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Evaluation of salinity-mediated end-point
cytogenotoxicity in Germinating Roots of
Lathyrus sativus L., Variety Mahatora.:
Bio-assay guided biomarker studies
Dipan Adhikari1, Rahul Ghosh2, Sagar Dig1
1 Plant Cell and Molecular Research Laboratory, Undergraduate and Post-Graduate Deptartment of Botany, Hooghly Mohsin College, Chinsurah, Hooghly
712101, India
2 Department of Botany, Memari College, Memari, Purba Bardhaman, West Bengal 713146, India
Corresponding author: Dipan Adhikari (dipanadhikari@gmail.com)
Academic editor: Robert Gabriel♦Received 11 December 2023♦Accepted 14 March 2024♦Published 30 April 2024
Abstract
Pulse crops are susceptible to salt stress as per dierent research reports but how far Lathyrus sativus L., responds to in-
creasing salinity has been taken up in this work. us, the harmful eects of increasing salinity on plant cells at various
phases of chromosomal integrity and nucleolus morphology have been evaluated in Lathyrus sativus L., variety Mahatora.
Lathyrus sativus variety Mahatora seeds were subjected to seed priming with serially diluted concentrations of NaCl
(500, 400, 300, 200 and 100 mM respectively) and germination percentage (72 hrs), root length inhibition (7 days) nor-
mal and abnormal MI (Mitotic Index) with 2% aceto-orcein staining, nucleolar morphometric cum frequency analysis
(0.05% hematoxylin staining), total soluble protein vs Peoxidase activity (POX), Electrolyte leakage (EL) from etiolated
roots and root metabolic activity/dehydrogenase activity were measured (TTC staining). From 200 mM onwards, sig-
nicant reduction in germination percentage and root length inhibition resulted and at 300 and 400 mM salt-priming
signicant reduction in normal MI%, increased Abnormal MI% showing both aneugenic and clastogenic responses were
accounted. At 500 mM pre-exposed root tip cells were found to develop gradual blackening and root tip death and very
less viable cells with highly necrotic, vacuolated with chromosomal erosions and nuclear dismantling and nuclear blob-
bing resulted apoptosis in addition to decreased POX and dehydrogenase activity (300–500 mM NaCl-treated test sets).
NaCl stands out as a potential cyto-genotoxicant in Lathyrus sativus L., variety Mahatora. e maximum tolerance level
(200–300 mM) and at 400–500 mM NaCl has been highly cytotoxic as per cytological and biochemical data. From 200
mM onwards, nucleolar volume and frequency were altered and at 500 mM pretreatment complete degradation of nuclear
machinery was encountered. Owing to high salinity signicant proportions of C-mitosis and polyploidy were accounted
which conclusively established that NaCl surely had a disruptive role to play during spindle bre formation process in
dividing root cells that in turn produced somatic diads and subsequent polyploidy formations (At 200 to 300 mM).
Keywords
Lathyrus sativus, Salt priming, germination, apoptosis, mitodepression
Introduction
Plants may suer harm that disturbs genomic stability be-
cause some ions produced by the buildup of sodium and
chloride in plant tissues as a result of either root uptake ulti-
mately resulting poisonous manifestations for normal phys-
iology. It is essential to comprehend the cytogenetic under-
pinnings of salt tolerance in order to create tolerant variants.
Rice, wheat, pigeon pea, and tomato are only a few of the
crops for which the genetics of salinity tolerance have been
Innovations in Agriculture 7: 1–17
doi: 10.3897/ia.2024.124263
RESEARCH PAPER
Copyright Adhikari, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited.
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus2
Innovations in Agriculture
studied around the world (Joshi 2007; Li and Xu 2007; Singh
et al. 2012; Chaudhary et al. 2013). ere are, however, few
data on the impact of salinity on the chromosomal levels of
various pulse crops. ere have been similar reports of ge-
netic regulation for several salinity tolerance traits in chick-
peas and soybeans. e salt stress tolerance of soybean was
discovered to be determined by salt tolerance rating (STR)
and ion buildup (Hamwieh and Xu 2008; Do et al. 2018),
but no such data are available from a climate-resilient pulse
crop, i.e. Lathyrus sativus. Salt aects the stability of pro-
teins (Ahmad et al. 2015) and the lipid composition of plant
membrane (Ibrahim et al. 2015) and causes asymmetric di-
vision of plant cells in dierent organs resulting in deregula-
tion of cellular homeostasis in higher plants (Baranova and
Gulevich 2021). However, the genotoxic consequences of
salt are poorly understood, and Teerarak et al. (2009), found
that few studies address the cytological harms to plant cells
when brought on by exposure to NaCl. By using plant root
tip cells as test models, which are both inexpensive and easy
to use, the present authors have tried to examine the geno-
toxic eects of toxicants i.e., increasing NaCl by calculating
the irregularity of the mitotic index. Numerous studies have
demonstrated the value of the mitotic Ana-telophase assay
as a tool for assessing the genotoxic eects of toxic chem-
icals. Lathyrus is widely used as cheap animal feed and a
source of protein for the underprivileged in India. Although
it has been suggested that salt damages the double helix of
DNA and contributes to chromosomal aberrations, no pre-
cise studies on how high salt concentrations (100–500 mM)
cause damage to Lathyrus sativus roots and chromosomal
aberrations have been published in recent years.
In this study, the focus has been laid on the architec-
ture of the nucleolus and the hazardous eects of salinity
on plant cells at dierent stages of mitosis. e authors
have also tried to look studied the development of cell
micronuclei, chromosomal abnormalities, and nuclear
anomalies in the roots of Lathyrus sativus seeds that were
germinating aer being pre-exposed to the salt regime.
Lathyrus sativus L. seeds are readily available, simple to
grow throughout the year in lab conditions, and have a
bimodal karyotype that displays good chromosome com-
plements (2n = 14) with equal spreads under microscopic
view, describing nearly the same types of clastogenic and
aneugenic eects as described in popular models like Al-
lium cepa L and Vicia faba L cells throughout the study
(Teerarak et al. 2009). Furthermore, there have been no
ndings published so far demonstrating the genotoxic ef-
fects of salinity on this plant’s enzymatic parameters. Lath-
yrus sativus is a fantastic model organism (Cavuşoğlu et al.
2017) for assessing all potential consequences, particularly
at the chromosomal level following various levels of stress
production caused by high salt (100–500 mM NaCl).
erefore, the aim of this research is to evaluate the im-
pact of salt stress over root cells through the study of mitotic
index (MI), and distinct chromosomal anomalies, utilizing
root tip cells of high yielding Mahatora variety. us, by an-
alysing the mitotic index (MI) and particular chromosomal
abnormalities in root tip cells from the Mahatora variety of
Lathyrus sativus L., the goal of this work is to ascertain how
salt stress aects germinating root cells. Additionally, as per
recent opinions prolonged salt stress produces severe met-
abolic and cytogentoxic stress in Allium cepa L., (Singh and
Roy 2016; Kielkowsla 2017). In this experimental process
the authors have also tried to opine that the study of nucle-
olar alterations as well as cytogenetic changes as a potential
cytological tool (Bio-marker) to evaluate the cytotoxicity of
tested chemicals may yield useful information relating to
the salt tolerance mechanism and suboptimal concentra-
tions of tolerance as of this crop is also concerned.
Materials and methods
Study area
e study had been carried out at the Plant Cell and
Molecular Research laboratory, Undergraduate and
Post-Graduate deptartment of Botany, Hooghly Mohsin
College, Chinsurah, Hooghly, starting from November
2021 to August 2022.
