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Allelopathy Journal 31 (1): 71-90 (2013) International Allelopathy Foundation 2013
Tables: 3, Figs : 7

Allelopathic effects of Lantana camara L. on Lathyrus

sativus L.: Oxidative imbalance and
cytogenetic consequences
DIBYENDU TALUKDAR

Department of Botany, R.P.M. College,

University of Calcutta, Uttarpara, Hooghly, 712258, West Bengal, India
E. Mail: dibyendutalukdar9@gmail.com

(Received in revised form: December 11, 2012)

ABSTRACT
Allelopathic effects of Lantana camara L. leaf aqueous extracts were
determined on Lathyrus sativus L. in Petri plate bioassay and pot experiments by
studying growth parameters, antioxidant defence responses and cytological anomalies
observed in root-tip mitosis and flower bud (anther) meiosis. Germination and dry
weight of the legume were reduced significantly in dose-dependent way. Activities of
superoxide dismutase and catalase increased, but these of ascorbate peroxidase
declined at higher doses (200-300 mg/mL). Significant loss of ascorbate redox pool
and decrease in ascorbate peroxidase activity in treated plants seriously impeded the
scavenging of hydrogen peroxide, leading to its over-accumulation and concomitant
rise in lipid peroxidation and membrane damage. At cytogenetic level, decrease in
mitotic activity was indicated by low mitotic index, abnormal transition of divisional
phases and even, complete halt in division. The chromotoxic effect of leaf extracts
was exhibited by diverse types of chromosomal abnormalities, (stickiness and
micronuclei formation in both divisions), while some phenomena like c-mitosis,
anomalous nucleolous, unequal/late separation were found stage specific. This study
strongly indicated the allelopathic potential of Lantana leaf extracts, particularly at
200 mg/mL and 300 mg/mL doses, on induction of oxidative stress and cytotoxicity in
L. sativus L.

Key words: Allelopathic effect, antioxidant defence, cytotoxicity, hydrogen peroxide,

micronuclei, oxidative stress, Lantana camara L, Lathyrus sativus L.

INTRODUCTION

Allelopathy, the inhibition of growth in one species of plants by chemicals

produced by another species, represents a form of chemical warfare between plants
competing for limited light, water, and nutrient resources (3,5). This biological
phenomenon has been considered one of the most potent weapons for the successful
invasion of many exotic plants (29). Lantana camara L. (family Verbenaceae) is a
common exotic invasive shrub in India (36) and is one of the 100-worst invasive plants in
the world (44). Its allelopathic effects were attributed to the presence of volatile essential
oils, phenolic compounds and triterpenoids in leaves (3,36), which suppresses the
germination, growth and development of recipient target species (14). Despite the
72 Talukdar

ecological and agronomic importance of allelopathy (29,31,48), relatively little is known

concerning the intrinsic biochemical defence mechanism adopted by plants in response to
allelopathic effect (5).
The allelochemicals interfere with several physiological processes including
antioxidant defence mechanism in receptor organism and can cause oxidative stress in
target plants by generating excess reactive oxygen species (ROS) (16,24,79,80). The mode
of antioxidant defence response to allelochemicals in these studies was shown by changes
in the hydrogen peroxide (H2O2)-scavenging mechanisms with membrane lipid
peroxidation and subsequent damage caused by it (5,7,17,46,79). Among the ROS-
scavenging antioxidant enzyme molecules, superoxide dismutase (SOD) constitutes the
first line of defence, but the enzymes converts one ROS (free radicals) to another (H2O2)
through dismutation (50). In contrast, both ascorbate peroxidase (APX) and catalase
(CAT) play pivotal role in scavenging the H2O2. While APX exclusively requires reduced
ascorbate as its co-factor to function normally, no reducing power is required for CAT to
decompose H2O2 (50). The H2O2 level is correlated with membrane lipid peroxidation and
subsequent membrane damage and hence, these three parameters are considered as
indication of oxidative stress in plants (6,70).
Cytogenetic analysis of plant chromosomes is widely used to monitor genotoxic
and/or chromotoxic effects of different toxic chemicals on living cell. Changes in the
mitotic activity in mitotic phase and individual cell aberrations of both mitotic and meiotic
stages are the key parameters to determine the cytotoxic effect of allelopathy on recipient
plant. The plant extracts causes chromosomal abnormalities due to allelotoxicity
(37,71,72) but the effects on legume chromosomes are rarely studied (28). Furthermore,
simultaneous responses of both mitotic and meiotic activity of recipient plant to single
plant extract are not known (27), it may provide more detailed cytological data about the
allelopathic effects of extracts on the first exposed organ (roots) than flowers of recipient
plant.
Legumes are major component of cropping systems due to their biological
nitrogen fixation capacity (49). but the decline in area of legumes and inhibition of their
nitrogen fixation due to allelopathic effects of weeds is major cause of concern
(24,49,52,77). Lathyrus sativus L. (Grass pea), an annual dual-purpose legume crop (food
and forage) is tolerant to biotic and abiotic stresses (65,66,68). It is an excellent
cytogenetic material to develop desirable mutants and tester stocks for trait mapping (62-
64). The responses of its genome to radiation and metal stress are chromosomal structural
and numerical aberrations (63,64) along with alterations in transcriptomes and antioxidant
defence responses (12,65), but the response of its antioxidant defence and cytogenetic
parameters in relation to allelopathy remains obscure. The crop was selected due to its
high seed germination, seedling growth, easy chromosome preparation and rapid screening
of karyological abnormalities hence, ideal for screening of allelopathic effects of leaves of
Lantana on it as recipient crop. This study aimed to study the effects of Lantana camara
L. leaf extracts on (i). Germination and seedling growth, (ii). Response of 3-prominent
H2O2-metabolizing enzymes-SOD, APX and CAT, (iii). Level of H2O2, MDA
(malondialdehyde) and membrane electrolyte leakage and (iv). Chromosome behavior of
root tip mitosis and flower bud meiosis in Lathyrus sativus L.
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 73

