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Cytological Changes in Trigonella

Foenum-graecum (l.) under the Cadmium Stress.

Kailash P. Patel and Kalpesh M. Patel Veer Narmad South Gujarat University, Surat, Gujarat, India- 395007.

Email: {kailash.patel, incredibleever}@yahoo.com

Abstract—The experiment was carried out to the study of

cytological change in Trigonella foenum-graecum under the

cadmium stress. Different kind of chromosomal

abnormalities like chromosome fragment, ring chromosome,

micronuclei, vagrant chromosome, C-metaphase etc. were

observed during cytological studies. The mitotic index was

frequently reduced and percentage of aberrant cells increase

due to the concentrations of cadmium was increase and

finally plant growth reduced.

Index Terms—Abnormalities, cadmium, chromosome,

mitotic index, micronuclei.

I. INTRODUCTION

Cadmium (Cd) is released into the environment by

human activities such as phosphate fertilizers, disposal of

household, municipal and industrial wastes. These sources

may cause enhanced soil and hence crop cadmium levels,

which may lead to increases in dietary cadmium exposure.

Cadmium is a particularly dangerous pollutant due to its

high toxicity and great solubility in water [1] and [2].

Exposures to high cadmium concentration have been

found to be carcinogenic, mutagenic and teratogenic for a

large number of animal species [3]. Characteristically

inhibits root growth and cell division in plants such as

onion, tradescantia, Vicia faba [4], Nicotiana tabacum [5]

and Allium sativum [6]. Numerous experimental studies

have shown the genotoxicity of cadmium salts [5] and [7].

The purpose of the present investigation was to evaluate

the influence of cadmium concentrations on somatic cells

of Trigonella foenum-graecum, since the most pronounced

effect of heavy metals on plant development is growth

inhibition, which is inseparably connected with cell

division.

Fenugreek (Trigonella foenum-graecum L.) is

undoubtedly one of the oldest cultivated medicinal plants.

It is an erect annual herb native to Southern Europe and

Asia, belonging to the family Leguminosae. Over 80% of

the total world's production of this seed is contributed by

India, one of the major producers and exporters of

fenugreek legume in the world. Fenugreek seeds can be a

good supplement to cereals because of its high protein

(25%), lysine (5.7 g / 16 g N), soluble (20%) and insoluble

Manuscript received October 16, 2012; revised Jan 28, 2013.

(28%) dietary fiber besides being rich in calcium, iron and

beta-carotene [8].

The climate of India being arid to semiarid necessitates

artificial irrigation for successful agriculture. There is very

less canal water available, which does not fulfill the need

for rising crops. Therefore, farmers near the cities use

industrial effluents for raising crops. Most of the sewage

wastewater contains heavy metal ions like Chromium,

Cadmium, Nickel, Mercury and Iron. Their concentrations

are variable in time and space. Therefore, soil near the

factories and cities are being contaminated the regular use

of effluent water for raising crops.

Keeping in view the above mentioned aspects of

industrial set up in India and its impact in creating stressful

environments for agriculture through industrial effluents

and the importance of Trigonella foenum-graecum as

nutritionally important crop, experiments were conducted

to evaluate the effects of cadmium toxicity on this crop.

II. MATERIALS AND METHODS

The germination was carried out in petri dishes. Seeds

were surface sterilized with H2O2 for the prevention of

surface fungal/bacterial contamination. Different ppm

solutions were prepared in pure distilled water in

laboratory by using Cd (NO3)2 (cadmium nitrate) pure

distilled water was used as control for the study. Ten seeds

were placed on cotton in each petri dish and 40 ml solution

of each concentration was supplied once for seed

germination. Distilled water was applied every alternate

day after this treatment. The petri dishes were monitored

daily for fungal and other inspections. The cytological

study was carried out control, 1ppm, 3ppm and 5ppm of

cadmium concentrations.

The staining procedure followed was that of [9]. The

staining with aceto orcine as well as 1:1 aceto orcine and

aceto carmine was found suitable for present study.