Certied Mahatora variety of Lathyrus sativus L., seeds
to conduct this research program was taken from State
Seed Testing Laboratory, Govt. of West Bengal, District
Agriculture Farm, Kalna Road, Burdawan 713101, India.
Lathyrus sativus L., seeds (Mahatora variety) were ster-
ilised with a 2% mercuric chloride solution for 10 minutes,
then repeatedly washed under running water. ree hours
later, the seeds were immersed in distilled water. e seeds
were then split into six groups (total 10 seed in each set was
employed in the study) and placed in petri plates with vari-
ous sodium chloride concentrations (500, 400, 300, 200, and
100 mM, respectively). Before the nal experimental setup,
the doses were tested and modied based on the morpho-
logical traits the germination of the seeds produced. Before
applying the aforementioned dosing regimen, a separate
500 mM NaCl treatment made the seeds black and prevent-
ed them from germinating. Even the few seeds germinated
aer 72 hours of incubation had turned dark brown- to-
black and neither any nuclear complement nor the chromo-
somal makeup could be seen under a microscope, Olympus
CH20i microscope, Japan); thereby these roots were reject-
ed. Following multiple trial runs, the salt dose for exposure
was decided upon, for the nal experiment. e seeds were
let to sprout for 24 hours aer exposure, and aer 72 hours
of root growth, the root tips were removed, soaked in 70%
ethanol for the night in Carnoy’s xation solution, which
contains ethanol and glacial acetic acid, and then hydro-
lyzed with 1N HCl,14.e root cap was removed, and the
root meristematic tissues were dyed with 2% aceto-orcein,
compressed onto slides, and then viewed with an Olympus
CH20i compound microscope (Japan) equipped with an
IS 500, 5.0 MP CMOS camera. Each duplicate had at least
three stained root meristems. A minimum of 500 cells were
subjected to each treatment (control and salt treatments)
for the analysis. A compound microscope (Olympus CH20i
microscope) equipped with CMOS Camera (IS 500, 5.0
Innov. Agric. ⋅ Volume 7 ⋅ 2024 3
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MP), attached to a PC, and VIEW 7 image analysis soware
were used to take pictures of the preparations.
Germination percentage
Every 24 hours up to 168 hours (7 days), the germination po-
tential of seeds that had been pre-treated with NaCl as well as
the radicle (embryonic root) length (measured using a mil-
limetre ruler) were examined. ree times the experiment
was conducted in identical circumstances. e proportion
of germination of seeds for Lathyrus sativus L. was calculated
(aer a 96-hour period). e rate of germination was calcu-
lated as % of seed germination = Total No of seeds germi-
nating (72 hrs)/total seeds taken X 100 (Ghosh et al. 2020).
Cytogenetic analysis: Determination of cytogen-
toxicity (mitotic inhibition by orcein staining)
e root tips of germination-tested seeds were utilised
as a source of mitotic cells to examine the cytogenetic
changes brought on by NaCl pre-treatment (in serially
diluted amounts) in the Lathyrus sativus L. root tips. A
minimum of 500 cells from each plate were scored, and
the mitotic index was computed. A minimum of 500 cells
per slide were examined, and the percentages of chromo-
somal abnormalities, both normal and abnormal (such as
Strap nucleus, disorganised metaphase, metaphase pung
C-metaphase, Star metaphase, and Scattered metaphase
Binucleus, telomere pung, tropokinesis, and bridges
during the anaphase and metaphase Dead cells, laggards,
and Lesions, polyploidy, and an elongated strap nucleus
Numerous abnormalities including Binucleus, Micro-
nucleus, Telomere pung, Metaphase, clumping, Diso-
riented metaphase, Tropokinesis, Disturbed Anaphase,
Disturbed Metaphase, and Metaphase pung) were seen
and manually recorded. In order to analyze all phases at
a magnication of 40× and under an oil immersion ob-
jective (100×), a compound microscope (Olympus CH20i
microscope, Japan) outtted with a CMOS Camera (IS
500, 5.0 MP) and its attachment to a computer with the
aid of VIEW 7 image processing soware was employed.
Images were acquired and cytotoxic and genotoxic
end-point parameters were calculated using the following
formulas:
Mitotic index % = (Number of dividing cells) ÷ (Total no
of cells) × 100 (Ghosh et al. 2020).
e Genotoxicity Index (GnI%) was calculated (aer de
Souza et al. 2022) (Adhikari et al. 2023). e genotoxici-
ty index (GenI) was calculated using the formula = (No of
cells showing abnormal chromosomal response + nuclear
buds+ nuclear breakage) ÷ (Total no of cells counted) × 100.
Percentage of % of Mitotic Inhibition = (Mitotic index
in Control-Abnormal Mitotic index aer treatment) ÷
(Mitotic index in Control) X 100 (Adhikari et al. 2021).
Detection of Morphological Characters for cell death:
Computation of the Percentage of Dying Cells: We chose
nucleus migration from centre to the margin of cell wall,
condensation, vacuolation of cytoplasm, and nuclear frag-
mentations as characteristic hallmarks of dying cells. Nu-
cleus margination is the displacement of the nucleus in a
cell wall margin. Percentage of Dying Cells = (No of cells
dying or dead cells) ÷ (Total no of cells counted) × 100.
Detection of morphometric changes in nucleo-
lus in germinating root tips of Lathyrus sativus
L. variety Mahatora using Hematoxylin staining
Seeds of Lathyrus sativus L., were allowed to germinate af-
ter 24 hrs aer priming with dierent concentrations of test
sample (500, 400, 300, 200 and 100 mM respectively) and
aer 72 hrs of germination the root tips were cut and xed
in FAA (4% formalin: Glacial Acetic Acid: Ethanol=1:2:7)
and kept overnight at 4 °C. e very next day the root tips
were hydrolyzed in 45% Acectic acid for 45 mints at water
bath not allowing the temperature to rise above 85 °C. Aer
acid hydrolysis the root tips were cooled, washed in distilled
water and incubated in saturated solutions of iron alum
(ferric ammounium sulphate) for 10 minutes followed by
staining in 0.5% aqueous hematoxylin solution for 45 min-
utes. e root tips were then washed and one drop of 0.2%
orcein was applied and squashed in 45% acetic acid and
observed under a compound microscope (Olympus CH20i
microscope, Japan) outtted with a CMOS Camera (IS 500,
5.0 MP) and its attachment to a computer with the aid of
VIEW 7 image processing soware.
Studies on nucleolar morphometric changes: treat-
ment groups having cells with dierent numbers of nu-
cleoli were manually scored and in dierent groups apart
from control groups showing dierent numbers of nucle-
oli with or without nuclear membranes tabulated. Nuclo-
lar volume was measured using the formula 4/3πr3 using
stage micrometer, Erma, Japan to measure the nuclear
and nucleolar diameters. Among dierent shaped nucle-
ar morphological alignments four distinct morphomet-
ric parameters were chosen as “cytological markers” of
endpoint cytotoxicity i.e., (i) big vacuolated nucleus with
translucent centres, (ii) Elongated nuclei, (iii) dumbbell
shaped nuclei, (iv) nuclei in chain, and (v) micronucleoli
(one fourth of diameter than control nuclei) in scattered
conditions throughout the cytoplasm.