MATERIALS AND METHODS

Leaves of Lantana camara L. plants were collected at early flowering stage from
the fields of Kalyani (22°59′N/ 88°29′E, Gangetic plain, clay-alluvial), West Bengal, India.
Collected samples were carefully cleaned and fully dried in shade for 96 h, ground and
stored at room temperature. The extracts were prepared by soaking 30 g crushed mass in
sterilized distilled water for 24 h at room temperature (24-28 °C), filtered through
Whatman No. 1 filter paper, made final volume to 100 mL (300 mg/mL w/v). The extract
was considered as stock solution (kept at 5 °C until used). The solution was further diluted
with distilled water to get test concentrations of 50, 100 and 200 mg/mL. The protocol and
concentration of extracts were standardized by preliminary experiments on survival of
Lathyrus sativus plants.

Petri Plate Lab bioassay

The L. sativus variety ‘BioL-212’ contains very low (< 0.1%) seed neurotoxin (9)
hence, selected for this study. Its 10 uniform sized seeds were surface sterilized with 10%
(v/v) sodium hypochlorite for 3 min and kept for germination in sterilized Petri dishes (9-
cm dia) lined with double blotting paper and moistened with 10 ml aqueous extracts (50,
100, 200 and 300 mg/mL) as per treatments. The treatments were replicated four times in
completely randomized design and distilled water served as control. Petri dishes were kept
in growth chamber (Day/night 20 °C /27 °C, photon-flux density of 200 µmol m-2 sec-1 and
14 h photoperiod humidity : 80%). Germination (%) was recorded at 7-days after sowing.

Pot Culture
The farm land soil (sandy-loam, pH 7.2) was passed through 2-mm sieve to
remove non-soil particles and filled (at 6.5 kg per pot) in earthen pots of 30 cm dia, then
irrigated with 100 mL leaf extracts of different concentrations as per treatment. The soil
irrigated with distilled water served as control. Fresh and healthy seeds of grass pea
(Lathyrus sativus L.) cv. ‘BioL-212’ were surface sterilized by 10% (v/v) sodium
hypochlorite for 3 min and allowed to germinate in conditions as stated above. Seven days
old germinated seedlings were transplanted in pots. Transferred seedlings were thinned to
one per pot after 10-days and the pots were kept under control conditions (temperature
day/night 20 °C /27 °C, photon-flux density of 200 µmol m-2 sec-1 and 14 h photoperiod,
humidity) 70%, for 30 d (onset of flowering) in randomized block design with 4-
replications. The pots were irrigated with test solutions on alternate days and with tap
water on other days.
The lengths of both root and shoot (cm plant-1) were measured at harvest (30 d
old plants). Dry weight (g plant-1) of shoot and root were recorded after oven drying at 60
°C for 48 h. Leaf injury level was ascertained with a scale of 0-4 standardized for grass pea
(66,68) as under: 0: No injury (without any damage), 1: Mild injury, indicated by small
area (approximately1/5) of leaflet apex and margin turning brownish yellow, 2: Moderate
injury, indicated by ½ of the leaflet turning whitish-yellow, 3: Severe injury, when over
80% of total leaflet area turned whitish yellow and very thin and 4: Extreme injury, when
leaflets became necrotic, crinkled, and finally fell off. Leaflets, borne on first formed
primary branches, were used for visual scorings.
74 Talukdar

Antioxidant enzyme activity assay

The mature fully expanded leaves (grown on primary branches) were collected
from control and treated seedlings (pot culture) at harvest (30 d old plant) for biochemical
studies. A pool of samples from six plants for each replication was collected and six
independent experiments were done. All operations were performed at 0-4 °C, except
mentioned otherwise. Leaf samples (1g) were homogenized in an extraction medium [50
mM K-phosphate buffer pH 7.8, 0.1 mM EDTA, 2 mM cysteine, 1% w/v PVP and 0.2%
v/v Triton X-100].