Mitotic index was calculated by observing the slides of

root tip of control and treated plants at 3rd, 5th and 7th days

of growth. Total 100 cells were observed on slide. The cell

in the stage of prophase, metaphase, anaphase and

telophase were counted. The mitotic index and percentage

of aberrant cells were calculated using following

formulae;

.

% 100.

No of dividing cellsMitotic index

Total no of cells studies (1)

10

Journal of Life Sciences and Technologies Vol. 1, No. 1, March 2013

©2013 Engineering and Technology Publishing doi: 10.12720/jolst.1.1.10-13

.100

.

No of aberrant cellsPercentage of aberrant cells

No of dividing cells (2)

Photomicrographs were taken on Carl-Zesis

photomicroscope with planophotochromatic objectives

using Kodak 100 ASA-35mm color film. Daylight, yellow

or green filter were used.

III. RESULTS

Trigonella foenum-graecum has 2n=16 number of

chromosomes. When the roots of Trigonella

foenum-graecum were treated with different concentration

of cadmium, the cells exhibited various abnormalities

showing its toxic effect on mitotic divisions. A number of

cytological abnormalities viz., chromosome fragments

(Fig.1), micronuclei (Fig. 1), vagrant chromosome (Fig. 1)

and ring chromosome etc. observed in the treated plants.

There was ‗stickiness of chromosomes‘ in the metaphase

some cells also seen with temporary chromosome bridge.

Difference and decline of mitotic index, compared to

controls were noticed in the cells of roots of treated plants.

The mitotic index and percentage of aberrant cells in

control and cadmium treated plants on 3rd, 5th and 7th day

of growth in main roots as well as in side roots (Table I,

Table II and Table III). At 3rd day mitotic index was

reduced in all treatment, compared to control. The

maximum reduction was observed in 5 ppm. At 7th day it

was almost nil in 1 ppm and 3 ppm treatment. The

percentage of aberrant cells was increased with increase in

the cadmium concentration.

Figure 1. Different kind of chromosomal abnormalities.

TABLE I. EFFECTS OF CADMIUM ON ROOT TIP CELLS OF TRIGONELLA FOENUM-GRAECUM ON 3RD DAY OF GROWTH.

Concentration of cadmium

Control 1 ppm 3 ppm 5 ppm

Main root Side root Main root Side root Main root Side root Main root Side root

Mitotic index

(%)

28.66

2.16 ND

15.33

1.25 ND

10.66

0.86 ND

8.33

0.56 ND

Aberrant

cell (%)

4.64

0.46 ND

26.09

2.18 ND

40.61

3.26 ND

48.02

4.14 ND

TABLE II. EFFECTS OF CADMIUM ON ROOT TIP CELLS OF TRIGONELLA FOENUM-GRAECUM ON 5TH DAY OF GROWTH.




Mitotic index

(%) 27.33 1.48 ND 8.66 0.32 ND 4.33 0.28 ND NS ND

Aberrant cell

(%) 4.86 0.60 ND 46.18 3.29 ND 61.43 4.43 ND NS ND

11


TABLE III. EFFECTS OF CADMIUM ON ROOT TIP CELLS OF TRIGONELLA FOENUM-GARECUM ON 7TH DAY OF GROWTH.




Mitotic index (%) 25.66 1.33 21.66 1.12 7.66 0.36 15.66 1.06 2.33 0.18 10.33 0.83 NS ND

Aberrant

cell (%) 2.57 0.42 1.52 0.28 52.21 4.56 27.65 2.68 57.08 4.88 35.43 3.59 NS ND

All the values are means S.D.

ND- Not developed during study period.

NS- Not studied due to bursting and decay.

IV. DISCUSSION

From these investigations it was revealed that cadmium

was mitodepressive and induced various types of

chromosomal aberrations in root tip cells of Trigonella

foenum-graecum. Different concentration of cadmium

solution induced different types of chromosomal

aberrations at various stages of cell division. Ring

formation, C-metaphase, vagrant chromosome,

micronuclei and chromosomal fragment were common

aberrations during cells division. Mitotic index was

decreased with the increase in concentration of cadmium.