Determination of total soluble soluble protein
e germinating roots (of all treatment groups and con-
trol aer 96 hr of germination) were cut with sharp razor
and were crushed with 10 mL of cold 0.05 M potassium
phosphate buer (pH 7.8) in a porcelain mortar that has
already been chilled for 10 minutes. e homogenate was
centrifuged at an ultracold 13,000 rpm for 30 minutes at
4 °C aer being ltered through Whatman’s No. 1 lter
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus4
Innovations in Agriculture
paper and transferred to an eppendorf tube. e superna-
tant from the centrifugation was then subjected to spec-
trophotometric analysis (O.D. changes in comparison to
blank and control) for biochemical analysis to determine
the amount of total soluble protein (Bradford 1976) anal-
ysis. All readings are made in triplicate under experiment
set ups, in the laboratory in vitro.
Determination of total soluble protein content
e total soluble protein content of the root homogenates of
the control and treatment sets were performed aer (Brad-
ford 1976) where bovine serum albumin (BSA) taken as a
standard. Triplicate trials of each set and control were done.
Determination of peroxidase (POX) activity
POX activity was measured (Özceylan and Aki 2020) us-
ing spectrophotometric analysis to measure variations in
POX activity in dierent treatment sets. For two minutes,
the spectrophotometer measured at 300 nm to determine
the POX kinetic reaction in SHIMADZU UV-1800 UV-
VIS Spectrophotometer. For each group, the largest chang-
es in the absorbance values taken every 10 seconds over a
2-minute period are identied. ese variations have been
calculated, converted to mg/mL/min POX enzyme activi-
ty, and given as mg/mg protein level. ree repetitions of
each POX activity measurement were carried out.
Determination of membrane permeability/
Electrolyte leakage after NaCl treatment on
etiolated roots
Ions that were leaking into deionized water from tissue
were used to measure membrane permeability or electro-
lyte leakage (EL). Test tubes containing 10 mL of deion-
ized water and segments of fresh root samples (processed
and controlled sets of 100 mg root tissues in each tube)
were used. e tubes were immersed in water that was
32 °C-heated for 6 hours. Following incubation, the bath-
ing solution’s electrical conductivity (EC1) was measured
using an electrical conductivity metre (Systronics M-308,
Kolkata, India). Aer that, the samples were autoclaved for
30 minutes at 121 °C to totally destroy the tissues and lib-
erate all electrolytes. e nal electrical conductivity (EC2)
of the samples was then calculated aer they had been
cooled to 25 °C. e formula EL%=EC1/EC2X100 was
used to convert the EL into a percentage (Adhikari 2021).
Evaluation of root metabolic/mitochondrial ac-
tivity
e best method for determining a cell’s viability is TTC
(2,3,5-Triphenyl tetrazolium chloride) staining. Lathyrus
sativus L. seeds were subjected to 24 hours of treatment with
various NaCl solution concentrations. e same procedure
was followed while using pure water as the positive control
and 0.1% hydrogen peroxide as the negative control. In 0.5%
(w/v) TTC stain for ve hours in the dark, all the roots were
submerged. Aer that, distilled water was used to cleanse
the roots. Using a spectrophotometer and 95% ethanol as a
blank, absorbance was measured at 490nm. e test O.D.s
had been translated into percentages representing the fol-
lowing rise or fall in metabolic activity, and the positive con-
trol (hydrogen peroxide O.D.) was taken to represent 100%
metabolic/respiratory activity (dehydrogenase) activity, out
of root mitochondrial activity (Ghosh et al. 2020).
Statistical analysis
All the values are presented as Mean±SD (standard de-
viation, n = 6). Statistical analyses were performed with
paired t-test and ANOVA is used rst, then, if necessary, a
post-hoc Dunnett’s multiple comparison test. P values be-
low 0.05 were regarded as signicant. Using the GRAPH
PAD PRIZM-version 6 computer program, analysis of
variance (ANOVA) was used to examine dierences be-
tween the groups in the statistical study.
Results
Effect of increasing molarity of NaCl priming
on germination and growth of Lathyrus sativus
seeds after 96 hrs
Evident from the gure plates it is evident that aer 96 hrs
of germination applying gradually increasing concentrations
of NaCl (seed priming for 24 hrs) there were qualitative and
quantitative inhibition of germination vis-a-vis embry-
onic root length inhibition with increasing salinity. In 500
mM NaCl primed seeds almost all the seed coats became
blackened showing signicant inhibition of radical forma-
tion; however, only a single seed with visible symptoms of
germination was seen and a stunted root with gradual wilt-
ing-like morphological signs was prominent. In 400 mM
NaCl primed seeds almost all the seed coats became black-
ened showing signicant inhibition of radical formation;
however, 4 seeds with visible symptoms of germination were
accounted but the root with gradual brownish to blackening
tips with morphologically stunted growth was prominent. In
300 mM NaCl primed seeds half of all the seed coats became
although a gradual increase in germination prole with an
increase in root length could be observed. However, the roots
here were with visible symptoms of less brownish tips were
visible. At 200 and 100 mM primed seeds there was marked
dierence could be accounted, whereasin the 200 mM treat-
ed group almost 85% seed germination with signicant root
length growth was seen. Interestingly 100 mM NaCl prim-
ing produced vigorous germination with a robust increase in
root length with shoot formation was accounted which was
almost similar to control sets (Fig. 1A–F).
Innov. Agric. ⋅ Volume 7 ⋅ 2024 5
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Effect of increasing molarity of NaCl priming on germination vs root length inhibition of Lathyrus
sativus seeds after 168 hrs (7 days) of germination
Figure 1. A–F. Plates showing the direct effect of increasing molarity of NaCl priming on germination and root shoot
length growth in Lathyrus sativus L., (variety Mahatora) after 96 hrs. A. Control seeds after 96 hrs of germination;
B. 500 mM salt primed seeds after 96 hrs of germination; C. 400 mM salt primed seeds after 96 hrs of germina-
tion; D. 300 mM salt primed seeds 96 hrs of germination; E. 200 mM salt primed seeds after 96 hrs of germination;
F. 100 mM salt primed seeds after 96 hrs of germination.
Figure 2. Line graphs (2a, b) showing the effect of increasing NaCl concentrations on seed germination percentage
(2a) and root length growth inhibition (2b) in Lathyrus sativus L., after 7 days in comparison to control groups.
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus6
Innovations in Agriculture
Effect of different salt concentrations on seed
germination Lathyrus sativus within a span of 7
days
In this assay in comparison to the control it was observed
that 500 and 400 mM salt perexposure signicantly re-
duced (40% and 65% respectively at 7 days’ intervals). In
comparison to control setups, 300 and 200 mM prexposed
seeds could augment the salt stress and come up with 70
and 80% germination percentages at 7 days of observa-
tion. In 100 mM salt-primed seeds reached up to 100%
germination eciency at 5th day as compared to control
seeds which attained 100% germination aer 48% hours
only. From the results, it might be deciphered that 500
and 400 mM salt exposure is growth inhibitory impart-
ing negative eects on seed germination. Interestingly 200
and 300 mM salt concentrations are tolerable concentra-
tions for the germinating seeds although seed germina-
tion percentage got delayed till 5th day which might be the
alterations in the cellular metabolic states and subsequent
metabolic adjustments of the germinating root tip cells
owing to abiotic stress formed within.
Effect of different salt concentrations on mean
root length Lathyrus sativus within a span of 7
days
is particular observation, in comparison to control
it was observed that 500 and 400 mM salt priming sig-
nicantly reduced the mean root length (less than 2 cm
in length) of germinating seeds of Lathyrus even aer 7
days’ intervals of observation. In comparison to con-
trol setups of 300 and 200, mM salt-primed seeds could
augment the salt stress and come up with less than 4 cm
roots aer 7 days of observation. In 100 mM pre-exposed
seeds reached up to 100% germination eciency on 5th
day as compared to control seeds attaining over 5 cm of
length nicely. I that 500 and 400 mM salt exposures are
truly growth inhibitory imparting negative eects on root
growth. Interestingly 200 and 300 mM salt concentrations
are tolerable concentrations for the germinating seeds al-
though the root length growth was hampered to attain up
to 4 cm in length till the 7th day which might be the altera-
tions in the cellular hormonal levels and subsequent met-
abolic adjustments of the germinating root tip cells owing
to abiotic stress formed within.