(i). SOD (EC 1.15.1.1) activity was determined by nitro-blue tetrazolium (NBT)
photochemical assay method as per Beyer and Fridovich (8). The reaction mixture (3
ml) contains 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 0.1
mM EDTA, 2µM riboflavin and 0.1 ml enzyme extract. One unit of SOD was defined
as the amount of protein causing a 50% NBT photoreduction.
(ii). For APX (EC1.11.1.11) activity, 20 mM ascorbate was added to the extraction buffer.
The extracts were filtered through two layers of cheesecloth, and the homogenate was
centrifuged at 14000 g for 20 min, at 4 °C. The supernatant fraction was filtered
through a column containing 1 mL of Sephadex G-50 equilibrated with the same
buffer used in homogenization. The hydrogen peroxide-dependent oxidation of
ascorbate was followed by a decrease in the absorbance at 290 nm with an extinction
constant of 2.8 mM-1 cm-1 (47).
(iii). For CAT (EC 1.11.1.6) activity, extraction was done in 50 mM K-phosphate buffer
(pH 7.0) and 0.5% PVP-10, and enzyme activity was assayed by measuring the
reduction of H2O2 at 240 nm (ε = 39.4 M-1 cm-1) for 1 min (1).
(iv). Reduced and oxidized forms of ascorbate were measured from leaves as per methods
of Law et al. (41).

V. Lipid peroxidation, H2O2 levels and electrolyte leakage

Lipid peroxidation was determined by measuring the malondialdehyde (MDA)
content at 532 nm with extinction coefficient of 155 mM-1 cm-1 (57). Hydrogen peroxide
(H2O2) content of leaves was measured as per method of Cheeseman (13). Tissue samples
were homogenized in the extraction medium 0.1 M K-phosphate (pH 6.4) supplemented
with 5 mM KCN. The assay mixture contained 250 µM ferrous ammonium sulphate, 100
µM sorbitol, 100 µM xylenol orange and 1% ethanol in 25 mM H2SO4. Changes in
absorbance were determined by the difference in absorbance between 550 nm and 800 nm
and H2O2 contents were calculated from standard curve. Membrane electrolyte leakage
(EL) was assayed by measuring the ions leaching from tissues into deionised water. It was
expressed as EL (%) = (EC1) / (EC2) ×100, where EC1 : Initial electrical conductivity and
EC2 : Final electrical conductivity (18).

Cytogenetic assay of toxicity

Mitotic activity and abnormalities

For cytogenetic assay of allelotoxicity induced by Lantana camara at mitosis,
seeds of grass pea plants were sterilized and placed in similar conditions as described
above. However, for mitotic assay the seeds were first germinated in Petri dishes lined
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 75

with two sheets of germination paper pre-moistened with 5 mL of distilled water, and
seedlings were transferred to Petri dishes (moistened with 10 mL aqueous extracts of
various concentrations (50 to 300 mg/ML) for 6, 12 and 18 h. At the end of each exposure
period, root tips (2-3 mm) were cut and washed thoroughly with distilled water. After
washing, the root-tips were fixed overnight in a freshly prepared mixture of absolute
ethanol and propionic acid (2:1 v/v) as per method of Talukdar (67,70).
To determine the mitotic index, mitotic phases and the presence of chromosomal
aberrations, root tips were hydrolyzed with 1(N) HCl for 10-12 min and stained in 2%
aceto-orcein (Hi-media) and kept for 45 min, and subsequently squashed in a drop of 45%
acetic acid. Treatments were replicated 4-times and scoring was determined from 5 roots
of each replicate. The mitotic index was calculated by dividing cells among the examined
total cells; at least 4000 cells (10 slides) were analyzed per treatment. The frequency of
each mitotic phase was calculated as the percentage in relation to dividing cells counted in
mitosis, whereas chromosomal abnormalities were calculated as the percentage in relation
to examined total cells. The chromosomal abnormalities observed in root-tip mitosis are
presented with photo-micrographs (× 1450, Olympus HB, equipped with a zoom digital
camera, Panasonic DMC-FH3).

