through chelation, and metal
exclusion to avoid chromium toxicity in rice varieties
However, limited activity of Fe transporters can reduce Fe uptake, which can enhance the tolerance level of rice
plants against Cr toxicity (Kabir 2016). But in the present study, Fe concentration in the root and shoot of these
four varieties was not followed when compared with the control plant, indicating that regulation of Fe transporter
was not involved with chromium tolerance.
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Oxidative stress generated from ROS is pronounced in the plant cells during metal toxicity, and activation of
antioxidant mechanisms is initiated (Ghori, 2019). Reduce protein content, lipids peroxidation, inefficient
enzyme activities, DNA damage and abnormal constituents of cells are the result of excessive accumulations of
reactive oxygen species (ROS) in plant. ROS also interacts with hormones and epigenetic modifiers to regulate
developmental processes and stress responses of plant (Kong et al., 2018). In this present investigation, under
chromium stress, ROS was found to be increased in the root of these varieties without any significant lipid
peroxidation compared with the control plant. Two types of mechanisms are present in the plant to scavenge the
ROS, and the first one is enzymatic and the other one is non-enzymatic. Hydrogen peroxide (H2O2) generated
first from oxidized metabolic and then convert into water by means of enzymatic activities while termination of
free radical chain reactions by the help of non-enzymatic antioxidants (Moussa Ziad 2019). Neutralization of
ROS by through enzymatic process enhance the plant tolerance level against diverse biotic and abiotic stresses
(Kanto et al. 2015). In the present study, GR activity was enhanced significantly in the root of varieties BR-58
and BR-68 and the shoot of varieties BR-58, BR-68, and BR-73 under chromium stress. Furthermore, , SOD
activity was found to be increased only in the root of variety BR-68 under same stress. Enhanced GR and SOD
activity reported in rice under Cd stress also (Bari, et.al., 2020)
Glutathione, a non-enzymatic antioxidant, is an efficient scavenger of O2, H2O2 and OH (Gill and Tuteja, 2010).
In our study, the amount of glutathione in the roots of varieties BR-58 and BR-68 as well as in the shoots of
varieties BR-58, BR-68 and BR-73 was significantly raised under chromium stress. However, our investigation
again notices that citric acid up regulate the antioxidant defense system as a secondary function along with
chromium avoidance mechanisms to keep down ROS levels.
Proline (Pro) an amino, responsible to mitigate biotic and abiotic stress in plant (Ágneset.al., 2018). It is also
reported as metals chelator (Gill and Tuteja, 2010). In the present investigation, proline content was found to be
enhanced significantly in the roots of BR-58, BR-63, and BR-68 to enhance the tolerance against Cr toxicity.
The findings of this study hold practical significance for both crop improvement and environmental remediation.
CONCLUSION
The exudation of citric acid has been shown to be the key physiological response against chromium toxicity in
rice plant. But the concentration of citric acid is deviated according to the genotypes of rice plants. The
assessment of the amount of citric acid exudates followed by rhizosphere pH reduction rate has allowed knowing
the potentiality of rice varieties to cope with chromium toxicity. Furthermore, rice genotypes that can reduce
their rhizosphere pH by 15% or more by releasing organic acid are chromium tolerant. However, the findings
offer an efficient screening technique for metal tolerant rice plants.
DECLARATION
Funding: This research did not receive any specific grant from funding agencies in public, commercial or not
for profit sectors.
Conflicts of interest/Competing interests: The authors declare that they have no conflict of interest.
Ethics approval: Not applicable
Consent to participate: All participants are in the list of authors and have full consent
Consent for publication: All participants are in the list of authors and have full consent
Availability of data and material: The datasets are available from the corresponding author on reasonable
request.
Generative AI: Not applicable
Acknowledgements: I am grateful to Bangladesh Rice Research Institute (BRRI) for providing seeds of rice
varieties. "The germplasm used in this study was obtained from the Bangladesh Rice Research Institute (BRRI)
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through their official procedure, which involved submitting a formal application stating the purpose of use. The
institute evaluated and approved the request prior to providing the seeds. No separate permission letter was
issued after seed distribution, as approval was granted at the initial stage
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