Effect of increasing NaCl concentrations on Mi-
totic index of Lathyrus sativus
Bar diagrams representing the toxic eect of gradually in-
creasing salt concentrations on normal Mitotic index and
induction of abnormal Mitotic index on Lathyrus sativus
L., root tip cells aer 72 hrs of germination. Dierent al-
phabets within a column represent signicant dierences
at p < 0.05 aer paired “t” test in comparison to respective
normal MIs.
e comparative normal and abnormal MI%s of dierent
treatments revealed an inverse dose-response relationship.
With increasing salt priming there was a gradual decline in
normal MI%s and a gradual rise in the abnormal MI%s. But
at 400 and 500 mM treated root tip cells both normal and
abnormal MI%s were declining. At 500 mM pretreated ger-
minating root tips very few diving cells and abnormal cells
could be accounted which were less than 10% in existence
and most of the giant strap cells were with multi-fragmenta-
tions with dismantled nuclear architectures having hyaline
cytoplasm showing nuclear blobs shied to corners repre-
sentative of the apoptotic cellular population.
Effect of increasing concentrations of NaCl con-
centrations on chromosomal division of Lathy-
rus sativus
Increasing salinity has a pronounced eect on chromosomal
morphology (induction of chromosomal aberrations) and
normal cell division (MI) of Lathyrus sativ us L., (Fig. 4A–O).
At 100 mM salt exposure in comparison to control there is
signicantly reduced abnormal MI%. Normal MI % was pre-
dominant showing very less chromosomal aberrations and
abnormal MI% showed only few metaphasic and anaphasic
clumping and stickiness. From 200 mM onwards there is a
proportional change in normal MI% and there is a relatively
high proportion of chromosomal aberrations (statistically
insignicant, aer paired t-test) showing early separation
and centric ssion at clumped metaphase. At 300 and 400
mM salt exposed root tips there was a signicant reduction
in the percentages of normally dividing cells showing up re-
duced normal MI% and signicantly increased abnormally
dividing cells i.e., high frequency of Abnormal MI% show-
ing C-mitosis, somatic diads, polyploidy (exceptionally high
in frequency), binucleate cells, Karyorrhexis and anaphasic
bridges. Giant strap cells with diminishing nucleus was also
observed. At 500 mM salt-primed root tip cells there was
0
5
10
15
20
CONTROL (MI)
CONTROL (AB MI%
)
100 mM (MI %)
100 mM (Ab mi%)
200 mM (MI %)
200 mM (Ab MI%)
300 MM (MI %)
300 mM (Ab MI%)
400 mM (MI%)
400 mM (Ab MI%)
500 mM (MI %)
500 mM (Ab MI%)
aa
a
a
a
Treatment (NaCl mM)
Mean MI % ± SD
Figure 3. Histograms representing the toxic effect of
gradually increasing salt concentrations on normal
Mitotic index (MI) and induction of abnormal Mitotic in-
dex (Ab MI) in Lathyrus sativus L., root tip cells after 72
hrs of germination. Different alphabets within in a col-
umn represent significant difference at P < 0.001 after
paired “t” test in comparison to respective normal MIs.
Innov. Agric. ⋅ Volume 7 ⋅ 2024 7
Innovations in Agriculture
Figure 4. A–O. Meristematic root tip cells of Lathyrus sativus L., representing chromosomal alterations after 72 hrs
of germination after increasing concentrations of salt priming. Photomicroplates (A–O) showing induction of chro-
mosomal abnormalities after varying concentrations of sodium chlorideon germinating root tips of lathyrus sativus
step by step induction of cellular death from early stages of cellular toxicity (from 100, 200,300,400 and 500 mM
doses respectively). A. Anaphasic clumping, metapahic bridges at 100 mM NaCl; B. Metaphasic stickiness and ball
metaphase at 100 mM NaCl; C. Asteroid like anaphase separation and multivacuolated nuclei at 200 mM NaCl;
D. C-mitosis with isochromosome formation at 200 mMNaCl; E. Polyploidy at 200 mMNaCl; F. Somatic diads with
Multilobed double nucleus at 300 mMNaCl; G. Isochromosomes showing pole to pole sticky methaphase at 300
mMNaCl; H. Micronuclei formation at 300 mM NaCl; I. Early decondensation at prophase with precocious chroma-
tin fragmentations at 400 mMNaCl; J. Karyorrhexis and tropokinesis at 400 mM; K. Hyperploidy polypoidy at 400
mM NaCl; L. Coagulated anaphase, laggard and late separation at 400 mM; M. Multifragmented nuclear lobes and
nuclear erosions at 500 mM Nacl; N. Vacuolated cytoplasm, and dislodged nucleus with karyorrhexis in giant strap
cells (500 mM); O. Translucent cytoplasm with karyolysis leading to apoptosis (500 mM).
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus8
Innovations in Agriculture
gradual blackening and root tip showing death symptoms
and the surviving roots ended up in very less viable cells
where almost all the cells were necrotic, highly vacuolated
with chromosomal erosions and nuclear dismantling with
nuclear blebbing showing apoptotic symptoms. In this high-
ly toxic dose, the normal and abnormal MI % are greatly
reduced as more than 50% of cells were enucleated or with
apoptotic signatures. is result correlated with the seed ger-
mination and root length inhibition assays where the nor-
mal growth was greatly halted and almost all the root tips
were blackened and dried up owing to the toxicity of high
slat stress in the meristematic tissue.
Effect of increasing NaCl concentrations on Nu-
cleolar dynamics of Lathyrus sativus
Figure 5. Meristematic root tip cells of Lathyrus sativus L., representing nucleolar alterations (frequency and vol-
ume) after 72 hrs of germination after increasing concentrations of salt priming. Photomicrophotograhs (A–L)
showing different shapes and states of nuclear morphometrics after different concentrations of NaCl priming in
germinating root tips of Lathyrus sativus L., A. Control root tip cells showing intact nuclear membranes with double
nuclei. B. Root cells after 100 mM NaCl pretreatment showing all double nuclei with disappearing nuclear mem-
brane. C. Root cells after 200 mM NaCl pretreatment showing no nuclear membranes with big round to oblate and
pear-shaped nucleoli showing translucent multiple lesions. D. Root cells after 200 mM NaCl pretreatment showing
round to oblate micronucleoli showing diplo to streptococci like appearance; E. Root cells after 200 mM NaCl pre-
treatment showing micronucleoli , 4-6 in numbers adhered together. F. Root cells after 300 mM NaCl pretreatment
showing reduced cellular volumes oblate-shaped micronucleoli with gradual comingling; G. Root cells after 400 mM
NaCl pretreatment showing altered cellular morphologies with pear to oblong shapes nucleoli; H. Root cells after
400 mM NaCl pretreatment showing pear-shaped, eye-shaped and dumbbell-shaped nucleoli, with diminishing
nucleolar volume; I. Root cells after 400 mM NaCl pretreatment showing dumbbell-shaped nucleoli with nulceolar
notches and fragmentations; J. Root cells after 400 mM NaCl pretreatment showing multiple micronucleoli forma-
tions with translucent cytotoplams; K. Root cells after 500 mM NaCl long strap cells with almost no-cytoplasm and
multifragmented micronucleoli scattered around; L. Root cells after 500 mM NaCl pretreatment showing no only
2-3 micronuceli scatted around corners with ruptured cell walls showing aopototic appearances.