Meiotic abnormalities
For meiotic studies of chromotoxic effect, suitable size young flower buds were
collected at 9-9.30 AM from 30 d old treated and control L. sativus L. plants and fixed
immediately in propionic-alcohol (1:2) at room temperature for 12 h and then preserved in
70% alcohol (60,70). Anthers were smeared taking one anther per slide in 1% propiono-
carmine solution and chromosomal anomalies were recorded from randomly chosen 200
flower buds/treatment. Pollen sterility was studied by staining anthers with 2% aceto-
carmine solution and considering fully stained as fertile and half-stained or unstained as
sterile grains. The results from the treated plants were compared with control plants. The
chromosomal abnormalities observed are presented with photo-micrographs.

Micronuclei (MN) study

For the analysis of MN, around 5000 cells were scored to calculate frequency as
per the criteria set for its analysis (75) with little modification for L. sativus L. MN should
be: (i). minimum one-third the diameter of main nucleus, (ii). on same plane focus, (iii)
oval or round-shaped and (iv) clearly separated from the main nucleus.

Statistical analysis
The data are means of 4-independent experiments. Two way (concentrations and
duration) analyses of variance were performed to determine significant differences (P <
0.05) among treatments for different parameters, using software STATISTICA 6.0
(StatSoft, Inc. Tulsa, U.S.A). Significance (P < 0.05) of differences between control and
each treatment was determined by student t-test (two-tailed) using Microsoft Excel tool
‘data analysis’ 2007. Simple correlation (n=10) between antioxidant enzyme activities and
dry weight as well as mitotic index was also carried out.
76 Talukdar

RESULTS AND DISCUSSION

Germination and seedling growth

The effect of Lantana camara L. leaf aqueous extracts (50-300 mg/mL) on seed
germination and seedling growth of Lathyrus sativus L. was studied in Petri plate and pot
bioassays methods, respectively. Germination was 99.5% in control, but decreased below
50% at 100 mg/mL concentration and there was no germination at 300 mg/mL. This
indicated toxic level of germinability of grass pea seeds at this concentration, which was in
accordance with previous studies (39,40). Compared to control, both shoot and root
lengths reduced to 57% and 79% at 100 mg/ mL and 200 mg/ mL, respectively, whereas
at 300 mg/ mL, shoot and root length were decreased by 89% and 93% respectively (Fig.
1). The dry weight of both shoot and root in Lathyrus seedlings also decreased with
increasing concentrations, exhibiting nearly 2-folds reduction at 200 mg/ mL for shoot and
at 100 mg/ mL for root (Fig. 1). Decreasing root length was accompanied with reduction in
lateral branches and conspicuous absence of nodulation at 200 mg/ mL and 300 mg/ mL
concentrations in treated plants (Fig. 2). No leaf injury was visible in control plants, but
was marked (level 2) at 100 mg/ mL and elevated to level 4 at 200 mg/ mL. Seedling
growth showed highest inhibition at 300 mg/ mL aqueous extract, but the root length and
its dry weight were reduced drastically than shoot (Fig. 1). These results agree with earlier
studies reporting that water extracts of allelopathic plants had more pronounced effects on
root growth than on shoot (7,25,58,59). It is likely that roots are the first organ to absorb
the allelochemicals from soil environment (73). In Pot Culture, apart from shoot growth
inhibition, Lantana leaf extract caused leaf injury in recipient grass pea seedlings and its
increase was dose-dependent, confirming easily identifiable marker of oxidative stress
(66,68) induced by allelopathy in grass pea. Lantana leaf extracts inhibited the seed
germination in Petri plate assay and growth of Lathyrus seedlings in pot culture, showing
the inhibitory potential of extract in both with soil and without soil conditions. Lantana
extract reduces the seedling growth of mung bean (59) due to allelopathic effect.
Lantana leaf extract at 200 mg/mL completely prevented nodulation on roots of
grass pea (Fig. 2), completely stopping its nitrogen-fixation ability. Ramanujam et al., (55)
reported reduction in nodule number and size in Vigna radiata plants treated with aqueous
leaf extract of tree species, Aglaia elaegnoidea, while Kharkwal et al., (35) observed both
inhibitory and stimulatory effect of allelopathy on nodule formation in different legumes.
Complete absence of nodulation in grass pea plants at 200 mg/ mL and 300 mg/ mL doses
showed the inhibitory effects of Lantana leaf extracts at higher concentrations on nodule
formation of L. sativus L.

Hydrogen peroxide metabolizing enzymes, lipid peroxidation and membrane leakage

Six biochemical parameters were studied to investigate the oxidative stress
responses of 30 d old Lathyrus seedlings to Lantana leaf extracts. Activities of SOD, APX
and CAT (Prominent H2O2-metabolizing antioxidant defence enzymes), were significantly
(P < 0.05) changed by the leaf extracts (Fig. 3). SOD activity increased significantly (1.5-
folds) at 100 mg/mL, but greatly increased (2.8-3.0-folds) at 200 mg/mL and 300 mg/mL
extract concentrations. APX activity enhanced markedly (1.6-folds) at 100 mg/mL extract,
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 77

Figure 1. Allelopathic effects of aqueous extracts of Lantana camara leaves on germination, root
length (RL), shoot length (SL), root dry weight (RDW) and shoot dry weight (SDW) of
Lathyrus sativus L. plants.