Effect of heavy metal on chromosomal aberration and

mitotic index was earlier studies by [10]-[16]. Reduction

in mitotic index by the treatment of copper chloride on the

root tips cells of Helianthus annus was reported by [17].

Presence of more than one nucleus in proliferating

following heavy metal treatment is a structural

abnormality [18]. In the present work the micronuclei

were observed in Trigonella foenum-graecum due to the

treatment of cadmium. These micronuclei arose from

acentric chromatid or chromosome fragments induced by

heavy metal cadmium. Evans et al. [19] studied the

production of micronuclei after irradiating the Vicia faba

roots. According to them all chromatid breaks,

chromosome breaks, isochromatid breaks, asymmetrical

exchanges and incomplete symmetrical exchanges would

give rise to acentric fragments at mitosis. At the later stage

of mitosis these fragments are frequently excluded from

the two daughter nuclei and in the following interphase

they appear as micronuclei in one or both the daughter

cells. The micronucleus test has been suggested by [20] as

an in vitro cytogenetic screening procedure for the

detection of freshly induced structural chromosomal

aberrations and for revealing chromosome loss due to

partial impairment of spindle apparatus. He had suggested

that micronuclei originate from chromatin lagging at

anaphase. In the course of telophase this material included

into one or the other daughter cell where it can either fuse

with the other daughter cell where it can either fuse with

the main nucleus or from one or several secondary nuclei.

Another interesting feature noticed after treatment with

cadmium was strong C-mitotic effect in the investigated

plants. The normal mitotic stages were modified in to

C-metaphase due to inactivation of spindle followed by

random scattering of chromosomes. Ring formation was

very rarely reported abnormalities in these studies. Younis

et al. [21] reported it in Vicia faba by the treatment of

nuvacron.

In Allium cepa vagrant was also noticed due to the

treatment of an herbicide avenoxan by [22]. The

abnormalities vagrant was results of impaired spindle

function [18]. Laggards may produce micronuclei if they

fail to reach the poles in time to be included in the main

nucleus [23].

In conclusion, as has been stated above, cadmium has

harmful effects on the root tip cells of Trigonella

foenum-graecum. In addition to these findings, the

increase in soil and water pollution can lead to certain

irreversible cytogenetic effects in plants and even in

higher organisms and reduced growth.

ACKNOWLEDGEMENT

We are acknowledges the University Grants

Commission, New Delhi, for granting minor research

project [F.No. 32-425-2006(SR)].

REFERENCES

12

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13

Dr. Kailash P. Patel was born in 1974 has done

his M.Sc. (Botany) at VNSG University, Gujarat,

India in 1997. She completed his B.Ed (Science

–Math) from same university in 1998. She also got

Ph.D. in 2004 from VNSG University, Gujarat,

India.

Presently she is working as a Assistant

Professor in Botany department of VNSG

University, Gujarat, India. She has published

about 12 research papers in various field like plant science and

environmental science, also she has published on book titled ―Effect of

Cd & Zn on seed germination and growth of Cajanus cajan & Trigonella

foenum and their removal by Various method‖ (LAP Lambert Academic

Publishing GmbH & Co. KG, Germany, 2011). Also 3 research students

have completed their M.Phil and 1 research student has completed his

Ph. D under her guidance.

Dr. Kalpesh Patel was born in 1983 has done his

M.Sc. (Botany) at VNSG University, Gujarat,

India in 2005. He completed his M. Phil from same

university in 2009. He also got Ph.D. in 2012 from

VNSG University, Gujarat, India.

Presently he is working as a Lecturer

In Botany department of VNSG University,

Gujarat, India. He has published about 6 research

papers in various field like plant science and

environmental science, he is also a co-author of a book titled ―Effect of

Cd & Zn on seed germination and growth of Cajanus cajan & Trigonella

foenum and their removal by Various method‖ (LAP Lambert Academic

Publishing GmbH & Co. KG, Germany, 2011)

Author‘s formal

photo

Author‘s formal

photo


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