Innov. Agric. ⋅ Volume 7 ⋅ 2024 9
Innovations in Agriculture
Changing percentages of root tip cells having different number of nucleoli (1–8) after pre-treat-
ment with increasing NaCl concentrations in Lathyrus sativus
Figure 6. Piecharts representing Meristematic root tip cells of Lathyrus sativus L., nucleolar alterations (frequency)
after 72 hrs of germination after increasing concentrations of salt priming. Photomicrophotograhs (A–F): These
relative pie charts are showing different stages, morphological types and numbers of nucleoli in different treat-
ments representing the morphometric and volumetric changes in the nucleus and nucleolus contents in root tip cells
of Lathyrus sativus L. A. Control cells showing the relative percentages of nulcoloar (mono, bi and tri nucleolate)
frequency. B. In 100 mM NaCl pretreated root tips percentages of tri-nucleolate populations reached nearly 29%
followed by C. 200 mM NaCl sets where tetranucleate conditions could be accounted in almost 18% of the cells and
pentanucleate condition could be seen in 19% of cellular populations. D. In 300 mM NaCl treated root tips tetra,
penta and hexanucleolate population accounted altogether of 63% of the overall cellular population. E. In 400 mM
NaCl treated root tip cells 5–6 nucleolated cells were 43% and 7–9 nucleolated cells were upto 20% of the whole
population. F. At 500 mM NaCl pretreated germinating root cells maximum up 60% of the population were giant
strap cells with no nuclear masses remaining with 19% of the cellular population contained 7–9 micronucleolate cells
with diminishing nucleolar volumes compared to control and lower treatment groups.
Determination of total soluble protein and POX
activity on etiolated roots of Lathyrus sativus
From the comparative bar diagram (total soluble protein
vs. POX activity) it was found that there had been a dif-
ferential expression in soluble protein concentrations in
comparison to control in all treatment groups (100, 200
and 300 mM NaCl treatments) and in treatment groups
having 400 and 500 mM NaCl pretreatments there a re-
versal of total soluble protein content could be accounted
and at 500 mM treated sets the amount of total soluble
protein was even less than control. In comparison to total
soluble protein vs POX activity, there had been a sharp in-
crease in POX activity in almost all the treatment groups
(7.5, 10.3, 11.3, 10.9 mg/mL/min respectively) in compar-
ison to control (6.6 mg/ml/min). But in the 500 mM treat-
ed sets there had been a sharp fall in POX activity (3.4 mg/
mL/min) which was almost half of the activity of the con-
trol group (6.6 mg/mL/min) (Fig. 7). is lessened trend
in both total soluble protein content and POX activity
could be due to the cellular poisoning of the highest dose
augmenting possible halting of both protein synthesis and
enzyme activity.
Determination of Membrane Permeability/
Electrolyte Leakage after NaCl treatment on
etiolated roots of Lathyrus sativus
Root electrolyte leakage from Lathyrus sativus L. seeds
that had not been treated was very low, at less than 10%
(Fig. 8). When the seeds germinated aer 72 hours of
incubation at various NaCl concentrations, the etiolat-
ed roots could exhibit a variable response in terms of
electrolyte leakage/membrane permeability, and 50% of
the electrolyte leakage could fall in the range of 200 to
300mM treatments. In comparison to the control, there
was considerable disruption of membrane leakage that
was concentration-dependent in the etiolated roots of
Lathyrus sativus L.
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus10
Innovations in Agriculture
Evaluation of alterations in root metabolic/mi-
tochondrial activity i.e., percentage of dehydro-
genase activity after NaCl treatment on etio-
lated roots of Lathyrus sativus
ere were variable responses in the metabolic prole of the
root mitochondrial system (dehydrogenase activity) with-
in positive control (2% H2O2), negative control (distilled
water) and salt-treated groups. In positive control groups
high colour formation (out of formazan complex forma-
tion) could be accounted followed by negative control sets
(distilled water). In treatment groups there were gradual
increase in dehydrogenase activity in 100 to 200mM NaCl
treatment groups in comparison to negative control and in-
hibition of root metabolic activity at 300 to 500 mM treat-
ment sets, which signied that in the higher (400 and 500
mM treatment) groups there was inhibition of dehydroge-
nase activity resulting root mitochondrial dysfunction in
comparison to Negative control (water) (Fig. 9).
Discussion
In this investigation pronounced increasing NaCl seed
priming produced a disruption of normal seed germina-
tion and root length inhibition (physiological biomarkers,
Figure 7. Histogram showing comparative concentrations of total soluble protein vs POX activity in all treatment
groups (after 72 hrs of germination) in addition to control groups in Lathyrus sativus L., germinating root tip cells.
Figure 8. Histogram showing differences in electrolyte leakage in etiolated roots of Lathyrus sativus L., after 72 hrs of
germination. Bars with letters within each panel are significantly different at P < 0.0001 according to one-way ANOVA
(control vs. treatment) followed by Dunnet’s multiple comparison test within treatment groups (100–500 mM NaCl).
Innov. Agric. ⋅ Volume 7 ⋅ 2024 11
Innovations in Agriculture
Figs 1, 2a, b), phase index changes coupled with a decrease
in normal MI% and an increased in abnormal MI% (Fig. 3)
coupled with both clastogenic and anegenic changes serv-
ing as a cytogenetic biomarker (Fig. 4A–O). Salt priming
at 400–500 mM produced total loss in chromosomal com-
plements coupled with cellular apoptosis leading to root
cell deaths. NaCl seed priming produced disruption of
normal nucleolar frequency and nucleolar volumes (Figs
5A–O, 6A–F) giving a clear indication that higher salt con-
centrations (from 300–500 mM) could augment severe
cytotoxicity. is increasing salinity also creates a menace
in germinating root tips metabolic pool in terms of a de-
crease in total soluble protein concentrations and relative
POX activity (Fig. 7) with higher levels of electrolyte leak-
age from roots (Fig. 8) resulting a possible mitochondrial
poisoning thereby; disrupting the mitochondrial respi-
ration cycle, which in turn decreased root metabolic ac-
tivity (Fig. 9) and possible factor for root cell apoptosis.
ese results are not only interesting but conclusively can
pinpoint that increasing salinity is not only genotoxic but
beyond suboptimal concentrations can disrupt several
biochemical cycles in germinating cells possibly through
membrane damage and mitochondrial deaths.
Many activities, including seed germination, vegetative
growth, and fruit setting, are inhibited by soil salinity be-
cause it lowers the water potential in plants and interferes
with cellular ion homeostasis. Seed germination and veg-
etative growth are just a few of the activities that are in-
hibited by increasing salinity, which also would lower the
water potential thus disrupting cellular ion homeostasis
(Katsuhara and Kawasaki 1996). e cytogenetic response
of cells exposed to 50 up to even 600 mM of NaCl was in-
vestigated in a brief time period from 0 up to 72 h in cereals
(Ogawa et al. 2006; Li et al. 2007; Yumurtaci et al. 2009).
e ndings demonstrated that during a salt shock, cells’
mitotic activity either rapidly declines or is completely halt-
ed, leading to cell death (Yumurtaci 2009; Tabu and Demir
2010; Deinlein et al. 2014). Increasing salt concentrations
had a pronounced eect on seed germination, radical
emergence, and root length growth in Lathyrus sativus L.,
variety Mahatora (Figs 1, 2a, b). In general, the reduction
in root emergence and length with increasing salt concen-
trations conrmed the previous ndings in cereals (West et
al. 2004; Stavridou et al. 2017). Reduced root elongation is
an outcome of altered levels of Abscisic acid, auxin, cytoki-
nin, brassinosteroid, gibberellin, and ethylene, which work
altogether to reduce cell cycle activity as a result of the sup-
pression of plant hormone signaling pathways. ese sub-
stances are thought to be essential for root growth because
they promote cell division, cell expansion and elongation,
and cell dierentiation (Demirkiran et al. 2013; Ryu and
Cho 2015; Majda and Robert 2018; Oh et al. 2020). Reac-
tive oxygen species production and calcium signaling path-
way inhibition cause oxidative damage to nucleic acid bases
that encourage single or double strand breaks in DNA, alter
cytosine methylation, and trigger programmed cell death,
which are all harmful eects of salt (Duan and Wang 1995;
Tuteja and Mahajan 2007; Hossain and Dietz 2016).