Figure 2. Allelopathic effects of Lantana camara leaf aqueous extracts on root length and nodulation
of 30-d-old Lathyrus sativus seedlings at (a) control, (b) 50 mg/mL, (c) and (d) 100
mg/mL, (e) 200 mg/mL and (f) 300 mg/mL extract concentrations.
Note: Nodulation decreased at 100 mg/mL, but no nodule formed at 200 and 300 mg/mL
extract conc. Root length decreased at higher concentrations. Arrow (→) indicates
nodulation, Scale = 12 cm
78 Talukdar

but then it declined sharply lower than control (Fig. 3). Significant increase in CAT
activity was observed at 100 mg/mL and the highest increase (10.6-folds) was at 200
mg/mL extract with a slight decline at 300 mg/mL (Fig. 3). Enzyme activity was strongly
correlated with both shoot and root dry weight from 100 mg/mL onwards. SOD and CAT
activity exhibited significant (P < 0.05, n=10) negative associations (r= -0.776- -0.837),
while activity of APX showed negative association (r= -0.689 for shoot and -0.653 for
root) with dry weights at 100 mg/mL. Significant positive correlation (r = 0.827-0.908)
between APX activity and dry weight was observed at 200 mg/mL and 300 mg/mL extract
concentrations.
Enzyme activity

Concentration (mg/mL)

Figure 3. Allelopathic effects of aqueous extracts of Lantana camara leaves on activities of

superoxide dismutase (SOD, U mg-1 protein), ascorbate peroxidase (APX, nmol min-1
mg-1 protein) and catalase (CAT, nmol min-1 mg-1 protein) in leaves of 30-d-old
Lathyrus sativus L. plants. Error bar represents standard error (n =6). Asterisk (*)
denotes Significant (P < 0.05) differences from control (0 mg/mL extract).

H2O2 accumulation increased from 100 mg/mL aqueous extract. Highest increase
(3.8-folds) was at 200 mg/mL and it remained unchanged at 300 mg/mL (Fig. 4), showing
a significant (P < 0.05, n=10) negative correlation (r = -0.677— -0.715) with APX
activity. Similar trend was noticed for MDA content and membrane electrolyte leakage in
leaves of treated seedlings (Fig. 4).
Reduced ascorbate (AsA) content in leaves of treated Lathyrus sativus L was
decreased steadily with increasing concentrations, the reduction was 1.6-folds, 6-folds and
6.2 folds over control at 100, 200 and 300 mg/mL extract concentrations, respectively
(Fig. 5). The dehydroascorbate or DHA (oxidized form of ascorbate), however, increased
concomitantly (Fig. 5). AsA redox, calculated as AsA/(AsA+DHA), was 0.95 at control
and 50 mg/mL treatment, reduced to 0.59 at 100 mg/mL and decreased further to 0.15 at
200 and 300 mg/mL treatment concentrations (data not shown).
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 79
Content

Concentration (mg/mL)

Figure 4. Allelopathic effects of aqueous extracts of Lantana camara leaves on hydrogen peroxide
(H2O2, µmol g-1 FW), malondealdehyde (MDA, nmol g-1 FW) and membrane electrolyte
leakage (EL%) in leaves of 30-d-old Lathyrus sativus L. seedlings. Error bar represents ±
Standard Error (n = 6). Asterisk (*) denotes significant (P < 0.05) differences with control
(0 mg/mL) treatment.
Content (nmol g-1 FW)

Concentration (mg/mL)

Figure 5. Allelopathic effects of aqueous extracts of Lantana camara leaves on reduced ascorbate
(AsA) and dehydro ascorbate (DHA) content in leaves of 30-d-old Lathyrus sativus. Error
bar represents ± standard error (n = 6). Asterisk (*) denotes the Significant (P < 0.05)
differences from control (0 mg/mL) treatment.
80 Talukdar

Lantana leaf extracts significantly increased the activities of SOD, APX (up to
100 mg/mL) and CAT in treated leaves of Lathyrus sativus L. strongly indicated
generation of ROS and alteration of antioxidant enzyme activities. However, their
responses were not similar to other recipient plants (15,45,53,79,80). As SOD is a
catalytic converter of superoxide radicals to H2O2, its enhanced activity level from 200
mg/mL extract, might lead to excess formation of H2O2 in treated cell. By contrast, APX
level was much higher than SOD at low concentrations, but the sudden fall in its activity
even below control level from 200 mg/mL extract concentration might be responsible for
steep rise in H2O2 level from this concentration onwards. Although, CAT activity reached
its highest peak at higher concentrations, it was not enough to cope with rising level of
H2O2. This indicated crippling of H2O2-scavenging machinery in treated seedlings and
failure of CAT-mediated compensatory defence mechanism to decompose H2O2, leading
to its over-accumulation in Lathyrus leaves. The importance of APX in H2O2-scavenging
and concomitant plant growth was evident from strong positive correlation of APX activity
with plant dry weight but negative association with H2O2, especially at 200 and 300
mg/mL treatments. The ineffective scavenging of H2O2 resulted in increasing level of
MDA, a cytotoxic aldehyde and a product of lipid peroxidation and concomitant increase
in electrolyte leakage in Lathyrus leaves. There is good correlation between H2O2 and
MDA levels and their enhanced level as the indication of oxidative stress on the structural
and functional integrity of biological membrane in plants (6,10,16,23,34,55,66,69,79).
Obviously, changes in antioxidant enzyme activities, over-accumulation of oxidative
species, lipid peroxidation and increased membrane leakage are strong indications of
biochemical effects of allelopathy exerted by Lantana leaf extracts on vital cellular
processes of present Lathyrus sativus plant. It was also revealed that biological membrane
is a potential target of allelopathy-induced ROS, although the mechanism of action of
different allelochemicals on it may be different (76).

Foliar AsA content and its redox pool

A search to identify the possible reasons for drop of APX level at 200 and 300
mg/mL concentrations revealed significant (P<0.05) deficiency of its co-factor, the
reduced ascorbate (AsA), in treated Lathyrus leaves. AsA serves pivotal role as cellular
antioxidants during metabolic processes (4). APX activity became subnormal in response
to rising H2O2 level coupled with AsA-deficiency (30). Low AsA level and rise of its
oxidized form, the dehydroascorbate (DHA) at higher concentrations in the present study
suggested increased consumption of AsA to meet the growing challenges of oxidative
stress at elevated doses, unbalancing redox (AsA/AsA+DHA) pool of AsA heavily in
favor of oxidative state in leaves of the treated seedlings. The depletion of AsA pool
seriously impeded normal functioning of APX and consequently resulted in onset of
oxidative stress in the recipient plant.

Mitotic index, transition of mitotic phases and cell division abnormalities

Significant (P < 0.05) differences in mitotic index (MI) values were observed
between control and Lantana leaf extract-treated Lathyrus sativus L. root in respect of both
concentrations and durations of treatment (Table 1). A concentration and duration-
dependent decrease in MI value was observed for 12 h and 18 h and the reduction became
statistically significant (P < 0.05) at 100-300 mg/mL extract treatments (Table 1). MI
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 81

values significantly (P < 0.05) and positively correlated (r = 0.774-0.818) with the three
enzyme activities at 50 mg/mL, but exhibited strong negative (r= -0.728 — -0.767)
relations at 100 mg/mL dose onwards. However, it showed a strong positive association
with APX at 200 (r = 0.688) and 300 mg/mL (r = 0.772) extract concentrations. For
transition of mitotic phases in treated plants, there was a marked increase (1.2-1.4-folds
across the treatments) in percentage of prophase cells between 100-300 mg/mL doses for 6
h and 12 h and of 50-300 mg/mL for 18 h treatment (Table 1). The increase in prophase
index was accompanied with reduction of other divisional phases, especially of metaphase.
Compared with control, it declined by about 2-fold at 6 h and 1.9-4.6-fold at 12 h, and the
maximal decline occurred at 300 mg/mL (18 h) where all the dividing cells were only in
prophase phase (Table 1). The increasing amount of cells in prophase resulted from total
inhibition of dividing cells to enter into the subsequent stages, indicating a complete
stoppage of cell cycle due to mito-toxic effect of leaf extract.

Table 1. Effects of Lantana camara L. leaf aqueous extracts on mitotic index (MI) and mitotic
phases of L. sativus L. for different concentrations and periods of treatment

Treatment Extract Total Cells in (MI) Mitotic phase index (%)