Cytological examinations showed a more dramatic
reduction in mitotic activity in the roots of Lathyrus sa-
tivus L. that had received NaCl treatment. e decrease
in mitotic activity under salt stress may be explained by
stopping mitosis in the interphase or lengthening the G2
phase (El – Ghamery et al. 2003; Yildiz et al. 2009; Chatter-
jee and Manjumdar 2010). e investigations that are now
available show that the disruption of cell divisions starts
very early, up to 24 hours aer exposure to chloride salts
(Grant1978; Katsuhara 1997; Atsushi et al. 2006) and that
P
C (2% H
2
O
2
)
N C (DW)
1
00 mM (NaCl)
2
00 mM (NaCl)
3
00 mM NaCl)
4
00 mM (NaCl)
5
00 mM (NaCl)
0.0
0.5
1.0
1.5
2.0
Mean O.D. of TTC ±
S.D. at 490 nm.
a
aa
a
a
a
ANOVA summary
F
P value
P value summary
Significant diff. among means (P < 0.05)?
R square
63.57
<0.0001
****
Yes
0.9646
Figure 9. Histogram showing effect of incresing concentrations of NaCl pertreatment in germinating root tips
(72 hrs) of Lathyrus sativus L., on root mitochondrial (dehydrogenase) activity. The Bars with letter within each
panel are significantly different at P < 0.0001 according to one way ANOVA (Positive control vs respective treat-
ment groups) followed by Dunnet’s multiple comparison tests within treatment groups.
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus12
Innovations in Agriculture
it advances concurrently with the duration of exposure to
mild and moderate stress, as revealed in the current work.
Application of salt resulted in a number of chromosom-
al aberrations at all stages of mitosis. Depending on the
dose, an increase in chromosomal aberrations was seen,
particularly aer treatment with NaCl. e CAs, such as
chromosomal bridges, fractures, and fragments report-
ed here in predominance, may have been brought on by
DNA breaks or suppression of DNA synthesis because
both chromosome fragments and complete chromosomes
cannot be absorbed into the main nucleus during the cell
cycle (West et al. 2004; Leme and Marin-Morales 2009).
CA’s such as chromosome adherence (Fig. 4A–C),
chromosome breaks and loss (Fig. 4E, I, L) and bridges
(Fig. 4A, C, J), were the major group of disturbances ob-
served aer salt treatment in Lathyrus in this present in-
vestigation. e highest frequency of CA’s was observed in
roots exposed to 300 to 400 mM of NaCl. NAs included
cells with double nucleus, chromatin fragmentation, nu-
cleus disintegration (nuclear budding, multilobulated nu-
clei; Fig. 4C, F, I, J), karyorrhexsis (Fig. 4M), karyolysis
(Fig. 4O) and changes in number of nucleoli, Fig. 5B–L).
Usually in interphase nuclei with one or two nucleo-
li were predominant (Fig. 6A); however in roots treated
with 200–400 mM NaCl as many as 3–8 micronucleoli
in squashed root tip cells were also observed (Fig. 5B–L).
Changes in nuclear morphology leaded to MN formation.
MNs were observed in interphase out of chromatin break-
age (Fig. 4H). At 200–300 mM concentration disturbances
in mitotic divisions resulting in binucleated cells were also
observed (Fig. 4C, F). Increase in the salts concentration
up to 400–500 mM signicantly blocked cell divisions
as evident from root tip growth inhibition (Fig. 1A–F),
what explains the low number of observed abnormalities
(Fig. 2a). In control roots and roots from pre-exposed to
100 mM of salts MIs were almost normal, however in-
creased in parallel to the salt concentrations aer 24 hrs
pretreatments which could be prominent aer 72 hrs of
germination under microscopic squashes.
e impact of salinity on cell demonstrated that a de-
crease in cell number and a shorter mature cell length
were responsible for the growth inhibition of Arabidopsis
primary roots under salt stress (Ding et al. 1960; West et
al. 2004). It was emphasized that salt’s ability to suppress
the activities of cyclin dependent kinases (CDKs). By
complexing with the cyclins, CDKs are regulatory pro-
teins that regulate transcription and control cellular divi-
sion in response to stressful circumstances (Bamum and
O’Connell 2014). In reality, checkpoints control how the
cell cycle, which is divided into the G1, S, G2, and M (mi-
tosis) stages, advances. Cell cycle checkpoints regulate the
mitotic spindle to control cell size, ensure accurate rep-
lication, and maintain the integrity of the chromosomes,
preventing cells with damaged or insuciently replicated
DNA from entering mitosis and promoting appropriate
segregation at mitosis (Tan 2010). ese array of clasto-
genic and aneugenic abnormalities (c-mitosis, polyploids
and somatic diads) observed under salinity conditions are
partially explained by disruption in checkpoints, CDKs,
and cyclin activity deciencies with decreased root growth
(Oztur et al. 2002; Utani et al. 2010; Qi and Zhang 2019).
Big and little MNs were the two types (as per nuclear
volume) that were present here. Acentric chromosomal
fragmentation (3a: E, G, and L) may be the root cause of
salt-treated small micornuclei, whereas chromosome loss
(3a: I) may be the root cause of large micronuclei (Leme et
al. 2008; Herrero et al. 2011). A distinguishing trait of NA
is interphasic nucleus morphological alterations (West et
al. 2004). Cells with nuclear buds or lobulated nuclei were
present in these cells as a result of the alterations. e pro-
cess of cell death may be triggered by nuclear anomalies,
which are an indication of DNA fragmentation, according
to various investigations (Katsuhara 1997; Zhu 2002). Cell
death induced on by salt stress was investigated in barley
roots (Demirkiran et al. 2013). Nuclear DNA cleavage was
observed one hour aer the addition of 500 mM NaCl,
and DNA fragmentation was identied eight hours later.
According to this ndings, high salt stress swily encourag-
es DNA deterioration, which causes cell death. Later studies
showed that excessive formation of reactive oxygen species
(ROS), which have a detrimental eect on DNA and cellular
structures, and an imbalance in ion homeostasis are both
responsible for cell death in response to salt stress (Aen-
zeller et al. 2009; Boulon et al. 2010). In this study, germi-
nating roots primed with NaCl concentrations of 400 and
500 mM showed higher frequencies of cell death (Figs 4M,
N, O, 5K–L). ese results suggest that DNA is negatively
aected by increased sodium ion concentrations have a neg-
ative impact on DNA stability and ion homeostasis, which
negatively aects Lathyrus sativus root meristem cells.
A site of ribosome synthesis, the nucleolus is a subnu-
clear structure which appears to be the primary structures
implicated in the activation of cellular stress responses
(Ohbayashi et al. 2018), is the only storehouse of rDNA
containing rRNA cistrons (Butorina and Kalaev 2000).