Duration conc. cells division mean (- Prophase Metaphase Anaphase-
(h) (mg/mL) scored /+ % of Telophase
control)
6 Control 4250 646 15.20 68.11 20.93 10.96
50 4510 599 13.28 74.57 16.37 9.06
100 5035 831 16.50 80.88* 10.40* 8.72
200 4672 582 12.47 79.33* 10.51* 10.16
300 4700 549 11.69 82.61* 10.88* 6.51*
CD at 5 % - - 2.23 7.17 5.01 2.65
12 Control 4380 631 14.40 70.50 19.67 9.83
50 4445 591 13.30 73.79 13.28* 12.93
100 5110 438 8.58 85.70* 6.65* 7.65
200 4800 318 6.63 87.15* 6.70* 6.15*
300 4190 130 3.10 94.00* 4.26* 1.74*
CD at 5 % - - 2.04 5.34 4.33 3.19
18 Control 5100 755 14.80 70.20 18.95 10.85
50 4435 477 10.75 82.69* 10.30* 7.01
100 4900 407 8.30 93.38* 4.88* 1.74*
200 4820 288 6.00 98.58* 1.42* 0.00
300 4500 115 2.55 100.00* 0.00 0.00
CD at 5 % - - 4.67 5.47 1.89 4.04
CD at 5 % (Duration) - - 2.22 4.14 2.19 1.77
Data represented are from four replicates per treatment, *Significantly different from respective
control treatment at P <0.05 by t-test, parameters varied significantly among treatments (ANOVA)
with CD at 5%.

The Lantana leaf extracts also induced a variety of mitotic abnormalities such as
clastogenic agent in root-tip cells of Lathyrus sativus L (Table 2). At metaphase,
chromosome stickiness and breaks, spindle disturbances and C-mitosis occurred at varying
frequencies (Figs. 6a-e), while at anaphase bridge formation (with or without fragments),
82 Talukdar

lagging, diagonal orientation, and multipolarity was observed (Figs. 6 f-i). Micronuclei
formed at telophase and even extended to next interphase (Figs. 6 j-k). The persistence of
micronuclei after division might be due to the time required for its degradation or they
might be resulted from lost fragments in anaphase or were extorted from the interphase
nucleus in diminution-like manner (11,72). Decrease in mitotic activities indicated
mitodepressive effect of Lantana leaf extract, while chromosomal abnormalities revealed
its chromotoxic potential on Lathyrus chromosomes in agreement with reported
clastogenic potentials of different plant extract on maize (73), Vicia faba (27,56) and other
plants (32,71).

Figure 6. Mitotic chromosome abnormalities of L. sativus root-meristem cells induced by Lantana camara
L. leaf aqueous extracts (a) Normal 2n=14 metaphase chromosomes at control (0 mg/mL), (b)
Sticky metaphase, (c) Chromosome breakage, (d) Spindle disturbances with irregular
chromosome arrangement, (e) c-mitosis, (f) anaphase bridge with lagging chromosome (bold
arrow) and a fragment (thin arrow), (g) sticky Anaphase with double bridge, (h) Diagonal
orientation at anaphase, (i) Multipolar configuration and micronuclei (MN) formation
(representative arrow) at (j) Telophase and in (k) Interphase. Scale bar 1SD = 10 µm.
Allelopathic effect of Lantana camara L. on Lathyrus sativus 83
84 Talukdar
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 85

Figure 7. Meiotic chromosomal aberrations of L. sativus L. flower bud microsporocytes induced by

Lantana camara L. leaf aqueous extracts. (a) Normal seven bivalents (7II, 2n=14) at metaphase
I at control (0 mg/mL) treatment, (b) Stickiness at metaphase I, (c) Two univalents (→) along
with six bivalents at metaphase I, (d) One quadrivalent ring (→) and five bivalents at metaphase
I, (e) Normal 7-7 separation at anaphase I at control, (f) Bridge formation at anaphase I, (g)
Multiple bridge (thin arrow) with lagging chromosome (bold arrow), (h) Unequal 8-6 separation
at anaphase I, (i) Late separation of chromosomes (→) at anaphase I, (j) Multipolar orientation,
(k) Tendency of non-disjunction towards pole, (l) Unoriented anaphase chromosomes and (m)
Micronuclei (MN) formation (representative →) at telophase II. Scale bar 1SD = 10 µm.

Meiotic chromosome associations

The chromotoxic effect of Lantana leaf extract was also found in flower bud
meiosis in 30 d old Lathyrus sativus seedlings in the form of chromosome stickiness (Fig.
7a, b), formation of univalent and multivalent (Figs. 7c,d), bridge formation (Figs. 7f, g),
laggard (Fig. 7 g), unequal separation (Fig. 7h), late separation towards pole (Fig. 7i),
multipolar anaphase (Fig. 7j), tendency of non-disjunction (Fig. 7 k), unoriented anaphase
chromosome in scattered (Fig. 7 l) and micronuclei formation at telophase II at varying
frequencies (Table 3; Fig. 7m). Chromosome stickiness has been considered as
chromosome agglutinations displaying a sticky appearance, and usually being irreversible
in nature, it is the most lethal type of cytotoxic effect in plants (21, 51). Anomalous
nucleolus (increasing nucleolus/cell from usual one to two in association with NOR
chromosome and bulging out of nucleolar boundary at one side in hypoploid cells) was
observed only at 300 mg/mL during diakinesis (Fig. 8a, b), representing role of nucleolus
in perception of diverse cellular stresses in plants (11,38,54,60,72). The observation of
86 Talukdar

spindle disturbances in late prophase, C-mitosis, non-oriented chromosomes, anaphase

bridge, lagging chromosomes, multipolar configurations, unequal separation, and diagonal
orientation reflected the toxic effect of Lantana extract on spindle formation of Lathyrus
cell divisions, as also reported in other plants including Lathyrus (60,64,70,71). Compared
with control, percentage of abnormal cells increased significantly at 100 mg/mL onwards,
but the higher effect was observed at 12 h and 18 h durations with significant differences
for concentrations and duration of treatments (Table 3). Pollen sterility was normal
(1.53%) at lower concentration, but it rose significantly at 100 mg/mL (31-fold)-300
mg/mL (66-fold) treated plants (data not in table) mainly due to abnormal disjunction at
meiotic anaphase I. The role of plant extracts as chromotoxic agents in induction of both
mitotic and meiotic chromosomal abnormalities has been observed in different plant
species treated with extracts (27, 28, 37, 71).

Figure 8. Anomalous nucleolar behaviour with hypoploidy in flower bud meiosis of 300
mg/mL extract-treated Lathyrus sativus L. plants at diakinesis; (a) Double
nucleoli (→) with 6 bivalents, two of which was associated with nucleolus as
NOR chromosomes and (b) Bulging out (→) of nucleolar material in hypoploid
(6 bivalents) cells. Scale bar 1SD = 10 µm.

Formation of micronuclei (MN) in mitotic telophase, interphase and meiotic

telophase II is another important event encountered in Lantana extract treated Lathyrus
root-tip and flower bud cell divisions, respectively (Figs. 6j, k, 7m). High MN frequency
was scored at 200 and 300 mg/mL treatments at 6 h and 12 h durations, and also at 18 h
treatment with 100 mg/mL at mitosis and 100-300 mg/mL for meiosis. MN results from
chromosome fragments or whole chromosome lagging during cell division (19, 64), and
was considered as an important biomarker for ascertaining chromotoxic/genotoxic damage
of plants (22, 43, 64, 75). Abnormal increase in univalent frequency at metaphase I cells
was one of the possible reasons for rise in laggard/fragments which failed in polar
movement at anaphase and remained within cell as MN. Remarkably, MN frequency in the
present Lathyrus sativus plant was nearly similar in both mitotic and meiotic cell divisions
in respect of treatment concentrations and durations, the specificity of which can certainly
be used as a cytogenetic parameter in assay of allelopathic effect in plants in line with MN
test for biomonitoring of ecotoxicological and environmental pollution.

CONCLUSIONS

Leaf aqueous extract of Lantana camara L. was inhibitory to both germination

and seedling growth of Lathyrus sativus L. plants. The inhibition in the target legume plant
was also manifested in induction of oxidative stress due to excessive accumulation of H2O2
and rise in lipid peroxidation level, resulting in oxidative imbalance and high magnitude of
 Allelopathic effect of Lantana camara L. on Lathyrus sativus 87

membrane electrolyte leakage. Obviously, the rise of SOD, APX and CAT was in response
of ROS accumulation, but the redox state of reduced ascorbate and its effect on APX
functioning holds the main key in ROS-scavenging. At cytogenetic level, the inhibitory
effect of Lantana leaf extract was exhibited by diverse types of chromosomal anomalies
encountered in both root-tip mitosis and flower bud meiosis of Lathyrus sativus. The
severity of the effect on roots was evidenced by low mitotic index and abnormal transition
of divisional phases with complete halt of division at higher treatment concentrations and
durations. It is worth noting that both mitosis and meiosis witnessed some common
anomalies such as MN formation and chromosome stickiness, whereas there were certain
abnormalities which found specific to either mitosis or meiosis only. Notable among these
are chromosomal breaks and C-mitosis at mitotic metaphase and double nucleoli and
unequal separation at meiosis I. Obviously, these karyomorphological abnormalities along
with alterations of H2O2-metabolism, AsA redox state and consequent oxidative imbalance
may be used as potential biomarkers of allelopathic effect of Lantana camara leaf extract
on Lathyrus sativus L., as suggested in other plants (16, 32, 79). The present
findings/parameters (morphological, biochemical and cytogenetic) can also be utilized to
assess the allelopathic effect of invasive weeds on other legume crops especially those
closely related with Lathyrus such as, Pisum, Lens, Phaseolus, Vicia, Cicer in a more
comprehensive way. Considering all the parameters it is concluded that 50 mg/mL
aqueous leaf extract of Lantana was not toxic, while 200 mg/mL and 300 mg/mL extract
concentrations were proved to be potent inhibitory or allelotoxic to Lathyrus sativus plants
at different stages of growth.

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