Stressful events can aect the morphology and function-
ality of the nucleolus in both plant and animal cells. Ac-
cording to reports (Boulon et al. 2010) these alterations are
connected to variations in the organism’s transcriptional
activity. Recent report said that (Mazzeo and Marin-Mo-
rales 2015) nucleolar activities is the most sensitive indi-
cator of cytotoxicity when compared to other tests like
chromosomal aberrations, mitotic index, and micronu-
cleus. e volumetric changes along with the frequency
of nucleoli per nucleus counted could be considered as
measureable cytogentic biomarkers (shapes like dumb-
bell, pear, eye-shaped, chain-like adherences, etc.) that are
most frequently detected in bioassays for the assessment
of the cytogenotoxicity of contaminants. e coordination
of processes for the interaction and modication of RNA
and proteins in proliferating cells may be aected by such
modications. e nucleoli should therefore receive spe-
cic consideration because changes to these structures can
act as strong cytological markers, which can be used as a
key parameter in investigations of environmental mon-
itoring (Lima et al. 2019). In this present investigation
morphological features like nucleolar number, volume and
shape etc., had been taken into consideration in the ger-
Innov. Agric. ⋅ Volume 7 ⋅ 2024 13
Innovations in Agriculture
minating root cells of Lathyrus sativus L., for evaluation of
salt-induced stresses (Figs 5B–L, 6). With increasing con-
centrations there was visible alterations in the nucleolar
number, shape and volume (Kalinina et al. 2018) which
would possibly arising out of adaptation strategies of the
germinating root tip cells of this test plant against increas-
ing ion-mediated ROS accumulation. Although in high-
er plant systems detailed link-up investigations depicting
nucleolar alterations vis-a-vis stress signaling pathways
are rare, but multiple studies could propound that plant
nucleolus has a direct sensing ability to counter stresses
such as increasing salinity through dierential responses
involving dierent biochemical pathways to quench salt
stress (Boulon et al. 2010; Kalinina et al. 2018). Investiga-
tions have revealed that stress can trigger dramatic mor-
phological alterations in plant nucleoli and protein content
in living cells which are direct outcomes of diverse nucle-
olar transcriptions under increasing stress (Yildiz and Aki
2019). Just like animals, plant don’t have p53 transcription
factor for genome stability, but plants have their unique
stress-sensing responses and genome-stability mainte-
nance machinery which are localised in plant nucleolus
(Yildiz and Aki 2019). At 400–500 mM NaCl pretreated
germinating root cells there were the formation of giant
strap cells which were showing translucent cytoplasm and
muti-fragmented micronucleoli within apoptotic/nectrot-
ic cells. Denitely, these numerous micronucleoli arose out
of salinity-driven ROS outburst and ROS-mediated stress
cellular stress which would have disrupted the total pool
of r-DNA cistrons thereby augmenting disruptive protein
synthesis and cellular metabolism. Possibly, here the cells
could not perform any transcriptional activity which was
reective in substantial decrease in soluble protein con-
tent and POX activity (Fig. 7). At the biochemical levels
of stress responses, total soluble protein content and POX
could be considered as natural elicitors within the cell cy-
toplasm (Anuradha and Rao 2001; Yildiz and Aki 2019)
that confer viable and variable protective tools against os-
molyte imbalance (increasing NaCl stress) for the plant
cell to survive. In this investigation NaCl pretreatment at
100, 200 and 300 mM resulted increased levels of soluble
protein with POX activity in germinating root tissues of
Lathyrus sativus L. Interestingly, this increasing salinity
suppressed POX activity, which easily could have also trig-
gered an upsurge of ROS propagation by local as well as
long-distance signalling cascades. is outburst of ROS in
germinating plant roots thus in turn could have stimulated
the plant’s metabolic system to change in such a fashion
that it might be taken into the possible consideration of a
potential signature of “biochemical biomarkers” owing to
disruptive cellular homeostasis and ROS imbalances. is
follows earlier reports (Anuradha and Rao 2001) where in-
creasing salinity root cells were trying to adjust with inher-
ent tolerance to salt. But the decrease in protein content is
due to the eects of sodium chloride on protein synthesis
(Wang et al. 2003), where above suboptimal levels of tol-
erance (here 400–500 mM) could aect protein synthesis
and provoke its decline (Wang et al. 2003). Protein content
in Vignia unguiculata (L) Walp., in comparison to control,
signicantly increased in the stems of plants grown with
100 mM of sodium chloride (Ravelombola et al. 2022). To
survive under stress, plants accumulate proteins that pro-
tect cells from stress eects (Ravelombola et al. 2022).
In earlier studies, increasing salt concentrations were
shown to disrupt membrane leakage caused by membrane
lipid peroxidation, which ultimately resulted in the loss of
cell electrolytes (Demidchik et al. 2019). Electrolyte leak-
age is a characteristic of the stress response in whole plant
cells. is phenomenon is widely used as a test for the
stress-induced damage of plant tissues because it serves as
a “biophysical marker” of plant stress tolerance (Demid-
chik et al. 2019). All primary stress factors, including
heavy metals (Hniličková et al. 2019) and oxidative stress
due to salinity (Demidchik et al. 2003; Demidchik et al.
2019) can cause electrolyte leakage, which aects a wide
range of species, tissues, and cell types. Following the ap-
plication of a stress factor, the electrolyte leakage is almost
immediately noticed and lasts for a few minutes to sever-
al hours. Even while electrolyte leakage has a signicant
physiological impact and a connection to stress tolerance,
the processes underlying this phenomenon are still poorly
understood. Eects of NaCl stress and membrane perme-
ability on Lathyrus sativus L., because it may regulate and
adapt the transport and exchange of intracellular chemi-
cals, as the root cell membrane is selective. It is the initial
site of stress injury at the cellular level. e plant cell is
most immediately harmed by ROS injury from increasing
NaCl pre-treatment in germination root tips through dis-
ruption of cell membrane structure and function, increase
in membrane permeability, decrease in membrane stabil-
ity, and enhancement of passive leaking of ion cells and
macromolecules. As result, enhanced membrane penetra-
bilitya biophysical indicator of cytotoxicity” is the clearest
sign that cell membranes have been damaged. Lathyrus
sativus L. roots that were germinating aer pre-treatment
with varying concentrations of NaCl, in this experimental
setup showed an almost dose-dependent increase in elec-
trolyte leakage (Fig. 8), which is consistent with the recent
ndings (Hniličková et al. 2019).
Another adverse eect of salinity is the build-up of salts
in the root apoplast, which can disrupt cellular water con-
nections and hinder growth as well as lead to wilting and
cell death. Later, sodium data were presented to support
this concept (Flowers et al. 1991). However, as NaCl salin-
ity also causes the apoplast to accumulate Cl- ions, which
act as an osmoticum (Shahzad et al. 2013), high Cl- con-
centrations in the apoplast may harm cells (Geilfus et al.
2018). In an in vitro investigation on barley, Yamashita et
al. (1994) discovered that when the roots are stressed with
NaCl, PM vesicles of the roots increase their permeability
to Cl- such membrane leakage may result from sodium’s
displacement of calcium, which on the other hand stabi-
lizes the membrane (De Costa et al. 2007). Furthermore,
under NaCl salinity, the thickness of membranes may
be impacted; although Cl- ions are anticipated to be ex-
posed to the aqueous phase because sodium is assumed
to be connected to the carbonyl and phosphate oxygen of
the membranes’ lipid head groups. According to dierent
Adhikari, et al.: Evaluation of salinity-mediated cytogenotoxicity in Lathyrus sativus14
Innovations in Agriculture
reports (Cordomi et al. 2008; Klasczyk et al. 2010), this
localization of the two ions creates a dipole moment to
the lipid head groups that is opposite from what polarized
water generally creates under non-stressed situations.
ough hypothetical, it’s possible that a thickening of the
PM under Cl- salinity could result in decreased metabolic
activity, which will inhibit growth. Changes in the cell’s
surface area to volume ratio have been observed to have
an impact on the electrostatic potential of the PM, which
has also been reported to be impacted by salt induced
changes (Klasczyk et al. 2010).
A higher diusion of oxygen from the roots is indicat-
ed by increased root oxidizability (RO), mostly to combat
the harmful substances nearby the site of action. When
TTC salt is used to detect RO, electrons from the mito-
chondrial transport are actually absorbed. In other words,
improved RO also signals increased ROS production. e
current investigation demonstrated that roots at higher
concentrations (500 mM NaCl; Fig. 8) had less root ox-
idizability as measured by TTC-reduction. e rate of
root respiration was found to have noticeably decreased,
and the root oxidizability indicates that mitochondrial
poisoning is likely what caused the cellular death (apop-
tosis) (Figs 3, 4M–O, 9). e greater quantity of NaCl
modied cellular stress in germination seeds via ROS
formation, according to a decline in root oxidizability.
According to Ghosh et al. (2023) when ROS damage or
rupture the root cell membrane, a signicant number of
intracellular ions and organic compounds leak out, which
causes physiological metabolic disturbance. It is obvious
that NaCl stress has an impact on the biochemical and
physiological features of Lathyrus roots. Lathyrus root
mitochondrial activity considerably increased from 100
to 300 mM NaCl pretreatments (in comparison to nega-
tive contol, 2% H2O2; a peroxyl radical genarator), with a
distinct downward trend in 400 and 500 mM treatments.
Additionally, we can deduce that exposure to 400 and
500 mM concentrations of NaCl caused deeper injury
vis-à-vis ROS generation augmenting severe membrane
and mitochondrial damage to newly sprouted roots that
would have hastened the physiological metabolism dis-
order resulting root cell death, a direct outcome of dis-
ruption in membrane architecture (owing to electrolyte
leaking). (Figs 8, 9). Reports already opined that, in re-
action to Cl- salinity, the pea (Pisum sativum) root’s mi-
tochondrial respiration had been specically inhibited,
and a switch from the glycolysis pathway to the pentose
phosphate pathway was evident (Hason-Porath and Pol-
jako-Mayber 1970). In wheat mitochondria under NaCl
stress (Jacoby et al. 2015), it was found that electron trans-
port routes were blocked and respiratory kinetics was dis-
rupted. is stress was also accompanied by an increase
in the amount of ROS produced by the mitochondria per
unit of oxygen consumed. However, in several plants the
benecial eects of salt stress enhance “epigenome” and
brings about epigenomic modications too (Adhikari and
Das 2023) that in long run could enhance salt mediated
stress adaptations in climate resilient crops. Till today the
bulk of research related to various regimes of salt treat-
ments are only restricted to monocots (Shan et al. 2024)
and species of Allium and very few dicot crops including
woody cash crops have been subjected to salinity induced
changes with detailed genotoxic proling studies (Dar-
wish et al. 2023). erefore, commercial cash crops need
to be brought into the process of elaborate investigative
bio-assays in greater numbers, to garner newer insights
into this complex process of salinity-induced ROS genera-
tion. Similarly, the ameliorative roles of dierent chemical
and phytoconstituents should be taken into more broader
applications to enhance the plants’ own internal resistance
against salinity-induced physiological stress which might
alter the drastic genomic loss or gross cytogenetic changes
(Omar et al. 2023; Tabur et al. 2023).
is climate resilience crop has a tremendous opportu-
nity to come out as the “future wonder crop” based upon
its readiness to get adopted to several ecological and an-
thropogenic stresses and these ndings might be insight-
ful to improve this crop with the help of state-of-the-art
biotechnological applications as a “wonder crop” with a
rich source of soluble proteins for readymade consump-
tion for domestic and human populations. So optimum
salt tolerance levels could be established primarily utiliz-
ing cytological bioassays during seed germination. How-
ever, it is not conclusive as the establishment of further
molecular categorization is needed to detect end-point
suboptimal salt tolerance mechanisms in crop plants.
Conclusion
e salt tolerance mechanism in plants is a very compli-
cated and unexplored area of plant breeding in consider-
ation of pulse crops, especially legume crops. is whole
experimental study for the rst time conclusively proved
that salt priming above the suboptimal level (100–200
mM) triggered severe cytogenotoxic responses in germi-
nating root tips of Lathyrus sativus L., variety Mahatora
from genomic (chromosomal and nucleolar) standpoint.
ere was a signicant increase in root metabolic activity
(dehydrogenase enzyme activity in root mitochondria),
increased production of soluble protein (as a biochemical
tool for osmolyte balance against stress) and increased
POX activity as an elicitor against ROS stress owing to
salt priming (100–300 mM). ese are important ndings
for the rst time in this pulse crop that might highlight
its resilience capacity against increasing salinity stress
and in vivo ROS outburst during the process of germina-
tion. According to this study, rising sodium and chloride
ions in the root cells combined with a severe cytological
and biochemical stress in the cellular microcosm had a
negative impact on the growth of Lathyrus sativus. is
work specically illustrates a wide range of biochemical
and cellular toxicity in roots that would eectively catch
scientists’ interest and motivate them to look into all po-
tential molecular pathways of salt tolerance in other key
types of pulse crops in the near future. is report can
demonstrate that, apart from conventional model plants
i.e., Vicia faba and Allium cepa L., which are the two most
Innov. Agric. ⋅ Volume 7 ⋅ 2024 15
Innovations in Agriculture
widely adopted plant-based assay systems worldwide,
Lathyrus sativus L., root tip cytogenetic biomarkers can
stand out rightly to elaborate all promising outcomes,
supporting it as an easy-to-handle, alternate, vis-à-vis a
cost-eective bioassay model for all plant scientists.
Competing Interests
e authors have no relevant nancial or non-nancial
interests to disclose.
Author’s contributions
All authors contributed to the study’s conception and de-
sign. Material preparation, data collection and analysis of
results were performed by [Dr. Dipan Adhikari], and [Mr.
Rahul Ghosh] and the work was performed manually by
Mr. Sagar Dig (P.G., Research Student) in the laboratory.
e rst dra of the manuscript was written by [Dr. Di-
pan Adhikari] and all authors commented on previous
versions of the manuscript. All authors read and approved
the nal manuscript
Availability of Data
All materials are reported in the text, and all the data col-
lected are reported in the manuscript.
Acknowledgements
e authors extend their heartfelt acknowledgement to
the corresponding mother departments i.e., the UG and
PG department of Botany Hooghly Mohsin College for
extending major support in the form of physical space,
and all other necessary instrumental facilities for car-
rying out the research work. e Authors sincerely and
gratefully acknowledge the nancial support accorded
by University Grants Commission (UGC Minor Re-
search Project No- F. PSW– 088/10 – 11(ERO)), Govt
of India. e authors convey their heartfelt thanks to
Dr. Chandrashekhar Chatterjee, Scientist, Oce of the
seed testing ocer, State Seed Testing Laboratory, Govt
of West Bengal, District Agriculture Farm, Kalna Road,
Burdawan 713101, for providing the certied varieties
of grass pea seeds, Lathyrus sativus variety Mahatora to
conduct this research program.
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